Erythrocytes of uranium miners: the activity of the pentose phosphate pathway

PubMed Central

Vích, Z.; Novosad, F.; Brychtová, V.

1970-01-01

Vích, Z., Novosad, F., and Brychtová, V. (1970).Brit. J. industr. Med.,27, 287-290. Erythrocytes of uranium miners: the activity of the pentose phosphate pathway. The functioning of erythrocytes was studied by determination of the activity of the pentose phosphate pathway in 431 individuals - 221 uranium miners, 42 employees of a uranium ore trimming station (30 of whom were exposed), 36 former uranium miners, 32 coal miners, and 100 persons not working in mines and with no previous exposure. In the groups exposed to long-term occupational radiation, the activity of the pentose phosphate cycle was found to be enhanced. This finding was interpreted as evidence for a change in the functional state of the erythrocytes in exposed persons due to the effects of radiation on the genesis of red cells in the bone marrow. PMID:5448126

An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver.

PubMed

Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

2016-09-02

Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The pentose phosphate pathway and cancer

PubMed Central

Patra, Krushna C.; Hay, Nissim

2015-01-01

The pentose phosphate pathway (PPP), which branches from glycolysis at the first committed step of glucose metabolism, is required for the synthesis of ribonucleotides and is a major source of NADPH. NADPH is required for and consumed during fatty acid synthesis and the scavenging of reactive oxygen species. Therefore, the PPP plays a pivotal role in helping glycolytic cancer cells to meet their anabolic demands and combat oxidative stress. Recently, several neoplastic lesions were shown to have evolved to facilitate the flux of glucose into the pentose phosphate pathway. This review summarizes the fundamental functions of the PPP, its regulation in cancer cells, and its importance in cancer cell metabolism and survival. PMID:25037503

Regulation of Mammary Tumor Formation and Lipid Biosynthesis by Spot14

DTIC Science & Technology

2014-06-01

consistent with these terms, showing enrichment in glycolysis/ gluconeogenesis , the pentose phosphate pathway, and the cell cycle in PyMT/S14-/- versus PyMT...Enrichment Glycolysis / Gluconeogenesis 0.0018 6.66 Pentose phosphate pathway 0.0045 11.62 Cell cycle 0.0254 3.54 Starch and sucrose metabolism 0.0799

Coutilization of D-Glucose, D-Xylose, and L-Arabinose in Saccharomyces cerevisiae by Coexpressing the Metabolic Pathways and Evolutionary Engineering

PubMed Central

Zhao, Jianzhi; Qiu, Chenxi; Wang, Shihao; Du, Binghai

2017-01-01

Efficient and cost-effective fuel ethanol production from lignocellulosic materials requires simultaneous cofermentation of all hydrolyzed sugars, mainly including D-glucose, D-xylose, and L-arabinose. Saccharomyces cerevisiae is a traditional D-glucose fermenting strain and could utilize D-xylose and L-arabinose after introducing the initial metabolic pathways. The efficiency and simultaneous coutilization of the two pentoses and D-glucose for ethanol production in S. cerevisiae still need to be optimized. Previously, we constructed an L-arabinose-utilizing S. cerevisiae BSW3AP. In this study, we further introduced the XI and XR-XDH metabolic pathways of D-xylose into BSW3AP to obtain D-glucose, D-xylose, and L-arabinose cofermenting strain. Benefits of evolutionary engineering: the resulting strain BSW4XA3 displayed a simultaneous coutilization of D-xylose and L-arabinose with similar consumption rates, and the D-glucose metabolic capacity was not decreased. After 120 h of fermentation on mixed D-glucose, D-xylose, and L-arabinose, BSW4XA3 consumed 24% more amounts of pentoses and the ethanol yield of mixed sugars was increased by 30% than that of BSW3AP. The resulting strain BSW4XA3 was a useful chassis for further enhancing the coutilization efficiency of mixed sugars for bioethanol production. PMID:28459063

Draft Genome Sequence of an Anaerobic and Extremophilic Bacterium, Caldanaerobacter yonseiensis, Isolated from a Geothermal Hot Stream

PubMed Central

Lee, Sang-Jae; Lee, Yong-Jik; Park, Gun-Seok; Kim, Byoung-Chan; Lee, Sang Jun; Shin, Jae-Ho

2013-01-01

Caldanaerobacter yonseiensis is a strictly anaerobic, thermophilic, spore-forming bacterium, which was isolated from a geothermal hot stream in Indonesia. This bacterium utilizes xylose and produces a variety of proteases. Here, we report the draft genome sequence of C. yonseiensis, which reveals insights into the pentose phosphate pathway and protein degradation metabolism in thermophilic microorganisms. PMID:24201201

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum.

PubMed

Gopinath, Vipin; Meiswinkel, Tobias M; Wendisch, Volker F; Nampoothiri, K Madhavan

2011-12-01

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the L-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more L-glutamate and L-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM L-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM L-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae.

PubMed

Madhavan, Anjali; Srivastava, Aradhana; Kondo, Akihiko; Bisaria, Virendra S

2012-03-01

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

Understanding D-Ribose and Mitochondrial Function.

PubMed

Mahoney, Diane E; Hiebert, John B; Thimmesch, Amanda; Pierce, John T; Vacek, James L; Clancy, Richard L; Sauer, Andrew J; Pierce, Janet D

2018-01-01

Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production. With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

Ethanol production from renewable resources.

PubMed

Gong, C S; Cao, N J; Du, J; Tsao, G T

1999-01-01

Vast amounts of renewable biomass are available for conversion to liquid fuel, ethanol. In order to convert biomass to ethanol, the efficient utilization of both cellulose-derived and hemicellulose-derived carbohydrates is essential. Six-carbon sugars are readily utilized for this purpose. Pentoses, on the other hand, are more difficult to convert. Several metabolic factors limit the efficient utilization of pentoses (xylose and arabinose). Recent developments in the improvement of microbial cultures provide the versatility of conversion of both hexoses and pentoses to ethanol more efficiently. In addition, novel bioprocess technologies offer a promising prospective for the efficient conversion of biomass and recovery of ethanol.

Carbohydrate and energy-yielding metabolism in non-conventional yeasts.

PubMed

Flores, C L; Rodríguez, C; Petit, T; Gancedo, C

2000-10-01

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses.

Continuous production of ethanol from hexoses and pentoses using immobilized mixed cultures of Escherichia coli strains

PubMed Central

Unrean, Pornkamol; Srienc, Friedrich

2010-01-01

We have developed highly efficient ethanologenic E. coli strains that selectively consume pentoses and/or hexoses. Mixed cultures of these strains can be used to selectively adjust the sugar utilization kinetics in ethanol fermentations. Based on the kinetics of sugar utilization, we have designed and implemented an immobilized cell system for the optimized continuous conversion of sugars into ethanol. The results confirm that immobilized mixed cultures support a simultaneous conversion of hexoses and pentoses into ethanol at high yield and at a faster rate than immobilized homogenous cells. Continuous ethanol production has been maintained for several weeks at high productivity with near complete sugar utilization. The control of sugar utilization using immobilized mixed cultures can be adapted to any composition of hexoses and pentoses by adjusting the strain distribution of immobilized cells. The approach, therefore, holds promise for ethanol fermentation from lignocellulosic hydrolysates where the feedstock varies in sugar composition. PMID:20699108

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells.

PubMed

Sun, Y; Gu, X; Zhang, E; Park, M-A; Pereira, A M; Wang, S; Morrison, T; Li, C; Blenis, J; Gerbaudo, V H; Henske, E P; Yu, J J

2014-05-15

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. (18)F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms.

Methods for the determination of intracellular levels of ribose phosphates.

PubMed

Camici, Marcella; Tozzi, Maria Grazia; Ipata, Piero Luigi

2006-10-31

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.

Untargeted Metabolic Profiling of Winery-Derived Biomass Waste Degradation by Penicillium chrysogenum.

PubMed

Karpe, Avinash V; Beale, David J; Godhani, Nainesh B; Morrison, Paul D; Harding, Ian H; Palombo, Enzo A

2015-12-16

Winery-derived biomass waste was degraded by Penicillium chrysogenum under solid state fermentation over 8 days in a (2)H2O-supplemented medium. Multivariate statistical analysis of the gas chromatography-mass spectrometry (GC-MS) data resulted in the identification of 94 significant metabolites, within 28 different metabolic pathways. The majority of biomass sugars were utilized by day 4 to yield products such as sugars, fatty acids, isoprenoids, and amino acids. The fungus was observed to metabolize xylose to xylitol, an intermediate of ethanol production. However, enzyme inhibition and autolysis were observed from day 6, indicating 5 days as the optimal time for fermentation. P. chrysogenum displayed metabolism of pentoses (to alcohols) and degraded tannins and lignins, properties that are lacking in other biomass-degrading ascomycetes. Rapid fermentation (3-5 days) may not only increase the pentose metabolizing efficiency but also increase the yield of medicinally important metabolites, such as syringate.

Pentose phosphates in nucleoside interconversion and catabolism.

PubMed

Tozzi, Maria G; Camici, Marcella; Mascia, Laura; Sgarrella, Francesco; Ipata, Piero L

2006-03-01

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.

Cross-regulation among arabinose, xylose and rhamnose utilization systems in E. coli.

PubMed

Choudhury, D; Saini, S

2018-02-01

Bacteria frequently encounter multiple sugars in their natural surroundings. While the dynamics of utilization of glucose-containing sugar mixtures have been well investigated, there are few reports addressing regulation of utilization of glucose-free mixtures particularly pentoses. These sugars comprise a considerable fraction in hemicellulose which can be converted by suitable biocatalysts to biofuels and other value-added products. Hence, understanding of transcriptional cross-regulation among different pentose sugar utilization systems is essential for successful development of industrial strains. In this work, we study mixed-sugar utilization with respect to three secondary carbon sources - arabinose, xylose and rhamnose at single-cell resolution in Escherichia coli. Our results reveal that hierarchical utilization among these systems is not strict but rather can be eliminated or reversed by altering the relative ratios of the preferred and nonpreferred sugars. Since transcriptional cross-regulation among pentose sugar systems operates through competitive binding of noncognate sugar-regulator complex, altering sugar concentrations is thought to eliminate nonspecific binding by affecting concentration of the regulator - sugar complexes. Plant biomass comprises of hexose and pentose sugar mixtures. These sugars are processed by micro-organisms to form products like biofuels, polymers etc. One of the major challenges with mixed-sugar processing by micro-organisms is hierarchical utilization of sugars due to cross-regulation among sugar systems. In this work, we discuss cross-regulation among three secondary carbon sources - arabinose, xylose and rhamnose. Our results show that cross-regulation between pentose sugars is complex with multiple layers of regulation. These aspects need to be addressed for effective design of processes to extract energy from biomass. © 2017 The Society for Applied Microbiology.

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells

PubMed Central

Sun, Y; Gu, X; Zhang, E; Park, M-A; Pereira, A M; Wang, S; Morrison, T; Li, C; Blenis, J; Gerbaudo, V H; Henske, E P; Yu, J J

2014-01-01

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. 18F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms. PMID:24832603

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

1998-03-10

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

Pentose fermentation by recombinant Zymomonas

DOEpatents

Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

1998-01-27

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

Pentose fermentation by recombinant zymomonas

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

1998-01-01

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

1998-01-01

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Metabolic changes associated with tumor metastasis, part 1: tumor pH, glycolysis and the pentose phosphate pathway.

PubMed

Payen, Valéry L; Porporato, Paolo E; Baselet, Bjorn; Sonveaux, Pierre

2016-04-01

Metabolic adaptations are intimately associated with changes in cell behavior. Cancers are characterized by a high metabolic plasticity resulting from mutations and the selection of metabolic phenotypes conferring growth and invasive advantages. While metabolic plasticity allows cancer cells to cope with various microenvironmental situations that can be encountered in a primary tumor, there is increasing evidence that metabolism is also a major driver of cancer metastasis. Rather than a general switch promoting metastasis as a whole, a succession of metabolic adaptations is more likely needed to promote different steps of the metastatic process. This review addresses the contribution of pH, glycolysis and the pentose phosphate pathway, and a companion paper summarizes current knowledge regarding the contribution of mitochondria, lipids and amino acid metabolism. Extracellular acidification, intracellular alkalinization, the glycolytic enzyme phosphoglucose isomerase acting as an autocrine cytokine, lactate and the pentose phosphate pathway are emerging as important factors controlling cancer metastasis.

The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes.

PubMed

Bradfield, Michael F A; Nicol, Willie

2016-11-01

Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady state. The results of the assays were coupled to a kinetic model that revealed an increase in oxidative pentose phosphate pathway (OPPP) flux relative to total substrate flux with increasing SA titre, for both substrates. Furthermore, applying metabolite concentration data to metabolic flux models that include the OPPP revealed similar flux relationships to those observed in the experimental kinetic analysis. A relative increase in OPPP flux produces additional reduction power that enables increased flux through the reductive branch of the TCA cycle, leading to increased SA yields, reduced by-product formation and complete closure of the overall redox balance.

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

DOE PAGES

Khosravi, Claire; Battaglia, Evy; Kun, Roland S.; ...

2018-03-22

Background: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involvedmore » in regulating preferential use of different carbon catabolic pathways. Results: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Conclusion: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.« less

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khosravi, Claire; Battaglia, Evy; Kun, Roland S.

Background: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involvedmore » in regulating preferential use of different carbon catabolic pathways. Results: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Conclusion: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.« less

Glucose Transporters in Cardiac Metabolism and Hypertrophy

PubMed Central

Shao, Dan; Tian, Rong

2016-01-01

The heart is adapted to utilize all classes of substrates to meet the high-energy demand, and it tightly regulates its substrate utilization in response to environmental changes. Although fatty acids are known as the predominant fuel for the adult heart at resting stage, the heart switches its substrate preference toward glucose during stress conditions such as ischemia and pathological hypertrophy. Notably, increasing evidence suggests that the loss of metabolic flexibility associated with increased reliance on glucose utilization contribute to the development of cardiac dysfunction. The changes in glucose metabolism in hypertrophied hearts include altered glucose transport and increased glycolysis. Despite the role of glucose as an energy source, changes in other nonenergy producing pathways related to glucose metabolism, such as hexosamine biosynthetic pathway and pentose phosphate pathway, are also observed in the diseased hearts. This article summarizes the current knowledge regarding the regulation of glucose transporter expression and translocation in the heart during physiological and pathological conditions. It also discusses the signaling mechanisms governing glucose uptake in cardiomyocytes, as well as the changes of cardiac glucose metabolism under disease conditions. PMID:26756635

Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli.

PubMed

Zhu, Xinna; Tan, Zaigao; Xu, Hongtao; Chen, Jing; Tang, Jinlei; Zhang, Xueli

2014-07-01

Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Understanding alternative fluxes/effluxes through comparative metabolic pathway analysis of phylum actinobacteria using a simplified approach.

PubMed

Verma, Mansi; Lal, Devi; Saxena, Anjali; Anand, Shailly; Kaur, Jasvinder; Kaur, Jaspreet; Lal, Rup

2013-12-01

Actinobacteria are known for their diverse metabolism and physiology. Some are dreadful human pathogens whereas some constitute the natural flora for human gut. Therefore, the understanding of metabolic pathways is a key feature for targeting the pathogenic bacteria without disturbing the symbiotic ones. A big challenge faced today is multiple drug resistance by Mycobacterium and other pathogens that utilize alternative fluxes/effluxes. With the availability of genome sequence, it is now feasible to conduct the comparative in silico analysis. Here we present a simplified approach to compare metabolic pathways so that the species specific enzyme may be traced and engineered for future therapeutics. The analyses of four key carbohydrate metabolic pathways, i.e., glycolysis, pyruvate metabolism, tri carboxylic acid cycle and pentose phosphate pathway suggest the presence of alternative fluxes. It was found that the upper pathway of glycolysis was highly variable in the actinobacterial genomes whereas lower glycolytic pathway was highly conserved. Likewise, pentose phosphate pathway was well conserved in contradiction to TCA cycle, which was found to be incomplete in majority of actinobacteria. The clustering based on presence and absence of genes of these metabolic pathways clearly revealed that members of different genera shared identical pathways and, therefore, provided an easy method to identify the metabolic similarities/differences between pathogenic and symbiotic organisms. The analyses could identify isoenzymes and some key enzymes that were found to be missing in some pathogenic actinobacteria. The present work defines a simple approach to explore the effluxes in four metabolic pathways within the phylum actinobacteria. The analysis clearly reflects that actinobacteria exhibit diverse routes for metabolizing substrates. The pathway comparison can help in finding the enzymes that can be used as drug targets for pathogens without effecting symbiotic organisms within the same host. This may help to prevail over the multiple drug resistance, for designing broad spectrum drugs, in food industries and other clinical research areas. © 2013.

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

PubMed

Larrabee, M G

1990-11-15

A classic equation that has frequently been used to estimate the fraction of glucose metabolized by the pentose phosphate pathway, using 14CO2 data, is more simply re-derived with careful consideration of the assumptions involved and the conditions under which it is applicable. The equation is shown to be unreliable for non-homogeneous tissues, depending on the fraction of triose phosphate converted to CO2. The formula in question is as follows: ([1]CO2/G-[6]CO2/G)/(1-[6]CO2/G) = 3Fmet./(1 + 2Fmet.) where [1]CO2 and [6]CO2 are output rates of carbons 1 and 6 of glucose respectively to CO2, G is the rate of glucose uptake and Fmet. is the fraction of the glucose that is metabolized to CO2 and triose phosphate by the pentose phosphate pathway, allowing for recycling of an appropriate fraction of the fructose-6-phosphate produced by the pathway. This analysis illustrates the importance of suitably testing any equation that assumes homogeneity before application to non-homogeneous tissues.

High-resolution structures of Lactobacillus salivarius transketolase in the presence and absence of thiamine pyrophosphate.

PubMed

Lukacik, Petra; Lobley, Carina M C; Bumann, Mario; Arena de Souza, Victoria; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

2015-10-01

Probiotic bacterial strains have been shown to enhance the health of the host through a range of mechanisms including colonization, resistance against pathogens, secretion of antimicrobial compounds and modulation of the activity of the innate immune system. Lactobacillus salivarius UCC118 is a well characterized probiotic strain which survives intestinal transit and has many desirable host-interaction properties. Probiotic bacteria display a wide range of catabolic activities, which determine their competitiveness in vivo. Some lactobacilli are heterofermentative and can metabolize pentoses, using a pathway in which transketolase and transaldolase are key enzymes. L. salivarius UCC118 is capable of pentose utilization because it encodes the key enzymes on a megaplasmid. The crystal structures of the megaplasmid-encoded transketolase with and without the enzyme cofactor thiamine pyrophosphate have been determined. Comparisons with other known transketolase structures reveal a high degree of structural conservation in both the catalytic site and the overall conformation. This work extends structural knowledge of the transketolases to the industrially and commercially important Lactobacillus genus.

The influence of different cultivation conditions on the metabolome of Fusarium oxysporum.

PubMed

Panagiotou, Gianni; Christakopoulos, Paul; Olsson, Lisbeth

2005-08-22

The two most widespread pentose sugars found in the biosphere are d-xylose and l-arabinose. They are both potential substrates for ethanol production. The purpose of this study was to better understand the redox constraints imposed to Fusarium oxysporum during utilization of pentoses. In order to increase ethanol yield and decrease by-product formation, nitrate was used as nitrogen source. The use of NADH, the cofactor in denitrification process when using nitrate as a nitrogen source, improved the ethanol yield on xylose to 0.89 mol mol(-1) compared to the ethanol yield achieved using ammonium as nitrogen source 0.44 mol mol(-1). The improved ethanol yield was followed by a 28% decrease in yield of the by-product xylitol. In order to investigate the metabolic pathway of arabinose and the metabolic limitations for the efficient ethanol production from this sugar, the extracellular and intracellular metabolite profiles were determined under aerobic and anaerobic cultivation conditions. The results of this study clearly show difficulties in channelling of glucose-1-P (G1P) to pentose phosphate pathway (PPP) and reduced NADPH regeneration, suggesting that NADPH becomes a limiting factor for arabinose conversion, resulting in excessive acetate production. Variations of the fungus intracellular amino and non-amino acid pool, under different culture conditions, were evaluated using principal component analysis (PCA). PCA projection of the metabolome data collected from F. oxysporum subjected to environmental perturbations succeeded to visualize different physiological states and the conclusions of this study were that the metabolite profile is unique according to: (1) the carbon source and (2) the oxygen supply, and to a lesser extent to the cultivation phase.

A reverse KREBS cycle in photosynthesis: consensus at last

NASA Technical Reports Server (NTRS)

Buchanan, B. B.; Arnon, D. I.

1990-01-01

The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It liberates CO2 and generates NADH whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation. In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al. 1966) (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the 'one important property ... shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged. It is only now that in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic CO2 assimilation pathway distinct from the pentose cycle. (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al. 1986). Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.

A reverse KREBS cycle in photosynthesis: consensus at last.

PubMed

Buchanan, B B; Arnon, D I

1990-01-01

The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It liberates CO2 and generates NADH whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation. In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al. 1966) (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the 'one important property ... shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged. It is only now that in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic CO2 assimilation pathway distinct from the pentose cycle. (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al. 1986). Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.

Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts.

PubMed

Fornalewicz, Karolina; Wieczorek, Aneta; Węgrzyn, Grzegorz; Łyżeń, Robert

2017-11-30

Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploring the potential of lactic acid production from lignocellulosic hydrolysates with various ratios of hexose versus pentose by Bacillus coagulans IPE22.

PubMed

Wang, Yujue; Cao, Weifeng; Luo, Jianquan; Wan, Yinhua

2018-08-01

The aim of this study was to investigate the feasibility of utilizing different lignocellulosic hydrolysates with various hexose versus pentose (H:P) ratios to produce lactic acid (LA) from Bacillus coagulans IPE22 by fermentations with single and mixed sugar. In single sugar utilization, glucose tended to promote LA production, and xylose preferred to enhance cell growth. In mixed sugar utilization, glucose and pentose were consumed simultaneously when glucose concentration was lower than 20 g/L, and almost the same concentration of LA (50 g/L) was obtained regardless of the differences of H:P values. Finally, LA production from corn cob hydrolysates (CCH) contained 60 g/L mixed sugar verified the mechanisms found in the fermentations with simulated sugar mixture. Comparing with single glucose utilization, CCH utilization was faster and the yield of LA was not significantly affected. Therefore, the great potential of producing LA with lignocellulosic materials by B. coagulans was proved. Copyright © 2018. Published by Elsevier Ltd.

Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose.

PubMed

Petersen, Kia Vest; Liu, Jianming; Chen, Jun; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

2017-08-01

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed-acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon-optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nuclear magnetic resonance studies of the regulation of the pentose phosphate pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolo, N.R.

1991-11-01

The goal of this work is to investigate the potential for and limitations of in vivo nuclear magnetic resonance (NMR) spectroscopy for quantitation of glucose flux through the pentose phosphate pathway (shunt). Interest in the shunt is motivated by the possibility that its activity may be greatly increased in cancer and in the pathological states of cardiac and cerebral ischemia. The ability to dynamically monitor flux through the pentose shunt can give new knowledge about metabolism in pathological states. {sup 13}C NMR spectroscopy was used to monitor shunt activity by determination of the ratios of ({sup 13}C-4) to ({sup 13}C-5)-glutamate,more » ({sup 13}C-3) to ({sup 13}C-2)-alanine or ({sup 13}C-3) to ({sup 13}C-2)-lactate produced when ({sup 13}C-2)-glucose is infused. These methods provide measures of the effect of oxidative stresses on shunt activity in systems ranging from cell free enzyme-substrate preparations to cell suspensions and whole animals. In anaerobic cell free preparations, the fraction of glucose flux through the shunt was monitored with a time resolution of 3 minutes. This work predicts the potential for in vivo human studies of pentose phosphate pathway activity based on the mathematical simulation of the {sup 13}C fractional enrichments of C4 and C5-glutamate as a function of shunt activity and on the signal-to- noise ratio acquired in {sup 13}C NMR human studies from the current literature.« less

Nuclear magnetic resonance studies of the regulation of the pentose phosphate pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolo, Nicolas Robin

1991-11-01

The goal of this work is to investigate the potential for and limitations of in vivo nuclear magnetic resonance (NMR) spectroscopy for quantitation of glucose flux through the pentose phosphate pathway (shunt). Interest in the shunt is motivated by the possibility that its activity may be greatly increased in cancer and in the pathological states of cardiac and cerebral ischemia. The ability to dynamically monitor flux through the pentose shunt can give new knowledge about metabolism in pathological states. 13C NMR spectroscopy was used to monitor shunt activity by determination of the ratios of [ 13C-4] to [ 13C-5]-glutamate, [more » 13C-3] to [ 13C-2]-alanine or [ 13C-3] to [ 13C-2]-lactate produced when [ 13C-2]-glucose is infused. These methods provide measures of the effect of oxidative stresses on shunt activity in systems ranging from cell free enzyme-substrate preparations to cell suspensions and whole animals. In anaerobic cell free preparations, the fraction of glucose flux through the shunt was monitored with a time resolution of 3 minutes. This work predicts the potential for in vivo human studies of pentose phosphate pathway activity based on the mathematical simulation of the 13C fractional enrichments of C4 and C5-glutamate as a function of shunt activity and on the signal-to- noise ratio acquired in 13C NMR human studies from the current literature.« less

Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production.

PubMed

Tan, Zaigao; Chen, Jing; Zhang, Xueli

2016-01-01

Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production. Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge. In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.

Quiescent Fibroblasts Exhibit High Metabolic Activity

PubMed Central

Lemons, Johanna M. S.; Feng, Xiao-Jiang; Bennett, Bryson D.; Legesse-Miller, Aster; Johnson, Elizabeth L.; Raitman, Irene; Pollina, Elizabeth A.; Rabitz, Herschel A.; Rabinowitz, Joshua D.; Coller, Hilary A.

2010-01-01

Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole. PMID:21049082

Process design considerations for optimal production of ethanol from lignocellulose using available yeasts, including natural pentose-fermenting yeasts, and their derivatives

USDA-ARS?s Scientific Manuscript database

To expand the biomass to fuel ethanol industry, process strategies are needed to foster the production and utilization of microorganisms which can survive and ferment both hexose (C6) and pentose (C5) sugars while exposed to inhibitors (such as ethanol, furfural, and hydroxymethylfurfural, or HMF). ...

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

PubMed Central

Matsushika, Akinori; Nagashima, Atsushi; Goshima, Tetsuya; Hoshino, Tamotsu

2013-01-01

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose. PMID:23874849

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

PubMed

Feng, Quanzhou; Liu, Z Lewis; Weber, Scott A; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

PubMed Central

Feng, Quanzhou; Weber, Scott A.; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production. PMID:29621349

Simultaneous utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform.

PubMed

Wei, Na; Oh, Eun Joong; Million, Gyver; Cate, Jamie H D; Jin, Yong-Su

2015-06-19

The inability of fermenting microorganisms to use mixed carbon components derived from lignocellulosic biomass is a major technical barrier that hinders the development of economically viable cellulosic biofuel production. In this study, we integrated the fermentation pathways of both hexose and pentose sugars and an acetic acid reduction pathway into one Saccharomyces cerevisiae strain for the first time using synthetic biology and metabolic engineering approaches. The engineered strain coutilized cellobiose, xylose, and acetic acid to produce ethanol with a substantially higher yield and productivity than the control strains, and the results showed the unique synergistic effects of pathway coexpression. The mixed substrate coutilization strategy is important for making complete and efficient use of cellulosic carbon and will contribute to the development of consolidated bioprocessing for cellulosic biofuel. The study also presents an innovative metabolic engineering approach whereby multiple substrate consumption pathways can be integrated in a synergistic way for enhanced bioconversion.

Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope-assisted labeling.

PubMed

Haviland, Julia A; Tonelli, Marco; Haughey, Dermot T; Porter, Warren P; Assadi-Porter, Fariba M

2012-08-01

Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, type 2 diabetes mellitus, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy and cavity ring-down spectroscopy to analyze serial plasma samples and real-time breath measurements following selective (13)C-isotope-assisted labeling. These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. This novel diagnostics approach is fast, noninvasive, and sensitive for determining specific pathway utilization, and provides a direct translational application in the health care field. Copyright © 2012 Elsevier Inc. All rights reserved.

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach.

PubMed

Akberdin, Ilya R; Thompson, Merlin; Hamilton, Richard; Desai, Nalini; Alexander, Danny; Henard, Calvin A; Guarnieri, Michael T; Kalyuzhnaya, Marina G

2018-02-06

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13 C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization in M. alcaliphilum 20Z R . A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. The computational framework of C 1 -metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.

Textbook Errors & Misconceptions in Biology: Cell Metabolism.

ERIC Educational Resources Information Center

Storey, Richard D.

1991-01-01

The idea that errors and misconceptions in biology textbooks are often slow to be discovered and corrected is discussed. Selected errors, misconceptions, and topics of confusion about cell metabolism are described. Fermentation, respiration, Krebs cycle, pentose phosphate pathway, uniformity of catabolism, and metabolic pathways as models are…

Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons.

PubMed

Sun, Shanshan; Hu, Fangyuan; Wu, Jihong; Zhang, Shenghai

2017-04-01

Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XF e 24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP + ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Glucose metabolism and astrocyte-neuron interactions in the neonatal brain.

PubMed

Brekke, Eva; Morken, Tora Sund; Sonnewald, Ursula

2015-03-01

Glucose is essentially the sole fuel for the adult brain and the mapping of its metabolism has been extensive in the adult but not in the neonatal brain, which is believed to rely mainly on ketone bodies for energy supply. However, glucose is absolutely indispensable for normal development and recent studies have shed light on glycolysis, the pentose phosphate pathway and metabolic interactions between astrocytes and neurons in the 7-day-old rat brain. Appropriately (13)C labeled glucose was used to distinguish between glycolysis and the pentose phosphate pathway during development. Experiments using (13)C labeled acetate provided insight into the GABA-glutamate-glutamine cycle between astrocytes and neurons. It could be shown that in the neonatal brain the part of this cycle that transfers glutamine from astrocytes to neurons is operating efficiently while, in contrast, little glutamate is shuttled from neurons to astrocytes. This lack of glutamate for glutamine synthesis is compensated for by anaplerosis via increased pyruvate carboxylation relative to that in the adult brain. Furthermore, compared to adults, relatively more glucose is prioritized to the pentose phosphate pathway than glycolysis and pyruvate dehydrogenase activity. The reported developmental differences in glucose metabolism and neurotransmitter synthesis may determine the ability of the brain at various ages to resist excitotoxic insults such as hypoxia-ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of Mammary Tumor Formation and Lipid Biosynthesis by Spot 14

DTIC Science & Technology

2013-10-01

1     Introduction: THRSP/Spot14/S14 is a small cytoplasmic protein that is highly expressed in tissues that synthesize fatty acids de novo...cycle regulation and also with various metabolic pathways, including glycolysis /gluconeogenesis and the pentose phosphate pathway (Table 1). The

In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability.

PubMed

Preller, A; Guixé, V; Ureta, T

1999-03-05

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO2 production from [1-(14)C] and [6-(14)C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP+. A high NADPH/NADP+ ratio of 4.3 was found in the cells, the intracellular concentration of NADP+ being 19 microM.

OXIDATIVE STRESS, INFLAMMATION AND CARCINOGENESIS ARE CONTROLLED THROUGH THE PENTOSE PHOSPHATE PATHWAY BY TRANSALDOLASE

PubMed Central

Perl, Andras; Hanczko, Robert; Telarico, Tiffany; Oaks, Zachary; Landas, Steve

2011-01-01

Metabolism of glucose through the pentose phosphate pathway (PPP) influences the development of diverse pathologies. Hemolytic anemia due to deficiency of PPP enzyme glucose 6-phosphate dehydrogenase is the most common genetic disease in humans. Recently, inactivation of another PPP enzyme, transaldolase (TAL), has been implicated in male infertility and fatty liver progressing to steatohepatitis and cancer. Hepatocarcinogenesis was associated with activation of aldose reductase and redox-sensitive transcription factors and prevented by N-acetylcysteine. Here, we discuss how alternative formulations of the PPP with and without TAL reflect cell type-specific metabolic control of oxidative stress, a critical source of inflammation and carcinogenesis. Ongoing studies of TAL deficiency will identify new molecular targets for diagnosis and treatment in clinical practice. PMID:21376665

Prediction of Metabolite Concentrations, Rate Constants and Post-Translational Regulation Using Maximum Entropy-Based Simulations with Application to Central Metabolism of Neurospora crassa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cannon, William; Zucker, Jeremy; Baxter, Douglas

We report the application of a recently proposed approach for modeling biological systems using a maximum entropy production rate principle in lieu of having in vivo rate constants. The method is applied in four steps: (1) a new ODE-based optimization approach based on Marcelin’s 1910 mass action equation is used to obtain the maximum entropy distribution, (2) the predicted metabolite concentrations are compared to those generally expected from experiment using a loss function from which post-translational regulation of enzymes is inferred, (3) the system is re-optimized with the inferred regulation from which rate constants are determined from the metabolite concentrationsmore » and reaction fluxes, and finally (4) a full ODE-based, mass action simulation with rate parameters and allosteric regulation is obtained. From the last step, the power characteristics and resistance of each reaction can be determined. The method is applied to the central metabolism of Neurospora crassa and the flow of material through the three competing pathways of upper glycolysis, the non-oxidative pentose phosphate pathway, and the oxidative pentose phosphate pathway are evaluated as a function of the NADP/NADPH ratio. It is predicted that regulation of phosphofructokinase (PFK) and flow through the pentose phosphate pathway are essential for preventing an extreme level of fructose 1, 6-bisphophate accumulation. Such an extreme level of fructose 1,6-bisphophate would otherwise result in a glassy cytoplasm with limited diffusion, dramatically decreasing the entropy and energy production rate and, consequently, biological competitiveness.« less

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

PubMed

Best, Sarah A; De Souza, David P; Kersbergen, Ariena; Policheni, Antonia N; Dayalan, Saravanan; Tull, Dedreia; Rathi, Vivek; Gray, Daniel H; Ritchie, Matthew E; McConville, Malcolm J; Sutherland, Kate D

2018-04-03

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1 f/f /Pten f/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations. Copyright © 2018 Elsevier Inc. All rights reserved.

Glucose and pyruvate metabolism in preimplantation blastocysts from normal and diabetic rats.

PubMed

Dufrasnes, E; Vanderheyden, I; Robin, D; Delcourt, J; Pampfer, S; De Hertogh, R

1993-05-01

Glucose metabolism was analysed in day-5 rat blastocysts incubated in the presence of [5-3H]-, [6-14C]- or [U-14C]glucose. Glycolysis, quantified by 3H2O recovery rate, was the main pathway of glucose utilization by fresh (11.5 +/- 0.36 pmol per embryo h-1) or cultured (24 h) blastocysts (20.4 +/- 0.6 pmol per embryo h-1). Glucose consumption rate was almost saturated at a medium glucose concentration of 0.28 mmol l-1 (Km: 0.17 mmol l-1; Vmax: 23 pmol per embryo h-1). A further 10% increase in glucose utilization was obtained with a tenfold higher glucose concentration (3 mmol l-1). Phloretin completely abolished the rapid component of glucose utilization kinetics, suggesting the existence of a Na(+)-independent glucose transport system. Less than 1% of [6-14C]glucose consumed by cultured blastocysts was oxidized through the Krebs cycle. [1-14C]pyruvate, however, was oxidized at a rate of 2 pmol per embryo h-1 by fresh blastocysts. The pentose-phosphate pathway accounted for about 2% of glucose utilization. One to two per cent of the total glucose metabolized in 24 h was retained in macromolecules. Insulin had no effect on glucose uptake, utilization, incorporation and turnover, or on pyruvate oxidation. Blastocysts from diabetic mothers utilized glucose at a rate similar to that of normal blastocysts. These results show that glucose is actively taken up by rat blastocysts and utilized mainly through the Embden-Meyerhof pathway, which is rapidly saturated at low glucose concentrations. Retention of glucose-derived products in macromolecules, although relatively small, may modulate the effect of high glucose concentrations on embryo growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Methane utilization in Methylomicrobium alcaliphilum 20Z R: a systems approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akberdin, Ilya R.; Thompson, Merlin; Hamilton, Richard

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization inmore » M. alcaliphilum 20Z R. A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. As a result, the computational framework of C 1-metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.« less

Methane utilization in Methylomicrobium alcaliphilum 20Z R: a systems approach

DOE PAGES

Akberdin, Ilya R.; Thompson, Merlin; Hamilton, Richard; ...

2018-02-06

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization inmore » M. alcaliphilum 20Z R. A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. As a result, the computational framework of C 1-metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.« less

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. Energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes.

PubMed

Schuster, R; Jacobasch, G; Holzhütter, H G

1989-07-01

The effects of various forms of glucose-6-phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism. The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose-6-phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed. It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose-6-phosphate dehydrogenase deficiency.

The coupling of glycolysis and the Rubisco-based pathway through the non-oxidative pentose phosphate pathway to achieve low carbon dioxide emission fermentation.

PubMed

Li, Ya-Han; Ou-Yang, Fan-Yu; Yang, Cheng-Han; Li, Si-Yu

2015-01-01

In this study, Rubisco-based engineered Escherichia coli, containing two heterologous enzymes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoribulokinase (PrkA), has been shown to be capable of the in situ recycling of carbon dioxide (CO2) during glycolysis. Two alternative approaches have been proposed to further enhance the carbon flow from glycolysis to a Rubisco-based pathway through the non-oxidative pentose phosphate pathway (NOPPP). The first is achieved by elevating the expression of transketolase I (TktA) and the second by blocking the native oxidation-decarboxylation reaction of E. coli by deleting the zwf gene from the chromosome (designated as JB/pTA and MZB, respectively). Decreases in the CO2 yield and the CO2 evolution per unit mole of ethanol production by at least 81% and 40% are observed. It is demonstrated in this study that the production of one mole of ethanol using E. coli strain MZB, the upper limit of CO2 emission is 0.052mol. Copyright © 2015 Elsevier Ltd. All rights reserved.

Curcumin improves alcoholic fatty liver by inhibiting fatty acid biosynthesis.

PubMed

Guo, Chang; Ma, Jingfan; Zhong, Qionghong; Zhao, Mengyuan; Hu, Tianxing; Chen, Tong; Qiu, Longxin; Wen, Longping

2017-08-01

Alcoholic fatty liver is a threat to human health. It has been long known that abstinence from alcohol is the most effective therapy, other effective therapies are not available for the treatment in humans. Curcumin has a great potential for anti-oxidation and anti-inflammation, but the effect on metabolic reconstruction remains little known. Here we performed metabolomic analysis by gas chromatography/mass spectrometry and explored ethanol pathogenic insight as well as curcumin action pattern. We identified seventy-one metabolites in mouse liver. Carbohydrates and lipids were characteristic categories. Pathway analysis results revealed that ethanol-induced pathways including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis and pentose and glucuronate interconversions were suppressed by curcumin. Additionally, ethanol enhanced galactose metabolism and pentose phosphate pathway. Glyoxylate and dicarboxylate metabolism and pyruvate metabolism were inhibited in mice fed ethanol diet plus curcumin. Stearic acid, oleic acid and linoleic acid were disease biomarkers and therapical biomarkers. These results reflect the landscape of hepatic metabolism regulation. Our findings illustrate ethanol pathological pathway and metabolic mechanism of curcumin therapy. Copyright © 2017. Published by Elsevier Inc.

Oxidative Pentose Phosphate Pathway Inhibition Is A Key Determinant of Antimalarial Induced Cancer Cell Death

PubMed Central

Salas, Eduardo; Roy, Srirupa; Marsh, Timothy; Rubin, Brian; Debnath, Jayanta

2015-01-01

Despite immense interest in employing antimalarials as autophagy inhibitors to treat cancer, it remains unclear if these agents act predominantly via autophagy inhibition or whether other pathways direct their anti-cancer properties. By comparing the treatment effects of the antimalarials chloroquine (CQ) and quinacrine (Q) on KRAS mutant lung cancer cells, we demonstrate that inhibition of the oxidative arm of the pentose phosphate pathway (oxPPP) is required for antimalarial induced apoptosis. Despite inhibiting autophagy, neither CQ treatment nor RNAi against autophagy regulators (ATGs) promote cell death. In contrast, Q triggers high levels of apoptosis, both in vitro and in vivo, and this phenotype requires both autophagy inhibition and p53-dependent inhibition of the oxPPP. Simultaneous genetic targeting of the oxPPP and autophagy is sufficient to trigger apoptosis in lung cancer cells, including cells lacking p53. Thus, in addition to reduced autophagy, oxPPP inhibition serves as an important determinant of antimalarial cytotoxicity in cancer cells. PMID:26434592

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons

PubMed Central

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L. Felipe

2016-01-01

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders. PMID:27688915

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons.

PubMed

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders.

Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae*

PubMed Central

Medeiros, Rychelle Clayde Affonso; Girardi, Karina do Carmo de Vasconcelos; Cardoso, Fernanda Karlla Luz; Mietto, Bruno de Siqueira; Pinto, Thiago Gomes de Toledo; Gomez, Lilian Sales; Rodrigues, Luciana Silva; Gandini, Mariana; Amaral, Julio Jablonski; Antunes, Sérgio Luiz Gomes; Corte-Real, Suzana; Rosa, Patricia Sammarco; Pessolani, Maria Cristina Vidal; Nery, José Augusto da Costa; Sarno, Euzenir Nunes; Batista-Silva, Leonardo Ribeiro; Sola-Penna, Mauro; Oliveira, Marcus Fernandes; Moraes, Milton Ozório; Lara, Flavio Alves

2016-01-01

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed. PMID:27555322

Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis*

PubMed Central

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L. Felipe; Inestrosa, Nibaldo C.

2016-01-01

The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells. PMID:27703002

Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis.

PubMed

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

2016-12-09

The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

PubMed

Seol, Eunhee; Sekar, Balaji Sundara; Raj, Subramanian Mohan; Park, Sunghoon

2016-02-01

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

PubMed

de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

2017-11-01

Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum.

PubMed

Verbeke, Tobin J; Giannone, Richard J; Klingeman, Dawn M; Engle, Nancy L; Rydzak, Thomas; Guss, Adam M; Tschaplinski, Timothy J; Brown, Steven D; Hettich, Robert L; Elkins, James G

2017-02-23

Clostridium thermocellum could potentially be used as a microbial biocatalyst to produce renewable fuels directly from lignocellulosic biomass due to its ability to rapidly solubilize plant cell walls. While the organism readily ferments sugars derived from cellulose, pentose sugars from xylan are not metabolized. Here, we show that non-fermentable pentoses inhibit growth and end-product formation during fermentation of cellulose-derived sugars. Metabolomic experiments confirmed that xylose is transported intracellularly and reduced to the dead-end metabolite xylitol. Comparative RNA-seq analysis of xylose-inhibited cultures revealed several up-regulated genes potentially involved in pentose transport and metabolism, which were targeted for disruption. Deletion of the ATP-dependent transporter, CbpD partially alleviated xylose inhibition. A putative xylitol dehydrogenase, encoded by Clo1313_0076, was also deleted resulting in decreased total xylitol production and yield by 41% and 46%, respectively. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of a cyclical AgrD-type, pentapeptide. Medium supplementation with the mature cyclical pentapeptide also inhibits bacterial growth. Together, these findings provide new foundational insights needed for engineering improved pentose utilizing strains of C. thermocellum and reveal the first functional Agr-type cyclic peptide to be produced by a thermophilic member of the Firmicutes.

Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum

PubMed Central

Verbeke, Tobin J.; Giannone, Richard J.; Klingeman, Dawn M.; Engle, Nancy L.; Rydzak, Thomas; Guss, Adam M.; Tschaplinski, Timothy J.; Brown, Steven D.; Hettich, Robert L.; Elkins, James G.

2017-01-01

Clostridium thermocellum could potentially be used as a microbial biocatalyst to produce renewable fuels directly from lignocellulosic biomass due to its ability to rapidly solubilize plant cell walls. While the organism readily ferments sugars derived from cellulose, pentose sugars from xylan are not metabolized. Here, we show that non-fermentable pentoses inhibit growth and end-product formation during fermentation of cellulose-derived sugars. Metabolomic experiments confirmed that xylose is transported intracellularly and reduced to the dead-end metabolite xylitol. Comparative RNA-seq analysis of xylose-inhibited cultures revealed several up-regulated genes potentially involved in pentose transport and metabolism, which were targeted for disruption. Deletion of the ATP-dependent transporter, CbpD partially alleviated xylose inhibition. A putative xylitol dehydrogenase, encoded by Clo1313_0076, was also deleted resulting in decreased total xylitol production and yield by 41% and 46%, respectively. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of a cyclical AgrD-type, pentapeptide. Medium supplementation with the mature cyclical pentapeptide also inhibits bacterial growth. Together, these findings provide new foundational insights needed for engineering improved pentose utilizing strains of C. thermocellum and reveal the first functional Agr-type cyclic peptide to be produced by a thermophilic member of the Firmicutes. PMID:28230109

A Genetic Locus Necessary for Rhamnose Uptake and Catabolism in Rhizobium leguminosarum bv. trifolii

PubMed Central

Richardson, Jason S.; Hynes, Michael F.; Oresnik, Ivan J.

2004-01-01

Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways. PMID:15576793

Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2.

PubMed

Nutt, Leta K; Margolis, Seth S; Jensen, Mette; Herman, Catherine E; Dunphy, William G; Rathmell, Jeffrey C; Kornbluth, Sally

2005-10-07

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.

Utilization of pentoses from sugarcane biomass: techno-economics of biogas vs. butanol production.

PubMed

Mariano, Adriano Pinto; Dias, Marina O S; Junqueira, Tassia L; Cunha, Marcelo P; Bonomi, Antonio; Filho, Rubens Maciel

2013-08-01

This paper presents the techno-economics of greenfield projects of an integrated first and second-generation sugarcane biorefinery in which pentose sugars obtained from sugarcane biomass are used either for biogas (consumed internally in the power boiler) or n-butanol production via the ABE batch fermentation process. The complete sugarcane biorefinery was simulated using Aspen Plus®. Although the pentoses stream available in the sugarcane biorefinery gives room for a relatively small biobutanol plant (7.1-12 thousand tonnes per year), the introduction of butanol and acetone to the product portfolio of the biorefinery increased and diversified its revenues. Whereas the IRR of the investment on a biorefinery with biogas production is 11.3%, IRR varied between 13.1% and 15.2% in the butanol production option, depending on technology (regular or engineered microorganism with improved butanol yield and pentoses conversion) and target market (chemicals or automotive fuels). Additional discussions include the effects of energy-efficient technologies for butanol processing on the profitability of the biorefinery. Copyright © 2013 Elsevier Ltd. All rights reserved.

Co-expression of TAL1 and ADH1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural.

PubMed

Hasunuma, Tomohisa; Ismail, Ku Syahidah Ku; Nambu, Yumiko; Kondo, Akihiko

2014-02-01

Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae.

PubMed

Medeiros, Rychelle Clayde Affonso; Girardi, Karina do Carmo de Vasconcelos; Cardoso, Fernanda Karlla Luz; Mietto, Bruno de Siqueira; Pinto, Thiago Gomes de Toledo; Gomez, Lilian Sales; Rodrigues, Luciana Silva; Gandini, Mariana; Amaral, Julio Jablonski; Antunes, Sérgio Luiz Gomes; Corte-Real, Suzana; Rosa, Patricia Sammarco; Pessolani, Maria Cristina Vidal; Nery, José Augusto da Costa; Sarno, Euzenir Nunes; Batista-Silva, Leonardo Ribeiro; Sola-Penna, Mauro; Oliveira, Marcus Fernandes; Moraes, Milton Ozório; Lara, Flavio Alves

2016-10-07

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy

PubMed Central

Cho, Eunae Sandra; Cha, Yong Hoon; Kim, Hyun Sil; Kim, Nam Hee; Yook, Jong In

2018-01-01

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeutic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphorylation required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progression as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress. PMID:29212304

Engineering Shewanella oneidensis enables xylose-fed microbial fuel cell.

PubMed

Li, Feng; Li, Yuanxiu; Sun, Liming; Li, Xiaofei; Yin, Changji; An, Xingjuan; Chen, Xiaoli; Tian, Yao; Song, Hao

2017-01-01

The microbial fuel cell (MFC) is a green and sustainable technology for electricity energy harvest from biomass, in which exoelectrogens use metabolism and extracellular electron transfer pathways for the conversion of chemical energy into electricity. However, Shewanella oneidensis MR-1, one of the most well-known exoelectrogens, could not use xylose (a key pentose derived from hydrolysis of lignocellulosic biomass) for cell growth and power generation, which limited greatly its practical applications. Herein, to enable S. oneidensis to directly utilize xylose as the sole carbon source for bioelectricity production in MFCs, we used synthetic biology strategies to successfully construct four genetically engineered S. oneidensis (namely XE, GE, XS, and GS) by assembling one of the xylose transporters (from Candida intermedia and Clostridium acetobutylicum ) with one of intracellular xylose metabolic pathways (the isomerase pathway from Escherichia coli and the oxidoreductase pathway from Scheffersomyces stipites ), respectively. We found that among these engineered S. oneidensis strains, the strain GS (i.e. harbouring Gxf1 gene encoding the xylose facilitator from C. intermedi , and XYL1 , XYL2 , and XKS1 genes encoding the xylose oxidoreductase pathway from S. stipites ) was able to generate the highest power density, enabling a maximum electricity power density of 2.1 ± 0.1 mW/m 2 . To the best of our knowledge, this was the first report on the rationally designed Shewanella that could use xylose as the sole carbon source and electron donor to produce electricity. The synthetic biology strategies developed in this study could be further extended to rationally engineer other exoelectrogens for lignocellulosic biomass utilization to generate electricity power.

Glucosamine Inhibits Decidualization of Human Endometrial Stromal Cells and Decreases Litter Sizes in Mice1

PubMed Central

Tsai, Jui-He; Schulte, Maureen; O'Neill, Kathleen; Chi, Maggie M.-Y.; Frolova, Antonina I.; Moley, Kelle H.

2013-01-01

ABSTRACT Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 μg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive. PMID:23718985

Handling Uncertainty in Dynamic Models: The Pentose Phosphate Pathway in Trypanosoma brucei

PubMed Central

Alibu, Vincent P.; Burchmore, Richard J.; Gilbert, Ian H.; Trybiło, Maciej; Driessen, Nicole N.; Gilbert, David; Breitling, Rainer; Bakker, Barbara M.; Barrett, Michael P.

2013-01-01

Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate “leak” must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally. PMID:24339766

Production of lactic acid from hemicellulose extracts by Bacillus coagulans MXL-9

USDA-ARS?s Scientific Manuscript database

Bacillus coagulans MXL-9 was found capable of growing on pre-pulping hemicellulose extracts, utilizing all of the principle monosugars found in woody biomass. This organism is a moderate thermophile isolated from compost for its pentose utilizing capabilities. It was found to have high tolerance f...

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

PubMed Central

Esser, Dominik; Rauch, Bernadette

2014-01-01

SUMMARY The metabolism of Archaea, the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya. However, this metabolic complexity in Archaea is accompanied by the absence of many “classical” pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of “new,” unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea. In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya. Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented. PMID:24600042

Carbon Metabolism of Soil microorganisms at Low Temperatures: Position-Specific 13C Labeled Glucose Reveals the Story

NASA Astrophysics Data System (ADS)

Apostel, C.; Bore, E. K.; Halicki, S.; Kuzyakov, Y.; Dippold, M.

2015-12-01

Metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze soil metabolism at low temperature, isotopomeres of position-specifically 13C labeled glucose were applied at three temperature levels; +5, -5 -20 oC. In additon, one sterilization treatment with sodium azide at +5 oC was also performed. Soils were incubated for 1, 3 and 10 days while soil samples at -20 oC were additionally sampled after 30 days. The 13C from individual molecule position in respired CO2 was quantifed. Incorporation of 13C in bulk soil, extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of different microbial communities classified by 13C phospholipid fatty acid analysis (PLFA) was carried out. Our 13CO2 data showed a dominance of C-1 respiration at +5 °C for treatments with and without sodium azide, but total respiration for sodium azide inhibited treatments increased by 14%. In contrast, at -5 and -20 oC metabolic behavior showed intermingling of preferential respiration of the glucose C-4 and C-1 positions. Therefore, at +5 °C, pentose phosphate pathway activity is a dominant metabolic pathway used by microorganisms to metabolize glucose. The respiration increase due to NaN3 inhibition was attributed to endoenzymes released from dead organisms that are stabilized at the soil matrix and have access to suitable substrate and co-factors to permit their funtions. Our PLFA analysis showed that incorporation of glucose 13C was higher in Gram negative bacteria than other microbial groups as they are most competitive for LMWOS. Only a limited amount of microbial groups maintained their glucose utilizing activity at -5 and -20 °C and they strongly shifted towards a metabolization of glucose via both glycolysis and pentose phosphate pathways indicating both growth and cellular maintenance. This study revealed a remarkable microbial acitivity at low temperatures which differs significantly from our observations at ambient temperature, which could be unraveled based on position-specific labeling.

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass.

PubMed

Kim, Jae-Han; Block, David E; Mills, David A

2010-11-01

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass

PubMed Central

Kim, Jae-Han; Block, David E.

2010-01-01

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy. PMID:20838789

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis.

PubMed

Guarnieri, Michael T; Chou, Yat-Chen; Salvachúa, Davinia; Mohagheghi, Ali; St John, Peter C; Peterson, Darren J; Bomble, Yannick J; Beckham, Gregg T

2017-09-01

Actinobacillus succinogenes , a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes , enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism. Copyright © 2017 American Society for Microbiology.

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

PubMed Central

Novello, F.; Gumaa, J. A.; McLean, Patricia

1969-01-01

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known `overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30–40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine–zinc–insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine–zinc–insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding. PMID:5791534

Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae.

PubMed

Garcia Sanchez, Rosa; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

2010-09-01

Overexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered Saccharomyces cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. In the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium. PGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.

Evidence for biochemical and physiological differences between enzyme genotypes in Drosophila melanogaster.

PubMed Central

Cavener, D R; Clegg, M T

1981-01-01

The in vivo flux of carbon through the pentose shunt is investigated as a function of different 6-phosphogluconate dehydrogenase (6Pgd) and glucose-6-phosphate dehydrogenase (G6pd) genotypes by using differential radioactive labeling of the C-1 and C-6 positions of glucose. Alternative 6Pgd-G6pd genotypes are shown to differ in relative in vivo carbon flux through the pentose shunt. The relative in vitro specific activity differences between the 6PgdSS and 6PgdFF genotypes appear to be primarily responsible for these differences. In addition, the pentose-shunt activity is correlated with the rate of lipid synthesis. This correlation is consistent with the major metabolic function of the pathway, which is to produce NADPH for lipid synthesis. Taken together, the results of these experiments show that different genotypes of 6Pgd are associated with measurable biochemical and physiological differences. Higher order phenotypic differences of this kind must be demonstrated to support the hypothesis that natural selection can discriminate among allozymes of a given genetic locus. PMID:6794031

Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.

PubMed

Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2009-08-01

Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose.

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis.

PubMed

Horal, Melissa; Zhang, Zhiquan; Stanton, Robert; Virkamäki, Antti; Loeken, Mary R

2004-08-01

Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.

Single Zymomonas mobilis strain for xylose and arabinose fermentation

DOEpatents

Zhang, M.; Chou, Y.C.; Picataggio, S.K.; Finkelstein, M.

1998-12-01

This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol. 6 figs.

Single zymomonas mobilis strain for xylose and arabinose fermentation

DOEpatents

Zhang, Min; Chou, Yat-Chen; Picataggio, Stephen K.; Finkelstein, Mark

1998-01-01

This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol.

Biotechnology to harness the benefits of dietary phenolics; focus on Lamiaceae.

PubMed

Shetty, K

1997-09-01

Phytochemicals from herbs and fermented legumes are excellent dietary sources of phenolic metabolites. These phenolics have importance not only as food preservatives but increasingly have therapeutic and pharmaceutical applications. The long-term research objecitves of the food biotechnology program at the University of Massachusetts are to elucidate the molecular and physiological mechanisms associated with synthesis of important health-related, therapeutic phenolic metabolites in food-related plants and fermented plant foods. Current efforts focus on elucidation of the role of the proline-linked pentose phosphate pathway in regulating the synthesis of anti-inflammatory compound, rosmarinic acid (RA). Specific aims of the current research efforts are: (i) To develop novel tissue culture-based selection techniques to isolate high RA-producing, shoot-based clonal lines from genetically heterogeneous, cross-pollinating species in the family Lamiaceae; (ii) To target genetically uniform, regenerated shoot-based clonal lines for: (a) preliminary characterization of key enzymes associated with the pentose phosphate pathway and linked to RA synthesis; (b) development of genetic transformation techniques for subsequent engineering of metabolic pathways associated with RA synthesis. These research objectives have substantial implications for harnessing the genetic and biochemical potential of genetically heterogeneous, food-related medicinal plant species. The success of this research also provides novel methods and strategies to gain access to metabolic pathways of pharmaceutically important metabolites from ginger, curcuma, chili peppers, melon or other food-related species with novel phenolics.

Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

PubMed

Nunn, Charlotte E M; Johnsen, Ulrike; Schönheit, Peter; Fuhrer, Tobias; Sauer, Uwe; Hough, David W; Danson, Michael J

2010-10-29

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

Quantitative Analysis of Energy Metabolic Pathways in MCF-7 Breast Cancer Cells by Selected Reaction Monitoring Assay*

PubMed Central

Drabovich, Andrei P.; Pavlou, Maria P.; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P.

2012-01-01

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells. PMID:22535206

Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

PubMed

Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

2016-08-10

Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization. Copyright © 2016 Elsevier B.V. All rights reserved.

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

USDA-ARS?s Scientific Manuscript database

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses nicotinamide adenine dinucl...

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains.

PubMed

Wang, Yipeng; San, Ka-Yiu; Bennett, George N

2013-08-01

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

PubMed

Tamano, Koichi; Miura, Ai

2016-09-01

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

PubMed Central

Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

2015-01-01

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

Improved Xylitol Production from D-Arabitol by Enhancing the Coenzyme Regeneration Efficiency of the Pentose Phosphate Pathway in Gluconobacter oxydans.

PubMed

Li, Sha; Zhang, Jinliang; Xu, Hong; Feng, Xiaohai

2016-02-10

Gluconobacter oxydans is used to produce xylitol from D-arabitol. This study aims to improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was obtained, with a yield of 73.2%, from 40 g/L d-arabitol in the batch biotransformation with the G. oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for five cycles without incurring a significant loss in productivity. These results indicate that the recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial bioconversion.

Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocysti s sp. PCC 6803.

PubMed

Lin, Po-Cheng; Saha, Rajib; Zhang, Fuzhong; Pakrasi, Himadri B

2017-12-13

Isoprenoids are diverse natural compounds, which have various applications as pharmaceuticals, fragrances, and solvents. The low yield of isoprenoids in plants makes them difficult for cost-effective production, and chemical synthesis of complex isoprenoids is impractical. Microbial production of isoprenoids has been considered as a promising approach to increase the yield. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 for sustainable production of a commercially valuable isoprenoid, limonene. Limonene synthases from the plants Mentha spicata and Citrus limon were expressed in cyanobacteria for limonene production. Production of limonene was two-fold higher with limonene synthase from M. spicata than that from C. limon. To enhance isoprenoid production, computational strain design was conducted by applying the OptForce strain design algorithm on Synechocystis 6803. Based on the metabolic interventions suggested by this algorithm, genes (ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase) in the pentose phosphate pathway were overexpressed, and a geranyl diphosphate synthase from the plant Abies grandis was expressed to optimize the limonene biosynthetic pathway. The optimized strain produced 6.7 mg/L of limonene, a 2.3-fold improvement in productivity. Thus, this study presents a feasible strategy to engineer cyanobacteria for photosynthetic production of isoprenoids.

Chemotaxis of Pseudomonas to furans

USDA-ARS?s Scientific Manuscript database

Background: Furfural (2-furaldehyde) is a furan compound formed by dehydration of pentose sugars. Furan molecules occur naturally in the environment and are also used industrially as solvents and chemical precursors. Although furan aldehydes are microbial inhibitors, a number of microbes can utilize...

13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses.

PubMed

Azizan, Kamalrul Azlan; Ressom, Habtom W; Mendoza, Eduardo R; Baharum, Syarul Nataqain

2017-01-01

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13 C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis ( r ) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis' central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis , in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis . Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of how L. lactis regulates central metabolism under various conditions.

Effects of long-term exposure to Cu2+ and Cd2+ on the pentose phosphate pathway dehydrogenase activities in the ovary of adult Bufo arenarum: possible role as biomarker for Cu2+ toxicity.

PubMed

Carattino, Marcelo D; Peralta, Susana; Pérez-Coll, Cristina; Naab, Fabián; Burlón, Alejandro; Kreiner, Andrés J; Preller, Ana F; de Schroeder, Teresa M Fonovich

2004-03-01

The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evaluated in Bufo arenarum toad ovary. The effects of the two metals on dehydrogenases from this pathway were evaluated by three experiments: (1) in samples obtained from control females with addition of the metals to the reaction mixture (in vitro), (2) in samples obtained from control females and after long-term exposure of females to 4 and 100 microg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) 14CO2 production through the pentose phosphate pathway was evaluated after [U-14C]glucose microinjection on ovulated oocytes (in vivo after microinjection of the metals). Results from (1) evidenced inhibition of both enzyme activities but only above 1.5 mM Cu and Cd added to the reaction mixture. In (2) both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities decreased in samples from the ovaries of females exposed in vivo to Cu, in a concentration-dependent manner (up to 90% in females exposed to 100 microg/L Cu: 2.12 +/- 1.57 NADPH micromol/min microg protein x 10(-5) vs 19.97 +/- 8.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 microg/L of the bivalent cation. (3) In vivo 14CO2 evolution significantly decreased in oocytes coinjected with 6.3 x 10(-3) mM Cu (calculated intracellular final concentration of the metal injected) and radioactive glucose. Cu and Cd concentration in samples from exposed females were always under detection limit by particle-induced X-ray emission. The results presented here are in agreement with a role for both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities determination as biomarkers of effect and exposure for Cu but not for Cd toxicity.

Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

PubMed

Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2009-12-01

The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

PubMed Central

Guarnieri, Michael T.; Chou, Yat-Chen; Salvachúa, Davinia; Mohagheghi, Ali; St. John, Peter C.; Peterson, Darren J.; Bomble, Yannick J.

2017-01-01

ABSTRACT Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism. PMID:28625987

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guarnieri, Michael T.; Chou, Yat -Chen; Salvachua, Davinia Rodriquez

Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of themore » key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Altogether, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.« less

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

DOE PAGES

Guarnieri, Michael T.; Chou, Yat -Chen; Salvachua, Davinia Rodriquez; ...

2017-06-16

Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of themore » key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Altogether, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.« less

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

PubMed

Lucena, Miguel C; Carvalho-Cruz, Patricia; Donadio, Joana L; Oliveira, Isadora A; de Queiroz, Rafaela M; Marinho-Carvalho, Monica M; Sola-Penna, Mauro; de Paula, Iron F; Gondim, Katia C; McComb, Mark E; Costello, Catherine E; Whelan, Stephen A; Todeschini, Adriane R; Dias, Wagner B

2016-06-17

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrated transcriptome sequencing and dynamic analysis reveal carbon source partitioning between terpenoid and oil accumulation in developing Lindera glauca fruits.

PubMed

Niu, Jun; Chen, Yinlei; An, Jiyong; Hou, Xinyu; Cai, Jian; Wang, Jia; Zhang, Zhixiang; Lin, Shanzhi

2015-10-08

Lindera glauca fruits (LGF) with the abundance of terpenoid and oil has emerged as a novel specific material for industrial and medicinal application in China, but the complex regulatory mechanisms of carbon source partitioning into terpenoid biosynthetic pathway (TBP) and oil biosynthetic pathway (OBP) in developing LGF is still unknown. Here we perform the analysis of contents and compositions of terpenoid and oil from 7 stages of developing LGF to characterize a dramatic difference in temporal accumulative patterns. The resulting 3 crucial samples at 50, 125 and 150 days after flowering (DAF) were selected for comparative deep transcriptome analysis. By Illumina sequencing, the obtained approximately 81 million reads are assembled into 69,160 unigenes, among which 174, 71, 81 and 155 unigenes are implicated in glycolysis, pentose phosphate pathway (PPP), TBP and OBP, respectively. Integrated differential expression profiling and qRT-PCR, we specifically characterize the key enzymes and transcription factors (TFs) involved in regulating carbon allocation ratios for terpenoid or oil accumulation in developing LGF. These results contribute to our understanding of the regulatory mechanisms of carbon source partitioning between terpenoid and oil in developing LGF, and to the improvement of resource utilization and molecular breeding for L. glauca.

The role of glucose-6-phosphate dehydrogenase in adipose tissue inflammation in obesity.

PubMed

Park, Yoon Jeong; Choe, Sung Sik; Sohn, Jee Hyung; Kim, Jae Bum

2017-04-03

Obesity is closely associated with metabolic diseases including type 2 diabetes. One hallmark characteristics of obesity is chronic inflammation that is coordinately controlled by complex signaling networks in adipose tissues. Compelling evidence indicates that reactive oxygen species (ROS) and its related signaling pathways play crucial roles in the progression of chronic inflammation in obesity. The pentose phosphate pathway (PPP) is an anabolic pathway that utilizes the glucoses to generate molecular building blocks and reducing equivalents in the form of NADPH. In particular, NADPH acts as one of the key modulators in the control of ROS through providing an electron for both ROS generation and scavenging. Recently, we have reported that glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the PPP, is implicated in adipose tissue inflammation and systemic insulin resistance in obesity. Mechanistically, G6PD potentiates generation of ROS that augments pro-inflammatory responses in adipose tissue macrophages, leading to systemic insulin resistance. Here, we provide an overview of cell type- specific roles of G6PD in the regulation of ROS balance as well as additional details on the significance of G6PD that contributes to pro-oxidant NADPH generation in obesity-related chronic inflammation and insulin resistance.

Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Yinjie J.; Sapra, Rajat; Joyner, Dominique

2009-01-20

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and istolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (includingmore » the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accuratelydetermined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yieldby approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.« less

Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates.

PubMed

Rosic, Nedeljka N

2012-04-01

Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310-362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

Effects of glucose metabolism pathways on sperm motility and oxidative status during long-term liquid storage of goat semen.

PubMed

Qiu, Jian-Hua; Li, You-Wei; Xie, Hong-Li; Li, Qing; Dong, Hai-Bo; Sun, Ming-Ju; Gao, Wei-Qiang; Tan, Jing-He

2016-08-01

Although great efforts were made to prolong the fertility of liquid-stored semen, limited improvements have been achieved in different species. Although it is expected that energy supply and the redox potential will play an essential role in sperm function, there are few reports on the impact of specific energy substrates on spermatozoa during liquid semen storage. Furthermore, although it is accepted that glucose metabolism through glycolysis provides energy, roles of pentose phosphate pathway (PPP) and tricarboxylic acid cycle remain to be unequivocally found in spermatozoa. We have studied the pathways by which spermatozoa metabolize glucose during long-term liquid storage of goat semen. The results indicated that among the substrates tested, glucose and pyruvate were better than lactate in maintaining goat sperm motility. Although both glycolysis and PPP were essential, PPP was more important than glycolysis to maintain sperm motility. Pentose phosphate pathway reduced oxidative stress and provided glycolysis with more intermediate products such as fructose-6-phosphate. Pyruvate entered goat spermatozoa through monocarboxylate transporters and was oxidized by the tricarboxylic acid cycle and electron transfer to sustain sperm motility. Long-term liquid semen storage can be used as a good model to study sperm glucose metabolism. The data are important for an optimal control of sperm survival during semen handling and preservation not only in the goat but also in other species. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel Insights into the Diversity of Catabolic Metabolism from Ten Haloarchaeal Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Iain; Scheuner, Carmen; Goker, Markus

2011-05-03

The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis. We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles weremore » isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses. These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.« less

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wohlbach, Dana J.; Kuo, Alan; Sato, Trey K.

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes,more » mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.« less

Proteomic analysis of pancreatic cancer stem cells: Functional role of fatty acid synthesis and mevalonate pathways.

PubMed

Brandi, Jessica; Dando, Ilaria; Pozza, Elisa Dalla; Biondani, Giulia; Jenkins, Rosalind; Elliott, Victoria; Park, Kevin; Fanelli, Giuseppina; Zolla, Lello; Costello, Eithne; Scarpa, Aldo; Cecconi, Daniela; Palmieri, Marta

2017-01-06

Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells. Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability. To better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

Preferential Use of Central Metabolism In Vivo Reveals a Nutritional Basis for Polymicrobial Infection

PubMed Central

Alteri, Christopher J.; Himpsl, Stephanie D.; Mobley, Harry L. T.

2015-01-01

The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche. PMID:25568946

Hydrothermal pentose to furfural conversion and simultaneous extraction with SC-CO2--kinetics and application to biomass hydrolysates.

PubMed

Gairola, Krishan; Smirnova, Irina

2012-11-01

This work explores hydrothermal d-xylose and hemicellulose to furfural conversion coupled with simultaneous furfural extraction by SC-CO(2) and the underlying reaction pathway. A maximum furfural yield of 68% was attained from d-xylose at 230°C and 12MPa. Additionally missing kinetic data for l-arabinose to furfural conversion was provided, showing close similarity to d-xylose. Furfural yields from straw and brewery waste hydrolysates were significantly lower than those obtained from model compounds, indicating side reactions with other hydrolysate components. Simultaneous furfural extraction by SC-CO(2) significantly increased extraction yield in all cases. The results indicate that furfural reacts with intermediates of pentose dehydration. The proposed processing route can be well integrated into existing lignocellulose biorefinery concepts. Copyright © 2012 Elsevier Ltd. All rights reserved.

Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114

PubMed Central

Tang, Kuo-Hsiang; Feng, Xueyang; Tang, Yinjie J.; Blankenship, Robert E.

2009-01-01

The Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO2, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO2 assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B12 (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of 13C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10–15% of protein carbon from CO2; (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source. PMID:19794911

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse

PubMed Central

Chao, Yu-Hsin; Giagtzoglou, Nikolaos; Putluri, Nagireddy; Coarfa, Cristian; Donti, Taraka; Faust, Joseph E.; McNew, James A.; Sardiello, Marco; Baes, Myriam; Bellen, Hugo J.

2017-01-01

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD. PMID:28640802

Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons.

PubMed

Brekke, Eva M F; Walls, Anne B; Schousboe, Arne; Waagepetersen, Helle S; Sonnewald, Ursula

2012-09-01

The brain is highly susceptible to oxidative injury, and the pentose phosphate pathway (PPP) has been shown to be affected by pathological conditions, such as Alzheimer's disease and traumatic brain injury. While this pathway has been investigated in the intact brain and in astrocytes, little is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-(13)C]glucose or [3-(13)C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of (13)C-labeled glucose via the PPP does not appear to contribute to the production of releasable lactate, it contributes to labeling of tricarboxylic acid (TCA) cycle intermediates and related amino acids. Based on glutamate isotopomers, it was calculated that PPP activity accounts for ~6% of glucose metabolism in cortical neurons and ~4% in cerebellar neurons. This is the first demonstration that pyruvate generated from glucose via the PPP contributes to the synthesis of acetyl CoA for oxidation in the TCA cycle. Moreover, the fact that (13)C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons.

Inhibition of the pentose phosphate pathway by dichloroacetate unravels a missing link between aerobic glycolysis and cancer cell proliferation.

PubMed

De Preter, Géraldine; Neveu, Marie-Aline; Danhier, Pierre; Brisson, Lucie; Payen, Valéry L; Porporato, Paolo E; Jordan, Bénédicte F; Sonveaux, Pierre; Gallez, Bernard

2016-01-19

Glucose fermentation through glycolysis even in the presence of oxygen (Warburg effect) is a common feature of cancer cells increasingly considered as an enticing target in clinical development. This study aimed to analyze the link between metabolism, energy stores and proliferation rates in cancer cells. We found that cell proliferation, evaluated by DNA synthesis quantification, is correlated to glycolytic efficiency in six cancer cell lines as well as in isogenic cancer cell lines. To further investigate the link between glycolysis and proliferation, a pharmacological inhibitor of the pentose phosphate pathway (PPP) was used. We demonstrated that reduction of PPP activity decreases cancer cells proliferation, with a profound effect in Warburg-phenotype cancer cells. The crucial role of the PPP in sustaining cancer cells proliferation was confirmed using siRNAs against glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the PPP. In addition, we found that dichloroacetate (DCA), a new clinically tested compound, induced a switch of glycolytic cancer cells to a more oxidative phenotype and decreased proliferation. By demonstrating that DCA decreased the activity of the PPP, we provide a new mechanism by which DCA controls cancer cells proliferation.

Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances

PubMed Central

Kilian, Stephanus G.; du Preez, James C.

2017-01-01

The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production. PMID:28542187

Culture conditions supporting inhibitor tolerance and rapid production of ethanol by P. stipitis NRRL Y-7124

USDA-ARS?s Scientific Manuscript database

To expand the biomass to fuel ethanol industry, process strategies are needed to foster the production and utilization of microorganisms which can survive and ferment hexose and pentose sugars while exposed to inhibitors (such as ethanol, furfural, and hydroxymethylfurfural (HMF)). Furfural and HMF...

How does metabolism affect cell death in cancer?

PubMed

Villa, Elodie; Ricci, Jean-Ehrland

2016-07-01

In cancer research, identifying a specificity of tumor cells compared with 'normal' proliferating cells for targeted therapy is often considered the Holy Grail for researchers and clinicians. Although diverse in origin, most cancer cells share characteristics including the ability to escape cell death mechanisms and the utilization of different methods of energy production. In the current paradigm, aerobic glycolysis is considered the central metabolic characteristic of cancer cells (Warburg effect). However, recent data indicate that cancer cells also show significant changes in other metabolic pathways. Indeed, it was recently suggested that Kreb's cycle, pentose phosphate pathway intermediates, and essential and nonessential amino acids have key roles. Renewed interest in the fact that cancer cells have to reprogram their metabolism in order to proliferate or resist treatment must take into consideration the ability of tumor cells to adapt their metabolism to the local microenvironment (low oxygen, low nutrients). This variety of metabolic sources might be either a strength, resulting in infinite possibilities for adaptation and increased ability to resist chemotherapy-induced death, or a weakness that could be targeted to kill cancer cells. Here, we discuss recent insights showing how energetic metabolism may regulate cell death and how this might be relevant for cancer treatment. © 2015 FEBS.

Utilization of xylose for growth by the eukaryotic alga, Chlorella.

PubMed

Hawkins, R L

1999-06-01

A green alga, Chlorella, was found to be capable of utilizing xylose or other pentose sugars (xylitol, arabinose) for enhanced growth rates when grown in the light, but not when grown heterotrophically in the dark. With selection for growth in xylose-containing medium, it was possible to improve dramatically the ability of selected Chlorella strains to grow on xylose mixotrophically. Growth on arabinose or xylitol was not changed in the xylose-selected strains.

Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

PubMed

Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

2015-10-23

The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux*

PubMed Central

Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A. M.; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric

2013-01-01

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host. PMID:23665470

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

PubMed Central

Stincone, Anna; Prigione, Alessandro; Cramer, Thorsten; Wamelink, Mirjam M. C.; Campbell, Kate; Cheung, Eric; Olin-Sandoval, Viridiana; Grüning, Nana-Maria; Krüger, Antje; Alam, Mohammad Tauqeer; Keller, Markus A.; Breitenbach, Michael; Brindle, Kevin M.; Rabinowitz, Joshua D.; Ralser, Markus

2015-01-01

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism. PMID:25243985

Ethanol production from lignocellulosic biomass by recombinant Escherichia coli strain FBR5

PubMed Central

Saha, Badal; Cotta, Michael A.

2012-01-01

Lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of hexose and pentose sugars such as glucose, xylose, arabinose and galactose. While Escherichia coli utilizes all these sugars it lacks the ability to produce ethanol from them. Recombinant ethanologenic E. coli strains have been created with a goal to produce ethanol from both hexose and pentose sugars. Herein, we review the current state of the art on the production of ethanol from lignocellulosic hydrolyzates by an ethanologenic recombinant E. coli strain (FBR5). The bacterium is stable without antibiotics and can tolerate ethanol up to 50 gL-1. It produces up to 45 g ethanol per L and has the potential to be used for industrial production of ethanol from lignocellulosic hydrolyzates. PMID:22705843

A transferable plasticity region in Campylobacter coli allows isolates of an otherwise non-glycolytic food-borne pathogen to catabolize glucose.

PubMed

Vorwerk, Hanne; Huber, Claudia; Mohr, Juliane; Bunk, Boyke; Bhuju, Sabin; Wensel, Olga; Spröer, Cathrin; Fruth, Angelika; Flieger, Antje; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar; Eisenreich, Wolfgang; Hofreuter, Dirk

2015-12-01

Thermophilic Campylobacter species colonize the intestine of agricultural and domestic animals commensally but cause severe gastroenteritis in humans. In contrast to other enteropathogenic bacteria, Campylobacter has been considered to be non-glycolytic, a metabolic property originally used for their taxonomic classification. Contrary to this dogma, we demonstrate that several Campylobacter coli strains are able to utilize glucose as a growth substrate. Isotopologue profiling experiments with (13) C-labeled glucose suggested that these strains catabolize glucose via the pentose phosphate and Entner-Doudoroff (ED) pathways and use glucose efficiently for de novo synthesis of amino acids and cell surface carbohydrates. Whole genome sequencing of glycolytic C. coli isolates identified a genomic island located within a ribosomal RNA gene cluster that encodes for all ED pathway enzymes and a glucose permease. We could show in vitro that a non-glycolytic C. coli strain could acquire glycolytic activity through natural transformation with chromosomal DNA of C. coli and C. jejuni subsp. doylei strains possessing the ED pathway encoding plasticity region. These results reveal for the first time the ability of a Campylobacter species to catabolize glucose and provide new insights into how genetic macrodiversity through intra- and interspecies gene transfer expand the metabolic capacity of this food-borne pathogen. © 2015 John Wiley & Sons Ltd.

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

PubMed Central

2015-01-01

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity. PMID:24555535

Production of a yeast artificial chromosome for stable expression of a synthetic xylose isomerase-xylulokinase polyprotein in a fuel ethanol yeast strain

USDA-ARS?s Scientific Manuscript database

Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. A yeast artificial chromosome (YAC) was engineered to contain a polyprotein gene construct expressing xylos...

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

PubMed

Nijland, Jeroen G; Shin, Hyun Yong; Boender, Leonie G M; de Waal, Paul P; Klaassen, Paul; Driessen, Arnold J M

2017-06-01

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8 , or SSN6 , which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10 , HXT13 , HXT15 , and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism ( V max ) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell. IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast. Copyright © 2017 American Society for Microbiology.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

PubMed

Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

PubMed Central

Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons

PubMed Central

Brekke, Eva M F; Walls, Anne B; Schousboe, Arne; Waagepetersen, Helle S; Sonnewald, Ursula

2012-01-01

The brain is highly susceptible to oxidative injury, and the pentose phosphate pathway (PPP) has been shown to be affected by pathological conditions, such as Alzheimer's disease and traumatic brain injury. While this pathway has been investigated in the intact brain and in astrocytes, little is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-13C]glucose or [3-13C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of 13C-labeled glucose via the PPP does not appear to contribute to the production of releasable lactate, it contributes to labeling of tricarboxylic acid (TCA) cycle intermediates and related amino acids. Based on glutamate isotopomers, it was calculated that PPP activity accounts for ∼6% of glucose metabolism in cortical neurons and ∼4% in cerebellar neurons. This is the first demonstration that pyruvate generated from glucose via the PPP contributes to the synthesis of acetyl CoA for oxidation in the TCA cycle. Moreover, the fact that 13C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons. PMID:22714050

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium.

PubMed

Smiri, Moêz; Chaoui, Abdelilah; El Ferjani, Ezzedine

2009-02-15

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production

PubMed Central

Li, Hongxing; Wu, Meiling; Xu, Lili; Hou, Jin; Guo, Ting; Bao, Xiaoming; Shen, Yu

2015-01-01

To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild-type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65 h−1 in yeast extract peptone dextrose medium, and the ethanol yield was 0.45 g g−1 consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production. PMID:25616171

The oxidized form of vitamin C, dehydroascorbic acid, regulates neuronal energy metabolism.

PubMed

Cisternas, Pedro; Silva-Alvarez, Carmen; Martínez, Fernando; Fernandez, Emilio; Ferrada, Luciano; Oyarce, Karina; Salazar, Katterine; Bolaños, Juan P; Nualart, Francisco

2014-05-01

Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time- and dose-dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes. © 2014 International Society for Neurochemistry.

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

PubMed

Jortzik, Esther; Mailu, Boniface M; Preuss, Janina; Fischer, Marina; Bode, Lars; Rahlfs, Stefan; Becker, Katja

2011-06-15

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

USDA-ARS?s Scientific Manuscript database

Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI...

Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia.

PubMed

Imahori, Taichiro; Hosoda, Kohkichi; Nakai, Tomoaki; Yamamoto, Yusuke; Irino, Yasuhiro; Shinohara, Masakazu; Sato, Naoko; Sasayama, Takashi; Tanaka, Kazuhiro; Nagashima, Hiroaki; Kohta, Masaaki; Kohmura, Eiji

2017-05-04

The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD + ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells*

PubMed Central

Shestov, Alexander A.; Mancuso, Anthony; Lee, Seung-Cheol; Guo, Lili; Nelson, David S.; Roman, Jeffrey C.; Henry, Pierre-Gilles; Leeper, Dennis B.; Blair, Ian A.; Glickson, Jerry D.

2016-01-01

A network model for the determination of tumor metabolic fluxes from 13C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-13C2]glucose under normoxic conditions at 37 °C and monitored by 13C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism. PMID:26703469

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides

PubMed Central

McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

2014-01-01

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain. PMID:24169853

Carbon Metabolism in Spores of the Arbuscular Mycorrhizal Fungus Glomus intraradices as Revealed by Nuclear Magnetic Resonance Spectroscopy1

PubMed Central

Bago, Berta; Pfeffer, Philip E.; Douds, David D.; Brouillette, Janine; Bécard, Guillaume; Shachar-Hill, Yair

1999-01-01

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments. Understanding why AM fungi do not complete their life cycle under free-living conditions has significant implications for the management of one of the world's most important symbioses. We used 13C-labeled substrates and nuclear magnetic resonance spectroscopy to study carbon fluxes during spore germination and the metabolic pathways by which these fluxes occur in the AM fungus Glomus intraradices. Our results indicate that during asymbiotic growth: (a) sugars are made from stored lipids; (b) trehalose (but not lipid) is synthesized as well as degraded; (c) glucose and fructose, but not mannitol, can be taken up and utilized; (d) dark fixation of CO2 is substantial; and (e) arginine and other amino acids are synthesized. The labeling patterns are consistent with significant carbon fluxes through gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, glycolysis, non-photosynthetic one-carbon metabolism, the pentose phosphate pathway, and most or all of the urea cycle. We also report the presence of an unidentified betaine-like compound. Carbon metabolism during asymbiotic growth has features in between those presented by intraradical and extraradical hyphae in the symbiotic state. PMID:10482682

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides.

PubMed

McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

2014-01-01

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain.

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.

PubMed

Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

2016-02-25

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

PubMed Central

Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

2016-01-01

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review. PMID:26927067

Enzymes of Glucose Catabolism in a Member of the Psittacosis Group

PubMed Central

Moulder, James W.; Grisso, Dorothy L.; Brubaker, Robert R.

1965-01-01

Moulder, James W. (University of Chicago, Chicago, Ill.), Dorothy L. Grisso, and Robert R. Brubaker. Enzymes of glucose catabolism in a member of the psittacosis group. J. Bacteriol. 89:810–812. 1965.—Extracts of preparations of the agent of meningopneumonitis made from infected chick-embryo allantoic fluid contained three enzymes of the pentose pathway of glucose degradation: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphoglucose isomerase. Vertical starch-gel electrophoresis showed that the two dehydrogenases were qualitatively different from the corresponding enzymes of the host. Enzymes of the Embden-Meyerhof and Entner-Doudoroff pathways were not found. Images PMID:14273665

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.

1996-05-07

The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 2 figs.

Recombinant zymomonas for pentose fermentation

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark

1996-01-01

The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Review: Metabolic Control of Immune System Activation in Rheumatic Diseases.

PubMed

Perl, Andras

2017-12-01

Metabolic pathways mediate lineage specification within the immune system through the regulation of glucose utilization, a process that generates energy in the form of ATP and synthesis of amino acids, nucleotides, and lipids to enable cell growth, proliferation, and survival. CD4+ T cells, a proinflammatory cell subset, preferentially produce ATP through glycolysis, whereas cells with an antiinflammatory lineage, such as memory and regulatory T cells, favor mitochondrial ATP generation. In conditions of metabolic stress or a shortage of nutrients, cells rely on autophagy to secure amino acids and other substrates, while survival depends on the sparing of mitochondria and maintenance of a reducing environment. The pentose phosphate pathway acts as a key gatekeeper of inflammation by supplying ribose-5-phosphate for cell proliferation and NADPH for antioxidant defenses. Increased lysosomal catabolism, accumulation of branched amino acids, glutamine, kynurenine, and histidine, and depletion of glutathione and cysteine activate the mechanistic target of rapamycin (mTOR), an arbiter of lineage development within the innate and adaptive immune systems. Mapping the impact of susceptibility genes to metabolic pathways allows for better understanding and therapeutic targeting of disease-specific expansion of proinflammatory cells. Therapeutic approaches aimed at glutathione depletion and mTOR pathway activation appear to be safe and effective for treating lupus, while an opposing intervention may be of benefit in rheumatoid arthritis. Environmental sources of origin for metabolites within immune cells may include microbiota and plants. Thus, a better understanding of the pathways of immunometabolism could provide new insights into the pathogenesis and treatment of the rheumatic diseases. © 2017 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.

Proteome Analyses of Hydrogen-producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1 in Different One-carbon Substrate Culture Conditions*

PubMed Central

Moon, Yoon-Jung; Kwon, Joseph; Yun, Sung-Ho; Lim, Hye Li; Kim, Min-Sik; Kang, Sung Gyun; Lee, Jung-Hyun; Choi, Jong-Soon; Kim, Seung Il; Chung, Young-Ho

2012-01-01

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, is capable of H2-producing growth, considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate can be used as its carbon source remains unknown. To obtain a global view of the metabolic characteristics of H2-producing growth, a quantitative proteome analysis of T. onnurineus NA1 grown on formate, CO, and starch was performed by combining one-dimensional SDS-PAGE with nano UPLC-MSE. A total of 587 proteins corresponding to 29.7% of the encoding genes were identified, and the major metabolic pathways (especially energy metabolism) were characterized at the protein level. Expression of glycolytic enzymes was common but more highly induced in starch-grown cells. In contrast, enzymes involved in key steps of the gluconeogenesis and pentose phosphate pathways were strongly up-regulated in formate-grown cells, suggesting that formate could be utilized as a carbon source by T. onnurineus NA1. In accordance with the genomic analysis, comprehensive proteomic analysis also revealed a number of hydrogenase clusters apparently associated with formate metabolism. On the other hand, CODH and CO-induced hydrogenases belonging to the Hyg4-II cluster, as well as sulfhydrogenase-I and Mbx, were prominently expressed during CO culture. Our data suggest that CO can be utilized as a sole energy source for H2 production via an electron transport mechanism and that CO2 produced from catabolism or CO oxidation by CODH and CO-induced hydrogenases may subsequently be assimilated into the organic carbon. Overall, proteomic comparison of formate- and CO-grown cells with starch-grown cells revealed that a single carbon compound, such as formate and CO, can be utilized as an efficient substrate to provide cellular carbon and/or energy by T. onnurineus NA1. PMID:22232491

Fructose Alters Intermediary Metabolism of Glucose in Human Adipocytes and Diverts Glucose to Serine Oxidation in the One–Carbon Cycle Energy Producing Pathway

PubMed Central

Varma, Vijayalakshmi; Boros, László G.; Nolen, Greg T.; Chang, Ching-Wei; Wabitsch, Martin; Beger, Richard D.; Kaput, Jim

2015-01-01

Increased consumption of sugar and fructose as sweeteners has resulted in the utilization of fructose as an alternative metabolic fuel that may compete with glucose and alter its metabolism. To explore this, human Simpson-Golabi-Behmel Syndrome (SGBS) preadipocytes were differentiated to adipocytes in the presence of 0, 1, 2.5, 5 or 10 mM of fructose added to a medium containing 5 mM of glucose representing the normal blood glucose concentration. Targeted tracer [1,2-13C2]-d-glucose fate association approach was employed to examine the influence of fructose on the intermediary metabolism of glucose. Increasing concentrations of fructose robustly increased the oxidation of [1,2-13C2]-d-glucose to 13CO2 (p < 0.000001). However, glucose-derived 13CO2 negatively correlated with 13C labeled glutamate, 13C palmitate, and M+1 labeled lactate. These are strong markers of limited tricarboxylic acid (TCA) cycle, fatty acid synthesis, pentose cycle fluxes, substrate turnover and NAD+/NADP+ or ATP production from glucose via complete oxidation, indicating diminished mitochondrial energy metabolism. Contrarily, a positive correlation was observed between glucose-derived 13CO2 formed and 13C oleate and doses of fructose which indicate the elongation and desaturation of palmitate to oleate for storage. Collectively, these results suggest that fructose preferentially drives glucose through serine oxidation glycine cleavage (SOGC pathway) one-carbon cycle for NAD+/NADP+ production that is utilized in fructose-induced lipogenesis and storage in adipocytes. PMID:26087138

3-Bromopyruvate treatment induces alterations of metabolic and stress-related pathways in glioblastoma cells.

PubMed

Chiasserini, Davide; Davidescu, Magdalena; Orvietani, Pier Luigi; Susta, Federica; Macchioni, Lara; Petricciuolo, Maya; Castigli, Emilia; Roberti, Rita; Binaglia, Luciano; Corazzi, Lanfranco

2017-01-30

Glioblastoma (GBM) is the most common and aggressive brain tumour of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Our results show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. Alteration of the glycolytic pathway characterizes glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We found that 3BP affected not only the glycolytic pathway, but also pathways sharing metabolic intermediates with glycolysis, such as the pentose phosphate pathway and aminoacid metabolism. Furthermore, changes in the expression of proteins linked to resistance to cell death and stress response were found. Our work is the first analysis on a global scale of the proteome changes induced by 3BP in a GBM model and may contribute to clarifying the anticancer potential of this drug. Copyright © 2016 Elsevier B.V. All rights reserved.

Aerobic glycolysis during brain activation: adrenergic regulation and influence of norepinephrine on astrocytic metabolism.

PubMed

Dienel, Gerald A; Cruz, Nancy F

2016-07-01

Aerobic glycolysis occurs during brain activation and is characterized by preferential up-regulation of glucose utilization compared with oxygen consumption even though oxygen level and delivery are adequate. Aerobic glycolysis is a widespread phenomenon that underlies energetics of diverse brain activities, such as alerting, sensory processing, cognition, memory, and pathophysiological conditions, but specific cellular functions fulfilled by aerobic glycolysis are poorly understood. Evaluation of evidence derived from different disciplines reveals that aerobic glycolysis is a complex, regulated phenomenon that is prevented by propranolol, a non-specific β-adrenoceptor antagonist. The metabolic pathways that contribute to excess utilization of glucose compared with oxygen include glycolysis, the pentose phosphate shunt pathway, the malate-aspartate shuttle, and astrocytic glycogen turnover. Increased lactate production by unidentified cells, and lactate dispersal from activated cells and lactate release from the brain, both facilitated by astrocytes, are major factors underlying aerobic glycolysis in subjects with low blood lactate levels. Astrocyte-neuron lactate shuttling with local oxidation is minor. Blockade of aerobic glycolysis by propranolol implicates adrenergic regulatory processes including adrenal release of epinephrine, signaling to brain via the vagus nerve, and increased norepinephrine release from the locus coeruleus. Norepinephrine has a powerful influence on astrocytic metabolism and glycogen turnover that can stimulate carbohydrate utilization more than oxygen consumption, whereas β-receptor blockade 're-balances' the stoichiometry of oxygen-glucose or -carbohydrate metabolism by suppressing glucose and glycogen utilization more than oxygen consumption. This conceptual framework may be helpful for design of future studies to elucidate functional roles of preferential non-oxidative glucose utilization and glycogen turnover during brain activation. Aerobic glycolysis, the preferential up-regulation of glucose utilization (CMRglc ) compared with oxygen consumption (CMRO2 ) during brain activation, is blocked by propranolol. Epinephrine release from the adrenal gland stimulates vagus nerve signaling to the locus coeruleus, enhancing norepinephrine release in the brain, and regulation of astrocytic and neuronal metabolism to stimulate CMRglc more than CMRO2 . Propranolol suppresses CMRglc more than CMRO2 . © 2016 International Society for Neurochemistry.

LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depuydt, Geert G.; Xie, Fang; Petyuk, Vladislav A.

2014-02-20

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity and metabolism in C. elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass-spectrometry (LC-MS) based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2); daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the up-regulation of many core intermediarymore » metabolic pathways. These include, glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complex I, II, III and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative for spatio-temporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves, possibly also shunting metabolites through alternative energy-generating pathways, in order to sustain longevity.« less

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.

2014-04-04

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediarymore » metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. Finally, this restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.« less

Low-energy collisionally activated dissociation of pentose-borate complexes

NASA Astrophysics Data System (ADS)

Pepi, Federico; Garzoli, Stefania; Tata, Alessandra; Giacomello, Pierluigi

2010-01-01

Pentose-borate 1:1 complexes were generated in the ESI source of a triple quadrupole and ion trap mass spectrometer by electrospray ionization of Na2B4O7 and pentose (arabinose, lyxose, ribose, xylose) 2:1 solution in CH3CN/H2O. The study of their low-energy collisionally activated dissociation (CAD) demonstrated that ribose and lyxose are preferentially complexed at the C2-C3 cis-diol function whereas arabinose and xylose are esterified at the C1-C2 hydroxyl groups. No evidence was found of the stronger affinity for ribose to borate. The ribose probiotic rule can be explained by considering its peculiar capability, among the investigated pentoses, to almost totally complex the borate anion at the C2-C3 hydroxyl group, thus enabling the subsequent stages of nucleotide assembly, such as phosphorylation and linkage to the nucleobases. Finally, the differences observed in the pentose-borate complex CAD spectra can be used for the mass spectrometric discrimination of isomeric pentoses in complex mixtures.

Melatonin Decreases Glucose Metabolism in Prostate Cancer Cells: A 13C Stable Isotope-Resolved Metabolomic Study.

PubMed

Hevia, David; Gonzalez-Menendez, Pedro; Fernandez-Fernandez, Mario; Cueto, Sergio; Rodriguez-Gonzalez, Pablo; Garcia-Alonso, Jose I; Mayo, Juan C; Sainz, Rosa M

2017-07-26

The pineal neuroindole melatonin exerts an exceptional variety of systemic functions. Some of them are exerted through its specific membrane receptors type 1 and type 2 (MT1 and MT2) while others are mediated by receptor-independent mechanisms. A potential transport of melatonin through facilitative glucose transporters (GLUT/ SLC2A ) was proposed in prostate cancer cells. The prostate cells have a particular metabolism that changes during tumor progression. During the first steps of carcinogenesis, oxidative phosphorylation is reactivated while the switch to the "Warburg effect" only occurs in advanced tumors and in the metastatic stage. Here, we investigated whether melatonin might change prostate cancer cell metabolism. To do so, 13 C stable isotope-resolved metabolomics in androgen sensitive LNCaP and insensitive PC-3 prostate cancer cells were employed. In addition to metabolite 13 C-labeling, ATP/AMP levels, and lactate dehydrogenase or pentose phosphate pathway activity were measured. Melatonin reduces lactate labeling in androgen-sensitive cells and it also lowers 13 C-labeling of tricarboxylic acid cycle metabolites and ATP production. In addition, melatonin reduces lactate 13 C-labeling in androgen insensitive prostate cancer cells. Results demonstrated that melatonin limits glycolysis as well as the tricarboxylic acid cycle and pentose phosphate pathway in prostate cancer cells, suggesting that the reduction of glucose uptake is a major target of the indole in this tumor type.

Metabolic Engineering of Fusarium oxysporum to Improve Its Ethanol-Producing Capability.

PubMed

Anasontzis, George E; Kourtoglou, Elisavet; Villas-Boâs, Silas G; Hatzinikolaou, Dimitris G; Christakopoulos, Paul

2016-01-01

Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharomyces cerevisiae is used-namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast-its low growth rate and low ethanol productivity-hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the γ-aminobutyric acid shunt, and an excess in NADPH regeneration.

Pentose Phosphate Pathway Function Affects Tolerance to the G-Quadruplex Binder TMPyP4

PubMed Central

Andrew, Elizabeth J.; Merchan, Stephanie; Lawless, Conor; Banks, A. Peter; Wilkinson, Darren J.; Lydall, David

2013-01-01

G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4. PMID:23776642

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

PubMed Central

Liu, Huaiwei; Sun, Yuanzhang; Ramos, Kristine Rose M.; Nisola, Grace M.; Valdehuesa, Kris Niño G.; Lee, Won–Keun; Park, Si Jae; Chung, Wook-Jin

2013-01-01

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways. PMID:24376679

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

PubMed Central

Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

2008-01-01

Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively. Conclusion The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme. PMID:18947407

Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803.

PubMed

Choi, Yun-Nam; Park, Jong Moon

2016-08-01

This study demonstrates that increased NADPH production can improve biomass and ethanol production in cyanobacteria. We over-expressed the endogenous zwf gene, which encodes glucose-6-phosphate dehydrogenase of pentose phosphate pathway, in the model cyanobacterium Synechocystis sp. PCC 6803. zwf over-expression resulted in increased NADPH production, and promoted biomass production compared to the wild type in both autotrophic and mixotrophic conditions. Ethanol production pathway including NADPH-dependent alcohol dehydrogenase was also integrated with and without zwf over-expression. Excessive NADPH production by zwf over-expression could improve both biomass and ethanol production in the autotrophic conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Warburg effect: persistence of stem-cell metabolism in cancers as a failure of differentiation.

PubMed

Riester, M; Xu, Q; Moreira, A; Zheng, J; Michor, F; Downey, R J

2018-01-01

Two recent observations regarding the Warburg effect are that (i) the metabolism of stem cells is constitutive (aerobic) glycolysis while normal cellular differentiation involves a transition to oxidative phosphorylation and (ii) the degree of glucose uptake of a malignancy as imaged by 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) is associated with histologic measures of tumor differentiation. Combining these observations, we hypothesized that the high levels of glucose uptake observed in poorly differentiated cancers may reflect persistence of the glycolytic metabolism of stem cells in malignant cells that fail to fully differentiate. Tumor glucose uptake was measured by FDG-PET in 552 patients with histologically diverse cancers. We used normal mixture modeling to explore FDG-PET standardized uptake value (SUV) distributions and tested for associations between glucose uptake and histological differentiation, risk of lymph node metastasis, and survival. Using RNA-seq data, we carried out pathway and transcription factor analyses to compare tumors with high and low levels of glucose uptake. We found that well-differentiated tumors had low FDG uptake, while moderately and poorly differentiated tumors had higher uptake. The distribution of SUV for each histology was bimodal, with a low peak around SUV 2-5 and a high peak at SUV 8-14. The cancers in the two modes were clinically distinct in terms of the risk of nodal metastases and death. Carbohydrate metabolism and the pentose-related pathway were elevated in the poorly differentiated/high SUV clusters. Embryonic stem cell-related signatures were activated in poorly differentiated/high SUV clusters. Our findings support the hypothesis that the biological basis for the Warburg effect is a persistence of stem cell metabolism (i.e. aerobic glycolysis) in cancers as a failure to transition from glycolysis-utilizing undifferentiated cells to oxidative phosphorylation-utilizing differentiated cells. We found that cancers cluster along the differentiation pathway into two groups, utilizing either glycolysis or oxidative phosphorylation. Our results have implications for multiple areas of clinical oncology. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Metabolomic and Proteomic Insights into Carbaryl Catabolism by Burkholderia sp. C3 and Degradation of Ten N-Methylcarbamates

PubMed Central

Seo, Jong-Su; Keum, Young-Soo; Li, Qing X.

2013-01-01

Burkholderia sp. C3, an efficient polycyclic aromatic hydrocarbon (PAH) degrader, can utilize 9 of the 10 N-methylcarbamate insecticides including carbaryl as a sole source of carbon. Rapid hydrolysis of carbaryl in C3 is followed by slow catabolism of the resulting 1-naphthol. This study focused on metabolomes and proteomes in C3 cells utilizing carbaryl in comparison to those using glucose or nutrient broth. Sixty of the 867 detected proteins were involved in primary metabolism, adaptive sensing and regulation, transport, stress response, and detoxification. Among the 41 proteins expressed in response to carbaryl were formate dehydrogenase, aldehyde-alcohol dehydrogenase and ethanolamine utilization protein involved in one carbon metabolism. Acetate kinase and phasin were 2 of the 19 proteins that were not detected in carbaryl-supported C3 cells, but detected in glucose-supported C3 cells. Down-production of phasin and polyhydroxyalkanoates in carbaryl-supported C3 cells suggests insufficient carbon sources and lower levels of primary metabolites to maintain an ordinary level of metabolism. Differential metabolomes (approximately 196 identified polar metabolites) showed up-production of metabolites in pentose phosphate pathways and metabolisms of cysteine, cystine and some other amino acids, disaccharides and nicotinate, in contract to down-production of most of the other amino acids and hexoses. The proteomic and metabolomic analyses showed that carbaryl-supported C3 cells experienced strong toxic effects, oxidative stresses, DNA/RNA damages and carbon nutrient deficiency. PMID:23463356

Glycolysis and the pentose phosphate pathway after human traumatic brain injury: microdialysis studies using 1,2-13C2 glucose

PubMed Central

Jalloh, Ibrahim; Carpenter, Keri L H; Grice, Peter; Howe, Duncan J; Mason, Andrew; Gallagher, Clare N; Helmy, Adel; Murphy, Michael P; Menon, David K; Carpenter, T Adrian; Pickard, John D; Hutchinson, Peter J

2015-01-01

Increased ‘anaerobic' glucose metabolism is observed after traumatic brain injury (TBI) attributed to increased glycolysis. An alternative route is the pentose phosphate pathway (PPP), which generates putatively protective and reparative molecules. To compare pathways we employed microdialysis to perfuse 1,2-13C2 glucose into the brains of 15 TBI patients and macroscopically normal brain in six patients undergoing surgery for benign tumors, and to simultaneously collect products for nuclear magnetic resonance (NMR) analysis. 13C enrichment for glycolytic 2,3-13C2 lactate was the median 5.4% (interquartile range (IQR) 4.6–7.5%) in TBI brain and 4.2% (2.4–4.4%) in ‘normal' brain (P<0.01). The ratio of PPP-derived 3-13C lactate to glycolytic 2,3-13C2 lactate was median 4.9% (3.6–8.2%) in TBI brain and 6.7% (6.3–8.9%) in ‘normal' brain. An inverse relationship was seen for PPP-glycolytic lactate ratio versus PbtO2 (r=−0.5, P=0.04) in TBI brain. Thus, glycolytic lactate production was significantly greater in TBI than ‘normal' brain. Several TBI patients exhibited PPP–lactate elevation above the ‘normal' range. There was proportionally greater PPP-derived lactate production with decreasing PbtO2. The study raises questions about the roles of the PPP and glycolysis after TBI, and whether they can be manipulated to achieve a better outcome. This study is the first direct comparison of glycolysis and PPP in human brain. PMID:25335801

Astroglial pentose phosphate pathway rates in response to high-glucose environments

PubMed Central

Takahashi, Shinichi; Izawa, Yoshikane; Suzuki, Norihiro

2012-01-01

ROS (reactive oxygen species) play an essential role in the pathophysiology of diabetes, stroke and neurodegenerative disorders. Hyperglycaemia associated with diabetes enhances ROS production and causes oxidative stress in vascular endothelial cells, but adverse effects of either acute or chronic high-glucose environments on brain parenchymal cells remain unclear. The PPP (pentose phosphate pathway) and GSH participate in a major defence mechanism against ROS in brain, and we explored the role and regulation of the astroglial PPP in response to acute and chronic high-glucose environments. PPP activity was measured in cultured neurons and astroglia by determining the difference in rate of 14CO2 production from [1-14C]glucose and [6-14C]glucose. ROS production, mainly H2O2, and GSH were also assessed. Acutely elevated glucose concentrations in the culture media increased PPP activity and GSH level in astroglia, decreasing ROS production. Chronically elevated glucose environments also induced PPP activation. Immunohistochemical analyses revealed that chronic high-glucose environments induced ER (endoplasmic reticulum) stress (presumably through increased hexosamine biosynthetic pathway flux). Nuclear translocation of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), which regulates G6PDH (glyceraldehyde-6-phosphate dehydrogenase) by enhancing transcription, was also observed in association with BiP (immunoglobulin heavy-chain-binding protein) expression. Acute and chronic high-glucose environments activated the PPP in astroglia, preventing ROS elevation. Therefore a rapid decrease in glucose level seems to enhance ROS toxicity, perhaps contributing to neural damage when insulin levels given to diabetic patients are not properly calibrated and plasma glucose levels are not adequately maintained. These findings may also explain the lack of evidence for clinical benefits from strict glycaemic control during the acute phase of stroke. PMID:22300409

Monitoring flux through the oxidative pentose phosphate pathway using [1-14C]gluconate.

PubMed

Garlick, Andrew P; Moore, Catherine; Kruger, Nicholas J

2002-12-01

The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh. Release of (14)CO(2) from [1-(14)C]gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component. At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells. There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either [1-(14)C]glucose or [6-(14)C]glucose by the culture. The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase. First, more than 95% of the label released from [1-(14)C]gluconate during metabolism by the cell culture was recovered as (14)CO(2). Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from [1-(14)C]gluconate relative to that from [1-(14)C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from [1-(14)C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from [1-(14)C]gluconate to a far greater extent than that from [1-(14)C]glucose. It is proposed that measurement of (14)CO(2) from [1-(14)C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.

Metabolic adaptation of Mycobacterium avium subsp. paratuberculosis to the gut environment.

PubMed

Weigoldt, Mathias; Meens, Jochen; Bange, Franz-Christoph; Pich, Andreas; Gerlach, Gerald F; Goethe, Ralph

2013-02-01

Knowledge on the proteome level about the adaptation of pathogenic mycobacteria to the environment in their natural hosts is limited. Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic and incurable granulomatous enteritis of ruminants, and has been suggested to be a putative aetiological agent of Crohn's disease in humans. Using a comprehensive LC-MS-MS and 2D difference gel electrophoresis (DIGE) approach, we compared the protein profiles of clinical strains of MAP prepared from the gastrointestinal tract of diseased cows with the protein profiles of the same strains after they were grown in vitro. LC-MS-MS analyses revealed that the principal enzymes for the central carbon metabolic pathways, including glycolysis, gluconeogenesis, the tricaboxylic acid cycle and the pentose phosphate pathway, were present under both conditions. Moreover, a broad spectrum of enzymes for β-oxidation of lipids, nine of which have been shown to be necessary for mycobacterial growth on cholesterol, were detected in vivo and in vitro. Using 2D-DIGE we found increased levels of several key enzymes that indicated adaptation of MAP to the host. Among these, FadE5, FadE25 and AdhB indicated that cholesterol is used as a carbon source in the bovine intestinal mucosa; the respiratory enzymes AtpA, NuoG and SdhA suggested increased respiration during infection. Furthermore higher levels of the pentose phosphate pathway enzymes Gnd2, Zwf and Tal as well as of KatG, SodA and GroEL indicated a vigorous stress response of MAP in vivo. In conclusion, our results provide novel insights into the metabolic adaptation of a pathogenic mycobacterium in its natural host.

Quantitative flux analysis reveals folate-dependent NADPH production

NASA Astrophysics Data System (ADS)

Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

2014-06-01

ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer *

PubMed Central

Hutton, Josiah E.; Wang, Xiaojing; Zimmerman, Lisa J.; Slebos, Robbert J. C.; Trenary, Irina A.; Young, Jamey D.; Li, Ming; Liebler, Daniel C.

2016-01-01

Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25–twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu. PMID:27340238

A possible role of NADPH-dependent cytochrome P450nor isozyme in glycolysis under denitrifying conditions.

PubMed

Watsuji, Tomo-o; Takaya, Naoki; Nakamura, Akira; Shoun, Hirofumi

2003-05-01

The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP+ for the pentose phosphate cycle.

13C-MFA delineates the photomixotrophic metabolism of Synechocystis sp. PCC 6803 under light- and carbon-sufficient conditions.

PubMed

You, Le; Berla, Bert; He, Lian; Pakrasi, Himadri B; Tang, Yinjie J

2014-05-01

The central carbon metabolism of cyanobacteria is under debate. For over 50 years, the lack of α-ketoglutarate dehydrogenase has led to the belief that cyanobacteria have an incomplete TCA cycle. Recent in vitro enzymatic experiments suggest that this cycle may in fact be closed. The current study employed (13) C isotopomers to delineate pathways in the cyanobacterium Synechocystis sp. PCC 6803. By tracing the incorporation of supplemented glutamate into the downstream metabolites in the TCA cycle, we observed a direct in vivo transformation of α-ketoglutarate to succinate. Additionally, isotopic tracing of glyoxylate did not show a functional glyoxylate shunt and glyoxylate was used for glycine synthesis. The photomixotrophic carbon metabolism was then profiled with (13) C-MFA under light and carbon-sufficient conditions. We observed that: (i) the in vivo flux through the TCA cycle reactions (α-ketoglutarate → succinate) was minimal (<2%); (ii) the flux ratio of CO2 fixation was six times higher than that of glucose utilization; (iii) the relative flux through the oxidative pentose phosphate pathway was low (<2%); (iv) high flux through malic enzyme served as a main route for pyruvate synthesis. Our results improve the understanding of the versatile metabolism in cyanobacteria and shed light on their application for photo-biorefineries. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zymomonas pentose-sugar fermenting strains and uses thereof

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Golden, CO; Howe, William [Golden, CO; Eddy, Christine [Golden, CO; Evans, Kent [Littleton, CO; Mohagheghi, Ali [Northglenn, CO

2007-05-29

Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a unique Zymomonas that may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using the Zymomonas integrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing a Zymomonas integrant as part of the present invention. The host Zymomonas strain found particularly useful in the creation of these compositions and methods is Zymomonas mobilis 31821.

Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.

PubMed

Oberg, Craig J; Oberg, Taylor S; Culumber, Michele D; Ortakci, Fatih; Broadbent, Jeffery R; McMahon, Donald J

2016-01-01

A Gram-stain positive, rod-shaped, non-spore-forming strain (WDC04T), which may be associated with late gas production in cheese, was isolated from aged Cheddar cheese following incubation on MRS agar (pH 5.2) at 6 °C for 35 days. Strain WDC04T had 97 % 16S rRNA gene sequence similarity with Lactobacillus hokkaidonensis DSM 26202T, Lactobacillus oligofermentans 533, 'Lactobacillus danicus' 9M3, Lactobacillus suebicus CCUG 32233T and Lactobacillus vaccinostercus DSM 20634T. API 50 CH carbohydrate fermentation panels indicated strain WDC04T could only utilize one of the 50 substrates tested, ribose, although it does slowly utilize galactose. In the API ZYM system, strain WDC04T was positive for leucine arylamidase, valine arylamidase, cysteine arylamidase (weakly), naphthol-AS-BI-phosphohydrolase and β-galactosidase activities. Total genomic DNA was sequenced from strain WDC04T using a whole-genome shotgun strategy on a 454 GS Titanium pyrosequencer. The sequence was assembled into a 1.90 Mbp draft genome consisting of 105 contigs with preliminary genome annotation performed using the RAST algorithm (rast.nmpdr.org). Genome analysis confirmed the pentose phosphate pathway for ribose metabolism as well as galactose, N-acetylglucosamine, and glycerol fermentation pathways. Genomic analysis places strain WDC04T in the obligately heterofermentative group of lactobacilli and metabolic results confirm this conclusion. The result of genome sequencing, along with 16S rRNA gene sequence analysis, indicates WDC04T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus wasatchensis sp. nov. is proposed. The type strain is WDC04T ( = DSM 29958T = LMG 28678T).

A Small System—High-Resolution Study of Metabolic Adaptation in the Central Metabolic Pathway to Temperate Climates in Drosophila melanogaster

PubMed Central

Lavington, Erik; Cogni, Rodrigo; Kuczynski, Caitlin; Koury, Spencer; Behrman, Emily L.; O’Brien, Katherine R.; Schmidt, Paul S.; Eanes, Walter F.

2014-01-01

In this article, we couple the geographic variation in 127 single-nucleotide polymorphism (SNP) frequencies in genes of 46 enzymes of central metabolism with their associated cis-expression variation to predict latitudinal or climatic-driven gene expression changes in the metabolic architecture of Drosophila melanogaster. Forty-two percent of the SNPs in 65% of the genes show statistically significant clines in frequency with latitude across the 20 local population samples collected from southern Florida to Ontario. A number of SNPs in the screened genes are also associated with significant expression variation within the Raleigh population from North Carolina. A principal component analysis of the full variance–covariance matrix of latitudinal changes in SNP-associated standardized gene expression allows us to identify those major genes in the pathway and its associated branches that are likely targets of natural selection. When embedded in a central metabolic context, we show that these apparent targets are concentrated in the genes of the upper glycolytic pathway and pentose shunt, those controlling glycerol shuttle activity, and finally those enzymes associated with the utilization of glutamate and pyruvate. These metabolites possess high connectivity and thus may be the points where flux balance can be best shifted. We also propose that these points are conserved points associated with coupling energy homeostasis and energy sensing in mammals. We speculate that the modulation of gene expression at specific points in central metabolism that are associated with shifting flux balance or possibly energy-state sensing plays a role in adaptation to climatic variation. PMID:24770333

A TIGAR-regulated metabolic pathway is critical for protection of brain ischemia.

PubMed

Li, Mei; Sun, Meiling; Cao, Lijuan; Gu, Jin-hua; Ge, Jianbin; Chen, Jieyu; Han, Rong; Qin, Yuan-Yuan; Zhou, Zhi-Peng; Ding, Yuqiang; Qin, Zheng-Hong

2014-05-28

TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits glycolysis and increases the flow of pentose phosphate pathway (PPP), which generates NADPH and pentose. We hypothesized that TIGAR plays a neuroprotective role in brain ischemia as neurons do not rely on glycolysis but are vulnerable to oxidative stress. We found that TIGAR was highly expressed in brain neurons and was rapidly upregulated in response to ischemia/reperfusion insult in a TP53-independent manner. Overexpression of TIGAR in normal mice with lentivirus reduced ischemic neuronal injury, whereas lentivirus-mediated TIGAR knockdown aggravated it. In cultured primary neurons, increasing TIGAR expression reduced oxygen and glucose deprivation (OGD)/reoxygenation-induced injury, whereas decreasing its expression worsened the injury. The glucose 6-phosphate dehydrogenase was upregulated in mouse and cellular models of stroke, and its upregulation was further enhanced by overexpression of TIGAR. Supplementation of NADPH also reduced ischemia/reperfusion brain injury and alleviated TIGAR knockdown-induced aggravation of ischemic injury. In animal and cellular stroke models, ischemia/reperfusion increased mitochondrial localization of TIGAR. OGD/reoxygenation-induced elevation of ROS, reduction of GSH, dysfunction of mitochondria, and activation of caspase-3 were rescued by overexpression of TIGAR or supplementation of NADPH, while knockdown of TIGAR aggravated these changes. Together, our results show that TIGAR protects ischemic brain injury via enhancing PPP flux and preserving mitochondria function, and thus may be a valuable therapeutic target for ischemic brain injury. Copyright © 2014 the authors 0270-6474/14/347458-14$15.00/0.

Meta-analysis and functional validation of nutritional requirements of solventogenic Clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose.

PubMed

Heluane, Humberto; Evans, Matthew R; Dagher, Sue F; Bruno-Bárcena, José M

2011-07-01

Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.

Nutrient starvation leading to triglyceride accumulation activates the Entner Doudoroff pathway in Rhodococcus jostii RHA1.

PubMed

Juarez, Antonio; Villa, Juan A; Lanza, Val F; Lázaro, Beatriz; de la Cruz, Fernando; Alvarez, Héctor M; Moncalián, Gabriel

2017-02-27

Rhodococcus jostii RHA1 and other actinobacteria accumulate triglycerides (TAG) under nutrient starvation. This property has an important biotechnological potential in the production of sustainable oils. To gain insight into the metabolic pathways involved in TAG accumulation, we analysed the transcriptome of R jostii RHA1 under nutrient-limiting conditions. We correlate these physiological conditions with significant changes in cell physiology. The main consequence was a global switch from catabolic to anabolic pathways. Interestingly, the Entner-Doudoroff (ED) pathway was upregulated in detriment of the glycolysis or pentose phosphate pathways. ED induction was independent of the carbon source (either gluconate or glucose). Some of the diacylglycerol acyltransferase genes involved in the last step of the Kennedy pathway were also upregulated. A common feature of the promoter region of most upregulated genes was the presence of a consensus binding sequence for the cAMP-dependent CRP regulator. This is the first experimental observation of an ED shift under nutrient starvation conditions. Knowledge of this switch could help in the design of metabolomic approaches to optimize carbon derivation for single cell oil production.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study.

PubMed

Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

2018-01-01

Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger . This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger , we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

PubMed Central

Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

2018-01-01

Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi. PMID:29623069

Transcriptional and functional analysis of galactooligosaccharide uptake by lacS in Lactobacillus acidophilus

PubMed Central

Andersen, Joakim M.; Barrangou, Rodolphe; Abou Hachem, Maher; Lahtinen, Sampo; Goh, Yong Jun; Svensson, Birte; Klaenhammer, Todd R.

2011-01-01

Probiotic microbes rely on their ability to survive in the gastrointestinal tract, adhere to mucosal surfaces, and metabolize available energy sources from dietary compounds, including prebiotics. Genome sequencing projects have proposed models for understanding prebiotic catabolism, but mechanisms remain to be elucidated for many prebiotic substrates. Although β-galactooligosaccharides (GOS) are documented prebiotic compounds, little is known about their utilization by lactobacilli. This study aimed to identify genetic loci in Lactobacillus acidophilus NCFM responsible for the transport and catabolism of GOS. Whole-genome oligonucleotide microarrays were used to survey the differential global transcriptome during logarithmic growth of L. acidophilus NCFM using GOS or glucose as a sole source of carbohydrate. Within the 16.6-kbp gal-lac gene cluster, lacS, a galactoside-pentose-hexuronide permease-encoding gene, was up-regulated 5.1-fold in the presence of GOS. In addition, two β-galactosidases, LacA and LacLM, and enzymes in the Leloir pathway were also encoded by genes within this locus and up-regulated by GOS stimulation. Generation of a lacS-deficient mutant enabled phenotypic confirmation of the functional LacS permease not only for the utilization of lactose and GOS but also lactitol, suggesting a prominent role of LacS in the metabolism of a broad range of prebiotic β-galactosides, known to selectively modulate the beneficial gut microbiota. PMID:22006318

Action of Cortisol on Sodium Transport in Canine Erythrocytes

PubMed Central

Streeten, David H. P.; Moses, Arnold M.

1968-01-01

Incubation of blood from deoxycorticosterone-treated, adrenalectomized dogs with glucose, 22NaCl, and cortisol, added in vitro, revealed log dose-related acceleration of sodium influx, of glucose utilization, and of lactate formation by cortisol in concentrations between 150 and 1000 µg/liter. Addition of 2-deoxyglucose, or preincubation of the blood until blood glucose concentration had fallen below 2.0 mg per 100 ml, reduced or abolished the acceleratory action of added cortisol on sodium influx but had no effect on sodium influx in the absence of added cortisol. Cortisol did not change the ATP or ATPase content of erythrocytes, or the metabolism of glucose via the pentose phosphate pathway, or the rate of efflux of 22Na from the erythrocytes. The acceleratory actions of cortisol on sodium, influx, glucose utilization, and lactate formation were significantly correlated. Cortisol (1000 µg/liter) enhanced sodium influx by approximately 8.7 mmole per liter erythrocytes per hour for each 1 mmole cortisol-induced increment in ATP production. It is concluded that sodium influx in canine erythrocytes comprises a passive component, unchanged by cellular metabolism, and a second component which is accelerated and inhibited in proportion to prevailing plasma concentrations of cortisol and aldosterone, and which (for cortisol) depends upon accelerated ATP production via glycolysis. These steroid actions probably result from effects on enzyme activity rather than on new enzyme induction. PMID:4233676

Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollinshead, Whitney D.; Rodriguez, Sarah; Martin, Hector Garcia

Background: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. Results: Overexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP bymore » phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium). Conclusions: We engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.« less

Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

DOE PAGES

Hollinshead, Whitney D.; Rodriguez, Sarah; Martin, Hector Garcia; ...

2016-10-10

Background: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. Results: Overexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP bymore » phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium). Conclusions: We engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.« less

Constructing xylose-assimilating pathways in Pediococcus acidilactici for high titer d-lactic acid fermentation from corn stover feedstock.

PubMed

Qiu, Zhongyang; Gao, Qiuqiang; Bao, Jie

2017-12-01

Xylose-assimilating pathway was constructed in a d-lactic acid producing Pediococcus acidilactici strain and evolutionary adapted to yield a co-fermentation strain P. acidilactici ZY15 with 97.3g/L of d-lactic acid and xylose conversion of 92.6% obtained in the high solids content simultaneous saccharification and co-fermentation (SSCF) of dry dilute acid pretreated and biodetoxified corn stover feedstock. The heterologous genes encoding xylose isomerase (xylA) and xylulokinase (xylB) were screened and integrated into the P. acidilactici chromosome. The metabolic flux to acetic acid in phosphoketolase pathway was re-directed to pentose phosphate pathway by substituting the endogenous phosphoketolase gene (pkt) with the heterologous transketolase (tkt) and transaldolase (tal) genes. The xylose-assimilating ability of the newly constructed P. acidilactici strain was significantly improved by adaptive evolution. This study provided an important strain and process prototype for high titer d-lactic acid production from lignocellulose feedstock with efficient xylose assimilation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mineral induced formation of pentose-2,4-bisphosphates

NASA Technical Reports Server (NTRS)

Krishnamurthy, R.; Pitsch, S.; Arrhenius, G.; Bada, J. L. (Principal Investigator)

1999-01-01

Formation of rac.-pentose-2,4-bisphosphates is demonstrated, starting from glycolaldehyde phosphate and glyceraldehyde-2-phosphate, and induced by mixed valence double layer metal hydroxide minerals. The reactions proceed from dilute aqueous reactant solutions (1.5 mM) at near neutral pH. Conditions have been established, where ribose-2,4-bisphosphate is the major product (approximately 48%) among the pentose-2,4-bisphosphates, which are formed with up to 25% yield.

A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation.

PubMed

Bera, Aloke K; Ho, Nancy W Y; Khan, Aftab; Sedlak, Miroslav

2011-05-01

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD(+)-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.

Lynn Westdal | NREL

Science.gov Websites

Process-relevant Biomass-derived Pentoses in a Biphasic Reaction System," ACS Sustainable Chem. Eng Biomass-derived Pentoses in a Biphasic Reaction System." ACS Sustainable Chem. Eng. (2017)

Production of Furfural from Process-Relevant Biomass-Derived Pentoses in a Biphasic Reaction System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mittal, Ashutosh; Black, Stuart K.; Vinzant, Todd B.

Furfural is an important fuel precursor which can be converted to hydrocarbon fuels and fuel intermediates. In this work, the production of furfural by dehydration of process-relevant pentose rich corn stover hydrolyzate using a biphasic batch reaction system has been investigated. Methyl isobutyl ketone (MIBK) and toluene have been used to extract furfural and enhance overall furfural yield by limiting its degradation to humins. The effects of reaction time, temperature, and acid concentration (H 2SO 4) on pentose conversion and furfural yield were investigated. For the dehydration of 8 wt % pentose-rich corn stover hydrolyzate under optimum reaction conditions, 0.05more » M H 2SO 4, 170 degrees C for 20 min with MIBK as the solvent, complete conversion of xylose (98-100%) and a furfural yield of 80% were obtained. Under these same conditions, except with toluene as the solvent, the furfural yield was 77%. Additionally, dehydration of process-relevant pentose rich corn stover hydrolyzate using solid acid ion-exchange resins under optimum reaction conditions has shown that Purolite CT275 is as effective as H 2SO 4 for obtaining furfural yields approaching 80% using a biphasic batch reaction system. In conclusion, this work has demonstrated that a biphasic reaction system can be used to process biomass-derived pentose rich sugar hydrolyzates to furfural in yields approaching 80%.« less

Production of Furfural from Process-Relevant Biomass-Derived Pentoses in a Biphasic Reaction System

DOE PAGES

Mittal, Ashutosh; Black, Stuart K.; Vinzant, Todd B.; ...

2017-05-16

Furfural is an important fuel precursor which can be converted to hydrocarbon fuels and fuel intermediates. In this work, the production of furfural by dehydration of process-relevant pentose rich corn stover hydrolyzate using a biphasic batch reaction system has been investigated. Methyl isobutyl ketone (MIBK) and toluene have been used to extract furfural and enhance overall furfural yield by limiting its degradation to humins. The effects of reaction time, temperature, and acid concentration (H 2SO 4) on pentose conversion and furfural yield were investigated. For the dehydration of 8 wt % pentose-rich corn stover hydrolyzate under optimum reaction conditions, 0.05more » M H 2SO 4, 170 degrees C for 20 min with MIBK as the solvent, complete conversion of xylose (98-100%) and a furfural yield of 80% were obtained. Under these same conditions, except with toluene as the solvent, the furfural yield was 77%. Additionally, dehydration of process-relevant pentose rich corn stover hydrolyzate using solid acid ion-exchange resins under optimum reaction conditions has shown that Purolite CT275 is as effective as H 2SO 4 for obtaining furfural yields approaching 80% using a biphasic batch reaction system. In conclusion, this work has demonstrated that a biphasic reaction system can be used to process biomass-derived pentose rich sugar hydrolyzates to furfural in yields approaching 80%.« less

Elucidation of the Metabolic Network of Helicobacter pylori J99 and Malaysian Clinical Strains by Phenotype Microarray.

PubMed

Lee, Woon Ching; Goh, Khean Lee; Loke, Mun Fai; Vadivelu, Jamuna

2017-02-01

Helicobacter pylori colonizes almost half of the human population worldwide. H. pylori strains are genetically diverse, and the specific genotypes are associated with various clinical manifestations including gastric adenocarcinoma, peptic ulcer disease (PUD), and nonulcer dyspepsia (NUD). However, our current knowledge of the H. pylori metabolism is limited. To understand the metabolic differences among H. pylori strains, we investigated four Malaysian H. pylori clinical strains, which had been previously sequenced, and a standard strain, H. pylori J99, at the phenotypic level. The phenotypes of the H. pylori strains were profiled using the Biolog Phenotype Microarray system to corroborate genomic data. We initiated the analyses by predicting carbon and nitrogen metabolic pathways from the H. pylori genomic data from the KEGG database. Biolog PM aided the validation of the prediction and provided a more intensive analysis of the H. pylori phenomes. We have identified a core set of metabolic nutrient sources that was utilized by all strains tested and another set that was differentially utilized by only the local strains. Pentose sugars are the preferred carbon nutrients utilized by H. pylori. The amino acids l-aspartic acid, d-alanine, and l-asparagine serve as both carbon and nitrogen sources in the metabolism of the bacterium. The phenotypic profile based on this study provides a better understanding on the survival of H. pylori in its natural host. Our data serve as a foundation for future challenges in correlating interstrain metabolic differences in H. pylori. © 2016 The Authors. Helicobacter Published by John Wiley & Sons Ltd.

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

PubMed

Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

2012-03-01

Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

Meta-Analysis and Functional Validation of Nutritional Requirements of Solventogenic Clostridia Growing under Butanol Stress Conditions and Coutilization of d-Glucose and d-Xylose ▿

PubMed Central

Heluane, Humberto; Evans, Matthew R.; Dagher, Sue F.; Bruno-Bárcena, José M.

2011-01-01

Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of d-glucose and d-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production. PMID:21602379

Metabolic profiling of lung granuloma in Mycobacterium tuberculosis infected guinea pigs: ex vivo 1H magic angle spinning NMR studies.

PubMed

Somashekar, B S; Amin, Anita G; Rithner, Christopher D; Troudt, JoLynn; Basaraba, Randall; Izzo, Angelo; Crick, Dean C; Chatterjee, Delphi

2011-09-02

A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Lian; Xiu, Yu; Jones, J. Andrew

Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this paper, 13C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37 °C in stage 1, and then the temperature was transitioned to 20 °C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (latemore » production phase) in the high producing strain, reaching a final concentration of 1.5 mmol/L. On the contrary, ~0.02 mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed 13C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Finally, interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).« less

Characterization of Central Carbon Metabolism of Streptococcus pneumoniae by Isotopologue Profiling*

PubMed Central

Härtel, Tobias; Eylert, Eva; Schulz, Christian; Petruschka, Lothar; Gierok, Philipp; Grubmüller, Stephanie; Lalk, Michael; Eisenreich, Wolfgang; Hammerschmidt, Sven

2012-01-01

The metabolism of Streptococcus pneumoniae was studied by isotopologue profiling after bacterial cultivation in chemically defined medium supplemented with [U-13C6]- or [1,2-13C2]glucose. GC/MS analysis of protein-derived amino acids showed lack of 13C label in amino acids that were also essential for pneumococcal growth. Ala, Ser, Asp, and Thr displayed high 13C enrichments, whereas Phe, Tyr, and Gly were only slightly labeled. The analysis of the labeling patterns showed formation of triose phosphate and pyruvate via the Embden-Meyerhof-Parnas pathway. The labeling patterns of Asp and Thr suggested formation of oxaloacetate exclusively via the phosphoenolpyruvate carboxylase reaction. Apparently, α-ketoglutarate was generated from unlabeled glutamate via the aspartate transaminase reaction. A fraction of Phe and Tyr obtained label via the chorismate route from erythrose 4-phosphate, generated via the pentose phosphate pathway, and phosphoenolpyruvate. Strikingly, the data revealed no significant flux from phosphoglycerate to Ser and Gly but showed formation of Ser via the reverse reaction, namely by hydroxymethylation of Gly. The essential Gly was acquired from the medium, and the biosynthesis pathway was confirmed in experiments using [U-13C2]glycine as a tracer. The hydroxymethyl group in Ser originated from formate, which was generated by the pyruvate formate-lyase. Highly similar isotopologue profiles were observed in corresponding experiments with pneumococcal mutants deficient in PavA, CodY, and glucose-6-phosphate dehydrogenase pointing to the robustness of the core metabolic network used by these facultative pathogenic bacteria. In conclusion, this study demonstrates the dual utilization of carbohydrates and amino acids under in vitro conditions and identifies the unconventional de novo biosynthesis of serine by pneumococci. PMID:22167202

Activation of neuronal nitric oxide synthase in cerebellum of chronic hepatic encephalopathy rats is associated with up-regulation of NADPH-producing pathway.

PubMed

Singh, Santosh; Trigun, Surendra K

2010-09-01

Cerebellum-associated functions get affected during mild hepatic encephalopathy (MHE) in patients with chronic liver failure (CLF). Involvement of nitrosative and antioxidant factors in the pathogenesis of chronic hepatic encephalopathy is an evolving concept and needs to be defined in a true CLF animal model. This article describes profiles of NADPH-dependent neuronal nitric oxide synthase (nNOS) and those of glutathione peroxidase and glutathione reductase (GR) vis-a-vis regulation of NADPH-producing pathway in the cerebellum of CLF rats induced by administration of thioacetamide (100 mg kg⁻¹ b.w., i.p.) up to 10 days and confirming MHE on Morris water maze tests. Significant increases in the expression of nNOS protein and nitric oxide (NOx) level coincided with a similar increment in NADPH-diaphorase activity in the cerebellum of CLF rats. Glutathione peroxidase and GR utilize NADPH to regenerate reduced glutathione (GSH) in the cells. Both these enzymes and GSH level were found to be static and thus suggested efficient turnover of GSH in the cerebellum of MHE rats. Relative levels of glucose-6-phosphate dehydrogenase (G6PD) vs. phosphofructokinase 2 (PFK2) determine the rate of pentose phosphate pathway (PPP) responsible to synthesize NADPH. The cerebellum of CLF rats showed overactivation of G6PD with a significant decline in the expression of PFK2 and thus suggested activation of PPP in the cerebellum during MHE. It is concluded that concordant activations of PPP and nNOS in cerebellum of MHE rats could be associated with the implication of NOx in the pathogenesis of MHE.

Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

PubMed

Rodriguez, Alberto; Martínez, Juan A; Millard, Pierre; Gosset, Guillermo; Portais, Jean-Charles; Létisse, Fabien; Bolivar, Francisco

2017-06-01

Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A small system--high-resolution study of metabolic adaptation in the central metabolic pathway to temperate climates in Drosophila melanogaster.

PubMed

Lavington, Erik; Cogni, Rodrigo; Kuczynski, Caitlin; Koury, Spencer; Behrman, Emily L; O'Brien, Katherine R; Schmidt, Paul S; Eanes, Walter F

2014-08-01

In this article, we couple the geographic variation in 127 single-nucleotide polymorphism (SNP) frequencies in genes of 46 enzymes of central metabolism with their associated cis-expression variation to predict latitudinal or climatic-driven gene expression changes in the metabolic architecture of Drosophila melanogaster. Forty-two percent of the SNPs in 65% of the genes show statistically significant clines in frequency with latitude across the 20 local population samples collected from southern Florida to Ontario. A number of SNPs in the screened genes are also associated with significant expression variation within the Raleigh population from North Carolina. A principal component analysis of the full variance-covariance matrix of latitudinal changes in SNP-associated standardized gene expression allows us to identify those major genes in the pathway and its associated branches that are likely targets of natural selection. When embedded in a central metabolic context, we show that these apparent targets are concentrated in the genes of the upper glycolytic pathway and pentose shunt, those controlling glycerol shuttle activity, and finally those enzymes associated with the utilization of glutamate and pyruvate. These metabolites possess high connectivity and thus may be the points where flux balance can be best shifted. We also propose that these points are conserved points associated with coupling energy homeostasis and energy sensing in mammals. We speculate that the modulation of gene expression at specific points in central metabolism that are associated with shifting flux balance or possibly energy-state sensing plays a role in adaptation to climatic variation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Furfural production from Eucalyptus wood using an Acidic Ionic Liquid.

PubMed

Peleteiro, Susana; Santos, Valentín; Garrote, Gil; Parajó, Juan Carlos

2016-08-01

Eucalyptus globulus wood samples were treated with hot, compressed water to separate hemicelluloses (as soluble saccharides) from a solid phase mainly made up of cellulose and lignin. The liquid phase was dehydrated, and the resulting solids (containing pentoses as well as poly- and oligo- saccharides made up of pentoses) were dissolved and reacted in media containing an Acidic Ionic Liquid (1-butyl-3-methylimidazolium hydrogen sulfate) and a co-solvent (dioxane). The effects of the reaction time on the product distribution were studied at temperatures in the range 120-170°C for reaction times up to 8h, and operational conditions leading to 59.1% conversion of the potential substrates (including pentoses and pentose structural units in oligo- and poly- saccharides) into furfural were identified. Copyright © 2016 Elsevier Ltd. All rights reserved.

End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen.

PubMed

Sell, D R; Monnier, V M

1990-02-01

Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.

Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

PubMed

Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J; Okada, Takao; Edwards, John G; Gupte, Sachin A

2012-01-01

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

PubMed

Dong, Li-Hua; Li, Liang; Song, Yu; Duan, Zhi-Li; Sun, Shao-Guang; Lin, Yan-Ling; Miao, Sui-Bing; Yin, Ya-Juan; Shu, Ya-Nan; Li, Huan; Chen, Peng; Zhao, Li-Li; Han, Mei

2015-09-25

Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates atherosclerotic plaque stability. © 2015 American Heart Association, Inc.

Shaofu Zhuyu decoction ameliorates obesity-mediated hepatic steatosis and systemic inflammation by regulating metabolic pathways

PubMed Central

Park, Hee-Sook; Lee, So Min; Jeong, Nam-Joo; Kim, Soon-Hee; Lee, Kyoung-Won; Lee, Ju-A

2017-01-01

Shaofu Zhuyu decoction (SFZYD, also known as Sobokchugeo-tang), a classical prescription drug in traditional East Asian medicine, has been used to treat blood stasis syndrome (BSS). Hepatic steatosis is the result of excess caloric intake, and its pathogenesis involves internal retention of phlegm and dampness, blood stasis, and liver Qi stagnation. To evaluate the effects of treatment with SFZYD on obesity-induced inflammation and hepatic steatosis, we fed male C57BL/6N mice a high fat diet (HFD) for 8 weeks and then treated them with SFZYD by oral gavage for an additional 4 weeks. The results of histological and biochemical examinations indicated that SFZYD treatment ameliorates systemic inflammation and hepatic steatosis. A partial least squares-discriminant analysis (PLS-DA) scores plot of serum metabolites showed that HFD mice began to produce metabolites similar to those of normal chow (NC) mice after SFZYD administration. We noted significant alterations in the levels of twenty-seven metabolites, alterations indicating that SFZYD regulates the TCA cycle, the pentose phosphate pathway and aromatic amino acid metabolism. Increases in the levels of TCA cycle intermediate metabolites, such as 2-oxoglutaric acid, isocitric acid, and malic acid, in the serum of obese mice were significantly reversed after SFZYD treatment. In addition to inducing changes in the above metabolites, treatment with SFZYD also recovered the expression of genes related to hepatic mitochondrial dysfunction, including Ucp2, Cpt1α, and Ppargc1α, as well as the expression of genes involved in lipid metabolism and inflammation, without affecting glucose uptake or insulin signaling. Taken together, these findings suggest that treatment with SFZYD ameliorated obesity-induced systemic inflammation and hepatic steatosis by regulating inflammatory cytokine and adipokine levels in the circulation and various tissues. Moreover, treatment with SFZYD also reversed alterations in the levels of metabolites of the TCA cycle, the pentose phosphate pathway and aromatic amino acid metabolism. PMID:28570676

Metabolic Reprogramming in Glioma

PubMed Central

Strickland, Marie; Stoll, Elizabeth A.

2017-01-01

Many cancers have long been thought to primarily metabolize glucose for energy production—a phenomenon known as the Warburg Effect, after the classic studies of Otto Warburg in the early twentieth century. Yet cancer cells also utilize other substrates, such as amino acids and fatty acids, to produce raw materials for cellular maintenance and energetic currency to accomplish cellular tasks. The contribution of these substrates is increasingly appreciated in the context of glioma, the most common form of malignant brain tumor. Multiple catabolic pathways are used for energy production within glioma cells, and are linked in many ways to anabolic pathways supporting cellular function. For example: glycolysis both supports energy production and provides carbon skeletons for the synthesis of nucleic acids; meanwhile fatty acids are used both as energetic substrates and as raw materials for lipid membranes. Furthermore, bio-energetic pathways are connected to pro-oncogenic signaling within glioma cells. For example: AMPK signaling links catabolism with cell cycle progression; mTOR signaling contributes to metabolic flexibility and cancer cell survival; the electron transport chain produces ATP and reactive oxygen species (ROS) which act as signaling molecules; Hypoxia Inducible Factors (HIFs) mediate interactions with cells and vasculature within the tumor environment. Mutations in the tumor suppressor p53, and the tricarboxylic acid cycle enzymes Isocitrate Dehydrogenase 1 and 2 have been implicated in oncogenic signaling as well as establishing metabolic phenotypes in genetically-defined subsets of malignant glioma. These pathways critically contribute to tumor biology. The aim of this review is two-fold. Firstly, we present the current state of knowledge regarding the metabolic strategies employed by malignant glioma cells, including aerobic glycolysis; the pentose phosphate pathway; one-carbon metabolism; the tricarboxylic acid cycle, which is central to amino acid metabolism; oxidative phosphorylation; and fatty acid metabolism, which significantly contributes to energy production in glioma cells. Secondly, we highlight processes (including the Randle Effect, AMPK signaling, mTOR activation, etc.) which are understood to link bio-energetic pathways with oncogenic signals, thereby allowing the glioma cell to achieve a pro-malignant state. PMID:28491867

Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mechanism of lipid accumulation at the proteomic level.

PubMed

Tang, Xin; Zan, Xinyi; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda; Ratledge, Colin

2016-02-11

The oleaginous fungus, Mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. Nitrogen (N) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. Although a simple pathway from N depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon N depletion have not been investigated. In this study, we have systematically analyzed the changes at the levels of protein expression in M. circinelloides WJ11, a high lipid-producing strain (36 %, lipid/cell dry weight), during lipid accumulation. Proteomic analysis demonstrated that N depletion increased the expression of glutamine synthetase, involved in ammonia assimilation, for the supply of cellular nitrogen but decreased the metabolism of amino acids. Upon N deficiency, many proteins (e.g., fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase) involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle (e.g., isocitrate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydratase) were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, glucose-6-phosphate dehydrogenase, transaldolase and transketolase, which participate in the pentose phosphate pathway, were up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency. N limitation increased those expressions of the proteins involved in ammonia assimilation but decreased that involved in the biosynthesis of amino acids. Upon N deprivation, the glycolytic pathway was up-regulated, while the activity of the tricarboxylic acid cycle was retarded, thus, leading more carbon flux to fatty acid biosynthesis. Moreover, the pentose phosphate pathway was up-regulated, then this would increase the production of NADPH. Together, coordinated regulation of central carbon metabolism upon N limitation, provides more carbon flux to acetyl-CoA and NADPH for fatty acid biosynthesis.

Serum Metabolomic Profiling in Acute Alcoholic Hepatitis Identifies Multiple Dysregulated Pathways

PubMed Central

Rachakonda, Vikrant; Gabbert, Charles; Raina, Amit; Bell, Lauren N.; Cooper, Sara; Malik, Shahid; Behari, Jaideep

2014-01-01

Background and Objectives While animal studies have implicated derangements of global energy homeostasis in the pathogenesis of acute alcoholic hepatitis (AAH), the relevance of these findings to the development of human AAH remains unclear. Using global, unbiased serum metabolomics analysis, we sought to characterize alterations in metabolic pathways associated with severe AAH and identify potential biomarkers for disease prognosis. Methods This prospective, case-control study design included 25 patients with severe AAH and 25 ambulatory patients with alcoholic cirrhosis. Serum samples were collected within 24 hours of the index clinical encounter. Global, unbiased metabolomics profiling was performed. Patients were followed for 180 days after enrollment to determine survival. Results Levels of 234 biochemicals were altered in subjects with severe AAH. Random-forest analysis, principal component analysis, and integrated hierarchical clustering methods demonstrated that metabolomics profiles separated the two cohorts with 100% accuracy. Severe AAH was associated with enhanced triglyceride lipolysis, impaired mitochondrial fatty acid beta oxidation, and upregulated omega oxidation. Low levels of multiple lysolipids and related metabolites suggested decreased plasma membrane remodeling in severe AAH. While most measured bile acids were increased in severe AAH, low deoxycholate and glycodeoxycholate levels indicated intestinal dysbiosis. Several changes in substrate utilization for energy homeostasis were identified in severe AAH, including increased glucose consumption by the pentose phosphate pathway, altered tricarboxylic acid (TCA) cycle activity, and enhanced peptide catabolism. Finally, altered levels of small molecules related to glutathione metabolism and antioxidant vitamin depletion were observed in patients with severe AAH. Univariable logistic regression revealed 15 metabolites associated with 180-day survival in severe AAH. Conclusion Severe AAH is characterized by a distinct metabolic phenotype spanning multiple pathways. Metabolomics profiling revealed a panel of biomarkers for disease prognosis, and future studies are planned to validate these findings in larger cohorts of patients with severe AAH. PMID:25461442

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

PubMed Central

Nunn, Brook L.; Faux, Jessica F.; Hippmann, Anna A.; Maldonado, Maria T.; Harvey, H. Rodger; Goodlett, David R.; Boyd, Philip W.; Strzepek, Robert F.

2013-01-01

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies. PMID:24146769

Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Rong; Pineda, Marco; Ajamian, Eunice

2009-01-15

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+},more » which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.« less

[Biosynthesis of indigo and indirubin by whole-cell catalyst designed by combination of protein engineering and metabolic engineering].

PubMed

Li, Yang; Zhu, Junge; Wang, Jianjun; Xia, Huanzhang; Wu, Sheng

2016-01-01

The phenylacetone monooxygenase, isolated from Thermobifida fusca, mainly catalyzes Baeyer-Villiger oxidation reaction towards aromatic compounds. Met446 plays a vital role in catalytic promiscuity, based on the structure and function of phenylacetone monooxygenase. Mutation in Met446 locus can offer enzyme new catalytic feature to activate C-H bond, oxidizing indole to finally generate indigo and indirubin, but the yield was only 1.89 mg/L. In order to further improve the biosynthesis efficiency of the whole-cell catalyst, metabolic engineering was applied to change glucose metabolism pathway of Escherichia coli. Blocking glucose isomerase gene pgi led to pentose phosphate pathway instead of the glycolytic pathway to become the major metabolic pathways of glucose, which provided more cofactor NADPH needed in enzymatic oxidation of indole. Engineering the host E. coli led to synthesis of indigo and indirubin efficiency further increased to 25 mg/L. Combination of protein and metabolic engineering to design efficient whole-cell catalysts not only improves the synthesis of indigo and indirubin, but also provides a novel strategy for whole-cell catalyst development.

Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

NASA Astrophysics Data System (ADS)

Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

2013-11-01

Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

Lignocellulosic ethanol: Technology design and its impact on process efficiency.

PubMed

Paulova, Leona; Patakova, Petra; Branska, Barbora; Rychtera, Mojmir; Melzoch, Karel

2015-11-01

This review provides current information on the production of ethanol from lignocellulosic biomass, with the main focus on relationships between process design and efficiency, expressed as ethanol concentration, yield and productivity. In spite of unquestionable advantages of lignocellulosic biomass as a feedstock for ethanol production (availability, price, non-competitiveness with food, waste material), many technological bottlenecks hinder its wide industrial application and competitiveness with 1st generation ethanol production. Among the main technological challenges are the recalcitrant structure of the material, and thus the need for extensive pretreatment (usually physico-chemical followed by enzymatic hydrolysis) to yield fermentable sugars, and a relatively low concentration of monosaccharides in the medium that hinder the achievement of ethanol concentrations comparable with those obtained using 1st generation feedstocks (e.g. corn or molasses). The presence of both pentose and hexose sugars in the fermentation broth, the price of cellulolytic enzymes, and the presence of toxic compounds that can inhibit cellulolytic enzymes and microbial producers of ethanol are major issues. In this review, different process configurations of the main technological steps (enzymatic hydrolysis, fermentation of hexose/and or pentose sugars) are discussed and their efficiencies are compared. The main features, benefits and drawbacks of simultaneous saccharification and fermentation (SSF), simultaneous saccharification and fermentation with delayed inoculation (dSSF), consolidated bioprocesses (CBP) combining production of cellulolytic enzymes, hydrolysis of biomass and fermentation into one step, together with an approach combining utilization of both pentose and hexose sugars are discussed and compared with separate hydrolysis and fermentation (SHF) processes. The impact of individual technological steps on final process efficiency is emphasized and the potential for use of immobilized biocatalysts is considered. Copyright © 2014 Elsevier Inc. All rights reserved.

13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

PubMed Central

Kleijn, Roelco J.; van Winden, Wouter A.; Ras, Cor; van Gulik, Walter M.; Schipper, Dick; Heijnen, Joseph J.

2006-01-01

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h−1 yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio. PMID:16820467

Modeling Central Carbon Metabolic Processes in Soil Microbial Communities: Comparing Measured With Modeled

NASA Astrophysics Data System (ADS)

Dijkstra, P.; Fairbanks, D.; Miller, E.; Salpas, E.; Hagerty, S.

2013-12-01

Understanding the mechanisms regulating C cycling is hindered by our inability to directly observe and measure the biochemical processes of glycolysis, pentose phosphate pathway, and TCA cycle in intact and complex microbial communities. Position-specific 13C labeled metabolic tracer probing is proposed as a new way to study microbial community energy production, biosynthesis, C use efficiency (the proportion of substrate incorporated into microbial biomass), and enables the quantification of C fluxes through the central C metabolic network processes (Dijkstra et al 2011a,b). We determined the 13CO2 production from U-13C, 1-13C, 2-13C, 3-13C, 4-13C, 5-13C, and 6-13C labeled glucose and 1-13C and 2,3-13C pyruvate in parallel incubations in three soils along an elevation gradient. Qualitative and quantitative interpretation of the results indicate a high pentose phosphate pathway activity in soils. Agreement between modeled and measured CO2 production rates for the six C-atoms of 13C-labeled glucose indicate that the metabolic model used is appropriate for soil community processes, but that improvements can be made. These labeling and modeling techniques may improve our ability to analyze the biochemistry and (eco)physiology of intact microbial communities. Dijkstra, P., Blankinship, J.C., Selmants, P.C., Hart, S.C., Koch, G.W., Schwartz, E., Hungate, B.A., 2011a. Probing C flux patterns of soil microbial metabolic networks using parallel position-specific tracer labeling. Soil Biology & Biochemistry 43, 126-132. Dijkstra, P., Dalder, J.J., Selmants, P.C., Hart, S.C., Koch, G.W., Schwartz, E., Hungate, B.A., 2011b. Modeling soil metabolic processes using isotopologue pairs of position-specific 13C-labeled glucose and pyruvate. Soil Biology & Biochemistry 43, 1848-1857.

Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes.

PubMed

Xiao, Wusheng; Sarsour, Ehab H; Wagner, Brett A; Doskey, Claire M; Buettner, Garry R; Domann, Frederick E; Goswami, Prabhat C

2016-02-01

Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of 4-monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ-treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ-induced toxicity.

Metabolic Flux Analysis of the Synechocystis sp. PCC 6803 ΔnrtABCD Mutant Reveals a Mechanism for Metabolic Adaptation to Nitrogen-Limited Conditions.

PubMed

Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

2017-03-01

Metabolic flux redirection during nitrogen-limited growth was investigated in the Synechocystis sp. PCC 6803 glucose-tolerant (GT) strain under photoautotrophic conditions by isotopically non-stationary metabolic flux analysis (INST-MFA). A ΔnrtABCD mutant of Synechocystis sp. PCC 6803 was constructed to reproduce phenotypes arising during nitrogen starvation. The ΔnrtABCD mutant and the wild-type GT strain were cultured under photoautotrophic conditions by a photobioreactor. Intracellular metabolites were labeled over a time course using NaH13CO3 as a carbon source. Based on these data, the metabolic flux distributions in the wild-type and ΔnrtABCD cells were estimated by INST-MFA. The wild-type GT and ΔnrtABCD strains displayed similar distribution patterns, although the absolute levels of metabolic flux were lower in ΔnrtABCD. Furthermore, the relative flux levels for glycogen metabolism, anaplerotic reactions and the oxidative pentose phosphate pathway were increased in ΔnrtABCD. This was probably due to the increased expression of enzyme genes that respond to nitrogen depletion. Additionally, we found that the ratio of ATP/NADPH demand increased slightly in the ΔnrtABCD mutant. These results indicated that futile ATP consumption increases under nitrogen-limited conditions because the Calvin-Benson cycle and the oxidative pentose phosphate pathway form a metabolic futile cycle that consumes ATP without CO2 fixation and NADPH regeneration. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Assessing Oxidative Stress in Tumors by Measuring the Rate of Hyperpolarized [1-13C]Dehydroascorbic Acid Reduction Using 13C Magnetic Resonance Spectroscopy*

PubMed Central

Timm, Kerstin N.; Hu, De-En; Williams, Michael; Wright, Alan J.; Kettunen, Mikko I.; Kennedy, Brett W. C.; Larkin, Timothy J.; Dzien, Piotr; Marco-Rius, Irene; Bohndiek, Sarah E.; Brindle, Kevin M.

2017-01-01

Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1-13C]dehydroascorbic acid (DHA) reduction, which can be measured in vivo using non-invasive 13C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1-13C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using 13C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-13C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo. However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic. PMID:27994059

Regional differences in brain glucose metabolism determined by imaging mass spectrometry.

PubMed

Kleinridders, André; Ferris, Heather A; Reyzer, Michelle L; Rath, Michaela; Soto, Marion; Manier, M Lisa; Spraggins, Jeffrey; Yang, Zhihong; Stanton, Robert C; Caprioli, Richard M; Kahn, C Ronald

2018-06-01

Glucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS). To examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 μm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism. In brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate. These data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

PubMed

Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R; Hetman, Michal

2016-08-01

The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. © The Author(s) 2016.

Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803.

PubMed

Ueda, Kentaro; Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

2018-02-27

The role of the oxidative pentose phosphate pathway (oxPPP) in Synechocystis sp. PCC 6803 under mixotrophic conditions was investigated by 13 C metabolic flux analysis. Cells were cultured under low (10 μmol m -2  s -1 ) and high light intensities (100 μmol m -2  s -1 ) in the presence of glucose. The flux of CO 2 fixation by ribulose bisphosphate carboxylase/oxygenase under the high light condition was approximately 3-fold higher than that under the low light condition. Although no flux of the oxPPP was observed under the high light condition, flux of 0.08-0.19 mmol gDCW -1  h -1 in the oxPPP was observed under the low light condition. The balance between the consumption and production of NADPH suggested that approximately 10% of the total NADPH production was generated by the oxPPP under the low light condition. The growth phenotype of a mutant with deleted zwf, which encodes glucose-6-phosphate dehydrogenase in the oxPPP, was compared to that of the parental strain under low and high light conditions. Growth of the Δzwf mutant nearly stopped during the late growth phase under the low light condition, whereas the growth rates of the two strains were identical under the high light condition. These results indicate that NADPH production in the oxPPP is essential for anabolism under low light conditions. The oxPPP appears to play an important role in producing NADPH from glucose and ATP to compensate for NADPH shortage under low light conditions. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations.

PubMed

Chin, Jonathan W; Cirino, Patrick C

2011-01-01

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Recombinant microorganisms for increased production of organic acids

DOEpatents

Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

2012-02-21

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

Recombinant microorganisms for increased production of organic acids

DOEpatents

Yi, Jian; Kleff, Susanne; Guettler, Michael V

2013-04-30

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability.

PubMed

Allaman, Igor; Gavillet, Mathilde; Bélanger, Mireille; Laroche, Thierry; Viertl, David; Lashuel, Hilal A; Magistretti, Pierre J

2010-03-03

Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism in cultured astrocytes. Following Abeta(25-35) exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Abeta increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Abeta on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Abeta impair neuronal viability. The effects of the Abeta(25-35) fragment were reproduced by Abeta(1-42) but not by Abeta(1-40). Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Abeta aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

PubMed

Alagesan, Swathi; Gaudana, Sandeep B; Sinha, Avinash; Wangikar, Pramod P

2013-11-01

Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

Highly efficient production of optically pure l-lactic acid from corn stover hydrolysate by thermophilic Bacillus coagulans.

PubMed

Ma, Kedong; Hu, Guoquan; Pan, Liwei; Wang, Zichao; Zhou, Yi; Wang, Yanwei; Ruan, Zhiyong; He, Mingxiong

2016-11-01

A thermophilic strain Bacillus coagulans (NBRC 12714) was employed to produce l-lactic acid from corn stover hydrolysate in membrane integrated continuous fermentation. The strain NBRC 12714 metabolized glucose and xylose by the Embden-Meyerhof-Parnas pathway (EMP) and the pentose phosphate pathway (PPP), producing l-lactic acid with optical purity >99.5%. The overall l-lactic acid titer of 92g/l with a yield of 0.91g/g and a productivity of 13.8g/l/h were achieved at a dilution rate of 0.15h(-1). The productivity obtained was 1.6-fold than that of conventional continuous fermentation without cell recycling, and also was the highest among the relevant studies ever reported. These results indicated that the process developed had great potential for economical industrial production of l-lactic acid from lignocellulosic biomass. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062].

PubMed

Zhang, Xiaomei; Dou, Wenfang; Xu, Hongyu; Xu, Zhenghong

2010-10-01

Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity.

Overexpression of hypoxia-inducible factor and metabolic pathways: possible targets of cancer.

PubMed

Singh, Davinder; Arora, Rohit; Kaur, Pardeep; Singh, Balbir; Mannan, Rahul; Arora, Saroj

2017-01-01

Cancer, the main cause of human deaths in the modern world is a group of diseases. Anticancer drug discovery is a challenge for scientists because of involvement of multiple survival pathways of cancer cells. An extensive study on the regulation of each step of these pathways may help find a potential cancer target. Up-regulated HIF-1 expression and altered metabolic pathways are two classical characteristics of cancer. Oxygen-dependent (through pVHL, PHDs, calcium-mediated) and independent (through growth factor signaling pathway, mdm2 pathway, HSP90) regulation of HIF-1α leads to angiogenesis, metastasis, and cell survival. The two subunits of HIF-1 regulates in the same fashion through different mechanisms. HIF-1α translation upregulates via mammalian target of rapamycin and mitogen-activated protein kinase signaling pathways, whereas HIF-1β through calmodulin kinase. Further, the stabilized interactions of these two subunits are important for proper functioning. Also, metabolic pathways crucial for the formation of building blocks (pentose phosphate pathway) and energy generation (glycolysis, TCA cycle and catabolism of glutamine) are altered in cancer cells to protect them from oxidative stress and to meet the reduced oxygen and nutrient supply. Up-regulated anaerobic metabolism occurs through enhanced expression of hexokinase, phosphofructokinase, triosephosphate isomerase, glucose 6-phosphate dehydrogenase and down-regulation of aerobic metabolism via pyruvate dehydrogenase kinase and lactate dehydrogenase which compensate energy requirements along with high glucose intake. Controlled expression of these two pathways through their common intermediate may serve as potent cancer target in future.

IKKβ promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3

PubMed Central

Reid, Michael A.; Lowman, Xazmin H.; Pan, Min; Tran, Thai Q.; Warmoes, Marc O.; Ishak Gabra, Mari B.; Yang, Ying; Locasale, Jason W.; Kong, Mei

2016-01-01

Glutamine is an essential nutrient for cancer cell survival and proliferation. Enhanced utilization of glutamine often depletes its local supply, yet how cancer cells adapt to low glutamine conditions is largely unknown. Here, we report that IκB kinase β (IKKβ) is activated upon glutamine deprivation and is required for cell survival independently of NF-κB transcription. We demonstrate that IKKβ directly interacts with and phosphorylates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major driver of aerobic glycolysis, at Ser269 upon glutamine deprivation to inhibit its activity, thereby down-regulating aerobic glycolysis when glutamine levels are low. Thus, due to lack of inhibition of PFKFB3, IKKβ-deficient cells exhibit elevated aerobic glycolysis and lactate production, leading to less glucose carbons contributing to tricarboxylic acid (TCA) cycle intermediates and the pentose phosphate pathway, which results in increased glutamine dependence for both TCA cycle intermediates and reactive oxygen species suppression. Therefore, coinhibition of IKKβ and glutamine metabolism results in dramatic synergistic killing of cancer cells both in vitro and in vivo. In all, our results uncover a previously unidentified role of IKKβ in regulating glycolysis, sensing low-glutamine-induced metabolic stress, and promoting cellular adaptation to nutrient availability. PMID:27585591

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains.

PubMed

Dos Santos, Leandro Vieira; Carazzolle, Marcelo Falsarella; Nagamatsu, Sheila Tiemi; Sampaio, Nádia Maria Vieira; Almeida, Ludimila Dias; Pirolla, Renan Augusto Siqueira; Borelli, Guilherme; Corrêa, Thamy Lívia Ribeiro; Argueso, Juan Lucas; Pereira, Gonçalo Amarante Guimarães

2016-12-21

The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

PubMed Central

dos Santos, Leandro Vieira; Carazzolle, Marcelo Falsarella; Nagamatsu, Sheila Tiemi; Sampaio, Nádia Maria Vieira; Almeida, Ludimila Dias; Pirolla, Renan Augusto Siqueira; Borelli, Guilherme; Corrêa, Thamy Lívia Ribeiro; Argueso, Juan Lucas; Pereira, Gonçalo Amarante Guimarães

2016-01-01

The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production. PMID:28000736

Metabolic response to MMS-mediated DNA damage in Saccharomyces cerevisiae is dependent on the glucose concentration in the medium.

PubMed

Kitanovic, Ana; Walther, Thomas; Loret, Marie Odile; Holzwarth, Jinda; Kitanovic, Igor; Bonowski, Felix; Van Bui, Ngoc; Francois, Jean Marie; Wölfl, Stefan

2009-06-01

Maintenance and adaptation of energy metabolism could play an important role in the cellular ability to respond to DNA damage. A large number of studies suggest that the sensitivity of cells to oxidants and oxidative stress depends on the activity of cellular metabolism and is dependent on the glucose concentration. In fact, yeast cells that utilize fermentative carbon sources and hence rely mainly on glycolysis for energy appear to be more sensitive to oxidative stress. Here we show that treatment of the yeast Saccharomyces cerevisiae growing on a glucose-rich medium with the DNA alkylating agent methyl methanesulphonate (MMS) triggers a rapid inhibition of respiration and enhances reactive oxygen species (ROS) production, which is accompanied by a strong suppression of glycolysis. Further, diminished activity of pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase upon MMS treatment leads to a diversion of glucose carbon to glycerol, trehalose and glycogen accumulation and an increased flux through the pentose-phosphate pathway. Such conditions finally result in a significant decline in the ATP level and energy charge. These effects are dependent on the glucose concentration in the medium. Our results clearly demonstrate that calorie restriction reduces MMS toxicity through increased respiration and reduced ROS accumulation, enhancing the survival and recovery of cells.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

Treesearch

Dana J. Wolbach; Alan Kuo; Trey K. Sato; Katlyn M. Potts; Asaf A. Salamov; Kurt M. LaButti; Hui Sun; Alicia Clum; Jasmyn L. Pangilinan; Erika A. Lindquist; Susan Lucas; Alla Lapidus; Mingjie Jin; Christa Gunawan; Venkatesh Balan; Bruce E. Dale; Thomas W. Jeffries; Robert Zinkel; Kerrie W. Barry; Igor V. Grigoriev; Audrey P. Gasch

2011-01-01

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative...

Production of lactic acid from hemicellulose extracts by Bacillus coagulans MXL-9.

PubMed

Walton, Sara L; Bischoff, Kenneth M; van Heiningen, Adriaan R P; van Walsum, G Peter

2010-08-01

Bacillus coagulans MXL-9 was found capable of growing on pre-pulping hemicellulose extracts, utilizing all of the principle monosugars found in woody biomass. This organism is a moderate thermophile isolated from compost for its pentose-utilizing capabilities. It was found to have high tolerance for inhibitors such as acetic acid and sodium, which are present in pre-pulping hemicellulose extracts. Fermentation of 20 g/l xylose in the presence of 30 g/l acetic acid required a longer lag phase but overall lactic acid yield was not diminished. Similarly, fermentation of xylose in the presence of 20 g/l sodium increased the lag time but did not affect overall product yield, though 30 g/l sodium proved completely inhibitory. Fermentation of hot water-extracted Siberian larch containing 45 g/l total monosaccharides, mainly galactose and arabinose, produced 33 g/l lactic acid in 60 h and completely consumed all sugars. Small amounts of co-products were formed, including acetic acid, formic acid, and ethanol. Hemicellulose extract formed during autohydrolysis of mixed hardwoods contained mainly xylose and was converted into lactic acid with a 94% yield. Green liquor-extracted hardwood hemicellulose containing 10 g/l acetic acid and 6 g/l sodium was also completely converted into lactic acid at a 72% yield. The Bacillus coagulans MXL-9 strain was found to be well suited to production of lactic acid from lignocellulosic biomass due to its compatibility with conditions favorable to industrial enzymes and its ability to withstand inhibitors while rapidly consuming all pentose and hexose sugars of interest at high product yields.

COMPREHENSIVE MOLECULAR CHARACTERIZATION OF CLEAR CELL RENAL CELL CARCINOMA

PubMed Central

2013-01-01

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (e.g. VHL) and the maintenance of chromatin states (e.g. PBRM1). We surveyed more than 400 tumors using different genomic platforms and identified 19 significantly mutated genes. The PI3K/Akt pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving down-regulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, up-regulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 and GRB10. Remodeling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumor stage and severity and offers new views on the opportunities for disease treatment. PMID:23792563

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

PubMed

Harner, Nicole K; Wen, Xin; Bajwa, Paramjit K; Austin, Glen D; Ho, Chi-Yip; Habash, Marc B; Trevors, Jack T; Lee, Hung

2015-01-01

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

PubMed

Papapetridis, Ioannis; van Dijk, Marlous; Dobbe, Arthur P A; Metz, Benjamin; Pronk, Jack T; van Maris, Antonius J A

2016-04-26

Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Replacement of the native NADP(+)-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD(+)-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15% increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP(+)-dependent acetaldehyde dehydrogenase, led to a 39% increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44% increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13% increase in the ethanol yield on glucose. The combination of NAD(+)-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.

Investigation of miR-136-5p key target genes and pathways in lung squamous cell cancer based on TCGA database and bioinformatics analysis.

PubMed

Xie, Zu-Cheng; Li, Tian-Tian; Gan, Bin-Liang; Gao, Xiang; Gao, Li; Chen, Gang; Hu, Xiao-Hua

2018-05-01

Lung squamous cell cancer (LUSC) is a common but challenging malignancy. It is important to illuminate the molecular mechanism of LUSC. Thus, we aim to explore the molecular mechanism of miR-136-5p in relation to LUSC. We used the Cancer Genome Atlas (TCGA) database to investigate the expression of miR-136-5p in relation to LUSC. Then, we identified the possible miR-136-5p target genes through intersection of the predicted miR-136-5p target genes and LUSC upregulated genes from TCGA. Bioinformatics analysis was performed to determine the key miR-136-5p targets and pathways associated with LUSC. Finally, the expression of hub genes, correlation between miR-136-5p and hub genes, and expected significance of hub genes were evaluated via the TCGA and Genotype-Tissue Expression (GTEx) project. MiR-136-5p was significantly downregulated in LUSC patients. Glucuronidation, glucuronosyltransferase, and the retinoic acid metabolic process were the most enriched metabolic interactions in LUSC patients. Ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism were identified as crucial pathways. Seven hub genes (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A10, SRD5A1, and ADH7) were found to be upregulated, and UGT1A1, UGT1A3, UGT1A6, UGT1A7, and ADH7 were negatively correlated with miR-136-5p. UGT1A7 and ADH7 were the most significantly involved miR-136-5p target genes, and high expression of these genes was correlated with better overall survival and disease-free survival of LUSC patients. Downregulated miR-136-5p may target UGT1A7 and ADH7 and participate in ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism. High expression of UGT1A7 and ADH7 may indicate better prognosis of LUSC patients. Copyright © 2018. Published by Elsevier GmbH.

Metabolomics-Based Elucidation of Active Metabolic Pathways in Erythrocytes and HSC-Derived Reticulocytes.

PubMed

Srivastava, Anubhav; Evans, Krystal J; Sexton, Anna E; Schofield, Louis; Creek, Darren J

2017-04-07

A detailed analysis of the metabolic state of human-stem-cell-derived erythrocytes allowed us to characterize the existence of active metabolic pathways in younger reticulocytes and compare them to mature erythrocytes. Using high-resolution LC-MS-based untargeted metabolomics, we found that reticulocytes had a comparatively much richer repertoire of metabolites, which spanned a range of metabolite classes. An untargeted metabolomics analysis using stable-isotope-labeled glucose showed that only glycolysis and the pentose phosphate pathway actively contributed to the biosynthesis of metabolites in erythrocytes, and these pathways were upregulated in reticulocytes. Most metabolite species found to be enriched in reticulocytes were residual pools of metabolites produced by earlier erythropoietic processes, and their systematic depletion in mature erythrocytes aligns with the simplification process, which is also seen at the cellular and the structural level. Our work shows that high-resolution LC-MS-based untargeted metabolomics provides a global coverage of the biochemical species that are present in erythrocytes. However, the incorporation of stable isotope labeling provides a more accurate description of the active metabolic processes that occur in each developmental stage. To our knowledge, this is the first detailed characterization of the active metabolic pathways of the erythroid lineage, and it provides a rich database for understanding the physiology of the maturation of reticulocytes into mature erythrocytes.

Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

PubMed Central

Rehberg, Markus; Ritter, Joachim B.; Reichl, Udo

2014-01-01

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model's predictive power supports the design of more efficient bioprocesses. PMID:25329309

Glycolysis is governed by growth regime and simple enzyme regulation in adherent MDCK cells.

PubMed

Rehberg, Markus; Ritter, Joachim B; Reichl, Udo

2014-10-01

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model's predictive power supports the design of more efficient bioprocesses.

Identification of Metabolic Pathways Expressed by Pichia anomala Kh6 in the Presence of the Pathogen Botrytis cinerea on Apple: New Possible Targets for Biocontrol Improvement

PubMed Central

Kwasiborski, Anthony; Bajji, Mohammed; Renaut, Jenny; Delaplace, Pierre; Jijakli, M. Haissam

2014-01-01

Yeast Pichia anomala strain Kh6 Kurtzman (Saccharomycetales: Endomycetaceae) exhibits biological control properties that provide an alternative to the chemical fungicides currently used by fruit or vegetable producers against main post-harvest pathogens, such as Botrytis cinerea (Helotiales: Sclerotiniaceae). Using an in situ model that takes into account interactions between organisms and a proteomic approach, we aimed to identify P. anomala metabolic pathways influenced by the presence of B. cinerea. A total of 105 and 60 P. anomala proteins were differentially represented in the exponential and stationary growth phases, respectively. In the exponential phase and in the presence of B. cinerea, the pentose phosphate pathway seems to be enhanced and would provide P. anomala with the needed nucleic acids and energy for the wound colonisation. In the stationary phase, P. anomala would use alcoholic fermentation both in the absence and presence of the pathogen. These results would suggest that the competitive colonisation of apple wounds could be implicated in the mode of action of P. anomala against B. cinerea. PMID:24614090

Using isotopic tracers to assess the impact of tillage and straw management on the microbial metabolic network in soil

NASA Astrophysics Data System (ADS)

Van Groenigen, K.; Forristal, D.; Jones, M. B.; Schwartz, E.; Hungate, B. A.; Dijkstra, P.

2013-12-01

By decomposing soil organic matter, microbes gain energy and building blocks for biosynthesis and release CO2 to the atmosphere. Therefore, insight into the effect of management practices on microbial metabolic pathways and C use efficiency (CUE; microbial C produced per substrate C utilized) may help to predict long term changes in soil C stocks. We studied the effects of reduced (RT) and conventional tillage (CT) on the microbial central C metabolic network, using soil samples from a 12-year-old field experiment in an Irish winter wheat cropping system. Each year after harvest, straw was removed from half of the RT and CT plots or incorporated into the soil in the other half, resulting in four treatment combinations. We added 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose as metabolic tracer isotopomers to composite soil samples taken at two depths (0-15 cm and 15-30 cm) from each treatment and used the rate of position-specific respired 13CO2 to parameterize a metabolic model. Model outcomes were then used to calculate CUE of the microbial community. We found that the composite samples differed in CUE, but the changes were small, with values ranging between 0.757-0.783 across treatments and soil depth. Increases in CUE were associated with a decrease in tricarboxylic acid cycle and reductive pentose phosphate pathway activity and increased consumption of metabolic intermediates for biosynthesis. Our results indicate that RT and straw incorporation promote soil C storage without substantially changing CUE or any of the microbial metabolic pathways. This suggests that at our site, RT and straw incorporation promote soil C storage mostly through direct effects such as increased soil C input and physical protection from decomposition, rather than by feedback responses of the microbial community.

The conversion of lignocellulosics to fermentable sugars - A survey of current research and applications to CELSS

NASA Technical Reports Server (NTRS)

Petersen, Gene R.; Baresi, Larry

1990-01-01

This report provides an overview options for converting lignocellulosics into fermentable sugars in CELSS. A requirement for pretreatment is shown. Physical-chemical and enzymatic hydrolysis processes for producing fermentable sugars are discussed. At present physical-chemical methods are the simplest and best characterized options, but enzymatic processes will be the likely method of choice in the future. The use of pentose sugars by microorganisms to produce edibles is possible. The use of mycelial food production on pretreated but not hydrolyzed lignocellulosics is also possible. Simple trade-off analyses to regenerate waste lignocellulosics for two pathways are made, one of which is compared to complete oxidation.

Isolation and characterization of a resident tolerant Saccharomyces cerevisiae strain from a spent sulfite liquor fermentation plant

PubMed Central

2012-01-01

Spent Sulfite Liquor (SSL) from wood pulping facilities is a sugar rich effluent that can be used as feedstock for ethanol production. However, depending on the pulping process conditions, the release of monosaccharides also generates a range of compounds that negatively affect microbial fermentation. In the present study, we investigated whether endogenous yeasts in SSL-based ethanol plant could represent a source of Saccharomyces cerevisiae strains with a naturally acquired tolerance towards this inhibitory environment. Two isolation processes were performed, before and after the re-inoculation of the plant with a commercial baker’s yeast strain. The isolates were clustered by DNA fingerprinting and a recurrent Saccharomyces cerevisiae strain, different from the inoculated commercial baker’s yeast strain, was isolated. The strain, named TMB3720, flocculated heavily and presented high furaldehyde reductase activity. During fermentation of undiluted SSL, TMB3720 displayed a 4-fold higher ethanol production rate and 1.8-fold higher ethanol yield as compared to the commercial baker’s yeast. Another non-Saccharomyces cerevisiae species, identified as the pentose utilizing Pichia galeiformis, was also recovered in the last tanks of the process where the hexose to pentose sugar ratio and the inhibitory pressure are expected to be the lowest. PMID:23237549

Evaluation of polymeric adsorbent resins for efficient detoxification of liquor generated during acid pretreatment of lignocellulosic biomass.

PubMed

Sandhya, Soolamkandath Variem; Kiran, Kumar; Kuttiraja, Mathiyazhakan; Preeti, Varghese Elizabeth; Sindhu, Raveendran; Vani, Sankar; Kumar, Sukumaran Rajeev; Pandey, Ashok; Binod, Parameswaran

2013-11-01

Production of fuel ethanol from lignocellulosic biomass conventionally includes biomass pretreatment, hydrolysis, and fermentation. The liquor generated during dilute acid pretreatment of biomass contains considerable quantities of pentose sugars as well as various degradation products of sugars and lignin, like furfural, hydroxymethyl furfural (HMF), organic acids, aldehydes and others, which are known to be inhibitory for microbial growth. This pentose rich liquor is a potent resource which can be used to produce alcohol or other value added metabolites by microbial fermentation. However, the presence of these inhibitory compounds is a major hindrance and their removal is essential for efficient utilization of this byproduct stream. In the present work, the polymeric adsorbent resins, XAD-4, XAD-7 and XAD-16 were evaluated for their ability to adsorb fermentation inhibitors like furfural and HMF from the acid pretreated liquor. These resins could remove 55-75% of furfural and 100% of HMF and more than 90% sugar remained un-adsorbed in the pretreated liquor. Desorption of furfural from stationary phase was evaluated by using ethanol and hot water. The results suggest that these polymeric resins may be used for detoxification of acid pretreatment liquor with selective removal of sugar degradation products without affecting the sugar content in the solution.

Differential Effects of Hormones on Cellular Metabolism in Keratoconus In Vitro

PubMed Central

McKay, Tina B.; Hjortdal, Jesper; Sejersen, Henrik; Karamichos, Dimitrios

2017-01-01

Keratoconus (KC) is a corneal thinning disease with an onset commonly immediately post-puberty and stabilization by 40 to 50 years of age. The role of hormones in regulating corneal tissue structure in homeostatic and pathological conditions is unknown. Our group recently linked altered hormone levels to KC. Our current study sought to investigate and delineate the effects of exogenous hormones, such as androgen, luteotropin, and estrogen, on corneal stroma bioenergetics. We utilized our established 3D in vitro model to characterize the effects of DHEA, prolactin, 17β-estradiol on insulin-growth factor-1 and -2 (IGF-1, -2) signaling and metabolic function in primary corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs). Our data showed that exogenous DHEA significantly downregulated IGF-1 and its receptor in both HCFs and HKCs with HKCs showing consistently lower basal pentose phosphate flux. Prolactin caused no significant change in IGF-1 levels and an increase in IGF-2 in HKCs correlating with an increase in ATP and NADH levels. 17β-estradiol led to a significant upregulation in pentose phosphate flux and glycolytic intermediates in HCFs. Our results identified hormone-specific responses regulated in HKCs compared to HCFs revealing a novel role for hormones on bioenergetics in KC. PMID:28211546

[Phenotypic properties of Sulfobacillus thermotolerans: comparative aspects].

PubMed

Tsaplina, I A; Krasil'nikova, E N; Zhuravleva, A E; Egorova, M A; Zakharchuk, L M; Suzina, N E; Duda, V I; Bogdanova, T I; Stadnichuk, I N; Kondrat'eva, T F

2008-01-01

The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1(T), as dependent on the cultivation conditions, are described in detail. High growth rates (0.22-0.30 h(-1)) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 degrees C on medium with inorganic (Fe(II), S(0), or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum conditions on medium with iron, hemes a, b, and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells. In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 degrees C, the enzymes of the three main pathways of carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth conditions. The growth rate in the regions of limiting temperatures (55 degrees C and 12-14 degrees C) decreased two- and tenfold, respectively; no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed. The rate of 14CO2 fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein), respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant sulfobacillus are compared versus moderately thermophilic sulfobacilli.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Enhancing ethanol yields through d-xylose and l-arabinose co-fermentation after construction of a novel high efficient l-arabinose-fermenting Saccharomyces cerevisiae strain.

PubMed

Caballero, Antonio; Ramos, Juan Luis

2017-04-01

Lignocellulose contains two pentose sugars, l-arabinose and d-xylose, neither of which is naturally fermented by first generation (1G) ethanol-producing Saccharomyces cerevisiae yeast. Since these sugars are inaccessible to 1G yeast, a significant percentage of the total carbon in bioethanol production from plant residues, which are used in second generation (2G) ethanol production, remains unused. Recombinant Saccharomyces cerevisiae strains capable of fermenting d-xylose are available on the market; however, there are few examples of l-arabinose-fermenting yeasts, and commercially, there are no strains capable of fermenting both d-xylose and l-arabinose because of metabolic incompatibilities when both metabolic pathways are expressed in the same cell. To attempt to solve this problem we have tested d-xylose and l-arabinose co-fermentation. To find efficient alternative l-arabinose utilization pathways to the few existing ones, we have used stringent methodology to screen for new genes (metabolic and transporter functions) to facilitate l-arabinose fermentation in recombinant yeast. We demonstrate the feasibility of this approach in a successfully constructed yeast strain capable of using l-arabinose as the sole carbon source and capable of fully transforming it to ethanol, reaching the maximum theoretical fermentation yield (0.43 g g-1). We demonstrate that efficient co-fermentation of d-xylose and l-arabinose is feasible using two different co-cultured strains, and observed no fermentation delays, yield drops or accumulation of undesired byproducts. In this study we have identified a technically efficient strategy to enhance ethanol yields by 10 % in 2G plants in a process based on C5 sugar co-fermentation.

Separation of Glucose and Pentose Sugars by Selective Enzyme Hydrolysis of AFEX-Treated Corn Fiber

NASA Astrophysics Data System (ADS)

Hanchar, Robert J.; Teymouri, Farzaneh; Nielson, Chandra D.; McCalla, Darold; Stowers, Mark D.

A process was developed to fractionate corn fiber into glucose- and pentose-rich fractions. Corn fiber was ammonia fiber explosion treated at 90°C, using 1 g anhydrous ammonia per gram of dry biomass, 60% moisture, and 30-min residence time. Twenty four hour hydrolysis of ammonia fiber explosion-treated corn fiber with cellulase converted 83% of available glucanto-glucose. In this hydrolysis the hemicellulose was partially broken down with 81% of the xylan and 68% of the arabinan being contained in the hydrolysate after filtration to remove lignin and other insoluble material. Addition of ethanol was used to precipitate and recover the solubilized hemicellulose from the hydrolysate, followed by hydrolysis with 2% (v/v) sulfuric acid to convert the recovered xylan and arabinan to monomeric sugars. Using this method, 57% of xylose and 54% of arabinose available in corn fiber were recovered in a pentose-rich stream. The carbohydrate composition of the pentose-enriched stream was 5% glucose, 57% xylose, 27% arabinose, and 11% galactose. The carbohydrate composition of the glucose-enriched stream was 87% glucose, 5% xylose, 6% arabinose, and 1% galactose, and contained 83% of glucose available from the corn fiber.

Targeting MUC1-C inhibits the AKT-S6K1-elF4A pathway regulating TIGAR translation in colorectal cancer.

PubMed

Ahmad, Rehan; Alam, Maroof; Hasegawa, Masanori; Uchida, Yasumitsu; Al-Obaid, Omar; Kharbanda, Surender; Kufe, Donald

2017-02-02

Colorectal cancer is third most common malignancy and is the second most common cause of cancer-related death. The MUC1 heterodimeric protein is aberrantly overexpressed in colorectal cancer and has been linked to poor outcomes in this disease. Here, we investigate the effects of the MUC1-C subunit inhibitor (GO-203), which disrupts MUC1-C homo-oligomerization, on human colorectal cancer cells. TIGAR mRNA level was determined using qRT-PCR. Western blotting was used to measure TIGAR protein level and AKT-mTOR-S6K1 pathways. Reactive oxygen species and apoptosis were measured by flow cytometry. Effect of MUC1-C peptide, GO-203 was studied on colorectal xenograft tumors. Immunohistochemistry was utilized for TIGAR staining. Treatment of MUC1-overexpressing SKCO-1 and Colo-205 colon cancer cells with GO-203 was associated with downregulation of the TP53-inducible glycolysis and apoptosis regulator (TIGAR) protein. TIGAR promotes the shunting of glycolytic intermediates into the pentose phosphate pathway and thus is of importance for maintaining redox balance. We show that GO-203-induced suppression of TIGAR is mediated by inhibition of AKT and the downstream mTOR pathway. The results also demonstrate that targeting MUC1-C blocks eIF4A cap-dependent translation of TIGAR. In concert with these results, GO-203-induced suppression of TIGAR was associated with decreases in GSH levels. GO-203 treatment also resulted in increases in reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. Consistent with these results, GO-203 inhibited the growth of colon cancer cells in vitro and as xenografts in nude mice. Inhibition of MUC1-C also downregulated TIGAR expression in xenograft tissues. These findings indicate that MUC1-C is a potential target for the treatment of colorectal cancer. Colorectal cancer patients who overexpress MUC1-C may be candidates for treatment with the MUC1-C inhibitor alone or in combination therapy with other agents.

Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

PubMed Central

2015-01-01

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression. PMID:24937102

Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain.

PubMed

Morken, Tora Sund; Brekke, Eva; Håberg, Asta; Widerøe, Marius; Brubakk, Ann-Mari; Sonnewald, Ursula

2014-01-01

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-(13)C]glucose and were sacrificed 30 min later. Brain extracts were analysed using (1)H- and (13)C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-(13)C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-(13)C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-(13)C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.

Glutathione-related enzymes and the eye.

PubMed

Ganea, Elena; Harding, John J

2006-01-01

Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and administration of some drugs.

Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors.

PubMed

Marin-Valencia, Isaac; Cho, Steve K; Rakheja, Dinesh; Hatanpaa, Kimmo J; Kapur, Payal; Mashimo, Tomoyuki; Jindal, Ashish; Vemireddy, Vamsidhara; Good, Levi B; Raisanen, Jack; Sun, Xiankai; Mickey, Bruce; Choi, Changho; Takahashi, Masaya; Togao, Osamu; Pascual, Juan M; Deberardinis, Ralph J; Maher, Elizabeth A; Malloy, Craig R; Bachoo, Robert M

2012-10-01

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-(13) C(2) ]glucose. The [3-(13) C]lactate/[2,3-(13) C(2) ]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, (13) C-(13) C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, (13) C multiplets of γ-aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that (13) C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Carbohydrate Analysis Experiment Involving Mono- and Disaccharides with a Twist of Glycobiology: Two New Tests for Distinguishing Pentoses and Glycosidic Bonds

ERIC Educational Resources Information Center

Herdman, Chelsea; Diop, Lamine; Dickman, Michael

2013-01-01

Carbohydrate analysis is an excellent way to expose second-year biochemistry majors to the subtlety and nuance of sugar chemistry. In one, 3-h practical period, students must identify an unknown mono- or disaccharide from a collection of three hexoses (glucose, galactose, and mannose), two pentoses (ribose and arabinose), and three disaccharides…

Comparative Proteome Analysis between High Lipid-Producing Strain Mucor circinelloides WJ11 and Low Lipid-Producing Strain CBS 277.49.

PubMed

Tang, Xin; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Song, Yuanda; Chen, Wei

2017-06-21

Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.

6-Phosphogluconate dehydrogenase links oxidative PPP, lipogenesis and tumour growth by inhibiting LKB1-AMPK signalling.

PubMed

Lin, Ruiting; Elf, Shannon; Shan, Changliang; Kang, Hee-Bum; Ji, Quanjiang; Zhou, Lu; Hitosugi, Taro; Zhang, Liang; Zhang, Shuai; Seo, Jae Ho; Xie, Jianxin; Tucker, Meghan; Gu, Ting-Lei; Sudderth, Jessica; Jiang, Lei; Mitsche, Matthew; DeBerardinis, Ralph J; Wu, Shaoxiong; Li, Yuancheng; Mao, Hui; Chen, Peng R; Wang, Dongsheng; Chen, Georgia Zhuo; Hurwitz, Selwyn J; Lonial, Sagar; Arellano, Martha L; Khoury, Hanna J; Khuri, Fadlo R; Lee, Benjamin H; Lei, Qunying; Brat, Daniel J; Ye, Keqiang; Boggon, Titus J; He, Chuan; Kang, Sumin; Fan, Jun; Chen, Jing

2015-11-01

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

High-quality draft genome sequence of the Thermus amyloliquefaciens type strain YIM 77409 T with an incomplete denitrification pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, En -Min; Murugapiran, Senthil K.; Mefferd, Chrisabelle C.

Thermus amyloliquefaciens type strain YIM 77409 T is a thermophilic, Gram-negative, non-motile and rod-shaped bacterium isolated from Niujie Hot Spring in Eryuan County, Yunnan Province, southwest China. In the present study we describe the features of strain YIM 77409 T together with its genome sequence and annotation. The genome is 2,160,855 bp long and consists of 6 scaffolds with 67.4 % average GC content. A total of 2,313 genes were predicted, comprising 2,257 protein-coding and 56 RNA genes. The genome is predicted to encode a complete glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle. Additionally, a large number of transportersmore » and enzymes for heterotrophy highlight the broad heterotrophic lifestyle of this organism. Furthermore, a denitrification gene cluster included genes predicted to encode enzymes for the sequential reduction of nitrate to nitrous oxide, consistent with the incomplete denitrification phenotype of this strain.« less

High-quality draft genome sequence of the Thermus amyloliquefaciens type strain YIM 77409 T with an incomplete denitrification pathway

DOE PAGES

Zhou, En -Min; Murugapiran, Senthil K.; Mefferd, Chrisabelle C.; ...

2016-02-27

Thermus amyloliquefaciens type strain YIM 77409 T is a thermophilic, Gram-negative, non-motile and rod-shaped bacterium isolated from Niujie Hot Spring in Eryuan County, Yunnan Province, southwest China. In the present study we describe the features of strain YIM 77409 T together with its genome sequence and annotation. The genome is 2,160,855 bp long and consists of 6 scaffolds with 67.4 % average GC content. A total of 2,313 genes were predicted, comprising 2,257 protein-coding and 56 RNA genes. The genome is predicted to encode a complete glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle. Additionally, a large number of transportersmore » and enzymes for heterotrophy highlight the broad heterotrophic lifestyle of this organism. Furthermore, a denitrification gene cluster included genes predicted to encode enzymes for the sequential reduction of nitrate to nitrous oxide, consistent with the incomplete denitrification phenotype of this strain.« less

The ALD6 gene product is indispensable for providing NADPH in yeast cells lacking glucose-6-phosphate dehydrogenase activity.

PubMed

Grabowska, Dorota; Chelstowska, Anna

2003-04-18

Reducing equivalents in the form of NADPH are essential for many enzymatic steps involved in the biosynthesis of cellular macromolecules. An adequate level of NADPH is also required to protect cells against oxidative stress. The major enzymatic source of NADPH in the cell is the reaction catalyzed by glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway. Disruption of the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae, results in methionine auxotrophy and increased sensitivity to oxidizing agents. It is assumed that both phenotypes are due to an NADPH deficiency in the zwf1Delta strain. We used a Met(-) phenotype displayed by the zwf1Delta strain to look for multicopy suppressors of this deletion. We found that overexpression of the ALD6 gene coding for cytosolic acetaldehyde dehydrogenase, which utilizes NADP(+) as its cofactor, restores the Met(+) phenotype of the zwf1Delta strain. Another multicopy suppressor identified in our screen, the ZMS1 gene encoding a putative transcription factor, regulates the level of ALD6 expression. A strain bearing a double ZWF1 ALD6 gene disruption is not viable. Thus, our results indicate the reaction catalyzed by Ald6p as an important source of reducing equivalents in the yeast cells.

Similar is not the same: differences in the function of the (hemi-)cellulolytic regulator XlnR (Xlr1/Xyr1) in filamentous fungi.

PubMed

Klaubauf, Sylvia; Narang, Hari Mander; Post, Harm; Zhou, Miaomiao; Brunner, Kurt; Mach-Aigner, Astrid R; Mach, Robert L; Heck, Albert J R; Altelaar, A F Maarten; de Vries, Ronald P

2014-11-01

The transcriptional activator XlnR (Xlr1/Xyr1) is a major regulator in fungal xylan and cellulose degradation as well as in the utilization of d-xylose via the pentose catabolic pathway. XlnR homologs are commonly found in filamentous ascomycetes and often assumed to have the same function in different fungi. However, a comparison of the saprobe Aspergillus niger and the plant pathogen Magnaporthe oryzae showed different phenotypes for deletion strains of XlnR. In this study wild type and xlnR/xlr1/xyr1 mutants of five fungi were compared: Fusarium graminearum, M. oryzae, Trichoderma reesei, A. niger and Aspergillus nidulans. Growth profiling on relevant substrates and a detailed analysis of the secretome as well as extracellular enzyme activities demonstrated a common role of this regulator in activating genes encoding the main xylanolytic enzymes. However, large differences were found in the set of genes that is controlled by XlnR in the different species, resulting in the production of different extracellular enzyme spectra by these fungi. This comparison emphasizes the functional diversity of a fine-tuned (hemi-)cellulolytic regulatory system in filamentous fungi, which might be related to the adaptation of fungi to their specific biotopes. Data are available via ProteomeXchange with identifier PXD001190. Copyright © 2014 Elsevier Inc. All rights reserved.

Signaling Pathways Regulating Redox Balance in Cancer Metabolism

PubMed Central

De Santis, Maria Chiara; Porporato, Paolo Ettore; Martini, Miriam; Morandi, Andrea

2018-01-01

The interplay between rewiring tumor metabolism and oncogenic driver mutations is only beginning to be appreciated. Metabolic deregulation has been described for decades as a bystander effect of genomic aberrations. However, for the biology of malignant cells, metabolic reprogramming is essential to tackle a harsh environment, including nutrient deprivation, reactive oxygen species production, and oxygen withdrawal. Besides the well-investigated glycolytic metabolism, it is emerging that several other metabolic fluxes are relevant for tumorigenesis in supporting redox balance, most notably pentose phosphate pathway, folate, and mitochondrial metabolism. The relationship between metabolic rewiring and mutant genes is still unclear and, therefore, we will discuss how metabolic needs and oncogene mutations influence each other to satisfy cancer cells’ demands. Mutations in oncogenes, i.e., PI3K/AKT/mTOR, RAS pathway, and MYC, and tumor suppressors, i.e., p53 and liver kinase B1, result in metabolic flexibility and may influence response to therapy. Since metabolic rewiring is shaped by oncogenic driver mutations, understanding how specific alterations in signaling pathways affect different metabolic fluxes will be instrumental for the development of novel targeted therapies. In the era of personalized medicine, the combination of driver mutations, metabolite levels, and tissue of origins will pave the way to innovative therapeutic interventions. PMID:29740540

Signaling Pathways Regulating Redox Balance in Cancer Metabolism.

PubMed

De Santis, Maria Chiara; Porporato, Paolo Ettore; Martini, Miriam; Morandi, Andrea

2018-01-01

The interplay between rewiring tumor metabolism and oncogenic driver mutations is only beginning to be appreciated. Metabolic deregulation has been described for decades as a bystander effect of genomic aberrations. However, for the biology of malignant cells, metabolic reprogramming is essential to tackle a harsh environment, including nutrient deprivation, reactive oxygen species production, and oxygen withdrawal. Besides the well-investigated glycolytic metabolism, it is emerging that several other metabolic fluxes are relevant for tumorigenesis in supporting redox balance, most notably pentose phosphate pathway, folate, and mitochondrial metabolism. The relationship between metabolic rewiring and mutant genes is still unclear and, therefore, we will discuss how metabolic needs and oncogene mutations influence each other to satisfy cancer cells' demands. Mutations in oncogenes, i.e., PI3K/AKT/mTOR, RAS pathway, and MYC, and tumor suppressors, i.e., p53 and liver kinase B1, result in metabolic flexibility and may influence response to therapy. Since metabolic rewiring is shaped by oncogenic driver mutations, understanding how specific alterations in signaling pathways affect different metabolic fluxes will be instrumental for the development of novel targeted therapies. In the era of personalized medicine, the combination of driver mutations, metabolite levels, and tissue of origins will pave the way to innovative therapeutic interventions.

Comparative genomics provides evidence for the 3-hydroxypropionate autotrophic pathway in filamentous anoxygenic phototrophic bacteria and in hot spring microbial mats.

PubMed

Klatt, Christian G; Bryant, Donald A; Ward, David M

2007-08-01

Stable carbon isotope signatures of diagnostic lipid biomarkers have suggested that Roseiflexus spp., the dominant filamentous anoxygenic phototrophic bacteria inhabiting microbial mats of alkaline siliceous hot springs, may be capable of fixing bicarbonate via the 3-hydroxypropionate pathway, which has been characterized in their distant relative, Chloroflexus aurantiacus. The genomes of three filamentous anoxygenic phototrophic Chloroflexi isolates (Roseiflexus sp. RS-1, Roseiflexus castenholzii and Chloroflexus aggregans), but not that of a non-photosynthetic Chloroflexi isolate (Herpetosiphon aurantiacus), were found to contain open reading frames that show a high degree of sequence similarity to genes encoding enzymes in the C. aurantiacus pathway. Metagenomic DNA sequences from the microbial mats of alkaline siliceous hot springs also contain homologues of these genes that are highly similar to genes in both Roseiflexus spp. and Chloroflexus spp. Thus, Roseiflexus spp. appear to have the genetic capacity for carbon dioxide reduction via the 3-hydroxypropionate pathway. This may contribute to heavier carbon isotopic signatures of the cell components of native Roseiflexus populations in mats compared with the signatures of cyanobacterial cell components, as a similar isotopic signature would be expected if Roseiflexus spp. were participating in photoheterotrophic uptake of cyanobacterial photosynthate produced by the reductive pentose phosphate cycle.

Transformation of oil palm fronds into pentose sugars using copper (II) sulfate pentahydrate with the assistance of chemical additive.

PubMed

Loow, Yu-Loong; Wu, Ta Yeong

2018-06-15

Among the chemical pretreatments available for pretreating biomass, the inorganic salt is considered to be a relatively new but simple reagent that offers comparable pentose (C5) sugar recoveries as the conventional dilute acid hydrolysis. This study investigated the effects of different concentrations (1.5-6.0% (v/v)) of H 2 O 2 or Na 2 S 2 O 8 in facilitating CuSO 4 ·5H 2 O pretreatment for improving pentose sugar recovery from oil palm fronds. The best result was observed when 0.2 mol/L of CuSO 4 ·5H 2 O was integrated with 4.5% (v/v) of Na 2 S 2 O 8 to recover 8.2 and 0.9 g/L of monomeric xylose and arabinose, respectively in the liquid fraction. On the other hand, an addition of 1.5% (v/v) of H 2 O 2 yielded approximately 74% lesser total pentose sugars as compared to using 4.5% (v/v) Na 2 S 2 O 8 . By using CuSO 4 ·5H 2 O alone (control), only 0.8 and 1.0 g/L xylose and arabinose, respectively could be achieved. The results mirrored the importance of using chemical additives together with the inorganic salt pretreatment of oil palm fronds. Thus, an addition of 4.5% (v/v) of Na 2 S 2 O 8 during CuSO 4 ·5H 2 O pretreatment of oil palm fronds at 120 °C and 30 min was able to attain a total pentose sugar yield up to ∼40%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis

PubMed Central

Liu, Wenlan; Sun, Zhirong; Qu, Jixu; Yang, Chunning; Zhang, Xiaomin; Wei, Xinxin

2017-01-01

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis. Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis. Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis. However, these results require verification in further studies. PMID:28962162

Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

PubMed

Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

2017-07-01

Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis.

PubMed

Liu, Wenlan; Sun, Zhirong; Qu, Jixu; Yang, Chunning; Zhang, Xiaomin; Wei, Xinxin

2017-09-01

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis . Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis . Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis . However, these results require verification in further studies.

Leukemia cells demonstrate a different metabolic perturbation provoked by 2-deoxyglucose.

PubMed

Miwa, Hiroshi; Shikami, Masato; Goto, Mineaki; Mizuno, Shohei; Takahashi, Miyuki; Tsunekawa-Imai, Norikazu; Ishikawa, Takamasa; Mizutani, Motonori; Horio, Tomohiro; Gotou, Mayuko; Yamamoto, Hidesuke; Wakabayashi, Motohiro; Watarai, Masaya; Hanamura, Ichiro; Imamura, Akira; Mihara, Hidetsugu; Nitta, Masakazu

2013-05-01

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid β-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast.

PubMed

Watanabe, Itsuki; Ando, Akira; Nakamura, Toshihide

2012-02-01

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (D-xylose and L-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ D-xylose and L-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ D-xylose, and 10 g l⁻¹ L-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both D-xylose and L-arabinose.

Glycolytic intermediates and adenosine phosphates in rat liver at high altitude /3,800 m/.

NASA Technical Reports Server (NTRS)

Cipriano, L. F.; Pace, N.

1973-01-01

Liver tissue obtained from adult rats exposed to 3800 m altitude for intervals ranging from 1.5 hr to 63 days was examined by enzymatic analysis. During the first 3 hr of exposure, an immediate decrease in rephosphorylation of high-energy phosphates led to reduced glycogenesis and eventual pileup of AMP, pyruvate, fructose 1,6-diphosphate, glucose 6-phosphate, and glucose. This was accompanied by a reduction of pentose phosphate pathway activity. After 3 to 6 hr, a secondary adjustment of substrate concentrations occurred along with the apparent facilitation of phosphofructokinase. This secondary adjustment appears to increase anaerobic production of ATP and represents a significant intracellular contribution to the acclimatization process at high altitude.

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5'-monophosphate by Saccharomyces cerevisiae.

PubMed

Chen, Yong; Li, Shuya; Xiong, Jian; Li, Zhenjiang; Bai, Jianxin; Zhang, Lei; Ye, Qi; Ouyang, Pingkai; Ying, Hanjie

2010-03-01

A whole cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l(-1) sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.

Glucose-6-Phosphate Dehydrogenase Deficiency.

PubMed

Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

2016-04-01

G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification and characterization of D-xylulokinase from the D-xylose-fermenting fungus, Mucor circinelloides.

PubMed

Komeda, Hidenobu; Yamasaki-Yashiki, Shino; Hoshino, Kazuhiro; Asano, Yasuhisa

2014-11-01

D-Xylulokinase catalyzes the phosphorylation of D-xylulose in the final step of the pentose catabolic pathway to form d-xylulose-5-phosphate. The D-xylulokinase activity was found to be induced by both D-xylose and L-arabinose, as well as some of the other enzymes involved in the pentose catabolism, in the D-xylose-fermenting zygomycetous fungus, Mucor circinelloides NBRC 4572. The putative gene, xyl3, which may encode D-xylulokinase, was detected in the genome sequence of this strain. The amino acid sequence deduced from the gene was more similar to D-xylulokinases from an animal origin than from other fungi. The recombinant enzyme was purified from the E. coli transformant expressing xyl3 and then characterized. The ATP-dependent phosphorylative activity of the enzyme was the highest toward D-xylulose. Its kinetic parameters were determined as Km (D-xylulose) = 0.29 mM and Km (ATP) = 0.51 mM, indicating that the xyl3 gene encoded D-xylulokinase (McXK). Western blot analysis revealed that McXK was induced by L-arabinose as well as D-xylose and the induction was repressed in the presence of D-glucose, suggesting that the enzyme may be involved in the catabolism of D-xylose and L-arabinose and is subject to carbon catabolite repression in this fungus. This is the first study on D-xylulokinase from zygomycetous fungi. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Responsive eLearning exercises to enhance student interaction with metabolic pathways.

PubMed

Roesler, William J; Dreaver-Charles, Kristine

2018-05-01

Successful learning of biochemistry requires students to engage with the material. In the past this often involved students writing out pathways by hand, and more recently directing students to online resources such as videos, songs, and animated slide presentations. However, even these latter resources do not really provide students an opportunity to engage with the material in an active fashion. As part of an online introductory metabolism course that was developed at our university, we created a series of twelve online interactive activities using Adobe Captivate 9. These activities targeted glycolysis, gluconeogenesis, the pentose phosphate pathway, glycogen metabolism, the citric acid cycle, and fatty acid oxidation. The interactive exercises consisted of two types. One involved dragging objects such as names of enzymes or allosteric modifiers to their correct drop locations such as a particular point in a metabolic pathway, a specific enzyme, and so forth. A second type involved clicking on objects, locations within a pathway, and so forth, in response to a particular question. In both types of exercises, students received feedback on their decisions in order to enhance learning. The student feedback received on these activities was very positive, and indicated that they found them to increase their confidence in the material and that they had learned the key principles of each pathway. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):223-229, 2018. © 2018 The International Union of Biochemistry and Molecular Biology.

Metabolic Mitigation of Staphylococcus aureus Vancomycin Intermediate-Level Susceptibility.

PubMed

Gardner, Stewart G; Marshall, Darrell D; Daum, Robert S; Powers, Robert; Somerville, Greg A

2018-01-01

Staphylococcus aureus is a major human pathogen whose infections are increasingly difficult to treat due to increased antibiotic resistance, including resistance to vancomycin. Vancomycin-intermediate S. aureus (VISA) strains develop resistance to vancomycin through adaptive changes that are incompletely understood. Central to this adaptation are metabolic changes that permit growth in the presence of vancomycin. To define the metabolic changes associated with adaptive resistance to vancomycin in S. aureus , the metabolomes of a vancomycin-sensitive and VISA strain pair isolated from the same patient shortly after vancomycin therapy began and following vancomycin treatment failure were analyzed. The metabolic adaptations included increases in acetogenesis, carbon flow through the pentose phosphate pathway, wall teichoic acid and peptidoglycan precursor biosynthesis, purine biosynthesis, and decreased tricarboxylic acid (TCA) cycle activity. The significance of these metabolic pathways for vancomycin-intermediate susceptibility was determined by assessing the synergistic potential of human-use-approved inhibitors of these pathways in combination with vancomycin against VISA strains. Importantly, inhibitors of amino sugar and purine biosynthesis acted synergistically with vancomycin to kill a diverse set of VISA strains, suggesting that combinatorial therapy could augment the efficacy of vancomycin even in patients infected with VISA strains. Copyright © 2017 American Society for Microbiology.

Isolation and characterization of yeasts capable of efficient utilization of hemicellulosic hydrolyzate as the carbon source.

PubMed

Cassa-Barbosa, L A; Procópio, R E L; Matos, I T S R; Filho, S A

2015-09-28

Few yeasts have shown the potential to efficiently utilize hemicellulosic hydrolyzate as the carbon source. In this study, microorganisms isolated from the Manaus region in Amazonas, Brazil, were characterized based on their utilization of the pentoses, xylose, and arabinose. The yeasts that showed a potential to assimilate these sugars were selected for the better utilization of lignocellulosic biomass. Two hundred and thirty seven colonies of unicellular microorganisms grown on hemicellulosic hydrolyzate, xylose, arabinose, and yeast nitrogen base selective medium were analyzed. Of these, 231 colonies were subjected to sugar assimilation tests. One hundred and twenty five of these were shown to utilize hydrolyzed hemicellulose, xylose, or arabinose as the carbon source for growth. The colonies that showed the best growth (N = 57) were selected, and their internal transcribed spacer-5.8S rDNA was sequenced. The sequenced strains formed four distinct groups in the phylogenetic tree, and showed a high percentage of similarity with Meyerozyma caribbica, Meyerozyma guilliermondii, Trichosporon mycotoxinivorans, Trichosporon loubieri, Pichia kudriavzevii, Candida lignohabitans, and Candida ethanolica. The discovery of these xylose-fermenting yeasts could attract widespread interest, as these can be used in the cost-effective production of liquid fuel from lignocellulosic materials.

Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing.

PubMed

Marx, A; de Graaf, A A; Wiechert, W; Eggeling, L; Sahm, H

1996-01-20

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. (c) 1996 John Wiley & Sons, Inc.

Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

PubMed Central

Driscoll, Timothy P.; Verhoeve, Victoria I.; Guillotte, Mark L.; Lehman, Stephanie S.; Rennoll, Sherri A.; Beier-Sexton, Magda; Rahman, M. Sayeedur; Azad, Abdu F.

2017-01-01

ABSTRACT Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. PMID:28951473

Metabolomic analysis of pancreatic β-cell insulin release in response to glucose.

PubMed

Huang, Mei; Joseph, Jamie W

2012-01-01

Defining the key metabolic pathways that are important for fuel-regulated insulin secretion is critical to providing a complete picture of how nutrients regulate insulin secretion. We have performed a detailed metabolomics study of the clonal β-cell line 832/13 using a gas chromatography-mass spectrometer (GC-MS) to investigate potential coupling factors that link metabolic pathways to insulin secretion. Mid-polar and polar metabolites, extracted from the 832/13 β-cells, were derivatized and then run on a GC/MS to identify and quantify metabolite concentrations. Three hundred fifty-five out of 527 chromatographic peaks could be identified as metabolites by our metabolomic platform. These identified metabolites allowed us to perform a systematic analysis of key pathways involved in glucose-stimulated insulin secretion (GSIS). Of these metabolites, 41 were consistently identified as biomarker for GSIS by orthogonal partial least-squares (OPLS). Most of the identified metabolites are from common metabolic pathways including glycolytic, sorbitol-aldose reductase pathway, pentose phosphate pathway, and the TCA cycle suggesting these pathways play an important role in GSIS. Lipids and related products were also shown to contribute to the clustering of high glucose sample groups. Amino acids lysine, tyrosine, alanine and serine were upregulated by glucose whereas aspartic acid was downregulated by glucose suggesting these amino acids might play a key role in GSIS. In summary, a coordinated signaling cascade elicited by glucose metabolism in pancreatic β-cells is revealed by our metabolomics platform providing a new conceptual framework for future research and/or drug discovery.

Furfural production using ionic liquids: A review.

PubMed

Peleteiro, Susana; Rivas, Sandra; Alonso, José Luis; Santos, Valentín; Parajó, Juan Carlos

2016-02-01

Furfural, a platform chemical with a bright future, is commercially obtained by acidic processing of xylan-containing biomass in aqueous media. Ionic liquids (ILs) can be employed in processed for furfural manufacture as additives, as catalysts and/or as reaction media. Depending on the IL utilized, externally added catalysts (usually, Lewis acids, Brönsted acids and/or solid acid catalysts) can be necessary to achieve high reaction yields. Oppositely, acidic ionic liquids (AILs) can perform as both solvents and catalysts, enabling the direct conversion of suitable substrates (pentoses, pentosans or xylan-containing biomass) into furfural. Operating in IL-containing media, the furfural yields can be improved when the product is continuously removed along the reaction (for example, by stripping or extraction), to avoid unwanted side-reactions leading to furfural consumption. These topics are reviewed, as well as the major challenges involved in the large scale utilization of ILs for furfural production. Copyright © 2015 Elsevier Ltd. All rights reserved.

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

PubMed Central

Johnsen, Ulrike; Dambeck, Michael; Zaiss, Henning; Fuhrer, Tobias; Soppa, Jörg; Sauer, Uwe; Schönheit, Peter

2009-01-01

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following 13C-labeling patterns of proteinogenic amino acids after growth on [13C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus. PMID:19584053

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

PubMed

Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

2014-09-01

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Screening the yeast genome for energetic metabolism pathways involved in a phenotypic response to the anti-cancer agent 3-bromopyruvate.

PubMed

Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2016-03-01

In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP.

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

PubMed

Zhu, Zhiguang; Zhang, Y-H Percival

2017-01-01

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm -2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Screening the yeast genome for energetic metabolism pathways involved in a phenotypic response to the anti-cancer agent 3-bromopyruvate

PubMed Central

Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H.; Pedersen, Peter L.; Goffeau, Andre; Ułaszewski, Stanisław

2016-01-01

In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP. PMID:26862728

High salt diet induces metabolic alterations in multiple biological processes of Dahl salt-sensitive rats.

PubMed

Wang, Yanjun; Liu, Xiangyang; Zhang, Chen; Wang, Zhengjun

2018-06-01

High salt induced renal disease is a condition resulting from the interactions of genetic and dietary factors causing multiple complications. To understand the metabolic alterations associated with renal disease, we comprehensively analyzed the metabonomic changes induced by high salt intake in Dahl salt-sensitive (SS) rats using GC-MS technology and biochemical analyses. Physiological features, serum chemistry, and histopathological data were obtained as complementary information. Our results showed that high salt (HS) intake for 16 weeks caused significant metabolic alterations in both the renal medulla and cortex involving a variety pathways involved in the metabolism of organic acids, amino acids, fatty acids, and purines. In addition, HS enhanced glycolysis (hexokinase, phosphofructokinase and pyruvate kinase) and amino acid metabolism and suppressed the TCA (citrate synthase and aconitase) cycle. Finally, HS intake caused up-regulation of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the ratio of NADPH/NADP + , NADPH oxidase activity and ROS production, suggesting that increased oxidative stress was associated with an altered PPP pathway. The metabolic pathways identified may serve as potential targets for the treatment of renal damage. Our findings provide comprehensive biochemical details about the metabolic responses to a high salt diet, which may contribute to the understanding of renal disease and salt-induced hypertension in SS rats. Copyright © 2018. Published by Elsevier Inc.

Acetobacter pasteurianus metabolic change induced by initial acetic acid to adapt to acetic acid fermentation conditions.

PubMed

Zheng, Yu; Zhang, Renkuan; Yin, Haisong; Bai, Xiaolei; Chang, Yangang; Xia, Menglei; Wang, Min

2017-09-01

Initial acetic acid can improve the ethanol oxidation rate of acetic acid bacteria for acetic acid fermentation. In this work, Acetobacter pasteurianus was cultured in ethanol-free medium, and energy production was found to increase by 150% through glucose consumption induced by initial acetic acid. However, oxidation of ethanol, instead of glucose, became the main energy production pathway when upon culturing ethanol containing medium. Proteome assay was used to analyze the metabolism change induced by initial acetic acid, which provided insight into carbon metabolic and energy regulation of A. pasteurianus to adapt to acetic acid fermentation conditions. Results were further confirmed by quantitative real-time PCR. In summary, decreased intracellular ATP as a result of initial acetic acid inhibition improved the energy metabolism to produce more energy and thus adapt to the acetic acid fermentation conditions. A. pasteurianus upregulated the expression of enzymes related to TCA and ethanol oxidation to improve the energy metabolism pathway upon the addition of initial acetic acid. However, enzymes involved in the pentose phosphate pathway, the main pathway of glucose metabolism, were downregulated to induce a change in carbon metabolism. Additionally, the enhancement of alcohol dehydrogenase expression promoted ethanol oxidation and strengthened the acetification rate, thereby producing a strong proton motive force that was necessary for energy production and cell tolerance to acetic acid.

Metabolism of halophilic archaea

PubMed Central

Falb, Michaela; Müller, Kerstin; Königsmaier, Lisa; Oberwinkler, Tanja; Horn, Patrick; von Gronau, Susanne; Gonzalez, Orland; Pfeiffer, Friedhelm; Bornberg-Bauer, Erich

2008-01-01

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature. Electronic supplementary material The online version of this article (doi:10.1007/s00792-008-0138-x) contains supplementary material, which is available to authorized users. PMID:18278431

TiO2-catalyzed synthesis of sugars from formaldehyde in extraterrestrial impacts on the early Earth.

PubMed

Civiš, Svatopluk; Szabla, Rafał; Szyja, Bartłomiej M; Smykowski, Daniel; Ivanek, Ondřej; Knížek, Antonín; Kubelík, Petr; Šponer, Jiří; Ferus, Martin; Šponer, Judit E

2016-03-16

Recent synthetic efforts aimed at reconstructing the beginning of life on our planet point at the plausibility of scenarios fueled by extraterrestrial energy sources. In the current work we show that beyond nucleobases the sugar components of the first informational polymers can be synthesized in this way. We demonstrate that a laser-induced high-energy chemistry combined with TiO2 catalysis readily produces a mixture of pentoses, among them ribose, arabinose and xylose. This chemistry might be highly relevant to the Late Heavy Bombardment period of Earth's history about 4-3.85 billion years ago. In addition, we present an in-depth theoretical analysis of the most challenging step of the reaction pathway, i.e., the TiO2-catalyzed dimerization of formaldehyde leading to glycolaldehyde.

Biosynthesis of butyric acid by Clostridium tyrobutyricum.

PubMed

Huang, Jin; Tang, Wan; Zhu, Shengquan; Du, Meini

2018-05-28

Butyric acid (C 3 H 7 COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid through large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production through genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disk-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks, and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.

Gut microbiome may contribute to insulin resistance and systemic inflammation in obese rodents: a meta-analysis.

PubMed

Jiao, Na; Baker, Susan S; Nugent, Colleen A; Tsompana, Maria; Cai, Liting; Wang, Yong; Buck, Michael J; Genco, Robert J; Baker, Robert D; Zhu, Ruixin; Zhu, Lixin

2018-04-01

A number of studies have associated obesity with altered gut microbiota, although results are discordant regarding compositional changes in the gut microbiota of obese animals. Herein we used a meta-analysis to obtain an unbiased evaluation of structural and functional changes of the gut microbiota in diet-induced obese rodents. The raw sequencing data of nine studies generated from high-fat diet (HFD)-induced obese rodent models were processed with QIIME to obtain gut microbiota compositions. Biological functions were predicted and annotated with KEGG pathways with PICRUSt. No significant difference was observed for alpha diversity and Bacteroidetes-to-Firmicutes ratio between obese and lean rodents. Bacteroidia, Clostridia, Bacilli, and Erysipelotrichi were dominant classes, but gut microbiota compositions varied among studies. Meta-analysis of the nine microbiome data sets identified 15 differential taxa and 57 differential pathways between obese and lean rodents. In obese rodents, increased abundance was observed for Dorea, Oscillospira, and Ruminococcus, known for fermenting polysaccharide into short chain fatty acids (SCFAs). Decreased Turicibacter and increased Lactococcus are consistent with elevated inflammation in the obese status. Differential functional pathways of the gut microbiome in obese rodents included enriched pyruvate metabolism, butanoate metabolism, propanoate metabolism, pentose phosphate pathway, fatty acid biosynthesis, and glycerolipid metabolism pathways. These pathways converge in the function of carbohydrate metabolism, SCFA metabolism, and biosynthesis of lipid. HFD-induced obesity results in structural and functional dysbiosis of gut microbiota. The altered gut microbiome may contribute to obesity development by promoting insulin resistance and systemic inflammation.

Global Metabolic Profiling of Infection by an Oncogenic Virus: KSHV Induces and Requires Lipogenesis for Survival of Latent Infection

PubMed Central

Delgado, Tracie; Sanchez, Erica L.; Camarda, Roman; Lagunoff, Michael

2012-01-01

Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi's Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors. PMID:22916018

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

PubMed

Xiong, Wei; Lee, Tai-Chi; Rommelfanger, Sarah; Gjersing, Erica; Cano, Melissa; Maness, Pin-Ching; Ghirardi, Maria; Yu, Jianping

2015-12-07

Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.

Glucose respiration in the intact chloroplast of Chlamydomonas reinhardtii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changguo Chen; Gibbs, M.

1991-01-01

Chloroplastic respiration was monitored by measuring {sup 14}CO{sub 2} from {sup 14}C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast, The patterns of {sup 14}CO{sub 2} evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolypyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K{sub m} for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of {sup 14}CO{sub 2} was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO{sub 2} evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1,more » C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO{sub 2} evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH{sub 4}Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolypyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to Co{sub 2} and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.« less

Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

PubMed Central

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima

2014-01-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and 13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products. PMID:24334667

Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

PubMed

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

2014-02-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

PubMed

Kumar, Arvind; Rai, Lal Chand

2015-01-01

Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Copper stress and filamentous fungus Humicola lutea 103 - ultrastructural changes and activities of key metabolic enzymes.

PubMed

Krumova, Ekaterina Ts; Stoitsova, Stoyanka R; Paunova-Krasteva, Tsvetelina S; Pashova, Svetlana B; Angelova, Maria B

2012-12-01

Humicola lutea 103 is a copper-tolerant fungal strain able to grow in the presence of 300 μg·mL(-1) Cu(2+) under submerged cultivation. To prevent the consequences of copper overload, microorganisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. In spite of this avoidance strategy, high heavy-metal concentrations caused distinct and widespread ultrastructural alterations in H. lutea. The mitochondria were the first and main target of the toxic action. The effect of copper on activities of the key enzymes (hexokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase) included in the 3 main metabolic pathways, glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, was investigated. High metal concentrations exhibited a dramatic negative effect on hexokinase, while the other 3 enzymes showed a significant and dose-dependent stimulation. On the basis of the present and previous results we concluded that the copper-induced oxidative stress plays an important role in the fungal tolerance to high Cu (2+) concentrations.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

PubMed

Messner, Christoph B; Driscoll, Paul C; Piedrafita, Gabriel; De Volder, Michael F L; Ralser, Markus

2017-07-11

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

[Chemical Potentials of Hydrothermal Systems and Formation of Coupled Modular Metabolic Pathways].

PubMed

Marakushev, S A; Belonogova, O V

2015-01-01

According to Gibbs J.W. the number of independent components is the least number of those chemical constituents, by combining which the compositions of all possible phases in the system can be obtained, and at the first stages of development of the primary metabolism of the three-component system C-H-O different hydrocarbons and molecular hydrogen were used as an energy source for, it. In the Archean hydrothermal conditions under the action of the phosphorus chemical potential the C-H-O system was transformed into a four-component system C-H-O-P setting up a gluconeogenic system, which became the basis of power supply for a protometabolism, and formation of a new cycle of CO2 fixation (reductive pentose phosphate pathway). It is shown that parageneses (association) of certain substances permitted the modular constructions of the central metabolism of the system C-H-O-P and the formed modules appear in association with each other in certain physicochemical hydrothermal conditions. Malate, oxaloacetate, pyruvate and phosphoenolpyruvate exhibit a turnstile-like mechanism of switching reaction directions.

Soil Fluxomics: Disentangling Microbial Group Specific Metabolism by Modeling of 13C-Incorporation into PLFAs

NASA Astrophysics Data System (ADS)

Apostel, C.; Kuzyakov, Y.; Dippold, M. A.

2016-12-01

Soils are the largest terrestrial C sinks and microorganisms are the most important drivers of organic matter (OM) dynamics in soils: C allocation to ana- or catabolism in microbial cells is the decisive step, whether C gets oxidized to CO2 or whether it is allocated to microbial biomass, which, after cell death can be stabilized in soils. The metabolic parameter describing the ratio between the two fluxes is the carbon use efficiency (CUE), which can be assessed by position-specific labeling followed by metabolic flux modelling. However, to disentangle the single microbial groups' contribution to the bulk soil CUE, a tracing of individual groups metabolism is necessary. We assessed short-term (3 and 10 days) transformations of monosaccharides by adding position-specifically 13C labeled glucose to soil in a field experiment. Incorporation of 13C in the microbial PLFAs enabled us to distinguish individual microbial groups metabolic fluxes and compare their C-utilization efficiency using a quantitative C-flux model. The position-specific pattern in PLFAs revealed two sets of microorganisms: one metabolized glucose mainly by glycolysis and the other mainly by the pentose-phosphate pathway, which results in a higher CUE. Both of those sets included prokaryotic as well as eukaryotic microorganisms. This demonstrates that phylogenetic grouping is not decisive for the metabolic behavior of a microbial group and that the contribution of individual group members to the soil C fluxes cannot be concluded from their phylogeny.

Thioredoxins in evolutionarily primitive organisms

NASA Technical Reports Server (NTRS)

Buchanan, B. B.

1986-01-01

Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

A method for accounting for maintenance costs in flux balance analysis improves the prediction of plant cell metabolic phenotypes under stress conditions.

PubMed

Cheung, C Y Maurice; Williams, Thomas C R; Poolman, Mark G; Fell, David A; Ratcliffe, R George; Sweetlove, Lee J

2013-09-01

Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade-off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper-osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper-osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Simultaneous Decolorization and Biohydrogen Production from Xylose by Klebsiella oxytoca GS-4-08 in the Presence of Azo Dyes with Sulfonate and Carboxyl Groups

PubMed Central

Cao, Ming-yue; Wang, Peng-tao; Wang, Shi; Yue, Ying-rong; Yuan, Wen-duo; Qiao, Wei-chuan; Wang, Fei

2017-01-01

ABSTRACT Biohydrogen production from the pulp and paper effluent containing rich lignocellulosic material could be achieved by the fermentation process. Xylose, an important hemicellulose hydrolysis product, is used less efficiently as a substrate for biohydrogen production. Moreover, azo dyes are usually added to fabricate anticounterfeiting paper, which further increases the complexity of wastewater. This study reports that xylose could serve as the sole carbon source for a pure culture of Klebsiella oxytoca GS-4-08 to achieve simultaneous decolorization and biohydrogen production. With 2 g liter−1 of xylose as the substrate, a maximum xylose utilization rate (URxyl) and a hydrogen molar yield (HMY) of 93.99% and 0.259 mol of H2 mol of xylose−1, respectively, were obtained. Biohydrogen kinetics and electron equivalent (e− equiv) balance calculations indicated that methyl red (MR) penetrates and intracellularly inhibits both the pentose phosphate pathway and pyruvate fermentation pathway, while methyl orange (MO) acted independently of the glycolysis and biohydrogen pathway. The data demonstrate that biohydrogen pathways in the presence of azo dyes with sulfonate and carboxyl groups were different, but the azo dyes could be completely reduced during the biohydrogen production period in the presence of MO or MR. The feasibility of hydrogen production from industrial pulp and paper effluent by the strain if the xylose is sufficient was also proved and was not affected by toxic substances which usually exist in such wastewater, except for chlorophenol. This study offers a promising energy-recycling strategy for treating pulp and paper wastewaters, especially for those containing azo dyes. IMPORTANCE The pulp and paper industry is a major industry in many developing countries, and the global market of pulp and paper wastewater treatment is expected to increase by 60% between 2012 and 2020. Such wastewater contains large amounts of refractory contaminants, such as lignin, whose reclamation is considered economically crucial and environmentally friendly. Furthermore, azo dyes are usually added in order to fabricate anticounterfeiting paper, which further increases the complexity of the pulp and paper wastewater. This work may offer a better understanding of biohydrogen production from xylose in the presence of azo dyes and provide a promising energy-recycling method for treating pulp and paper wastewater, especially for those containing azo dyes. PMID:28283518

Altered metabolic pathways in clear cell renal cell carcinoma: A meta-analysis and validation study focused on the deregulated genes and their associated networks

PubMed Central

Zaravinos, Apostolos; Pieri, Myrtani; Mourmouras, Nikos; Anastasiadou, Natassa; Zouvani, Ioanna; Delakas, Dimitris; Deltas, Constantinos

2014-01-01

Clear cell renal cell carcinoma (ccRCC) is the predominant subtype of renal cell carcinoma (RCC). It is one of the most therapy-resistant carcinomas, responding very poorly or not at all to radiotherapy, hormonal therapy and chemotherapy. A more comprehensive understanding of the deregulated pathways in ccRCC can lead to the development of new therapies and prognostic markers. We performed a meta- analysis of 5 publicly available gene expression datasets and identified a list of co- deregulated genes, for which we performed extensive bioinformatic analysis coupled with experimental validation on the mRNA level. Gene ontology enrichment showed that many proteins are involved in response to hypoxia/oxygen levels and positive regulation of the VEGFR signaling pathway. KEGG analysis revealed that metabolic pathways are mostly altered in ccRCC. Similarly, Ingenuity Pathway Analysis showed that the antigen presentation, inositol metabolism, pentose phosphate, glycolysis/gluconeogenesis and fructose/mannose metabolism pathways are altered in the disease. Cellular growth, proliferation and carbohydrate metabolism, were among the top molecular and cellular functions of the co-deregulated genes. qRT-PCR validated the deregulated expression of several genes in Caki-2 and ACHN cell lines and in a cohort of ccRCC tissues. NNMT and NR3C1 increased expression was evident in ccRCC biopsies from patients using immunohistochemistry. ROC curves evaluated the diagnostic performance of the top deregulated genes in each dataset. We show that metabolic pathways are mostly deregulated in ccRCC and we highlight those being most responsible in its formation. We suggest that these genes are candidate predictive markers of the disease. PMID:25594006

Anaplasma phagocytophilum Infection Subverts Carbohydrate Metabolic Pathways in the Tick Vector, Ixodes scapularis.

PubMed

Cabezas-Cruz, Alejandro; Alberdi, Pilar; Valdés, James J; Villar, Margarita; de la Fuente, José

2017-01-01

The obligate intracellular pathogen, Anaplasma phagocytophilum , is the causative agent of human, equine, and canine granulocytic anaplasmosis and tick-borne fever (TBF) in ruminants. A. phagocytophilum has become an emerging tick-borne pathogen in the United States, Europe, Africa, and Asia, with increasing numbers of infected people and animals every year. It has been recognized that intracellular pathogens manipulate host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. However, our current knowledge on how A. phagocytophilum affect these processes in the tick vector, Ixodes scapularis is limited. In this study, a genome-wide search for components of major carbohydrate metabolic pathways was performed in I. scapularis ticks for which the genome was recently published. The enzymes involved in the seven major carbohydrate metabolic pathways glycolysis, gluconeogenesis, pentose phosphate, tricarboxylic acid cycle (TCA), glyceroneogenesis, and mitochondrial oxidative phosphorylation and β-oxidation were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis major carbohydrate metabolic pathway components in response to A. phagocytophilum infection of tick tissues and cultured cells. The results showed that major carbohydrate metabolic pathways are conserved in ticks. A. phagocytophilum infection inhibits gluconeogenesis and mitochondrial metabolism, but increases the expression of glycolytic genes. A model was proposed to explain how A. phagocytophilum could simultaneously control tick cell glucose metabolism and cytoskeleton organization, which may be achieved in part by up-regulating and stabilizing hypoxia inducible factor 1 alpha in a hypoxia-independent manner. The present work provides a more comprehensive view of the major carbohydrate metabolic pathways involved in the response to A. phagocytophilum infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

Engineering of Corynebacterium glutamicum for xylitol production from lignocellulosic pentose sugars.

PubMed

Dhar, Kiran S; Wendisch, Volker F; Nampoothiri, Kesavan Madhavan

2016-07-20

Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L). Copyright © 2016 Elsevier B.V. All rights reserved.

A "footprint" of plant carbon fixation cycle functions during the development of a heterotrophic fungus.

PubMed

Lyu, Xueliang; Shen, Cuicui; Xie, Jiatao; Fu, Yanping; Jiang, Daohong; Hu, Zijin; Tang, Lihua; Tang, Liguang; Ding, Feng; Li, Kunfei; Wu, Song; Hu, Yanping; Luo, Lilian; Li, Yuanhao; Wang, Qihua; Li, Guoqing; Cheng, Jiasen

2015-08-11

Carbon fixation pathway of plants (CFPP) in photosynthesis converts solar energy to biomass, bio-products and biofuel. Intriguingly, a large number of heterotrophic fungi also possess enzymes functionally associated with CFPP, raising the questions about their roles in fungal development and in evolution. Here, we report on the presence of 17 CFPP associated enzymes (ten in Calvin-Benson-Basham reductive pentose phosphate pathway and seven in C4-dicarboxylic acid cycle) in the genome of Sclerotinia sclerotiorum, a heterotrophic phytopathogenic fungus, and only two unique enzymes: ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) and phosphoribulokinase (PRK) were absent. This data suggested an incomplete CFPP-like pathway (CLP) in fungi. Functional profile analysis demonstrated that the activity of the incomplete CLP was dramatically regulated during different developmental stages of S. sclerotiorum. Subsequent experiments confirmed that many of them were essential to the virulence and/or sclerotial formation. Most of the CLP associated genes are conserved in fungi. Phylogenetic analysis showed that many of them have undergone gene duplication, gene acquisition or loss and functional diversification in evolutionary history. These findings showed an evolutionary links in the carbon fixation processes of autotrophs and heterotrophs and implicated the functions of related genes were in course of continuous change in different organisms in evolution.

Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae.

PubMed

Xia, Peng-Fei; Zhang, Guo-Chang; Walker, Berkley; Seo, Seung-Oh; Kwak, Suryang; Liu, Jing-Jing; Kim, Heejin; Ort, Donald R; Wang, Shu-Guang; Jin, Yong-Su

2017-02-17

Global climate change caused by the emission of anthropogenic greenhouse gases (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and phosphoribulokinase from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with the synthetic rPPP was able to exhibit a higher yield of ethanol and lower yields of byproducts (xylitol and glycerol) than a control strain. In addition, the reduced release of carbon dioxide by the engineered strain was observed during xylose fermentation, suggesting that the carbon dioxide generated by pyruvate decarboxylase was partially reassimilated through the synthetic rPPP. These results demonstrated that recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative metabolic pathway. This strategy has a great potential to alleviate GHG emissions during the production of second-generation ethanol.

High-quality-draft genome sequence of the fermenting bacterium Anaerobium acetethylicum type strain GluBS11T (DSM 29698)

DOE PAGES

Patil, Yogita; Müller, Nicolai; Schink, Bernhard; ...

2017-02-20

Anaerobium acetethylicum strain GluBS11 T belongs to the family Lachnospiraceae within the order Clostridiales. It is a Gram-positive, non-motile and strictly anaerobic bacterium isolated from biogas slurry that was originally enriched with gluconate as carbon source (Patil, et al., Int J Syst Evol Microbiol 65:3289-3296, 2015). Here we describe the draft genome sequence of strain GluBS11 T and provide a detailed insight into its physiological and metabolic features. The draft genome sequence generated 4,609,043 bp, distributed among 105 scaffolds assembled using the SPAdes genome assembler method. It comprises in total 4,132 genes, of which 4,008 were predicted to be proteinmore » coding genes, 124 RNA genes and 867 pseudogenes. The content was 43.51 mol %. The annotated genome of strain GluBS11 T contains putative genes coding for the pentose phosphate pathway, the Embden-Meyerhoff-Parnas pathway, the Entner-Doudoroff pathway and the tricarboxylic acid cycle. The genome revealed the presence of most of the necessary genes required for the fermentation of glucose and gluconate to acetate, ethanol, and hydrogen gas. However, a candidate gene for production of formate was not identified.« less

High-quality-draft genome sequence of the fermenting bacterium Anaerobium acetethylicum type strain GluBS11T (DSM 29698)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patil, Yogita; Müller, Nicolai; Schink, Bernhard

Anaerobium acetethylicum strain GluBS11 T belongs to the family Lachnospiraceae within the order Clostridiales. It is a Gram-positive, non-motile and strictly anaerobic bacterium isolated from biogas slurry that was originally enriched with gluconate as carbon source (Patil, et al., Int J Syst Evol Microbiol 65:3289-3296, 2015). Here we describe the draft genome sequence of strain GluBS11 T and provide a detailed insight into its physiological and metabolic features. The draft genome sequence generated 4,609,043 bp, distributed among 105 scaffolds assembled using the SPAdes genome assembler method. It comprises in total 4,132 genes, of which 4,008 were predicted to be proteinmore » coding genes, 124 RNA genes and 867 pseudogenes. The content was 43.51 mol %. The annotated genome of strain GluBS11 T contains putative genes coding for the pentose phosphate pathway, the Embden-Meyerhoff-Parnas pathway, the Entner-Doudoroff pathway and the tricarboxylic acid cycle. The genome revealed the presence of most of the necessary genes required for the fermentation of glucose and gluconate to acetate, ethanol, and hydrogen gas. However, a candidate gene for production of formate was not identified.« less

Properties of anaerobic fungi isolated from several habitats: complexity of phenotypes.

PubMed

Zelená, Viera; Birošová, Lucia; Olejníková, Petra; Polák, Martin; Lakatoš, Boris; Varečka, Ľudovít

2016-01-01

Isolates of anaerobic fungi from rumen, animal faeces and compost displayed morphological similarity with known anaerobic fungi. According to their ITS sequences, species were related to Neocallimastix and Piromyces. Rumen fungi tolerated exposure to an aerobic atmosphere for at least four days. Under anaerobic conditions, they could grow on both, defined or complex substrates. Growth in liquid media was monitored by the continuous measurement of metabolic gases (O2, CO2, H2, CO, H2S, CH4). Monitored metabolism was complex, showed that both CO2 and H2 were produced and subsequently consumed by yet unknown metabolic pathway(s). CO and H2S were evolved similarly, but not identically with the generation of CO2 and H2 suggesting their connection with energetic metabolism. Anaerobic fungi from snail faeces and compost produced concentrations of H2S, H2, CO near the lower limit of detection. The rumen isolates produced cellulases and xylanases with similar pH and temperature optima. Proteolytic enzymes were secreted as well. Activities of some enzymes of the main catabolic pathways were found in cell-free homogenates of mycelia. The results indicate the presence of the pentose cycle, the glyoxylate cycle and an incomplete citrate cycle in these fungi. Differences between isolates indicate phenotypic variability between anaerobic fungi.

Type 2 Diabetes Dysregulates Glucose Metabolism in Cardiac Progenitor Cells*

PubMed Central

Salabei, Joshua K.; Lorkiewicz, Pawel K.; Mehra, Parul; Gibb, Andrew A.; Haberzettl, Petra; Hong, Kyung U.; Wei, Xiaoli; Zhang, Xiang; Li, Qianhong; Wysoczynski, Marcin; Bolli, Roberto; Bhatnagar, Aruni; Hill, Bradford G.

2016-01-01

Type 2 diabetes is associated with increased mortality and progression to heart failure. Recent studies suggest that diabetes also impairs reparative responses after cell therapy. In this study, we examined potential mechanisms by which diabetes affects cardiac progenitor cells (CPCs). CPCs isolated from the diabetic heart showed diminished proliferation, a propensity for cell death, and a pro-adipogenic phenotype. The diabetic CPCs were insulin-resistant, and they showed higher energetic reliance on glycolysis, which was associated with up-regulation of the pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). In WT CPCs, expression of a mutant form of PFKFB, which mimics PFKFB3 activity and increases glycolytic rate, was sufficient to phenocopy the mitochondrial and proliferative deficiencies found in diabetic cells. Consistent with activation of phosphofructokinase in diabetic cells, stable isotope carbon tracing in diabetic CPCs showed dysregulation of the pentose phosphate and glycero(phospho)lipid synthesis pathways. We describe diabetes-induced dysregulation of carbon partitioning using stable isotope metabolomics-based coupling quotients, which relate relative flux values between metabolic pathways. These findings suggest that diabetes causes an imbalance in glucose carbon allocation by uncoupling biosynthetic pathway activity, which could diminish the efficacy of CPCs for myocardial repair. PMID:27151219

Positive selection in glycolysis among Australasian stick insects

PubMed Central

2013-01-01

Background The glycolytic pathway is central to cellular energy production. Selection on individual enzymes within glycolysis, particularly phosphoglucose isomerase (Pgi), has been associated with metabolic performance in numerous organisms. Nonetheless, how whole energy-producing pathways evolve to allow organisms to thrive in different environments and adopt new lifestyles remains little explored. The Lanceocercata radiation of Australasian stick insects includes transitions from tropical to temperate climates, lowland to alpine habitats, and winged to wingless forms. This permits a broad investigation to determine which steps within glycolysis and what sites within enzymes are the targets of positive selection. To address these questions we obtained transcript sequences from seven core glycolysis enzymes, including two Pgi paralogues, from 29 Lanceocercata species. Results Using maximum likelihood methods a signature of positive selection was inferred in two core glycolysis enzymes. Pgi and Glyceraldehyde 3-phosphate dehydrogenase (Gaphd) genes both encode enzymes linking glycolysis to the pentose phosphate pathway. Positive selection among Pgi paralogues and orthologues predominately targets amino acids with residues exposed to the protein’s surface, where changes in physical properties may alter enzyme performance. Conclusion Our results suggest that, for Lancerocercata stick insects, adaptation to new stressful lifestyles requires a balance between maintaining cellular energy production, efficiently exploiting different energy storage pools and compensating for stress-induced oxidative damage. PMID:24079656

The effect of 2,4-dinitrophenol on adipose-tissue metabolism

PubMed Central

Rognstad, Robert; Katz, Joseph

1969-01-01

1. The effect of dinitrophenol on the metabolism of glucose labelled with 14C and tritium by epididymal fat-pad segments from fed rats was studied. Dinitrophenol at concentrations of 0·1–0·3mm: (a) had little effect on glucose utilization; (b) depressed synthesis of fatty acids and greatly increased that of lactate; (c) increased the T/14C ratio in fatty acids synthesized from [U-14C,3-T]glucose and decreased that in fatty acids synthesized from [U-14C,4-T]glucose; (d) abolished randomization of 14C from [6-14C]glucose in lactate. 2. Dinitrophenol stimulated oxidation of pyruvate and greatly inhibited the oxidation of lactate. It inhibited lipogenesis from pyruvate and lactate. 3. From the isotope data it was calculated that: (a) dinitrophenol stimulates oxidation via the tricarboxylic acid cycle three- to six-fold; (b) dinitrophenol depresses markedly the operation of the pentose cycle; (c) in the presence of dinitrophenol, NADPH formed in the pentose cycle provides all the hydrogen equivalents for fatty acid reduction, whereas, in its absence, NADPH provides 50–70% of the hydrogen equivalents; (d) in the presence of dinitrophenol, there is an excess of ATP produced in the cytoplasm, which flows into the mitochondria. A reverse flow operates in the absence of dinitrophenol. 4. A balance of formation and utilization of reduced nicotinamide nucleotides in the cytoplasm was established. With dinitrophenol there is some excess of NADH. There are indications that this excess may be transferred into mitochondria in the form of malate. 5. Our results are interpreted to indicate the absence from adipose tissue of the α-glycerophosphate shuttle for transferring reducing equivalents from the cytoplasm to mitochondria. 6. The effects of dinitrophenol are accounted for in terms of decreased ATP concentrations in the cells, leading to marked decrease in pyruvate carboxylation in the mitochondria and depression of fatty acid synthesis in the cytoplasm. PMID:4388239

Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction.

PubMed

Gupte, Rakhee S; Ata, Hirotaka; Rawat, Dhawjbahadur; Abe, Madoka; Taylor, Mark S; Ochi, Rikuo; Gupte, Sachin A

2011-02-15

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 μM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 μM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 μM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

Improvement of neuronal differentiation by carbon monoxide: Role of pentose phosphate pathway.

PubMed

Almeida, Ana S; Soares, Nuno L; Sequeira, Catarina O; Pereira, Sofia A; Sonnewald, Ursula; Vieira, Helena L A

2018-05-15

Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system. A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Secretome protein signature of human pancreatic cancer stem-like cells.

PubMed

Brandi, Jessica; Dalla Pozza, Elisa; Dando, Ilaria; Biondani, Giulia; Robotti, Elisa; Jenkins, Rosalind; Elliott, Victoria; Park, Kevin; Marengo, Emilio; Costello, Eithne; Scarpa, Aldo; Palmieri, Marta; Cecconi, Daniela

2016-03-16

Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified. The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9. We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered. Copyright © 2016 Elsevier B.V. All rights reserved.

A structural examination and collision cross section database for over 500 metabolites and xenobiotics using drift tube ion mobility spectrometry† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03464d

PubMed Central

Zheng, Xueyun; Aly, Noor A.; Zhou, Yuxuan; Dupuis, Kevin T.; Bilbao, Aivett; Paurus, Vanessa L.; Orton, Daniel J.; Wilson, Ryan; Payne, Samuel H.; Smith, Richard D.

2017-01-01

The confident identification of metabolites and xenobiotics in biological and environmental studies is an analytical challenge due to their immense dynamic range, vast chemical space and structural diversity. Ion mobility spectrometry (IMS) is widely used for small molecule analyses since it can separate isomeric species and be easily coupled with front end separations and mass spectrometry for multidimensional characterizations. However, to date IMS metabolomic and exposomic studies have been limited by an inadequate number of accurate collision cross section (CCS) values for small molecules, causing features to be detected but not confidently identified. In this work, we utilized drift tube IMS (DTIMS) to directly measure CCS values for over 500 small molecules including primary metabolites, secondary metabolites and xenobiotics. Since DTIMS measurements do not need calibrant ions or calibration like some other IMS techniques, they avoid calibration errors which can cause problems in distinguishing structurally similar molecules. All measurements were performed in triplicate in both positive and negative polarities with nitrogen gas and seven different electric fields, so that relative standard deviations (RSD) could be assessed for each molecule and structural differences studied. The primary metabolites analyzed to date have come from key metabolism pathways such as glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle, while the secondary metabolites consisted of classes such as terpenes and flavonoids, and the xenobiotics represented a range of molecules from antibiotics to polycyclic aromatic hydrocarbons. Different CCS trends were observed for several of the diverse small molecule classes and when urine features were matched to the database, the addition of the IMS dimension greatly reduced the possible number of candidate molecules. This CCS database and structural information are freely available for download at http://panomics.pnnl.gov/metabolites/ with new molecules being added frequently. PMID:29568436

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass.

PubMed

Poudel, Suresh; Giannone, Richard J; Rodriguez, Miguel; Raman, Babu; Martin, Madhavi Z; Engle, Nancy L; Mielenz, Jonathan R; Nookaew, Intawat; Brown, Steven D; Tschaplinski, Timothy J; Ussery, David; Hettich, Robert L

2017-01-01

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Although much of the work-to-date has centered on characterizing this microbe's growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand its metabolism on more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum's cellular membrane as the culture progresses. This is undoubtedly a response to the gradual accumulation of lignocellulose-derived inhibitory compounds as the organism deconstructs the switchgrass to access the embedded cellulose. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in various enzymes, including those involved in the interconversion of branched amino acids valine, leucine, and isoleucine to iso- and anteiso-fatty acid precursors. Additionally, the metabolic accumulation of hemicellulose-derived sugars and sugar alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism/pentose phosphate pathway indicates that C. thermocellum shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux in response to C5 sugar metabolites that increase during lignocellulose deconstruction. Integrated omic platforms provided complementary systems biological information that highlight C. thermocellum 's specific response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. These additional viewpoints allowed us to fully realize the level to which the organism adapts to an increasingly challenging culture environment-information that will prove critical to C. thermocellum 's industrial efficacy.

Enhanced uridine 5'-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux.

PubMed

Liu, Dong; Chen, Yong; Li, An; Xie, Jingjing; Xiong, Jian; Bai, Jianxin; Chen, Xiaochun; Niu, Huanqing; Zhou, Tao; Ying, Hanjie

2012-06-01

A whole-cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH(2)PO(4), MgCl(2) and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH(2)PO(4) 22.1 g/L; MgCl(2) 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.

Potential toxicity of nonregulated asbestiform minerals: balangeroite from the western Alps. Part 3: Depletion of antioxidant defenses.

PubMed

Gazzano, Elena; Riganti, Chiara; Tomatis, Maura; Turci, Francesco; Bosia, Amalia; Fubini, Bice; Ghigo, Dario

2005-01-08

The asbestiform fibrous silicate balangeroite exhibits cytotoxic and oxidative properties similar to those exerted by crocidolite asbestos. In human lung epithelial cells A549, balangeroite, like crocidolite, inhibited the pentose phosphate pathway (PPP), one of the main antioxidant intracellular tools; this inhibition was exerted also when PPP was activated by the redox-cycling compound menadione. PPP inhibition may be accounted for by the inhibition of its rate-limiting enzyme, glucose-6-phosphate dehydrogenase (G6PD). Reduced glutathione (GSH), the most important intracellular antioxidant molecule, was decreased by both balangeroite and crocidolite incubation. This effect was not related to any increased content of oxidized glutathione, or to any enhanced efflux of glutathione, suggesting that balangeroite fibers, like crocidolite, might favor the reaction of GSH with other molecules.

Hawkmoths use nectar sugar to reduce oxidative damage from flight.

PubMed

Levin, E; Lopez-Martinez, G; Fane, B; Davidowitz, G

2017-02-17

Nectar-feeding animals have among the highest recorded metabolic rates. High aerobic performance is linked to oxidative damage in muscles. Antioxidants in nectar are scarce to nonexistent. We propose that nectarivores use nectar sugar to mitigate the oxidative damage caused by the muscular demands of flight. We found that sugar-fed moths had lower oxidative damage to their flight muscle membranes than unfed moths. Using respirometry coupled with δ 13 C analyses, we showed that moths generate antioxidant potential by shunting nectar glucose to the pentose phosphate pathway (PPP), resulting in a reduction in oxidative damage to the flight muscles. We suggest that nectar feeding, the use of PPP, and intense exercise are causally linked and have allowed the evolution of powerful fliers that feed on nectar. Copyright © 2017, American Association for the Advancement of Science.

Chiral Sugars Drive Enantioenrichment in Prebiotic Amino Acid Synthesis.

PubMed

Wagner, Alexander J; Zubarev, Dmitry Yu; Aspuru-Guzik, Alán; Blackmond, Donna G

2017-04-26

Chiral pentose sugars mediate the enantioselective synthesis of amino acid precursors, with the magnitude of the chiral induction dictated by a subtle cooperativity between sugar hydroxyl groups. Ribose and lyxose give opposite chiral preferences, and theoretical calculations reveal the pseudoenantiomeric nature of transition state structures from the two sugars. Prebiotically plausible mixtures of natural d-sugars lead to enantioenrichment of natural l-amino acid precursors. Temporal monitoring and kinetic modeling of the reaction reveal an unusual dynamic kinetic resolution that shifts toward an enantioselective pathway over time, providing an amplification mechanism for the transfer of chiral information. This work adds to growing evidence for synergy in the etiology of the single chirality of the two most important classes of biological molecules, the sugars that make up DNA and RNA and the amino acids that form proteins.

Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis.

PubMed

Nakano, Haruko; Minami, Itsunari; Braas, Daniel; Pappoe, Herman; Wu, Xiuju; Sagadevan, Addelynn; Vergnes, Laurent; Fu, Kai; Morselli, Marco; Dunham, Christopher; Ding, Xueqin; Stieg, Adam Z; Gimzewski, James K; Pellegrini, Matteo; Clark, Peter M; Reue, Karen; Lusis, Aldons J; Ribalet, Bernard; Kurdistani, Siavash K; Christofk, Heather; Nakatsuji, Norio; Nakano, Atsushi

2017-12-12

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy.

Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis

PubMed Central

Nakano, Haruko; Minami, Itsunari; Braas, Daniel; Pappoe, Herman; Wu, Xiuju; Sagadevan, Addelynn; Vergnes, Laurent; Fu, Kai; Morselli, Marco; Dunham, Christopher; Ding, Xueqin; Stieg, Adam Z; Gimzewski, James K; Pellegrini, Matteo; Clark, Peter M; Reue, Karen; Lusis, Aldons J; Ribalet, Bernard; Kurdistani, Siavash K; Christofk, Heather; Nakatsuji, Norio

2017-01-01

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy. PMID:29231167

Metabolism of the hexose monophosphate shunt in glucose-6-phosphate dehydrogenase deficiency and closely interrelated reactions.

PubMed

Jacobasch, G; Bleiber, R; Schönian, G

1982-12-01

The metabolic changes of red blood cells from 25 patients with chronic hemolytic anemia caused by G6PD deficiency were investigated. The average G6PD activity exhibited 5 per cent of the normal control. The glucose oxidation was in most cases reduced even by 50 per cent. Three groups could be distinguished according to their degree of methylene blue stimulation of the oxidative pentose phosphate pathway. These results are in agreement with changes of the kinetic constants for NADP, NADPH and G6P, respectively. The filtrability of red blood cells decreased in all cases of G6PD deficiency but no correlation was found with the survival time. First results of a preventive medication with D-L-alpha-tocopherol let assume a reduction of chronic hemolysis.

Chiral Sugars Drive Enantioenrichment in Prebiotic Amino Acid Synthesis

PubMed Central

2017-01-01

Chiral pentose sugars mediate the enantioselective synthesis of amino acid precursors, with the magnitude of the chiral induction dictated by a subtle cooperativity between sugar hydroxyl groups. Ribose and lyxose give opposite chiral preferences, and theoretical calculations reveal the pseudoenantiomeric nature of transition state structures from the two sugars. Prebiotically plausible mixtures of natural d-sugars lead to enantioenrichment of natural l-amino acid precursors. Temporal monitoring and kinetic modeling of the reaction reveal an unusual dynamic kinetic resolution that shifts toward an enantioselective pathway over time, providing an amplification mechanism for the transfer of chiral information. This work adds to growing evidence for synergy in the etiology of the single chirality of the two most important classes of biological molecules, the sugars that make up DNA and RNA and the amino acids that form proteins. PMID:28470050

TiO2-catalyzed synthesis of sugars from formaldehyde in extraterrestrial impacts on the early Earth

PubMed Central

Civiš, Svatopluk; Szabla, Rafał; Szyja, Bartłomiej M.; Smykowski, Daniel; Ivanek, Ondřej; Knížek, Antonín; Kubelík, Petr; Šponer, Jiří; Ferus, Martin; Šponer, Judit E.

2016-01-01

Recent synthetic efforts aimed at reconstructing the beginning of life on our planet point at the plausibility of scenarios fueled by extraterrestrial energy sources. In the current work we show that beyond nucleobases the sugar components of the first informational polymers can be synthesized in this way. We demonstrate that a laser-induced high-energy chemistry combined with TiO2 catalysis readily produces a mixture of pentoses, among them ribose, arabinose and xylose. This chemistry might be highly relevant to the Late Heavy Bombardment period of Earth’s history about 4–3.85 billion years ago. In addition, we present an in-depth theoretical analysis of the most challenging step of the reaction pathway, i.e., the TiO2-catalyzed dimerization of formaldehyde leading to glycolaldehyde. PMID:26979666

Differential Substrate Usage and Metabolic Fluxes in Francisella tularensis Subspecies holarctica and Francisella novicida

PubMed Central

Chen, Fan; Rydzewski, Kerstin; Kutzner, Erika; Häuslein, Ina; Schunder, Eva; Wang, Xinzhe; Meighen-Berger, Kevin; Grunow, Roland; Eisenreich, Wolfgang; Heuner, Klaus

2017-01-01

Francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas F. tularensis subsp. holarctica is highly pathogenic for humans, F. novicida is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed 13C-labeling experiments with F. tularensis subsp. holarctica wild type (beaver isolate), F. tularensis subsp. holarctica strain LVS, or F. novicida strain U112 in complex media containing either [U-13C6]glucose, [1,2-13C2]glucose, [U-13C3]serine, or [U-13C3]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the F. tularensis subsp. holarctica wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the F. novicida strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in F. tularensis subsp. holarctica or metabolized, with higher rates in F. novicida, via glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose via gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of F. tularensis and F. novicida. Glycerol efficiently served as a gluconeogenetic substrate in F. novicida, but only less in the F. tularensis subsp. holarctica strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in downstream metabolic processes, e.g., via acetyl-CoA in the citrate cycle and for fatty acid biosynthesis, especially in the F. tularensis subsp. holarctica strains. In summary, the data reflect differential metabolite fluxes in F. tularensis subsp. holarctica and F. novicida suggesting that the different utilization of substrates could be related to host specificity and virulence of Francisella. PMID:28680859

Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway

PubMed Central

Oracz, Krystyna; El-Maarouf-Bouteau, Hayat; Bogatek, Renata; Bailly, Christophe

2008-01-01

Freshly harvested sunflower (Helianthus annuus L.) seeds are considered to be dormant because they fail to germinate at relatively low temperatures (10 °C). This dormancy results mainly from an embryo dormancy and disappears during dry storage. Although endogenous ethylene is known to be involved in sunflower seed alleviation of dormancy, little attention had been paid to the possible role of cyanide, which is produced by the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene, in this process. The aims of this work were to investigate whether exogenous cyanide could improve the germination of dormant sunflower seeds and to elucidate its putative mechanisms of action. Naked dormant seeds became able to germinate at 10 °C when they were incubated in the presence of 1 mM gaseous cyanide. Other respiratory inhibitors showed that this effect did not result from an activation of the pentose phosphate pathway or the cyanide-insensitive pathway. Cyanide stimulated germination of dormant seeds in the presence of inhibitors of ethylene biosynthesis, but its improving effect required functional ethylene receptors. It did not significantly affect ethylene production and the expression of genes involved in ethylene biosynthesis or in the first steps of ethylene signalling pathway. However, the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was markedly stimulated in the presence of gaseous cyanide. It is proposed that the mode of action of cyanide in sunflower seed dormancy alleviation does not involve ethylene production and that ERF1 is a common component of the ethylene and cyanide signalling pathways. PMID:18448476

Simulation of the pentose cycle in lactating rat mammary gland

PubMed Central

Haut, Michael J.; London, Jack W.; Garfinkel, David

1974-01-01

A computer model representing the pentose cycle, the tricarboxylic acid cycle and glycolysis in slices of lactating rat mammary glands has been constructed. This model is based primarily on the studies, with radioactive chemicals, of Abraham & Chaikoff (1959) [although some of the discrepant data of Katz & Wals (1972) could be accommodated by changing one enzyme activity]. Data obtained by using [1-14C]-, [6-14C]- and [3,4-14C]-glucose were simulated, as well as data obtained by using unlabelled glucose (for which some new experimental data are presented). Much past work on the pentose cycle has been mainly concerned with the division of glucose flow between the pentose cycle and glycolysis, and has relied on the assumption that the system is in steady state (both labelled and unlabelled). This assumption may not apply to lactating rat mammary glands, since the model shows that the percentage flow through the shunt progressively decreased for the first 2h of a 3h experiment, and we were unable to construct a completely steady-state model. The model allows examination of many quantitative features of the system, especially the amount of material passing through key enzymes, some of which appear to be regulated by NADP+ concentrations as proposed by McLean (1960). Supplementary information for this paper has been deposited as Supplementary Publication SUP 50023 at the British Museum (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. PMID:4154746

Data mining and pathway analysis of glucose-6-phosphate dehydrogenase with natural language processing.

PubMed

Chen, Long; Zhang, Chunhua; Wang, Yanling; Li, Yuqian; Han, Qiaoqiao; Yang, Huixin; Zhu, Yuechun

2017-08-01

Human glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway, and serves an important role in biosynthesis and the redox balance. G6PD deficiency is a major cause of neonatal jaundice and acute hemolyticanemia, and recently, G6PD has been associated with diseases including inflammation and cancer. The aim of the present study was to conduct a search of the National Center for Biotechnology Information PubMed library for articles discussing G6PD. Genes that were identified to be associated with G6PD were recorded, and the frequency at which each gene appeared was calculated. Gene ontology (GO), pathway and network analyses were then performed. A total of 98 G6PD‑associated genes and 33 microRNAs (miRNAs) that potentially regulate G6PD were identified. The 98 G6PD‑associated genes were then sub‑classified into three functional groups by GO analysis, followed by analysis of function, pathway, network, and disease association. Out of the 47 signaling pathways identified, seven were significantly correlated with G6PD‑associated genes. At least two out of four independent programs identified the 33 miRNAs that were predicted to target G6PD. miR‑1207‑5P, miR‑1 and miR‑125a‑5p were predicted by all four software programs to target G6PD. The results of the present study revealed that dysregulation of G6PD was associated with cancer, autoimmune diseases, and oxidative stress‑induced disorders. These results revealed the potential roles of G6PD‑regulated signaling and metabolic pathways in the etiology of these diseases.

Comparative genome analysis of the Flavobacteriales bacterium strain UJ101, isolated from the gut of Atergatis reticulatus.

PubMed

Yang, Jhung-Ahn; Yang, Sung-Hyun; Kim, Junghee; Kwon, Kae Kyoung; Oh, Hyun-Myung

2017-07-01

Here we report the comparative genomic analysis of strain UJ101 with 15 strains from the family Flavobacteriaceae, using the CGExplorer program. Flavobacteriales bacterium strain UJ101 was isolated from a xanthid crab, Atergatis reticulatus, from the East Sea near Korea. The complete genome of strain UJ101 is a 3,074,209 bp, single, circular chromosome with 30.74% GC content. While the UJ101 genome contains a number of annotated genes for many metabolic pathways, such as the Embden-Meyerhof pathway, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, genes for the Entner-Douddoroff pathway are not found in the UJ101 genome. Overall, carbon fixation processes were absent but nitrate reduction and denitrification pathways were conserved. The UJ101 genome was compared to genomes from other marine animals (three invertebrate strains and 5 fish strains) and other marine animal- derived genera. Notable results by genome comparisons showed that UJ101 is capable of denitrification and nitrate reduction, and that biotin-thiamine pathway participation varies among marine bacteria; fish-dwelling bacteria, freeliving bacteria, invertebrate-dwelling bacteria, and strain UJ101. Pan-genome analysis of the 16 strains in this study included 7,220 non-redundant genes that covered 62% of the pan-genome. A core-genome of 994 genes was present and consisted of 8% of the genes from the pan-genome. Strain UJ101 is a symbiotic hetero-organotroph isolated from xanthid crab, and is a metabolic generalist with nitrate-reducing abilities but without the ability to synthesize biotin. There is a general tendency of UJ101 and some fish pathogens to prefer thiamine-dependent glycolysis to gluconeogenesis. Biotin and thiamine auxotrophy or prototrophy may be used as important markers in microbial community studies.

Data mining and pathway analysis of glucose-6-phosphate dehydrogenase with natural language processing

PubMed Central

Chen, Long; Zhang, Chunhua; Wang, Yanling; Li, Yuqian; Han, Qiaoqiao; Yang, Huixin; Zhu, Yuechun

2017-01-01

Human glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway, and serves an important role in biosynthesis and the redox balance. G6PD deficiency is a major cause of neonatal jaundice and acute hemolyticanemia, and recently, G6PD has been associated with diseases including inflammation and cancer. The aim of the present study was to conduct a search of the National Center for Biotechnology Information PubMed library for articles discussing G6PD. Genes that were identified to be associated with G6PD were recorded, and the frequency at which each gene appeared was calculated. Gene ontology (GO), pathway and network analyses were then performed. A total of 98 G6PD-associated genes and 33 microRNAs (miRNAs) that potentially regulate G6PD were identified. The 98 G6PD-associated genes were then sub-classified into three functional groups by GO analysis, followed by analysis of function, pathway, network, and disease association. Out of the 47 signaling pathways identified, seven were significantly correlated with G6PD-associated genes. At least two out of four independent programs identified the 33 miRNAs that were predicted to target G6PD. miR-1207-5P, miR-1 and miR-125a-5p were predicted by all four software programs to target G6PD. The results of the present study revealed that dysregulation of G6PD was associated with cancer, autoimmune diseases, and oxidative stress-induced disorders. These results revealed the potential roles of G6PD-regulated signaling and metabolic pathways in the etiology of these diseases. PMID:28627690

Systematic engineering of the central metabolism in Escherichia coli for effective production of n-butanol.

PubMed

Saini, Mukesh; Li, Si-Yu; Wang, Ze Win; Chiang, Chung-Jen; Chao, Yun-Peng

2016-01-01

Microbes have been extensively explored for production of environment-friendly fuels and chemicals. The microbial fermentation pathways leading to these commodities usually involve many redox reactions. This makes the fermentative production of highly reduced products challenging, because there is a limited NADH output from glucose catabolism. Microbial production of n-butanol apparently represents one typical example. In this study, we addressed the issue by adjustment of the intracellular redox state in Escherichia coli. This was initiated with strain BuT-8 which carries the clostridial CoA-dependent synthetic pathway. Three metabolite nodes in the central metabolism of the strain were targeted for engineering. First, the pyruvate node was manipulated by enhancement of pyruvate decarboxylation in the oxidative pathway. Subsequently, the pentose phosphate (PP) pathway was amplified at the glucose-6-phosphate (G6P) node. The pathway for G6P isomerization was further blocked to force the glycolytic flux through the PP pathway. It resulted in a growth defect, and the cell growth was later recovered by limiting the tricarboxylic acid cycle at the acetyl-CoA node. Finally, the resulting strain exhibited a high NADH level and enabled production of 6.1 g/L n-butanol with a yield of 0.31 g/g-glucose and a productivity of 0.21 g/L/h. The production efficiency of fermentative products in microbes strongly depends on the intracellular redox state. This work illustrates the flexibility of pyruvate, G6P, and acetyl-CoA nodes at the junction of the central metabolism for engineering. In principle, high production of reduced products of interest can be achieved by individual or coordinated modulation of these metabolite nodes.

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

PubMed

Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

CcpN Controls Central Carbon Fluxes in Bacillus subtilis▿ ‡

PubMed Central

Tännler, Simon; Fischer, Eliane; Le Coq, Dominique; Doan, Thierry; Jamet, Emmanuel; Sauer, Uwe; Aymerich, Stéphane

2008-01-01

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes. PMID:18586936

Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress.

PubMed

Spencer, Jennifer; Phister, Trevor G; Smart, Katherine A; Greetham, Darren

2014-03-17

Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress.

Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress

PubMed Central

2014-01-01

Background Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. Results Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. Conclusions Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress. PMID:24636079

Tandem catalysis for the production of alkyl lactates from ketohexoses at moderate temperatures

DOE PAGES

Orazov, Marat; Davis, Mark E.

2015-09-08

Retro-aldol reactions have been implicated as the limiting steps in catalytic routes to convert biomass-derived hexoses and pentoses into valuable C2, C3, and C4 products such as glycolic acid, lactic acid, 2-hydroxy-3-butenoic acid, 2,4-dihydroxybutanoic acid, and alkyl esters thereof. Due to a lack of efficient retro-aldol catalysts, most previous investigations of catalytic pathways involving these reactions were conducted at high temperatures (≥160 °C). Here, we report moderate-temperature (around 100 °C) retro-aldol reactions of various hexoses in aqueous and alcoholic media with catalysts traditionally known for their capacity to catalyze 1,2-intramolecular carbon shift (1,2-CS) reactions of aldoses, i.e., various molybdenum oxidemore » and molybdate species, nickel(II) diamine complexes, alkali-exchanged stannosilicate molecular sieves, and amorphous TiO2–SiO2 coprecipitates. Solid Lewis acid cocatalysts that are known to catalyze 1,2-intramolecular hydride shift (1,2-HS) reactions that enable the formation of α-hydroxy carboxylic acids from tetroses, trioses, and glycolaldehyde, but cannot readily catalyze retro-aldol reactions of hexoses and pentoses at these moderate temperatures, are shown to be compatible with the aforementioned retro-aldol catalysts. The combination of a distinct retro-aldol catalyst with a 1,2-HS catalyst enables lactic acid and alkyl lactate formation from ketohexoses at moderate temperatures (around 100 °C), with yields comparable to best-reported chemocatalytic examples at high temperature conditions (≥160 °C). The use of moderate temperatures enables numerous desirable features such as lower pressure and significantly less catalyst deactivation.« less

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.1

PubMed Central

Fan, Jianhua; Ning, Kang; Zeng, Xiaowei; Luo, Yuanchan; Wang, Dongmei; Hu, Jianqiang; Li, Jing; Xu, Hui; Huang, Jianke; Wan, Minxi; Wang, Weiliang; Zhang, Daojing; Shen, Guomin; Run, Conglin; Liao, Junjie; Fang, Lei; Huang, Shi; Jing, Xiaoyan; Su, Xiaoquan; Wang, Anhui; Bai, Lili; Hu, Zanmin; Xu, Jian; Li, Yuanguang

2015-01-01

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels. PMID:26486592

Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus.

PubMed

Rodrussamee, Nadchanok; Lertwattanasakul, Noppon; Hirata, Katsushi; Suprayogi; Limtong, Savitree; Kosaka, Tomoyuki; Yamada, Mamoru

2011-05-01

Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

A first immunohistochemistry study of transketolase and transketolase-like 1 expression in canine hyperplastic and neoplastic mammary lesions.

PubMed

Burrai, Giovanni Pietro; Tanca, Alessandro; Cubeddu, Tiziana; Abbondio, Marcello; Polinas, Marta; Addis, Maria Filippa; Antuofermo, Elisabetta

2017-01-31

Canine mammary tumors represent the most common neoplasm in female dogs, and the discovery of cancer biomarkers and their translation to clinical relevant assays is a key requirement in the war on cancer. Since the description of the 'Warburg effect', the reprogramming of metabolic pathways is considered a hallmark of pathological changes in cancer cells. In this study, we investigate the expression of two cancer-related metabolic enzymes, transketolase (TKT) and transketolase-like 1 (TKTL1), involved in the pentose phosphate pathway (PPP), an alternative metabolic pathway for glucose breakdown that could promote cancer by providing the precursors and energy required for rapidly growing cells. TKT and TKTL1 protein expression was investigated by immunohistochemistry in canine normal (N = 6) and hyperplastic glands (N = 3), as well as in benign (N = 11) and malignant mammary tumors (N = 17). TKT expression was higher in hyperplastic lesions and in both benign and malignant tumors compared to the normal mammary gland, while TKTL1 levels were remarkably higher in hyperplastic lesions, simple adenomas and simple carcinomas than in the normal mammary glands (P < 0.05). This study reveals that the expression of a key PPP enzyme varies along the evolution of canine mammary neoplastic lesions, and supports a role of metabolic changes in the development of canine mammary tumors.

Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein.

PubMed

Rodrigues, André L; Trachtmann, Nathalie; Becker, Judith; Lohanatha, Ananta F; Blotenberg, Jana; Bolten, Christoph J; Korneli, Claudia; de Souza Lima, André O; Porto, Luismar M; Sprenger, Georg A; Wittmann, Christoph

2013-11-01

Violacein and deoxyviolacein are interesting therapeutics against pathogenic bacteria and viruses as well as tumor cells. In the present work, systems-wide metabolic engineering was applied to target Escherichia coli, a widely accepted recombinant host in pharmaceutical biotechnology, for production of these high-value products. The basic producer, E. coli dVio-1, that expressed the vioABCE cluster from Chromobacterium violaceum under control of the inducible araC system, accumulated 180 mg L(-1) of deoxyviolacein. Targeted intracellular metabolite analysis then identified bottlenecks in tryptophan supporting pathways, the major product building block. This was used for comprehensive engineering of serine, chorismate and tryptophan biosynthesis and the non-oxidative pentose-phosphate pathway. The final strain, E. coli dVio-6, accumulated 320 mg L(-1) deoxyviolacein in shake flask cultures. The created chassis of a high-flux tryptophan pathway was complemented by genomic integration of the vioD gene of Janthinobacterium lividum, which enabled exclusive production of violacein. In a fed-batch process, the resulting producer E. coli Vio-4 accumulated 710 mg L(-1) of the desired product. With straightforward broth extraction and subsequent crystallization, violacein could be obtained with 99.8% purity. This demonstrates the potential of E. coli as a platform for production of tryptophan based therapeutics. Copyright © 2013 Elsevier Inc. All rights reserved.

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

PubMed Central

Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

2015-01-01

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

O-GlcNAc in cancer: An Oncometabolism-fueled vicious cycle.

PubMed

Hanover, John A; Chen, Weiping; Bond, Michelle R

2018-06-01

Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key 'hallmarks of cancer'. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb's cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a 'vicious cycle'. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

PubMed

Creek, Darren J; Mazet, Muriel; Achcar, Fiona; Anderson, Jana; Kim, Dong-Hyun; Kamour, Ruwida; Morand, Pauline; Millerioux, Yoann; Biran, Marc; Kerkhoven, Eduard J; Chokkathukalam, Achuthanunni; Weidt, Stefan K; Burgess, Karl E V; Breitling, Rainer; Watson, David G; Bringaud, Frédéric; Barrett, Michael P

2015-03-01

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum

DOE PAGES

Verbeke, Tobin J.; Giannone, Richard J.; Klingeman, Dawn M.; ...

2017-02-23

Significant hurdles exist in efforts to domesticate and industrialize a microbial species for biotechnological application as specific metabolic functions found in natural communities disappear in axenic cultures. For the lignocellulose-deconstructing specialist Clostridium thermocellum, the catabolism of hemicellulose-derived pentoses, which the bacterium cannot ferment, is one such function. Here, we report that various xylo-oligomers significantly inhibit C. thermocellum metabolism and growth and that microbe-sugar interactions occur across multiple dimensions. First, stable isotope metabolomics confirmed C. thermocellum s ability to transport and metabolize pentose sugars. This transport occurs, at least in part, through the ATP-dependent transporter, CbpD. Secondly, xylose is an electronmore » sink for C. thermocellum metabolism leading to the production of xylitol. Deletion of Clo1313_0076, annotated as a xylitol dehydrogenase, reduced the total production and molar xylitol yields by 41% and 46%, respectively. However, it also altered the relative end-product distribution patterns confirming that external electron acceptors may influence the bacterium s redox metabolism to a greater extent than previously considered. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of an AgrD-type, lactone cyclized pentapeptide signaling molecule; which is the first report of an AgrD-type signaling peptide in any thermophile. Addition of synthetic versions of the cyclic peptide inhibited cultures grown in the absence of xylose, but had no effect on cultures already inhibited by the pentose sugar. Together, our findings identify that C. thermocellum has evolved previously unrecognized strategies to cope with C5-sugars, but the absence of a native catabolic sink negatively affects strain metabolism and growth.« less

Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verbeke, Tobin J.; Giannone, Richard J.; Klingeman, Dawn M.

Significant hurdles exist in efforts to domesticate and industrialize a microbial species for biotechnological application as specific metabolic functions found in natural communities disappear in axenic cultures. For the lignocellulose-deconstructing specialist Clostridium thermocellum, the catabolism of hemicellulose-derived pentoses, which the bacterium cannot ferment, is one such function. Here, we report that various xylo-oligomers significantly inhibit C. thermocellum metabolism and growth and that microbe-sugar interactions occur across multiple dimensions. First, stable isotope metabolomics confirmed C. thermocellum s ability to transport and metabolize pentose sugars. This transport occurs, at least in part, through the ATP-dependent transporter, CbpD. Secondly, xylose is an electronmore » sink for C. thermocellum metabolism leading to the production of xylitol. Deletion of Clo1313_0076, annotated as a xylitol dehydrogenase, reduced the total production and molar xylitol yields by 41% and 46%, respectively. However, it also altered the relative end-product distribution patterns confirming that external electron acceptors may influence the bacterium s redox metabolism to a greater extent than previously considered. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of an AgrD-type, lactone cyclized pentapeptide signaling molecule; which is the first report of an AgrD-type signaling peptide in any thermophile. Addition of synthetic versions of the cyclic peptide inhibited cultures grown in the absence of xylose, but had no effect on cultures already inhibited by the pentose sugar. Together, our findings identify that C. thermocellum has evolved previously unrecognized strategies to cope with C5-sugars, but the absence of a native catabolic sink negatively affects strain metabolism and growth.« less

Glucose pathways adaptation supports acquisition of activated microglia phenotype.

PubMed

Gimeno-Bayón, J; López-López, A; Rodríguez, M J; Mahy, N

2014-06-01

With its capacity to survey the environment and phagocyte debris, microglia assume a diversity of phenotypes to respond specifically through neurotrophic and toxic effects. Although these roles are well accepted, the underlying energetic mechanisms associated with microglial activation remain largely unclear. This study investigates microglia metabolic adaptation to ATP, NADPH, H(+) , and reactive oxygen species production. To this end, in vitro studies were performed with BV-2 cells before and after activation with lipopolysaccharide + interferon-γ. Nitric oxide (NO) was measured as a marker of cell activation. Our results show that microglial activation triggers a metabolic reprogramming based on an increased glucose uptake and a strengthening of anaerobic glycolysis, as well as of the pentose pathway oxidative branch, while retaining the mitochondrial activity. Based on this energy commitment, microglial defense capacity increases rapidly as well as ribose-5-phosphate and nucleic acid formation for gene transcription, essential to ensure the newly acquired functions demanded by central nervous system signaling. We also review the role of NO in this microglial energy commitment that positions cytotoxic microglia within the energetics of the astrocyte-neuron lactate shuttle. Copyright © 2014 Wiley Periodicals, Inc.

Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

PubMed Central

Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

2007-01-01

In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

¹³C-based metabolic flux analysis of Saccharomyces cerevisiae with a reduced Crabtree effect.

PubMed

Kajihata, Shuichi; Matsuda, Fumio; Yoshimi, Mika; Hayakawa, Kenshi; Furusawa, Chikara; Kanda, Akihisa; Shimizu, Hiroshi

2015-08-01

Saccharomyces cerevisiae shows a Crabtree effect that produces ethanol in a high glucose concentration even under fully aerobic condition. For efficient production of cake yeast or compressed yeast for baking, ethanol by-production is not desired since glucose limited chemostat or fed-batch cultivations are performed to suppress the Crabtree effect. In this study, the (13)C-based metabolic flux analysis ((13)C-MFA) was performed for the S288C derived S. cerevisiae strain to characterize a metabolic state under the reduced Crabtree effect. S. cerevisiae cells were cultured at a low dilution rate (0.1 h(-1)) under the glucose-limited chemostat condition. The estimated metabolic flux distribution showed that the acetyl-CoA in mitochondria was mainly produced from pyruvate by pyruvate dehydrogenase (PDH) reaction and that the level of the metabolic flux through the pentose phosphate pathway was much higher than that of the Embden-Meyerhof-Parnas pathway, which contributes to high biomass yield at low dilution rate by supplying NADPH required for cell growth. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Global microbialization of coral reefs.

PubMed

Haas, Andreas F; Fairoz, Mohamed F M; Kelly, Linda W; Nelson, Craig E; Dinsdale, Elizabeth A; Edwards, Robert A; Giles, Steve; Hatay, Mark; Hisakawa, Nao; Knowles, Ben; Lim, Yan Wei; Maughan, Heather; Pantos, Olga; Roach, Ty N F; Sanchez, Savannah E; Silveira, Cynthia B; Sandin, Stuart; Smith, Jennifer E; Rohwer, Forest

2016-04-25

Microbialization refers to the observed shift in ecosystem trophic structure towards higher microbial biomass and energy use. On coral reefs, the proximal causes of microbialization are overfishing and eutrophication, both of which facilitate enhanced growth of fleshy algae, conferring a competitive advantage over calcifying corals and coralline algae. The proposed mechanism for this competitive advantage is the DDAM positive feedback loop (dissolved organic carbon (DOC), disease, algae, microorganism), where DOC released by ungrazed fleshy algae supports copiotrophic, potentially pathogenic bacterial communities, ultimately harming corals and maintaining algal competitive dominance. Using an unprecedented data set of >400 samples from 60 coral reef sites, we show that the central DDAM predictions are consistent across three ocean basins. Reef algal cover is positively correlated with lower concentrations of DOC and higher microbial abundances. On turf and fleshy macroalgal-rich reefs, higher relative abundances of copiotrophic microbial taxa were identified. These microbial communities shift their metabolic potential for carbohydrate degradation from the more energy efficient Embden-Meyerhof-Parnas pathway on coral-dominated reefs to the less efficient Entner-Doudoroff and pentose phosphate pathways on algal-dominated reefs. This 'yield-to-power' switch by microorganism directly threatens reefs via increased hypoxia and greater CO2 release from the microbial respiration of DOC.

Linking the morphological and metabolomic response of Lactuca sativa L exposed to emerging contaminants using GC × GC-MS and chemometric tools.

PubMed

Hurtado, Carlos; Parastar, Hadi; Matamoros, Víctor; Piña, Benjamín; Tauler, Romà; Bayona, Josep M

2017-07-26

The occurrence of contaminants of emerging concern (CECs) in irrigation waters (up to low μg L -1 ) and irrigated crops (ng g -1 in dry weight) has been reported, but the linkage between plant morphological changes and plant metabolomic response has not yet been addressed. In this study, a non-targeted metabolomic analysis was performed on lettuce (Lactuca sativa L) exposed to 11 CECs (pharmaceuticals, personal care products, anticorrosive agents and surfactants) by irrigation. The plants were watered with different CEC concentrations (0-50 µg L -1 ) for 34 days under controlled conditions and then harvested, extracted, derivatised and analysed by comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometer (GC × GC-TOFMS). The resulting raw data were analysed using multivariate curve resolution (MCR) and partial least squares (PLS) methods. The metabolic response indicates that exposure to CECs at environmentally relevant concentrations (0.05 µg L -1 ) can cause significant metabolic alterations in plants (carbohydrate metabolism, the citric acid cycle, pentose phosphate pathway and glutathione pathway) linked to changes in morphological parameters (leaf height, stem width) and chlorophyll content.

Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS.

PubMed

Yuan, Wei; Li, Shuwei; Edwards, James L

2015-08-04

The extraction and quantitation of carbonyl metabolites from cell lysate was accomplished using a carbonyl-reactive fluorous tag and capillary liquid chromatography coupled to mass spectrometry (capLC-MS). Selective fluorous tagging for ketones and aldehydes provided a 30-fold increase in sensitivity using electrospray ionization MS. Separation of fluorous tagged carbonyl resulted in good separation of all components, and tandem MS was able to differentiate structural carbonyl isomers. The average limit of detection for carbonyl standards was 37 nM (range 1.5-250 nM), with linearity of R(2) > 0.99. Reproducibility for metabolites in cell lysate averaged 9% RSD. Human aortic endothelial cells (HAECs) were exposed to varying levels of glucose, and their carbonyl metabolite levels were quantified. Significant metabolite changes were seen in glycolysis and the propanoate pathway from a glucose challenge. Using an untargeted approach, 120 carbonyl metabolites were found to change in hyperglycemic HAECs. From this list of compounds, multiple metabolites from the pentose phosphate and tryptophan metabolic pathways were discovered. This system provides excellent sensitivity and quantitation of carbonyl metabolites without the need for isotope standards or labels.

Metabolic responses in Candida tropicalis to complex inhibitors during xylitol bioconversion.

PubMed

Wang, Shizeng; Li, Hao; Fan, Xiaoguang; Zhang, Jingkun; Tang, Pingwah; Yuan, Qipeng

2015-09-01

During xylitol fermentation, Candida tropicalis is often inhibited by inhibitors in hemicellulose hydrolysate. The mechanisms involved in the metabolic responses to inhibitor stress and the resistances to inhibitors are still not clear. To understand the inhibition mechanisms and the metabolic responses to inhibitors, a GC/MS-based metabolomics approach was performed on C. tropicalis treated with and without complex inhibitors (CI, including furfural, phenol and acetic acid). Partial least squares discriminant analysis was used to determine the metabolic variability between CI-treated groups and control groups, and 25 metabolites were identified as possible entities responsible for the discrimination caused by inhibitors. We found that xylose uptake rate and xylitol oxidation rate were promoted by CI treatment. Metabolomics analysis showed that the flux from xylulose to pentose phosphate pathway increased, and tricarboxylic acid cycle was disturbed by CI. Moreover, the changes in levels of 1,3-propanediol, trehalose, saturated fatty acids and amino acids showed different mechanisms involved in metabolic responses to inhibitor stress. The increase of 1,3-propanediol was considered to be correlated with regulating redox balance and osmoregulation. The increase of trehalose might play a role in protein stabilization and cellular membranes protection. Saturated fatty acids could cause the decrease of membrane fluidity and make the plasma membrane rigid to maintain the integrity of plasma membrane. The deeper understanding of the inhibition mechanisms and the metabolic responses to inhibitors will provide us with more information on the metabolism regulation during xylitol bioconversion and the construction of industrial strains with inhibitor tolerance for better utilization of bioresource. Copyright © 2015 Elsevier Inc. All rights reserved.

Fueling and imaging brain activation

PubMed Central

Dienel, Gerald A

2012-01-01

Metabolic signals are used for imaging and spectroscopic studies of brain function and disease and to elucidate the cellular basis of neuroenergetics. The major fuel for activated neurons and the models for neuron–astrocyte interactions have been controversial because discordant results are obtained in different experimental systems, some of which do not correspond to adult brain. In rats, the infrastructure to support the high energetic demands of adult brain is acquired during postnatal development and matures after weaning. The brain's capacity to supply and metabolize glucose and oxygen exceeds demand over a wide range of rates, and the hyperaemic response to functional activation is rapid. Oxidative metabolism provides most ATP, but glycolysis is frequently preferentially up-regulated during activation. Underestimation of glucose utilization rates with labelled glucose arises from increased lactate production, lactate diffusion via transporters and astrocytic gap junctions, and lactate release to blood and perivascular drainage. Increased pentose shunt pathway flux also causes label loss from C1 of glucose. Glucose analogues are used to assay cellular activities, but interpretation of results is uncertain due to insufficient characterization of transport and phosphorylation kinetics. Brain activation in subjects with low blood-lactate levels causes a brain-to-blood lactate gradient, with rapid lactate release. In contrast, lactate flooding of brain during physical activity or infusion provides an opportunistic, supplemental fuel. Available evidence indicates that lactate shuttling coupled to its local oxidation during activation is a small fraction of glucose oxidation. Developmental, experimental, and physiological context is critical for interpretation of metabolic studies in terms of theoretical models. PMID:22612861

High Carbon Use Efficiency is Not Explained by Production of Storage Compounds

NASA Astrophysics Data System (ADS)

Dijkstra, Paul; van Groenigen, Kees-Jan

2015-04-01

The efficiency with which microbes use substrate to make new microbial biomass (Carbon Use Efficiency or CUE; mol C / mol C) is an important variable in soil and ecosystem C cycling models. Estimates of CUE in soil microbial communities vary widely. It has been hypothesized that high values of CUE are associated with production of storage compounds following a sudden increases in substrate availability during CUE measurements. In that case, these high CUE values would not be representative for balanced microbial growth (i.e. the production of all compounds needed to make new microbial cells). To test this hypothesis, we added position-specific 13C-labeled glucose isotopomers in parallel incubations of a ponderosa pine and piñon-juniper soil. We compared the measured pattern of CO2 release for the six glucose C atoms with patterns of CO2 production expected for balanced growth with a low, medium, or high CUE, and with CO2 production patterns associated with production of storage compounds (glycogen, lipids, or polyhydroxybutyrate). The measured position-specific CO2 production did not match that for production of glycogen, lipids, or polyhydroxybutyrate, but agreed closely with that expected for balanced growth at high CUE and high pentose phosphate pathway activity. We conclude that soil microbial communities utilize glucose substrate for biomass growth with high CUE, and that addition of small amounts of 13C-labeled glucose tracers do not affect CUE or induce storage compounds production. We submit that the measurement of position-specific CO2 production offers a quick and easy way to test biochemically explicit hypotheses concerning microbial growth metabolism.

Pectin-rich biomass as feedstock for fuel ethanol production.

PubMed

Edwards, Meredith C; Doran-Peterson, Joy

2012-08-01

The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes.

Deletion or Inhibition of the Oxygen Sensor PHD1 Protects against Ischemic Stroke via Reprogramming of Neuronal Metabolism.

PubMed

Quaegebeur, Annelies; Segura, Inmaculada; Schmieder, Roberta; Verdegem, Dries; Decimo, Ilaria; Bifari, Francesco; Dresselaers, Tom; Eelen, Guy; Ghosh, Debapriva; Davidson, Shawn M; Schoors, Sandra; Broekaert, Dorien; Cruys, Bert; Govaerts, Kristof; De Legher, Carla; Bouché, Ann; Schoonjans, Luc; Ramer, Matt S; Hung, Gene; Bossaert, Goele; Cleveland, Don W; Himmelreich, Uwe; Voets, Thomas; Lemmens, Robin; Bennett, C Frank; Robberecht, Wim; De Bock, Katrien; Dewerchin, Mieke; Ghesquière, Bart; Fendt, Sarah-Maria; Carmeliet, Peter

2016-02-09

The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network. Instead, PHD1(-/-) neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1(-/-) neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose away from glycolysis. As a result, PHD1(-/-) neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

NASA Astrophysics Data System (ADS)

Adem, Şevki; Kayhan, Naciye

2016-04-01

6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Zhenhai, E-mail: tomsyu@163.com; Huang, Liangqian; Qiao, Pengyun

Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. Thesemore » findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer.« less

PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells.

PubMed

Yu, Zhenhai; Huang, Liangqian; Qiao, Pengyun; Jiang, Aifang; Wang, Li; Yang, Tingting; Tang, Shengjian; Zhang, Wei; Ren, Chune

2016-05-13

Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. These findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Improved health-relevant functionality in dark germinated Mucuna pruriens sprouts by elicitation with peptide and phytochemical elicitors.

PubMed

Randhir, Reena; Kwon, Young-In; Shetty, Kalidas

2009-10-01

The health-relevant functionality of Mucuna pruriens was improved by priming the seeds with elicitors of the pentose phosphate pathway (PPP) such as fish protein hydrolysates (FPHs), lactoferrin (LF) and oregano extract (OE) followed by dark germination. FPH elicited the highest phenolic content of 19 mg/g FW on day 1, which was 38% higher than control sprouts. OE enhanced Parkinson's disease-relevant L-DOPA content by 33% on day 1 compared to control sprouts. Anti-diabetes-relevant alpha-amylase inhibition percent (AIP) and alpha-glucosidase inhibition percent (GIP) were high in the cotyledons and decreased following elicitation and sprouting. For potential anti-diabetic applications, low AIP and high GIP with moderate L-DOPA content on day 4 of dark germination could be optimal. Improved L-DOPA concentrations in a soluble phenolic and antioxidant-rich M. pruriens background on day 1 sprouts have potential for Parkinson's disease management.

Lethal Dysregulation of Energy Metabolism During Embryonic Vitamin E Deficiency

PubMed Central

McDougall, Melissa; Choi, Jaewoo; Kim, Hye-Kyeong; Bobe, Gerd; Stevens, J. Frederik; Cadenas, Enrique; Tanguay, Robert; Traber, Maret G.

2017-01-01

Vitamin E (α-tocopherol, VitE) was discovered in 1922 for its role in preventing embryonic mortality. We investigated the underlying mechanisms causing lethality using targeted metabolomics analyses of zebrafish VitE-deficient embryos over five days of development, which coincided with their increased morbidity and mortality. VitE deficiency resulted in peroxidation of docosahexaenoic acid (DHA), depleting DHA-containing phospholipids, especially phosphatidylcholine, which also caused choline depletion. This increased lipid peroxidation also increased NADPH oxidation, which depleted glucose by shunting it to the pentose phosphate pathway. VitE deficiency was associated with mitochondrial dysfunction with concomitant impairment of energy homeostasis. The observed morbidity and mortality outcomes could be attenuated, but not fully reversed, by glucose injection into VitE-deficient embryos at developmental day one. Thus, embryonic VitE deficiency in vertebrates leads to a metabolic reprogramming that adversely affects methyl donor status and cellular energy homeostasis with lethal outcomes. PMID:28095320

Phosphoglycerate Mutase 1 Coordinates Glycolysis and Biosynthesis to Promote Tumor Growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hitosugi, Taro; Zhou, Lu; Elf, Shannon

2012-11-12

It is unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation partially by controlling intracellular levels of its substrate, 3-phosphoglycerate (3-PG), and product, 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancermore » cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth.« less

DELETION OR INHIBITION OF THE OXYGEN SENSOR PHD1 PROTECTS AGAINST ISCHEMIC STROKE VIA REPROGRAMMING OF NEURONAL METABOLISM

PubMed Central

Quaegebeur, Annelies; Segura, Inmaculada; Schmieder, Roberta; Verdegem, Dries; Decimo, Ilaria; Bifari, Francesco; Dresselaers, Tom; Eelen, Guy; Ghosh, Debapriva; Schoors, Sandra; Janaki Raman, Sudha Rani; Cruys, Bert; Govaerts, Kristof; De Legher, Carla; Bouché, Ann; Schoonjans, Luc; Ramer, Matt S.; Hung, Gene; Bossaert, Goele; Cleveland, Don W.; Himmelreich, Uwe; Voets, Thomas; Lemmens, Robin; Bennett, C. Frank; Robberecht, Wim; De Bock, Katrien; Dewerchin, Mieke; Fendt, Sarah-Maria; Ghesquière, Bart; Carmeliet, Peter

2016-01-01

Summary The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network, nor to enhanced neurotrophin expression. Instead, PHD1−/− neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1−/− neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose from glycolysis. As a result, PHD1−/− neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a novel regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke. PMID:26774962

Ribose in the heart.

PubMed

Herrick, James; St Cyr, John

2008-01-01

Every cell needs energy, i.e., adenosine triphosphate (ATP), to carry out its function. Decreased oxygen levels, decreased blood flow, and other stressful conditions can drastically effect the intracellular concentrations of these energy compounds. Skeletal muscle, unlike the heart, can address this drop in ATP by employing the myokinase reaction, ultimately producing ATP with a subsequent elevation in adenosine monophosphate (AMP). Ribose, a naturally occurring 5-carbon monosaccharide, is a key component of RNA, DNA (which has deoxyribose), acetyl coenzyme A, and ATP. Each cell produces its own ribose, involved in the pentose phosphate pathway (PPP), to aid in ATP production. States of ischemia and/or hypoxia can severely lower levels of cellular energy compounds in the heart, with an associated compromise in cellular processes, ultimately reflected in altered function. Ribose appears to provide a solution to the problem in replenishing the depressed ATP levels and improving functional status of patients afflicted with cardiovascular diseases.

Viscosity dictates metabolic activity of Vibrio ruber

PubMed Central

Borić, Maja; Danevčič, Tjaša; Stopar, David

2012-01-01

Little is known about metabolic activity of bacteria, when viscosity of their environment changes. In this work, bacterial metabolic activity in media with viscosity ranging from 0.8 to 29.4 mPas was studied. Viscosities up to 2.4 mPas did not affect metabolic activity of Vibrio ruber. On the other hand, at 29.4 mPas respiration rate and total dehydrogenase activity increased 8 and 4-fold, respectively. The activity of glucose-6-phosphate dehydrogenase (GPD) increased up to 13-fold at higher viscosities. However, intensified metabolic activity did not result in faster growth rate. Increased viscosity delayed the onset as well as the duration of biosynthesis of prodigiosin. As an adaptation to viscous environment V. ruber increased metabolic flux through the pentose phosphate pathway and reduced synthesis of a secondary metabolite. In addition, V. ruber was able to modify the viscosity of its environment. PMID:22826705

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

An Overview of Plant Phenolic Compounds and Their Importance in Human Nutrition and Management of Type 2 Diabetes.

PubMed

Lin, Derong; Xiao, Mengshi; Zhao, Jingjing; Li, Zhuohao; Xing, Baoshan; Li, Xindan; Kong, Maozhu; Li, Liangyu; Zhang, Qing; Liu, Yaowen; Chen, Hong; Qin, Wen; Wu, Hejun; Chen, Saiyan

2016-10-15

In this paper, the biosynthesis process of phenolic compounds in plants is summarized, which include the shikimate, pentose phosphate and phenylpropanoid pathways. Plant phenolic compounds can act as antioxidants, structural polymers (lignin), attractants (flavonoids and carotenoids), UV screens (flavonoids), signal compounds (salicylic acid, flavonoids) and defense response chemicals (tannins, phytoalexins). From a human physiological standpoint, phenolic compounds are vital in defense responses, such as anti-aging, anti-inflammatory, antioxidant and anti-proliferative activities. Therefore, it is beneficial to eat such plant foods that have a high antioxidant compound content, which will cut down the incidence of certain chronic diseases, for instance diabetes, cancers and cardiovascular diseases, through the management of oxidative stress. Furthermore, berries and other fruits with low-amylase and high-glucosidase inhibitory activities could be thought of as candidate food items in the control of the early stages of hyperglycemia associated with type 2 diabetes.

HIV-1 Vpr modulates macrophage metabolic pathways: a SILAC-based quantitative analysis.

PubMed

Barrero, Carlos A; Datta, Prasun K; Sen, Satarupa; Deshmane, Satish; Amini, Shohreh; Khalili, Kamel; Merali, Salim

2013-01-01

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

Liver glucose metabolism in humans

PubMed Central

Adeva-Andany, María M.; Pérez-Felpete, Noemi; Fernández-Fernández, Carlos; Donapetry-García, Cristóbal; Pazos-García, Cristina

2016-01-01

Information about normal hepatic glucose metabolism may help to understand pathogenic mechanisms underlying obesity and diabetes mellitus. In addition, liver glucose metabolism is involved in glycosylation reactions and connected with fatty acid metabolism. The liver receives dietary carbohydrates directly from the intestine via the portal vein. Glucokinase phosphorylates glucose to glucose 6-phosphate inside the hepatocyte, ensuring that an adequate flow of glucose enters the cell to be metabolized. Glucose 6-phosphate may proceed to several metabolic pathways. During the post-prandial period, most glucose 6-phosphate is used to synthesize glycogen via the formation of glucose 1-phosphate and UDP–glucose. Minor amounts of UDP–glucose are used to form UDP–glucuronate and UDP–galactose, which are donors of monosaccharide units used in glycosylation. A second pathway of glucose 6-phosphate metabolism is the formation of fructose 6-phosphate, which may either start the hexosamine pathway to produce UDP-N-acetylglucosamine or follow the glycolytic pathway to generate pyruvate and then acetyl-CoA. Acetyl-CoA may enter the tricarboxylic acid (TCA) cycle to be oxidized or may be exported to the cytosol to synthesize fatty acids, when excess glucose is present within the hepatocyte. Finally, glucose 6-phosphate may produce NADPH and ribose 5-phosphate through the pentose phosphate pathway. Glucose metabolism supplies intermediates for glycosylation, a post-translational modification of proteins and lipids that modulates their activity. Congenital deficiency of phosphoglucomutase (PGM)-1 and PGM-3 is associated with impaired glycosylation. In addition to metabolize carbohydrates, the liver produces glucose to be used by other tissues, from glycogen breakdown or from de novo synthesis using primarily lactate and alanine (gluconeogenesis). PMID:27707936

Developmental control of apiogalacturonan biosynthesis and UDP-apiose production in a duckweed. [Spirodela polyrrhiza

DOE Office of Scientific and Technical Information (OSTI.GOV)

Longland, J.M.; Fry, S.C.; Trewavas, A.J.

1989-07-01

Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, D-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed D-({sup 3}H)glucuronic acid for 30 minutes, the accumulated UDP-({sup 3}H)apiose pool accounted for about 27% of the total phosphorylated ({sup 3}H)pentose derivatives; in turions, the UDP({sup 3}H)apiose pool accounted for only about 4% of the total phosphorylated ({sup 3}H)pentose derivatives.more » They conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDP-apiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase.« less

Improvements In Ethanologenic Escherichia Coli and Klebsiella Oxytoca

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dr. David Nunn

2010-09-30

The current Verenium cellulosic ethanol process is based on the dilute-acid pretreatment of a biomass feedstock, followed by a two-stage fermentation of the pentose sugar-containing hydrolysate by a genetically modified ethanologenic Escherichia coli strain and a separate simultaneous saccharification-fermentation (SSF) of the cellulosic fraction by a genetically modified ethanologenic Klebsiella oxytoca strain and a fungal enzyme cocktail. In order to reduce unit operations and produce a fermentation beer with higher ethanol concentrations to reduce distillation costs, we have proposed to develop a simultaneous saccharification co-fermentation (SScF) process, where the fermentation of the pentose-containing hydrolysate and cellulosic fraction occurs within themore » same fermentation vessel. In order to accomplish this goal, improvements in the ethanologens must be made to address a number of issues that arise, including improved hydrolysate tolerance, co-fermentation of the pentose and hexose sugars and increased ethanol tolerance. Using a variety of approaches, including transcriptomics, strain adaptation, metagenomics and directed evolution, this work describes the efforts of a team of scientists from Verenium, University of Florida, Massachusetts Institute of Technology and Genomatica to improve the E. coli and K. oxytoca ethanologens to meet these requirements.« less

Microbial production, ultrasound-assisted extraction and characterization of biopolymer polyhydroxybutyrate (PHB) from terrestrial (P. hysterophorus) and aquatic (E. crassipes) invasive weeds.

PubMed

Pradhan, Sushobhan; Borah, Arup Jyoti; Poddar, Maneesh Kumar; Dikshit, Pritam Kumar; Rohidas, Lilendar; Moholkar, Vijayanand S

2017-10-01

This study reports synthesis of biodegradable poly(3-hydroxybutyrate) (PHB) polymer from two invasive weeds, viz. P. hysterophorus and E. crassipes. The pentose and hexose-rich hydrolyzates obtained from acid pretreatment and enzymatic hydrolysis of two biomasses were separately fermented using Ralstonia eutropha MTCC 8320 sp. PHB was extracted using sonication and was characterized using FTIR, 1 H and 13 C NMR and XRD. PHB content of dry cell mass was 8.1-21.6% w/w, and the PHB yield was 6.85×10 -3 -36.41×10 -3 % w/w raw biomass. Thermal properties of PHB were determined by TGA, DTG and DSC analysis. PHB obtained from pentose-hydrolyzate had glass transition temperatures of 6°-9°C, while PHB from hexose-rich hydrolyzate had maximum thermal degradation temperatures of 370°-389°C. These thermal properties were comparable to the properties of commercial PHB. Probable causes leading to differences in thermal properties of pentose and hexose-derived PHB are: extent of crystallinity and presence of impurity in the polymer matrix. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differentiation-inducing and anti-proliferative activities of isoliquiritigenin and all-trans-retinoic acid on B16F0 melanoma cells: Mechanisms profiling by RNA-seq.

PubMed

Chen, Xiaoyu; Yang, Ming; Hao, Wenjin; Han, Jichun; Ma, Jun; Wang, Caixia; Sun, Shiguo; Zheng, Qiusheng

2016-10-30

Melanoma is a cancer that arises from melanocytes, specialized pigmented cells that are found predominantly in the skin. The incidence of malignant melanoma has significantly increased over the last decade. With the development of therapy, the survival rate of some kind of cancer has been improved greatly. But the treatment of melanoma remains unsatisfactory. Much of melanoma's resistance to traditional chemotherapy is believed to arise intrinsically, by virtue of potent growth and cell survival-promoting genetic alteration. Therefore, significant attention has recently been focused on differentiation therapy, as well as differentiation inducer compounds. In previous study, we found isoliquiritigenin (ISL), a natural product extracted from licorice, could induce B16F0 melanoma cell differentiation. Here we investigated the transcriptional response of melanoma differentiation process induced by ISL and all-trans-retinoic acid (RA). Results showed that 390 genes involves in 201 biochemical pathways were differentially expressed in ISL treatment and 304 genes in 193 pathways in RA treatment. Differential expressed genes (DGEs, fold-change (FC)≥10) with the function of anti-proliferative and differentiation inducing indicated a loss of grade malignancy characteristic. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated glutathione metabolism, glycolysis/gluconeogenesis and pentose phosphate pathway were the top three relative pathway perturbed by ISL, and mitogen-activated protein kinase (MAPK) signaling pathway was the most important pathway in RA treatment. In the analysis of hierarchical clustering of DEGs, we discovered 72 DEGs involved in the process of drug action. We thought Cited1, Tgm2, Xaf1, Cd59a, Fbxo2, Adh7 may have critical role in the differentiation of melanoma. The evidence displayed herein confirms the critical role of reactive oxygen species (ROS) in melanoma pathobiology and provides evidence for future targets in the development of next-generation biomarkers and therapeutics. Copyright © 2016 Elsevier B.V. All rights reserved.

Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

PubMed

Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

2015-12-01

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma*

PubMed Central

Makinoshima, Hideki; Takita, Masahiro; Saruwatari, Koichi; Umemura, Shigeki; Obata, Yuuki; Ishii, Genichiro; Matsumoto, Shingo; Sugiyama, Eri; Ochiai, Atsushi; Abe, Ryo; Goto, Koichi; Esumi, Hiroyasu; Tsuchihara, Katsuya

2015-01-01

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells. PMID:26023239

Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodrigues, Juan, E-mail: juanricardorodrigues@gmail.com; Laboratory of Biochemistry, Faculty of Pharmacy, Central University of Venezuela; Branco, Vasco

Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibitedmore » the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates the inhibitory effects of TM/EtHg on TrxR and G6PDH. • EtHg is more toxic than the parent compound TM.« less

Metabolic alterations without metal accumulation in the ovary of adult Bufo arenarum females, observed after long-term exposure to Zn(2+), followed by toxicity to embryos.

PubMed

Naab, F; Volcomirsky, M; Burlón, A; Caraballo, M E; Debray, M; Kesque, J M; Kreiner, A J; Ozafrán, M J; Schuff, J A; Stoliar, P; Vázquez, M E; Davidson, J; Davidson, M; Fonovich de Schroeder, T M

2001-08-01

Long-term exposure of aquatic organisms to metals, even those considered micronutrients, may affect their metabolism and produce sublethal effects. We evaluated the effects of long-term exposure of adult amphibian (Bufo arenarum) females to 4 microg/L of Zn(2+) (ZnSO(4) x H(2)O) in Ringer solution on the concentration of Zn and Fe, the activity of the key enzyme of the pentose phosphate pathway glucose 6-phosphate dehydrogenase, and glutathione content, both in the liver and ovary of these animals. We also performed early embryonic development studies by in vitro insemination from control and treated females. Zn exposure rendered lower Zn concentrations in the ovaries than did exposure of animals to Ringer solution without metal addition (97 +/- 50 versus 149 +/- 46 Zn microg/wet tissue g). Zn and Fe concentration correlation was positive and linear in the ovary, but was negative and nonlinear in the liver of the studied females. The activity of the enzyme glucose 6-phosphate dehydrogenase decreased (0.0599 +/- 0.0109 versus 0.0776 +/- 0.0263 micromol of NADPH/min x mg of proteins) and the endogenous glutathione content increased (0.027 +/- 0.005 versus 0.018 +/- 0.007 mg/10 mg of proteins) in the ovary but remained unaltered in the liver as a consequence of Zn treatment. Our results suggest the existence of different mechanisms of regulation of Zn and Fe concentrations in the ovary and in the liver of adult B. arenarum females. Binding of Zn to low-molecular-weight proteins, as metallothioneins, may occur in the liver, thus protecting this organ from toxic effects. In the ovary high-molecular-weight proteins, like glucose-6-phosphate dehydrogenase, should be able to bind Zn, leading to oxidative stress responsible for the observed increase in endogenous glutathione content. Inhibition of the pentose phosphate pathway in the ovary by Zn can be responsible for the reproductive failure that we detected through embryos survival studies during early life stages: 81.3 +/- 6.3% of embryos from control females survived versus 63.1 +/- 13.8% of embryos from Zn-treated females at the branchial circulation stage of development.

Synthesis of Furfural from Water Hyacinth (Eichornia croassipes)

NASA Astrophysics Data System (ADS)

Ismiyarto; Ngadiwiyana; windarti, T.; Purbowatiningrum, RS; Hapsari, M.; Rafi'ah, FH; Suyanti; Haq, MS

2017-02-01

Furfural has been prepared from hydrolysis of dried biomass of water hyacinth (Eichornia crassipes) by using diluted hydrochloric acid and sulphuric acid as catalysts. This process involved the conversion of the pentosane fraction in water hyacinth into pentose, and then pentose was cyclodehydrated into furfural. The reaction was conducted in a distillation set with receiving the flask that contains chloroform. Furfural was identified by fehling test which was then characterized using Fourier Transform Infra Red (FTIR) and Proton Nuclear Magnetic Resonance (1H-NMR), followed by Gas Chromatography with Mass Spectroscopy (GC-MS). The yield of furfural obtained using sulphuric acid catalyst was 0.38% and hydrochloric acid catalyst was 0.01% of dried biomass.

Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

PubMed

Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

1983-07-01

Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

Contrasting Changes Caused by Drought and Submergence Stresses in Bermudagrass (Cynodon dactylon)

PubMed Central

Ye, Tiantian; Shi, Haitao; Wang, Yanping; Chan, Zhulong

2015-01-01

In this study, we investigated the mechanisms by which bermudagrass withstands the drought and submergence stresses through physiological, proteomic and metabolomic approaches. The results showed that significant physiological changes were observed after drought treatment, while only slight changes after submergence treatment, including compatible solute contents, ROS levels and antioxidant enzyme activities. Proteomics results showed that 81 proteins regulated by drought or submergence treatment were identified by MALDI-TOF-MS. Among them, 76 proteins were modulated by drought stress with 46 increased abundance and 30 decreased abundance. Forty-five showed abundance changes after submergence treatment with 10 increased and 35 decreased. Pathway enrichment analysis revealed that pathways of amino acid metabolism and mitochondrial electron transport/ATP synthesis were only enriched by drought treatment, while other pathways including photosynthesis, biodegradation of xenobiotics, oxidative pentose phosphate, glycolysis and redox were commonly over-represented after both drought and submergence treatments. Metabolomic analysis indicated that most of the metabolites were up-regulated by drought stress, while 34 of 40 metabolites contents exhibited down-regulation or no significant changes when exposed to submergence stress, including sugars and sugar alcohols. These data indicated that drought stress extensively promoted photosynthesis and redox metabolisms while submergence stress caused declined metabolisms and dormancy in Cynodon dactylon. Taken together, the quiescence strategy with retarded growth might allow bermudagrass to be adaptive to long-term submerged environment, while activation of photosynthesis and redox, and accumulation of compatible solutes and molecular chaperones increased bermudagrass tolerance to drought stress. PMID:26617615

Urinary metabolomics analysis identifies key biomarkers of different stages of nonalcoholic fatty liver disease

PubMed Central

Dong, Shu; Zhan, Zong-Ying; Cao, Hong-Yan; Wu, Chao; Bian, Yan-Qin; Li, Jian-Yuan; Cheng, Gen-Hong; Liu, Ping; Sun, Ming-Yu

2017-01-01

AIM To identify a panel of biomarkers that can distinguish between non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), and explore molecular mechanism involved in the process of developing NASH from NAFLD. METHODS Biomarkers may differ during stages of NAFLD. Urine and blood were obtained from non-diabetic subjects with NAFLD and steatosis, with normal liver function (n = 33), from patients with NASH, with abnormal liver function (n = 45), and from healthy age and sex-matched controls (n = 30). Samples were subjected to metabolomic analysis to identify potential non-invasive biomarkers. Differences in urinary metabolic profiles were analyzed using liquid chromatography tandem mass spectrometry with principal component analysis and partial least squares-discriminate analysis. RESULTS Compared with NAFLD patients, patients with NASH had abnormal liver function and high serum lipid concentrations. Urinary metabonomics found differences in 31 metabolites between these two groups, including differences in nucleic acids and amino acids. Pathway analysis based on overlapping metabolites showed that pathways of energy and amino acid metabolism, as well as the pentose phosphate pathway, were closely associated with pathological processes in NAFLD and NASH. CONCLUSION These findings suggested that a panel of biomarkers could distinguish between NAFLD and NASH, and could help to determine the molecular mechanism involved in the process of developing NASH from NAFLD. Urinary biomarkers may be diagnostic in these patients and could be used to assess responses to therapeutic interventions. PMID:28487615

Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure.

PubMed

Vigentini, Ileana; Joseph, C M Lucy; Picozzi, Claudia; Foschino, Roberto; Bisson, Linda F

2013-11-01

Brettanomyces bruxellensis displays a high degree of genotypic and phenotypic polymorphism and is the main yeast species involved in wine spoilage. The innate resistance of 108 B. bruxellensis strains to the antimicrobial agent SO2 used in winemaking was investigated. Nineteen strains (17.6%) were sensitive to SO2 , failing to grow at the lowest concentration tested (0.1 mg L(-1) molecular SO2). Twenty-nine strains (26.8%) grew at 0.1 mg L(-1), 42 strains (38.9%) grew at 0.2 mg L(-1) , and 16 strains (14.8%) were able to grow as high as 0.4 mg L(-1) mol. SO2. Two strains able to grow in the presence of 0.6 mg L(-1) mol. SO2 were further studied by GCMS-TOF analysis to define the metabolic response to SO2 treatment. Two hundred and fifty-three intracellular metabolites were detected. The main effect observed was a decrease in cytoplasmic levels of polyols and an increase in levels of some amino acids, alanine, glutamic acid, glycine, proline, 5-oxoproline, serine and valine, which were significantly accumulated in the presence of SO2. No alteration in the pentose phosphate pathway was observed, suggesting NADPH usage could be diverted to other pathways. Finally, a change in metabolites involved in the glycerophospholipid pathway (glycerol-3-phosphate and myo-inositol) was also found. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Sublethal Effects of the Neonicotinoid Insecticide Thiamethoxam on the Transcriptome of the Honey Bees (Hymenoptera: Apidae).

PubMed

Shi, Teng-Fei; Wang, Yu-Fei; Liu, Fang; Qi, Lei; Yu, Lin-Sheng

2017-12-05

Neonicotinoid insecticides are now the most widely used insecticides in the world. Previous studies have indicated that sublethal doses of neonicotinoids impair learning, memory capacity, foraging, and immunocompetence in honey bees (Apis mellifera, Linnaeus) (Hymenoptera: Apidae). Despite these, few studies have been carried out on the molecular effects of neonicotinoids. In this study, we focus on the second-generation neonicotinoid thiamethoxam, which is currently widely used in agriculture to protect crops. Using high-throughput RNA-Seq, we investigated the transcriptome profile of honey bees after subchronic exposure to 10 ppb thiamethoxam over 10 d. In total, 609 differentially expressed genes (DEGs) were identified, of which 225 were upregulated and 384 were downregulated. Several genes, including vitellogenin, CSP3, defensin-1, Mrjp1, and Cyp6as5 were selected and further validated using real-time quantitative polymerase chain reaction assays. The functions of some DEGs were identified, and Gene Ontology-enrichment analysis showed that the enriched DEGs were mainly linked to metabolism, biosynthesis, and translation. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that thiamethoxam affected biological processes including ribosomes, the oxidative phosphorylation pathway, tyrosine metabolism pathway, pentose and glucuronate interconversions, and drug metabolism. Overall, our results provide a basis for understanding the molecular mechanisms of the complex interactions between neonicotinoid insecticides and honey bees. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

PubMed Central

Hottes, Alison K.; Meewan, Maliwan; Yang, Desiree; Arana, Naomi; Romero, Pedro; McAdams, Harley H.; Stephens, Craig

2004-01-01

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. PMID:14973021

Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis.

PubMed

Gu, Yang; Deng, Jieying; Liu, Yanfeng; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Liu, Long

2017-10-01

N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. In a previous study, the recombinant Bacillus subtilis strain BSGN6-P xylA -glmS-pP43NMK-GNA1 (BN0-GNA1) had been constructed for microbial production of GlcNAc by pathway design and modular optimization. Here, the production of GlcNAc is further improved by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) is blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit IIA YyzE), ypqE (encoding the PTS system transporter subunit IIA YpqE), and ptsG (encoding the PTS system glucose-specific EIICBA component), resulting in 47.6% increase in the GlcNAc titer (from 6.5 ± 0.25 to 9.6 ± 0.16 g L -1 ) in shake flasks. Then, reinforcement of the expression of the glcP and glcK genes and optimization of glucose facilitator proteins are performed to promote glucose import and phosphorylation. Next, the competitive pathways for GlcNAc synthesis, namely glycolysis, peptidoglycan synthesis pathway, pentose phosphate pathway, and tricarboxylic acid cycle, are repressed by initiation codon-optimization strategies, and the GlcNAc titer in shake flasks is improved from 10.8 ± 0.25 to 13.2 ± 0.31 g L -1 . Finally, the GlcNAc titer is further increased to 42.1 ± 1.1 g L -1 in a 3-L fed-batch bioreactor, which is 1.72-fold that of the original strain, BN0-GNA1. This study shows considerably enhanced GlcNAc production, and the metabolic engineering strategy described here will be useful for engineering other prokaryotic microorganisms for the production of GlcNAc and related molecules. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermophilic Gram-Positive Biocatalysts for Biomass Conversion to Ethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shanmugam, K.T.; Ingram, L.O.; Maupin-Furlow, J.A.

2003-12-01

Production of energy from renewable sources is receiving increased attention due to the finite nature of fossil fuels and the environmental impact associated with the continued large scale use of fossil energy sources. Biomass, a CO2-neutral abundant resource, is an attractive alternate source of energy. Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals. Extracellular cellulases produced by fungi are commercially developed for depolymerization of cellulose in biomass to glucose for fermentation by appropriate biocatalysts in a simultaneous saccharification and fermentation (SSF) process. Due to the differences in themore » optimum conditions for the activity of the fungal cellulases and the growth and fermentation characteristics of the current industrial biocatalysts, SSF of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity leading to higher than required cost of cellulase in SSF. We have isolated bacterial biocatalysts whose growth and fermentation requirements match the optimum conditions for commercial fungal cellulase activity (pH 5.0 and 50 deg. C). These isolates fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(+)-lactic acid. Xylose was metabolized through the pentose-phosphate pathway by these organisms as evidenced by the fermentation profile and analysis of the fermentation products of 13C1-xylose by NMR. As expected for the metabolism of xylose by the pentose-phosphate pathway, 13C-lactate accounted for more than 90% of the total 13C-labeled products. All three strains fermented crystalline cellulose to lactic acid with the addition of fungal cellulase (Spezyme CE) (SSF) at an optimum of about 10 FPU/g cellulose. These isolates also fermented cellulose and sugar cane bagasse hemicellulose acid hydrolysate simultaneously. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans although B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. For successful production of ethanol from pyruvate, both pyruvate decarboxylase (PDC) and alcohol dehydrogenase (AHD) need to be produced at optimal levels in these biocatalysts. A plasmid containing the S. ventriculi pdc gene and the adh gene from geobacillus stearothermophilus was constructed using plasmid pWH1520 that was successfully used for expression of pdc in B. megaterium. The resulting portable ethanol (PET) plasmid, pJAM423, was transformed into B. megaterium. After xylose induction, a significant fraction of cell cytoplasm was composed of the S. ventriculi PDC and G. stearothermophilus ADH proteins. In preliminary experiments, the amount of ethanol produced by b. megaterium with plasmid pJAM423 was about twice (20 mM) of the bacterium without the plasmid. These results show that the PET operon is functional in B. megaterium but high level ethanol production needs further genetic and metabolic engineering. A genetic transfer system for the second generation biocatalysts needs to be developed for transferring the plasmid pJAM423 and its derivatives for engineering these organisms for ethanol production from biomass derived sugars and cellulose to ethanol. One of the new biocatalysts, strain P4-102B was found to be transformable with plasmids and the method for introducing plasmid pJAM423 into this strain and expression of the encoded DNA is being optimized. These new second generation biocatalysts have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource for production of fuels and chemicals.« less

Plasma metabolomic profiling of dairy cows affected with ketosis using gas chromatography/mass spectrometry.

PubMed

Zhang, Hongyou; Wu, Ling; Xu, Chuang; Xia, Cheng; Sun, Lingwei; Shu, Shi

2013-09-26

Ketosis is an important problem for dairy cows` production performance. However, it is still little known about plasma metabolomics details of dairy ketosis. A gas chromatography/mass spectrometry (GC/MS) technique was used to investigate plasma metabolic differences in cows that had clinical ketosis (CK, n=22), subclinical ketosis (SK, n=32), or were clinically normal controls (NC, n=22). The endogenous plasma metabolome was measured by chemical derivatization followed by GC/MS, which led to the detection of 267 variables. A two-sample t-test of 30, 32, and 13 metabolites showed statistically significant differences between SK and NC, CK and NC, and CK and SK, respectively. Orthogonal signal correction-partial least-square discriminant analysis (OPLS-DA) revealed that the metabolic patterns of both CK and SK were mostly similar, with the exception of a few differences. The development of CK and SK involved disturbances in many metabolic pathways, mainly including fatty acid metabolism, amino acid metabolism, glycolysis, gluconeogenesis, and the pentose phosphate pathway. A diagnostic model arbitrary two groups was constructed using OPLS-DA and receiver-operator characteristic curves (ROC). Multivariate statistical diagnostics yielded the 19 potential biomarkers for SK and NC, 31 for CK and NC, and 8 for CK and SK with area under the curve (AUC) values. Our results showed the potential biomarkers from CK, SK, and NC, including carbohydrates, fatty acids, amino acids, even sitosterol and vitamin E isomers, etc. 2-piperidinecarboxylic acid and cis-9-hexadecenoic acid were closely associated with metabolic perturbations in ketosis as Glc, BHBA and NEFA for dealing with metabolic disturbances of ketosis in clinical practice. However, further research is needed to explain changes of 2,3,4-trihydroxybutyric acid, 3,4-dihydroxybutyric acid, α-aminobutyric acid, methylmalonic acid, sitosterol and α-tocopherol in CK and SK, and to reveal differences between CK and SK. Our study shows that some new biomarkers of ketosis from plasma may find new metabolic changes to have clinically new utility and significance in diagnosis, prognosis, and prevention of ketosis in the future.

Plasma metabolomic profiling of dairy cows affected with ketosis using gas chromatography/mass spectrometry

PubMed Central

2013-01-01

Background Ketosis is an important problem for dairy cows` production performance. However, it is still little known about plasma metabolomics details of dairy ketosis. Results A gas chromatography/mass spectrometry (GC/MS) technique was used to investigate plasma metabolic differences in cows that had clinical ketosis (CK, n=22), subclinical ketosis (SK, n=32), or were clinically normal controls (NC, n=22). The endogenous plasma metabolome was measured by chemical derivatization followed by GC/MS, which led to the detection of 267 variables. A two-sample t-test of 30, 32, and 13 metabolites showed statistically significant differences between SK and NC, CK and NC, and CK and SK, respectively. Orthogonal signal correction-partial least-square discriminant analysis (OPLS-DA) revealed that the metabolic patterns of both CK and SK were mostly similar, with the exception of a few differences. The development of CK and SK involved disturbances in many metabolic pathways, mainly including fatty acid metabolism, amino acid metabolism, glycolysis, gluconeogenesis, and the pentose phosphate pathway. A diagnostic model arbitrary two groups was constructed using OPLS-DA and receiver–operator characteristic curves (ROC). Multivariate statistical diagnostics yielded the 19 potential biomarkers for SK and NC, 31 for CK and NC, and 8 for CK and SK with area under the curve (AUC) values. Our results showed the potential biomarkers from CK, SK, and NC, including carbohydrates, fatty acids, amino acids, even sitosterol and vitamin E isomers, etc. 2-piperidinecarboxylic acid and cis-9-hexadecenoic acid were closely associated with metabolic perturbations in ketosis as Glc, BHBA and NEFA for dealing with metabolic disturbances of ketosis in clinical practice. However, further research is needed to explain changes of 2,3,4-trihydroxybutyric acid, 3,4-dihydroxybutyric acid, α-aminobutyric acid, methylmalonic acid, sitosterol and α-tocopherol in CK and SK, and to reveal differences between CK and SK. Conclusion Our study shows that some new biomarkers of ketosis from plasma may find new metabolic changes to have clinically new utility and significance in diagnosis, prognosis, and prevention of ketosis in the future. PMID:24070026

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

PubMed

Welkie, David; Zhang, Xiaohui; Markillie, Meng Lye; Taylor, Ronald; Orr, Galya; Jacobs, Jon; Bhide, Ketaki; Thimmapuram, Jyothi; Gritsenko, Marina; Mitchell, Hugh; Smith, Richard D; Sherman, Louis A

2014-12-29

Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light-dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions. By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light-dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid. This investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salminen, S.O.; Streeter, J.G.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with /sup 14/C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O/sub 2/ (2% in the gas phase). Uptake and conversion of /sup 14/C to CO/sub 2/ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO/sub 2/, and fructose was respired at a rate of at least double that of glucose. Sucrose and glucose were converted to CO/sub 2/ at a very low but measurable rate (<0.1more » nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO/sub 2/ at a rate 30 times greater than the conversion of carbon Number 6 to CO/sub 2/, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.« less

Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

NASA Astrophysics Data System (ADS)

Zhang, Zhihua; Qu, Yinbo; Zhang, Xiao; Lin, Jianqiang

The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h-1 when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.

Microbiome-Metabolome Responses in the Cecum and Colon of Pig to a High Resistant Starch Diet.

PubMed

Sun, Yue; Su, Yong; Zhu, Weiyun

2016-01-01

Currently, knowledge about the impact of long-term intake of high resistant starch diet on pig hindgut microbiota and metabolite profile is limited. In this study, a combination of the pyrosequencing and the mass spectrometry (MS)-based metabolomics techniques were used to investigate the effects of a raw potato starch (RPS, high in resistant starch) diet on microbial composition and microbial metabolites in the hindgut of pig. The results showed that Coprococcus, Ruminococcus, and Turicibacter increased significantly, while Sarcina and Clostridium decreased in relative abundances in the hindgut of pigs fed RPS. The metabolimic analysis revealed that RPS significantly affected starch and sucrose metabolites, amino acid turnover or protein biosynthesis, lipid metabolites, glycolysis, the pentose phosphate pathway, inositol phosphate metabolism, and nucleotide metabolism. Furthermore, a Pearson's correlation analysis showed that Ruminococcus and Coprococcus were positively correlated with glucose-6-phosphate, maltose, arachidonic acid, 9, 12-octadecadienoic acid, oleic acid, phosphate, but negatively correlated with α-aminobutyric acid. However, the correlation of Clostridium and Sarcina with these compounds was in the opposite direction. The results suggest that RPS not only alters the composition of the gut microbial community but also modulates the metabolic pathway of microbial metabolism, which may further affect the hindgut health of the host.

Microbiome-Metabolome Responses in the Cecum and Colon of Pig to a High Resistant Starch Diet

PubMed Central

Sun, Yue; Su, Yong; Zhu, Weiyun

2016-01-01

Currently, knowledge about the impact of long-term intake of high resistant starch diet on pig hindgut microbiota and metabolite profile is limited. In this study, a combination of the pyrosequencing and the mass spectrometry (MS)-based metabolomics techniques were used to investigate the effects of a raw potato starch (RPS, high in resistant starch) diet on microbial composition and microbial metabolites in the hindgut of pig. The results showed that Coprococcus, Ruminococcus, and Turicibacter increased significantly, while Sarcina and Clostridium decreased in relative abundances in the hindgut of pigs fed RPS. The metabolimic analysis revealed that RPS significantly affected starch and sucrose metabolites, amino acid turnover or protein biosynthesis, lipid metabolites, glycolysis, the pentose phosphate pathway, inositol phosphate metabolism, and nucleotide metabolism. Furthermore, a Pearson's correlation analysis showed that Ruminococcus and Coprococcus were positively correlated with glucose-6-phosphate, maltose, arachidonic acid, 9, 12-octadecadienoic acid, oleic acid, phosphate, but negatively correlated with α-aminobutyric acid. However, the correlation of Clostridium and Sarcina with these compounds was in the opposite direction. The results suggest that RPS not only alters the composition of the gut microbial community but also modulates the metabolic pathway of microbial metabolism, which may further affect the hindgut health of the host. PMID:27303373

Quantitative proteomics links metabolic pathways to specific developmental stages of the plant-pathogenic oomycete Phytophthora capsici.

PubMed

Pang, Zhili; Srivastava, Vaibhav; Liu, Xili; Bulone, Vincent

2017-04-01

The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Analysis of the core genome and pangenome of Pseudomonas putida.

PubMed

Udaondo, Zulema; Molina, Lázaro; Segura, Ana; Duque, Estrella; Ramos, Juan L

2016-10-01

Pseudomonas putida are strict aerobes that proliferate in a range of temperate niches and are of interest for environmental applications due to their capacity to degrade pollutants and ability to promote plant growth. Furthermore solvent-tolerant strains are useful for biosynthesis of added-value chemicals. We present a comprehensive comparative analysis of nine strains and the first characterization of the Pseudomonas putida pangenome. The core genome of P. putida comprises approximately 3386 genes. The most abundant genes within the core genome are those that encode nutrient transporters. Other conserved genes include those for central carbon metabolism through the Entner-Doudoroff pathway, the pentose phosphate cycle, arginine and proline metabolism, and pathways for degradation of aromatic chemicals. Genes that encode transporters, enzymes and regulators for amino acid metabolism (synthesis and degradation) are all part of the core genome, as well as various electron transporters, which enable aerobic metabolism under different oxygen regimes. Within the core genome are 30 genes for flagella biosynthesis and 12 key genes for biofilm formation. Pseudomonas putida strains share 85% of the coding regions with Pseudomonas aeruginosa; however, in P. putida, virulence factors such as exotoxins and type III secretion systems are absent. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues.

PubMed

Mangeot-Peter, Lauralie; Legay, Sylvain; Hausman, Jean-Francois; Esposito, Sergio; Guerriero, Gea

2016-09-15

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

PubMed

Mukherjee, Joy; Ow, Saw Yen; Noirel, Josselin; Biggs, Catherine A

2011-02-01

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzymic activities of cadmium- and zinc-tolerant strains of Klebsiella (Aerobacter) aerogenes growing in glucose-limited chemostats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickett, A.W.; Carter, I.S.; Dean, A.C.R.

The activities of phosphatases and some enzymes of glucose metabolism were determined in K. aerogenes NCIB 418 and in two strains derived from it, resistant to 50 ..mu..g Cd/sup 2 +/ ml/sup -1/ and 16..mu..g Zn/sup 2 +/ ml/sup -1/ respectively, during growth at D = 0.38 h/sup -1/ in medium containing ..beta..-glycerophosphate as sole phosphorus source and supplemented with Cd/sup 2 +/ and Zn/sup 2 +/, as appropriate for the resistant strains. The pH-activity profiles of the phosphatases differed from strain to strain but all showed maximum activity at an acid pH and this activity was very much lowermore » in the Zn/sup 2 +/-resistant strain than in the control and even lower in the Cd/sup 2 +/-resistant strain. Resistance to either metal was associated with decreased glucose-6-phosphate dehydrogenase activity and increased phosphoglucose isomerase activity, suggesting an increased flow of carbon through the Embden-Meyerhof pathway relative to the pentose phosphate pathway, but the efficiency of the conversion of glucose into biomass was largely unaffected. Glucose phosphoenolpyruvate phosphotransferase activity was also lower in the resistant strains. 25 references, 1 figure, 1 table.« less

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

PubMed

Xiao, Yang; Kwong, Mandy; Daemen, Anneleen; Belvin, Marcia; Liang, Xiaorong; Hatzivassiliou, Georgia; O'Brien, Thomas

2016-01-01

Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

The fate of glucose during the period of decreased metabolism after fluid percussion injury: a 13C NMR study.

PubMed

Bartnik, Brenda L; Lee, Stefan M; Hovda, David A; Sutton, Richard L

2007-07-01

The present study determined the metabolic fate of [1, 2 13C2] glucose in male control rats and in rats with moderate lateral fluid percussion injured (FPI) at 3.5 h and 24 h post-surgery. After a 3-h infusion, the amount of 13C-labeled glucose increased bilaterally (26% in left/injured cerebral cortex and 45% in right cerebral cortex) at 3.5 h after FPI and in injured cortex (45%) at 24 h after injury, indicating an accumulation of unmetabolised glucose not seen in controls. No evidence of an increase in anaerobic glycolysis above control levels was found after FPI, as 13C-labeled lactate tended to decrease at both time points and was significantly reduced (33%) in the injured cortex at 24 h post-FPI. A bilateral decrease in the 13C-labeling of both glutamate and glutamine was observed in the FPI rats at 3.5 h and the glutamine pool remained significantly decreased in the injured cortex at 24 h, suggesting reduced oxidative metabolism in both neuronal and astrocyte compartments after injury. The percentage of glucose metabolism through the pentose phosphate pathway (PPP) increased in the injured (13%) and contralateral (11%) cortex at 3.5 h post-FPI and in the injured cortex (9%) at 24 h post-injury. Based upon the changes in metabolite pools, our results show an injury-induced decrease in glucose utilization and oxidation within the first 24 h after FPI. Increased metabolism through the PPP would result in increased NADPH synthesis, suggesting a need for reducing equivalents after FPI to help restore the intracellular redox state and/or in response to free radical stress.

Use of a Tn5-based transposon system to create a cost-effective Zymomonas mobilis for ethanol production from lignocelluloses

PubMed Central

2013-01-01

Background Current methods of ethanol production from lignocelluloses generate a mixture of sugars, primarily glucose and xylose; the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions that affect their growth and productivity. Stress-tolerant strains capable of using both glucose and xylose to produce ethanol with high yield are highly desirable. Results A recombinant Zymomonas mobilis (Z. mobilis) designated as HYMX was constructed by integrating seven genes (Pfu-sHSP, yfdZ, metB, xylA, xylB, tktA and talB) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furious (P. furious) was used to increase the heat-tolerance, the yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. To overcome the bottleneck of CP4 being unable to use pentose, xylose catabolic genes (xylA, xylB, tktA and talB) from E. coli were integrated into CP4 also for construction of the xylose utilizing metabolic pathway. Conclusions The genomic integration confers on Z. mobilis the ability to grow in medium containing xylose as the only carbon source, and to grow in simple chemical defined medium without addition of amino acid. The HYMX demonstrated not only the high tolerance to unfavorable stresses like high temperature and low nutrient, but also the capability of converting both glucose and xylose to ethanol with high yield at high temperature. What’s more, these genetic characteristics were stable up to 100 generations on nonselective medium. Although significant improvements were achieved, yeast extract is needed for ethanol production. PMID:23635356

Processing of waste material of radix physochlainae for preparation of fine chemicals after extraction

NASA Astrophysics Data System (ADS)

He, A.; Yohannes, A.; Feng, X. T.; Yao, S.

2017-02-01

Waste residues of Chinese traditional medicine radix physochlainae (Huashanshen) contain a large amount of hemicelluloses after extraction. After the removal of the cellulose and lignin, main components of the solution are different degree of hydrolysis products of hemicelluloses. In the degradation process, hemicelluloses firstly become pentose, and then pentose loses 3 molecules of water and turns into furfural. This study explored a series of conditions of the method; finally the yield of furfural can reach 8.5% (calculated with the weight of raw residues) under the condition of pH of 0.2-0.3, temperature of 104-106°C, hydrolysis duration for 10 minutes. Furfural can be further processed to be resin materials.

Metabolic evolution of Escherichia coli strains that produce organic acids

DOEpatents

Grabar, Tammy; Gong, Wei; Yocum, R Rogers

2014-10-28

This invention relates to the metabolic evolution of a microbial organism previously optimized for producing an organic acid in commercially significant quantities under fermentative conditions using a hexose sugar as sole source of carbon in a minimal mineral medium. As a result of this metabolic evolution, the microbial organism acquires the ability to use pentose sugars derived from cellulosic materials for its growth while retaining the original growth kinetics, the rate of organic acid production and the ability to use hexose sugars as a source of carbon. This invention also discloses the genetic change in the microorganism that confers the ability to use both the hexose and pentose sugars simultaneously in the production of commercially significant quantities of organic acids.

Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

PubMed Central

Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

2013-01-01

Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

Metabolomic studies identify changes in transmethylation and polyamine metabolism in a brain-specific mouse model of tuberous sclerosis complex.

PubMed

McKenna, James; Kapfhamer, David; Kinchen, Jason M; Wasek, Brandi; Dunworth, Matthew; Murray-Stewart, Tracy; Bottiglieri, Teodoro; Casero, Robert A; Gambello, Michael J

2018-06-15

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mechanistic Target of Rapamycin Complex 1 (mTORC1) dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/tricarboxylic acid cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM-the major methyl donor), reduced SAM/S-adenosylhomocysteine ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A and cystathionine beta synthase. We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2+/- mice with the ornithine decarboxylase inhibitor α-difluoromethylornithine, to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

PubMed

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways

PubMed Central

Ma, Menggen; Song, Mingzhou

2010-01-01

Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)+ for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress. PMID:19517136

Metabolic pathways recruited in the production of a recombinant enveloped virus: mining targets for process and cell engineering.

PubMed

Rodrigues, A F; Formas-Oliveira, A S; Bandeira, V S; Alves, P M; Hu, W S; Coroadinha, A S

2013-11-01

Biopharmaceuticals derived from enveloped virus comprise an expanding market of vaccines, oncolytic vectors and gene therapy products. Thus, increased attention is given to the development of robust high-titer cell hosts for their manufacture. However, the knowledge on the physiological constraints modulating virus production is still scarce and the use of integrated strategies to improve hosts productivity and upstream bioprocess an under-explored territory. In this work, we conducted a functional genomics study, including the transcriptional profiling and central carbon metabolism analysis, following the metabolic changes in the transition 'parental-to-producer' of two human cell lines producing recombinant retrovirus. Results were gathered into three comprehensive metabolic maps, providing a broad and integrated overview of gene expression changes for both cell lines. Eight pathways were identified to be recruited in the virus production state: amino acid catabolism, carbohydrate catabolism and integration of the energy metabolism, nucleotide metabolism, glutathione metabolism, pentose phosphate pathway, polyamines biosynthesis and lipid metabolism. Their ability to modulate viral titers was experimentally challenged, leading to improved specific productivities of recombinant retrovirus up to 6-fold. Within recruited pathways in the virus production state, we sought for metabolic engineering gene targets in the low producing phenotypes. A mining strategy was used alternative to the traditional approach 'high vs. low producer' clonal comparison. Instead, 'high vs. low producer' from different genetic backgrounds (i.e. cell origins) were compared. Several genes were identified as limiting in the low-production phenotype, including two enzymes from cholesterol biosynthesis, two enzymes from glutathione biosynthesis and the regulatory machinery of polyamines biosynthesis. This is thus a frontier work, bridging fundamentals to technological research and contributing to enlarge our understanding of enveloped virus production dynamics in mammalian cell hosts. © 2013 Published by Elsevier Inc.

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma.

PubMed

Makinoshima, Hideki; Takita, Masahiro; Saruwatari, Koichi; Umemura, Shigeki; Obata, Yuuki; Ishii, Genichiro; Matsumoto, Shingo; Sugiyama, Eri; Ochiai, Atsushi; Abe, Ryo; Goto, Koichi; Esumi, Hiroyasu; Tsuchihara, Katsuya

2015-07-10

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence: Cyanobacterial Carbon Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Ni; DeLorenzo, Drew M.; He, Lian

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner–Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed 13C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me)more » corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593–1602. © 2017 Wiley Periodicals, Inc.« less

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

PubMed

Kamikawa, Ryoma; Moog, Daniel; Zauner, Stefan; Tanifuji, Goro; Ishida, Ken-Ichiro; Miyashita, Hideaki; Mayama, Shigeki; Hashimoto, Tetsuo; Maier, Uwe G; Archibald, John M; Inagaki, Yuji

2017-09-01

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.

PubMed

Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, Kathryn L; Oppermann, Udo; Brecker, Lothar; Nidetzky, Bernd

2012-09-07

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aklujkar, Muktak; Krushkal, Julia; DiBartolo, Genevieve

Background. The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results. The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recentlymore » in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion. The genomic evidence suggests that metabolism, physiology Background. The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results. The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion. The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.« less

Epigenomic reprogramming during pancreatic cancer progression links anabolic glucose metabolism to distant metastasis.

PubMed

McDonald, Oliver G; Li, Xin; Saunders, Tyler; Tryggvadottir, Rakel; Mentch, Samantha J; Warmoes, Marc O; Word, Anna E; Carrer, Alessandro; Salz, Tal H; Natsume, Sonoko; Stauffer, Kimberly M; Makohon-Moore, Alvin; Zhong, Yi; Wu, Hao; Wellen, Kathryn E; Locasale, Jason W; Iacobuzio-Donahue, Christine A; Feinberg, Andrew P

2017-03-01

During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.

Nicotinamide Improves Aspects of Healthspan, but Not Lifespan, in Mice.

PubMed

Mitchell, Sarah J; Bernier, Michel; Aon, Miguel A; Cortassa, Sonia; Kim, Eun Young; Fang, Evandro F; Palacios, Hector H; Ali, Ahmed; Navas-Enamorado, Ignacio; Di Francesco, Andrea; Kaiser, Tamzin A; Waltz, Tyler B; Zhang, Ning; Ellis, James L; Elliott, Peter J; Frederick, David W; Bohr, Vilhelm A; Schmidt, Mark S; Brenner, Charles; Sinclair, David A; Sauve, Anthony A; Baur, Joseph A; de Cabo, Rafael

2018-03-06

The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD + , is elusive. Here, we report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Untargeted metabolite profiling of the liver and metabolic flux analysis of liver-derived cells revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Targeted NAD metabolome analysis in liver revealed depressed expression of NAM salvage in NAM-treated mice, an effect counteracted by higher expression of de novo NAD biosynthetic enzymes. Although neither hepatic NAD + nor NADP + was boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in NAM-supplemented HFD-fed mice in the absence of survival effects. Published by Elsevier Inc.

(13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

PubMed

Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2015-12-01

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain

PubMed Central

Chen, Jinyu; Lee, Ho-Jeong; Wu, Xuefeng; Huo, Lei; Kim, Sun-Jin; Xu, Lei; Wang, Yan; He, Junqing; Bollu, Lakshmi Reddy; Gao, Guang; Su, Fei; Briggs, James; Liu, Xiaojing; Melman, Tamar; Asara, John M.; Fidler, Isaiah J.; Cantley, Lewis C.; Locasale, Jason W.; Weihua, Zhang

2014-01-01

Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain-metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the non-oxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBPs) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis, and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients. PMID:25511375

NADPH-generating systems in bacteria and archaea

PubMed Central

Spaans, Sebastiaan K.; Weusthuis, Ruud A.; van der Oost, John; Kengen, Servé W. M.

2015-01-01

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided. PMID:26284036

Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain.

PubMed

Chen, Jinyu; Lee, Ho-Jeong; Wu, Xuefeng; Huo, Lei; Kim, Sun-Jin; Xu, Lei; Wang, Yan; He, Junqing; Bollu, Lakshmi R; Gao, Guang; Su, Fei; Briggs, James; Liu, Xiaojing; Melman, Tamar; Asara, John M; Fidler, Isaiah J; Cantley, Lewis C; Locasale, Jason W; Weihua, Zhang

2015-02-01

Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the nonoxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBP) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients. ©2014 American Association for Cancer Research.

Integration of carbohydrate metabolism and redox state controls dauer larva formation in Caenorhabditis elegans.

PubMed

Penkov, Sider; Kaptan, Damla; Erkut, Cihan; Sarov, Mihail; Mende, Fanny; Kurzchalia, Teymuras V

2015-08-20

Under adverse conditions, Caenorhabditis elegans enters a diapause stage called the dauer larva. External cues signal the nuclear hormone receptor DAF-12, the activity of which is regulated by its ligands: dafachronic acids (DAs). DAs are synthesized from cholesterol, with the last synthesis step requiring NADPH, and their absence stimulates dauer formation. Here we show that NADPH levels determine dauer formation in a regulatory mechanism involving key carbohydrate and redox metabolic enzymes. Elevated trehalose biosynthesis diverts glucose-6-phosphate from the pentose phosphate pathway, which is the major source of cellular NADPH. This enhances dauer formation due to the decrease in the DA level. Moreover, DAF-12, in cooperation with DAF-16/FoxO, induces negative feedback of DA synthesis via activation of the trehalose-producing enzymes TPS-1/2 and inhibition of the NADPH-producing enzyme IDH-1. Thus, the dauer developmental decision is controlled by integration of the metabolic flux of carbohydrates and cellular redox potential.

Upgrading Lignocellulosic Products to Drop-In Biofuels via Dehydrogenative Cross-Coupling and Hydrodeoxygenation Sequence.

PubMed

Sreekumar, Sanil; Balakrishnan, Madhesan; Goulas, Konstantinos; Gunbas, Gorkem; Gokhale, Amit A; Louie, Lin; Grippo, Adam; Scown, Corinne D; Bell, Alexis T; Toste, F Dean

2015-08-24

Life-cycle analysis (LCA) allows the scientific community to identify the sources of greenhouse gas (GHG) emissions of novel routes to produce renewable fuels. Herein, we integrate LCA into our investigations of a new route to produce drop-in diesel/jet fuel by combining furfural, obtained from the catalytic dehydration of lignocellulosic pentose sugars, with alcohols that can be derived from a variety of bio- or petroleum-based feedstocks. As a key innovation, we developed recyclable transition-metal-free hydrotalcite catalysts to promote the dehydrogenative cross-coupling reaction of furfural and alcohols to give high molecular weight adducts via a transfer hydrogenation-aldol condensation pathway. Subsequent hydrodeoxygenation of adducts over Pt/NbOPO4 yields alkanes. Implemented in a Brazilian sugarcane biorefinery such a process could result in a 53-79% reduction in life-cycle GHG emissions relative to conventional petroleum fuels and provide a sustainable source of low carbon diesel/jet fuel. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous succinic acid production from xylose by Actinobacillus succinogenes.

PubMed

Bradfield, Michael F A; Nicol, Willie

2016-02-01

Continuous, anaerobic fermentations of D-xylose were performed by Actinobacillus succinogenes 130Z in a custom, biofilm reactor at dilution rates of 0.05, 0.10 and 0.30 h(-1). Succinic acid yields on xylose (0.55-0.68 g g(-1)), titres (10.9-29.4 g L(-1)) and productivities (1.5-3.4 g L(-1) h(-1)) were lower than those of a previous study on glucose, but product ratios (succinic acid/acetic acid = 3.0-5.0 g g(-1)) and carbohydrate consumption rates were similar. Also, mass balance closures on xylose were up to 18.2 % lower than those on glucose. A modified HPLC method revealed pyruvic acid excretion at appreciable concentrations (1.2-1.9 g L(-1)) which improved the mass balance closure by up to 16.8 %. Furthermore, redox balances based on the accounted xylose consumed and the excreted metabolites, indicated an overproduction of reducing power. The oxidative pentose phosphate pathway was shown to be a plausible source of the additional reducing power.

BAG3 elevation inhibits cell proliferation via direct interaction with G6PD in hepatocellular carcinomas

PubMed Central

Kong, De-Hui; Li, Si; Du, Zhen-Xian; Liu, Chuan; Liu, Bao-Qin; Li, Chao; Zong, Zhi-Hong; Wang, Hua-Qin

2016-01-01

Bcl-2 associated athanogene 3 (BAG3) contains multiple protein-binding motifs to mediate potential interactions with chaperons and/or other proteins, which is possibly ascribed to the multifaceted functions assigned to BAG3. The current study demonstrated that BAG3 directly interacted with glucose 6 phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). BAG3 suppressed the PPP flux, de novo DNA synthesis and cell growth in hepatocellular carcinomas (HCCs). The growth defect of HCCs with forced BAG3 expression can be rescued by enforced G6PD expression. However, BAG3 elevation did not cause a reduction in cellular NADPH concentrations, another main product of G6PD. In addition, supplement of nucleosides alone was sufficient to recover the growth defect mediated by BAG3 elevation. Collectively, the current study established a tumor suppressor-like function of BAG3 via direct interaction with G6PD in HCCs at the cellular level. PMID:26621836

BAG3 elevation inhibits cell proliferation via direct interaction with G6PD in hepatocellular carcinomas.

PubMed

Kong, De-Hui; Li, Si; Du, Zhen-Xian; Liu, Chuan; Liu, Bao-Qin; Li, Chao; Zong, Zhi-Hong; Wang, Hua-Qin

2016-01-05

Bcl-2 associated athanogene 3 (BAG3) contains multiple protein-binding motifs to mediate potential interactions with chaperons and/or other proteins, which is possibly ascribed to the multifaceted functions assigned to BAG3. The current study demonstrated that BAG3 directly interacted with glucose 6 phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). BAG3 suppressed the PPP flux, de novo DNA synthesis and cell growth in hepatocellular carcinomas (HCCs). The growth defect of HCCs with forced BAG3 expression can be rescued by enforced G6PD expression. However, BAG3 elevation did not cause a reduction in cellular NADPH concentrations, another main product of G6PD. In addition, supplement of nucleosides alone was sufficient to recover the growth defect mediated by BAG3 elevation. Collectively, the current study established a tumor suppressor-like function of BAG3 via direct interaction with G6PD in HCCs at the cellular level.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

PubMed Central

Hawkins, Kate E.; Joy, Shona; Delhove, Juliette M.K.M.; Kotiadis, Vassilios N.; Fernandez, Emilio; Fitzpatrick, Lorna M.; Whiteford, James R.; King, Peter J.; Bolanos, Juan P.; Duchen, Michael R.; Waddington, Simon N.; McKay, Tristan R.

2016-01-01

Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

The conversion of lignocellulosics to fermentable sugars: A survey of current research and application to CELSS

NASA Technical Reports Server (NTRS)

Petersen, Gene R.; Baresi, Larry

1990-01-01

An overview of the options for converting lignocellulosics into fermentable sugars as applied to the Closed Ecological Life Support System (CELSS) is given. A requirement for pretreatment is shown as well as the many available options. At present, physical/chemical methods are the simplest and best characterized options, but enzymatic processes will likely be the method of choice in the future. The use of pentose sugars by microorganisms to produce edibles at levels comparable to conventional plants is shown. The possible use of mycelial food production on pretreated but not hydrolyzed lignocelluloscis is also presented. Simple tradeoff analysis among some of the many possible biological pathways to regeneration of waste lignocellulosics was undertaken. Comparisons with complete oxidation processes were made. It is suggested that the NASA Life Sciences CELSS program maintain relationships with other government agencies involved in lignocellulosic conversions and use their expertise when the actual need for such conversion technology arises rather than develop this expertise within NASA.

Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations.

PubMed Central

Griffiths, W T

1975-01-01

1. Chlorophyll (ide) formation from protochlorophyll (ide) that is normally inactive was demonstrated in etioplast membranes isolated from maize and barlley plants, the process being dependent on intermittent illumination and the addition of NADPH. 2. The addition of NADPH to the membranes was shown to result in the conversion of inactive protochlorophyll (ide) absorbing at about 630 nm into a form(s) with light-absorption maxima at about 640 and 652 nm, both of which disappear when chlorophyll (ide) is formed on illumination. 3. The temperature-dependence of the activation process and its response to a variety of reagents were examined. From these, the conclusion is drawn that -SH groups are involved in the activation but in the active complex these are unavailable for reaction with -SH reagents. 4. Evidence is presented for the occurrence of glucose 6-phosphate dehydrogenase activity within etioplasts and the suggestion is made that the oxidative pentose phosphate pathway can provide the NADPH required for chlorophyll biosynthesis during the early stages of greening. PMID:5998

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses.

PubMed

Sabra, Wael; Bommareddy, Rajesh Reddy; Maheshwari, Garima; Papanikolaou, Seraphim; Zeng, An-Ping

2017-05-08

Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate. Controlling the dissolved oxygen concentration (pO 2 ) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO 2 was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO 2 control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/L*h in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/L*h) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO 2 -limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and 13 C-based fluxomics levels. Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO 2 control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in dual substrate fermentations. This study provides interesting targets for metabolic engineering of this industrial yeast.

G6pd Deficiency Does Not Affect the Cytosolic Glutathione or Thioredoxin Antioxidant Defense in Mouse Cochlea.

PubMed

White, Karessa; Kim, Mi-Jung; Ding, Dalian; Han, Chul; Park, Hyo-Jin; Meneses, Zaimary; Tanokura, Masaru; Linser, Paul; Salvi, Richard; Someya, Shinichi

2017-06-07

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP + to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd -deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP + to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1 , but not G6pd , decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea. SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP + to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea. Copyright © 2017 the authors 0270-6474/17/375770-12$15.00/0.

Scheffersomyces stipitis: a comparative systems biology study with the Crabtree positive yeast Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background Scheffersomyces stipitis is a Crabtree negative yeast, commonly known for its capacity to ferment pentose sugars. Differently from Crabtree positive yeasts such as Saccharomyces cerevisiae, the onset of fermentation in S. stipitis is not dependent on the sugar concentration, but is regulated by a decrease in oxygen levels. Even though S. stipitis has been extensively studied due to its potential application in pentoses fermentation, a limited amount of information is available about its metabolism during aerobic growth on glucose. Here, we provide a systems biology based comparison between the two yeasts, uncovering the metabolism of S. stipitis during aerobic growth on glucose under batch and chemostat cultivations. Results Starting from the analysis of physiological data, we confirmed through 13C-based flux analysis the fully respiratory metabolism of S. stipitis when growing both under glucose limited or glucose excess conditions. The patterns observed showed similarity to the fully respiratory metabolism observed for S. cerevisiae under chemostat cultivations however, intracellular metabolome analysis uncovered the presence of several differences in metabolite patterns. To describe gene expression levels under the two conditions, we performed RNA sequencing and the results were used to quantify transcript abundances of genes from the central carbon metabolism and compared with those obtained with S. cerevisiae. Interestingly, genes involved in central pathways showed different patterns of expression, suggesting different regulatory networks between the two yeasts. Efforts were focused on identifying shared and unique families of transcription factors between the two yeasts through in silico transcription factors analysis, suggesting a different regulation of glycolytic and glucoenogenic pathways. Conclusions The work presented addresses the impact of high-throughput methods in describing and comparing the physiology of Crabtree positive and Crabtree negative yeasts. Based on physiological data and flux analysis we identified the presence of one metabolic condition for S. stipitis under aerobic batch and chemostat cultivations, which shows similarities to the oxidative metabolism observed for S. cerevisiae under chemostat cultivations. Through metabolome analysis and genome-wide transcriptomic analysis several differences were identified. Interestingly, in silico analysis of transciption factors was useful to address a different regulation of mRNAs of genes involved in the central carbon metabolism. To our knowledge, this is the first time that the metabolism of S. stiptis is investigated in details and is compared to S. cerevisiae. Our study provides useful results and allows for the possibility to incorporate these data into recently developed genome-scaled metabolic, thus contributing to improve future industrial applications of S. stipitis as cell factory. PMID:23043429

Global Transcriptomic Effects of Environmentally Relevant Concentrations of the Neonicotinoids Clothianidin, Imidacloprid, and Thiamethoxam in the Brain of Honey Bees ( Apis mellifera).

PubMed

Christen, Verena; Schirrmann, Melanie; Frey, Juerg E; Fent, Karl

2018-06-14

Neonicotinoids are implicated in the decline of honey bees, but the molecular basis underlying adverse effects is poorly known. Here we describe global transcriptomic profiles in the brain of honey bee workers exposed for 48 h at one environmentally realistic and one sublethal concentration of 0.3 and 3.0 ng/bee clothianidin and imidacloprid, respectively, and 0.1 and 1.0 ng/bee thiamethoxam (1-30 ng/mL sucrose solution) by high-throughput RNA-sequencing (RNA-seq). All neonicotinoids led to significant alteration (mainly down-regulation) of gene expression, generally with a concentration-dependent effect. Among many others, genes related to metabolism and detoxification were differently expressed. Gene ontology (GO) enrichment analysis of biological processes revealed catabolic carbohydrate metabolism (regulation of enzyme activities such as amylase), lipid metabolism, and transport mechanisms as shared terms between all neonicotinoids at high concentrations. KEGG pathway analysis indicated that at least two neonicotinoids induced changes in expression of various metabolic pathways: pentose phosphate pathways, starch and sucrose metabolism, and sulfur metabolism, in which glucose 1-dehydrogenase and alpha-amylase were down-regulated and 3'(2'), 5'-bisphosphate nucleotidase was up-regulated. RT-qPCR analysis confirmed the down-regulation of major royal jelly proteins, hbg3, and cyp9e2 found by RNA-seq. Our study highlights the comparative molecular effects of neonicotinoid exposure to bees. Further studies should link these effects with physiological outcomes for a better understanding of effects of neonicotinoids.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hay, J.; Schwender, J.

Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large-scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior 13C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from themore » available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH4+ assimilation. Analysis of the light-dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO2 efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the 13C flux studies, ribulose-1,5-bisphosphate carboxylase activity is predicted to account fully for the light-dependent changes in carbon balance.« less

Metabolomics Analysis of Metabolic Effects of Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibition on Human Cancer Cells

PubMed Central

Tolstikov, Vladimir; Nikolayev, Alexander; Dong, Sucai; Zhao, Genshi; Kuo, Ming-Shang

2014-01-01

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide–consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry–based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level. PMID:25486521

Modeling Neisseria meningitidis metabolism: from genome to metabolic fluxes

PubMed Central

Baart, Gino JE; Zomer, Bert; de Haan, Alex; van der Pol, Leo A; Beuvery, E Coen; Tramper, Johannes; Martens, Dirk E

2007-01-01

Background Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years. Results Using the genomic database of N. meningitidis serogroup B together with biochemical and physiological information in the literature we constructed a genome-scale flux model for the primary metabolism of N. meningitidis. The validity of a simplified metabolic network derived from the genome-scale metabolic network was checked using flux-balance analysis in chemostat cultures. Several useful predictions were obtained from in silico experiments, including substrate preference. A minimal medium for growth of N. meningitidis was designed and tested succesfully in batch and chemostat cultures. Conclusion The verified metabolic model describes the primary metabolism of N. meningitidis in a chemostat in steady state. The genome-scale model is valuable because it offers a framework to study N. meningitidis metabolism as a whole, or certain aspects of it, and it can also be used for the purpose of vaccine process development (for example, the design of growth media). The flux distribution of the main metabolic pathways (that is, the pentose phosphate pathway and the Entner-Douderoff pathway) indicates that the major part of pyruvate (69%) is synthesized through the ED-cleavage, a finding that is in good agreement with literature. PMID:17617894

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

NASA Astrophysics Data System (ADS)

Campos, Christiane Gonçalves; Veras, Henrique César Teixeira; de Aquino Ribeiro, José Antônio; Costa, Patrícia Pinto Kalil Gonçalves; Araújo, Katiúscia Pereira; Rodrigues, Clenilson Martins; de Almeida, João Ricardo Moreira; Abdelnur, Patrícia Verardi

2017-12-01

Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. [Figure not available: see fulltext.

Metabolite fingerprinting of pennycress (Thlaspi arvense L.) embryos to assess active pathways during oil synthesis

PubMed Central

Tsogtbaatar, Enkhtuul; Cocuron, Jean-Christophe; Sonera, Marcos Corchado; Alonso, Ana Paula

2015-01-01

Pennycress (Thlaspi arvense L.), a plant naturalized to North America, accumulates high levels of erucic acid in its seeds, which makes it a promising biodiesel and industrial crop. The main carbon sinks in pennycress embryos were found to be proteins, fatty acids, and cell wall, which respectively represented 38.5, 33.2, and 27.0% of the biomass at 21 days after pollination. Erucic acid reached a maximum of 36% of the total fatty acids. Together these results indicate that total oil and erucic acid contents could be increased to boost the economic competitiveness of this crop. Understanding the biochemical basis of oil synthesis in pennycress embryos is therefore timely and relevant to guide future breeding and/or metabolic engineering efforts. For this purpose, a combination of metabolomics approaches was conducted to assess the active biochemical pathways during oil synthesis. First, gas chromatography–mass spectrometry (GC-MS) profiling of intracellular metabolites highlighted three main families of compounds: organic acids, amino acids, and sugars/sugar alcohols. Secondly, these intermediates were quantified in developing pennycress embryos by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Finally, partitional clustering analysis grouped the intracellular metabolites that shared a similar pattern of accumulation over time into eight clusters. This study underlined that: (i) sucrose might be stored rather than cleaved into hexoses; (ii) glucose and glutamine would be the main sources of carbon and nitrogen, respectively; and (iii) glycolysis, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and the Calvin cycle were active in developing pennycress embryos. PMID:25711705

Identification and Metabolite Profiling of Chemical Activators of Lipid Accumulation in Green Algae1[OPEN

PubMed Central

2017-01-01

Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii, and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity, and 15 were selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using gas chromatography-mass spectrometry quantification of total fatty acids, and targeted TAG and galactolipid measurements were performed using liquid chromatography-multiple reaction monitoring/mass spectrometry. These results demonstrated that TAG accumulation does not necessarily proceed at the expense of galactolipid. Untargeted metabolite profiling provided important insights into pathway shifts due to five different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle, and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds also was demonstrated in three other algal species. These lipid-inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation. PMID:28652262

Carbon Acquisition by Cyanobacteria: Mechanisms, Comparative Genomics, and Evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplan, Aaron; Hagemann, Martin; Bauwe, Hermann

2008-01-01

In this chapter we mainly focus on the mechanisms of inorganic carbon uptake, photorespiration, and the regulation between the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth. We identify the genes involved, their regulation and phylogeny. Living in an environment where the CO₂ concentration is considerably lower than required to saturate their carboxylating enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), cyanobacteria acquired the CO₂ concentrating mechanism (CCM) that enables them to accumulate CO₂ at the carboxylation site. All the cyanobacteria examined to date are able to fix CO₂ into carbohydrates. However, in addition to variance in the range of physical growthmore » conditions, cyanobacteria also vary substantially in their ability to consume organic carbon from their surroundings. Many strains are obligate photoautotrophs where the sole carbon source is CO₂, while others are able to perform photomixotrophic or even heterotrophic growth using a wide variety of organic substances (c.f. Rippka et al., 1979; Stal and Moezelaar, 1997b). Cyanobacteria constitute a unique case where the anabolic and catabolic carbohydrate metabolisms function in the same cellular compartment. In addition, the photosynthetic and respiratory electron transport pathways share components in the thylakoid membranes. Despite its importance to our understanding of cyanobacterial metabolism, little is known about the mechanisms involved in the shifts between photoautotrophic, heterotrophic and photomixotrophic modes of growth, and their regulation; between the different pathways of carbohydrate breakdown- glycolysis, fermentation, the oxidative pentose phosphate, the Krebs cycle and the photorespiratory pathways. In this chapter we shall briefly focus on recent advances in our understanding of the CCM and carbon metabolism in cyanobacteria.« less

Carbon acquisition by Cyanobacteria: Mechanisms, Comparative Genomics and Evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplan, Aaron; Hagemann, Martin; Bauwe, Hermann

2008-01-01

In this chapter we mainly focus on the mechanisms of inorganic carbon uptake, photorespiration, and the regulation between the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth. We identify the genes involved, their regulation and phylogeny. Living in an environment where the CO₂ concentration is considerably lower than required to saturate their carboxylating enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), cyanobacteria acquired the CO₂ concentrating mechanism (CCM) that enables them to accumulate CO₂ at the carboxylation site. All the cyanobacteria examined to date are able to fix CO₂ into carbohydrates. However, in addition to variance in the range of physical growthmore » conditions, cyanobacteria also vary substantially in their ability to consume organic carbon from their surroundings. Many strains are obligate photoautotrophs where the sole carbon source is CO₂, while others are able to perform photomixotrophic or even heterotrophic growth using a wide variety of organic substances (c.f. Rippka et al., 1979; Stal and Moezelaar, 1997b). Cyanobacteria constitute a unique case where the anabolic and catabolic carbohydrate metabolisms function in the same cellular compartment. In addition, the photosynthetic and respiratory electron transport pathways share components in the thylakoid membranes. Despite its importance to our understanding of cyanobacterial metabolism, little is known about the mechanisms involved in the shifts between photoautotrophic, heterotrophic and photomixotrophic modes of growth, and their regulation; between the different pathways of carbohydrate breakdown- glycolysis, fermentation, the oxidative pentose phosphate, the Krebs cycle and the photorespiratory pathways. In this chapter we shall briefly focus on recent advances in our understanding of the CCM and carbon metabolism in cyanobacteria.« less

HORSE SPECIES SYMPOSIUM: Canine intestinal microbiology and metagenomics: From phylogeny to function.

PubMed

Guard, B C; Suchodolski, J S

2016-06-01

Recent molecular studies have revealed a complex microbiota in the dog intestine. Convincing evidence has been reported linking changes in microbial communities to acute and chronic gastrointestinal inflammation, especially in canine inflammatory bowel disease (IBD). The most common microbial changes observed in intestinal inflammation are decreases in the bacterial phyla Firmicutes (i.e., Lachnospiraceae, Ruminococcaceae, and ) and Bacteroidetes, with concurrent increases in Proteobacteria (i.e., ). Due to the important role of microbial-derived metabolites for host health, it is important to elucidate the metabolic consequences of gastrointestinal dysbiosis and physiological pathways implicated in specific disease phenotypes. Metagenomic studies have used shotgun sequencing of DNA as well as phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) to characterize functional changes in the bacterial metagenome in gastrointestinal disease. Furthermore, wide-scale and untargeted measurements of metabolic products derived by the host and the microbiota in intestinal samples allow a better understanding of the functional alterations that occur in gastrointestinal disease. For example, changes in bile acid metabolism and tryptophan catabolism recently have been reported in humans and dogs. Also, metabolites associated with the pentose phosphate pathway were significantly altered in chronic gastrointestinal inflammation and indicate the presence of oxidative stress in dogs with IBD. This review focuses on the advancements made in canine metagenomics and metabolomics and their implications in understanding gastrointestinal disease as well as the development of better treatment approaches.

Proteomic analysis of the compatible interaction of wheat and powdery mildew (Blumeria graminis f. sp. tritici).

PubMed

Li, Jie; Yang, Xiwen; Liu, Xinhao; Yu, Haibo; Du, Congyang; Li, Mengda; He, Dexian

2017-02-01

Proteome characteristics of wheat leaves with the powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt) infection were investigated by two-dimensional electrophoresis and tandem MALDI-TOF/TOF-MS. We identified 46 unique proteins which were differentially expressed at 24, 48, and 72 h post-inoculation. The functional classification of these proteins showed that most of them were involved in photosynthesis, carbohydrate and nitrogen metabolism, defense responses, and signal transduction. Upregulated proteins included primary metabolism pathways and defense responses, while proteins related to photosynthesis and signal transduction were mostly downregulated. As expected, more antioxidative proteins were activated at the later infection stage than the earlier stage, suggesting that the antioxidative system of host plays a role in maintaining the compatible interaction between wheat and powdery mildew. A high accumulation of 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase in infected leaves indicated the regulation of the TCA cycle and pentose phosphate pathway in parallel to the activation of host defenses. The downregulation of MAPK5 could be facilitated for the compatible interaction of wheat plants and Bgt. qRT-PCR analysis supported the data of protein expression profiles. Our results reveal the relevance of primary plant metabolism and defense responses during compatible interaction, and provide new insights into the biology of susceptible wheat in response to Bgt infection. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Global metabolic analyses identify key differences in metabolite levels between polymyxin-susceptible and polymyxin-resistant Acinetobacter baumannii

PubMed Central

Mahamad Maifiah, Mohd Hafidz; Cheah, Soon-Ee; Johnson, Matthew D.; Han, Mei-Ling; Boyce, John D.; Thamlikitkul, Visanu; Forrest, Alan; Kaye, Keith S.; Hertzog, Paul; Purcell, Anthony W.; Song, Jiangning; Velkov, Tony; Creek, Darren J.; Li, Jian

2016-01-01

Multidrug-resistant Acinetobacter baumannii presents a global medical crisis and polymyxins are used as the last-line therapy. This study aimed to identify metabolic differences between polymyxin-susceptible and polymyxin-resistant A. baumannii using untargeted metabolomics. The metabolome of each A. baumannii strain was measured using liquid chromatography-mass spectrometry. Multivariate and univariate statistics and pathway analyses were employed to elucidate metabolic differences between the polymyxin-susceptible and -resistant A. baumannii strains. Significant differences were identified between the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii strains. The lipopolysaccharide (LPS) deficient, polymyxin-resistant 19606R showed perturbation in specific amino acid and carbohydrate metabolites, particularly pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle intermediates. Levels of nucleotides were lower in the LPS-deficient 19606R. Furthermore, 19606R exhibited a shift in its glycerophospholipid profile towards increased abundance of short-chain lipids compared to the parent polymyxin-susceptible ATCC 19606. In contrast, in a pair of clinical isolates 03–149.1 (polymyxin-susceptible) and 03–149.2 (polymyxin-resistant, due to modification of lipid A), minor metabolic differences were identified. Notably, peptidoglycan biosynthesis metabolites were significantly depleted in both of the aforementioned polymyxin-resistant strains. This is the first comparative untargeted metabolomics study to show substantial differences in the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii. PMID:26924392

Global metabolic analyses identify key differences in metabolite levels between polymyxin-susceptible and polymyxin-resistant Acinetobacter baumannii.

PubMed

Maifiah, Mohd Hafidz Mahamad; Cheah, Soon-Ee; Johnson, Matthew D; Han, Mei-Ling; Boyce, John D; Thamlikitkul, Visanu; Forrest, Alan; Kaye, Keith S; Hertzog, Paul; Purcell, Anthony W; Song, Jiangning; Velkov, Tony; Creek, Darren J; Li, Jian

2016-02-29

Multidrug-resistant Acinetobacter baumannii presents a global medical crisis and polymyxins are used as the last-line therapy. This study aimed to identify metabolic differences between polymyxin-susceptible and polymyxin-resistant A. baumannii using untargeted metabolomics. The metabolome of each A. baumannii strain was measured using liquid chromatography-mass spectrometry. Multivariate and univariate statistics and pathway analyses were employed to elucidate metabolic differences between the polymyxin-susceptible and -resistant A. baumannii strains. Significant differences were identified between the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii strains. The lipopolysaccharide (LPS) deficient, polymyxin-resistant 19606R showed perturbation in specific amino acid and carbohydrate metabolites, particularly pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle intermediates. Levels of nucleotides were lower in the LPS-deficient 19606R. Furthermore, 19606R exhibited a shift in its glycerophospholipid profile towards increased abundance of short-chain lipids compared to the parent polymyxin-susceptible ATCC 19606. In contrast, in a pair of clinical isolates 03-149.1 (polymyxin-susceptible) and 03-149.2 (polymyxin-resistant, due to modification of lipid A), minor metabolic differences were identified. Notably, peptidoglycan biosynthesis metabolites were significantly depleted in both of the aforementioned polymyxin-resistant strains. This is the first comparative untargeted metabolomics study to show substantial differences in the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii.

13C-labelled microdialysis studies of cerebral metabolism in TBI patients☆

PubMed Central

Carpenter, Keri L.H.; Jalloh, Ibrahim; Gallagher, Clare N.; Grice, Peter; Howe, Duncan J.; Mason, Andrew; Timofeev, Ivan; Helmy, Adel; Murphy, Michael P.; Menon, David K.; Kirkpatrick, Peter J.; Carpenter, T. Adrian; Sutherland, Garnette R.; Pickard, John D.; Hutchinson, Peter J.

2014-01-01

Human brain chemistry is incompletely understood and better methodologies are needed. Traumatic brain injury (TBI) causes metabolic perturbations, one result of which includes increased brain lactate levels. Attention has largely focussed on glycolysis, whereby glucose is converted to pyruvate and lactate, and is proposed to act as an energy source by feeding into neurons’ tricarboxylic acid (TCA) cycle, generating ATP. Also reportedly upregulated by TBI is the pentose phosphate pathway (PPP) that does not generate ATP but produces various molecules that are putatively neuroprotective, antioxidant and reparative, in addition to lactate among the end products. We have developed a novel combination of 13C-labelled cerebral microdialysis both to deliver 13C-labelled substrates into brains of TBI patients and recover the 13C-labelled metabolites, with high-resolution 13C NMR analysis of the microdialysates. This methodology has enabled us to achieve the first direct demonstration in humans that the brain can utilise lactate via the TCA cycle. We are currently using this methodology to make the first direct comparison of glycolysis and the PPP in human brain. In this article, we consider the application of 13C-labelled cerebral microdialysis for studying brain energy metabolism in patients. We set this methodology within the context of metabolic pathways in the brain, and 13C research modalities addressing them. PMID:24361470

Advancing biopharmaceutical process development by system-level data analysis and integration of omics data.

PubMed

Schaub, Jochen; Clemens, Christoph; Kaufmann, Hitto; Schulz, Torsten W

2012-01-01

Development of efficient bioprocesses is essential for cost-effective manufacturing of recombinant therapeutic proteins. To achieve further process improvement and process rationalization comprehensive data analysis of both process data and phenotypic cell-level data is essential. Here, we present a framework for advanced bioprocess data analysis consisting of multivariate data analysis (MVDA), metabolic flux analysis (MFA), and pathway analysis for mapping of large-scale gene expression data sets. This data analysis platform was applied in a process development project with an IgG-producing Chinese hamster ovary (CHO) cell line in which the maximal product titer could be increased from about 5 to 8 g/L.Principal component analysis (PCA), k-means clustering, and partial least-squares (PLS) models were applied to analyze the macroscopic bioprocess data. MFA and gene expression analysis revealed intracellular information on the characteristics of high-performance cell cultivations. By MVDA, for example, correlations between several essential amino acids and the product concentration were observed. Also, a grouping into rather cell specific productivity-driven and process control-driven processes could be unraveled. By MFA, phenotypic characteristics in glycolysis, glutaminolysis, pentose phosphate pathway, citrate cycle, coupling of amino acid metabolism to citrate cycle, and in the energy yield could be identified. By gene expression analysis 247 deregulated metabolic genes were identified which are involved, inter alia, in amino acid metabolism, transport, and protein synthesis.

Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

PubMed

Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

2017-02-01

Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Effects of cationic species on visual color formation in model Maillard reactions of pentose sugars and amino acids.

PubMed

Rizzi, George P

2008-08-27

Effects of cationic species on Maillard browning were examined after heating (ca. 100 degrees C) aqueous pH 7.2 buffered solutions of amino acids and pentose sugars. Metallic ions of Group I metals (Li, Na, K, Rb and Cs) produced a small increase in browning (A420), but somewhat greater effects were observed with ions of Group II metals Ca and Mg. Browning was suppressed by triethylammonium ion, but unaffected by a salt of the stronger base, guanidine. The quaternary amine salt choline chloride produced enhanced browning and served as a model for phospholipid involvement in Maillard reactions. With alpha,omega-diamino acids increases in browning were observed which related to lowered pK2 values resulting from positively charged omega-substituents in these molecules.

Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose.

PubMed

Lawford, Hugh G; Rousseau, Joyce D

2002-01-01

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN's biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

James C. Liao

This project is a collaboration with F. R. Tabita of Ohio State. Our major goal is to understand the factors and regulatory mechanisms that influence hydrogen production. The organisms to be utilized in this study, phototrophic microorganisms, in particular nonsulfur purple (NSP) bacteria, catalyze many significant processes including the assimilation of carbon dioxide into organic carbon, nitrogen fixation, sulfur oxidation, aromatic acid degradation, and hydrogen oxidation/evolution. Our part of the project was to develop a modeling technique to investigate the metabolic network in connection to hydrogen production and regulation. Organisms must balance the pathways that generate and consume reducing powermore » in order to maintain redox homeostasis to achieve growth. Maintaining this homeostasis in the nonsulfur purple photosynthetic bacteria is a complex feat with many avenues that can lead to balance, as these organisms possess versatile metabolic capabilities including anoxygenic photosynthesis, aerobic or anaerobic respiration, and fermentation. Growth is achieved by using H{sub 2} as an electron donor and CO{sub 2} as a carbon source during photoautotrophic and chemoautotrophic growth, where CO{sub 2} is fixed via the Calvin-Benson-Bassham (CBB) cycle. Photoheterotrophic growth can also occur when alternative organic carbon compounds are utilized as both the carbon source and electron donor. Regardless of the growth mode, excess reducing equivalents generated as a result of oxidative processes, must be transferred to terminal electron acceptors, thus insuring that redox homeostasis is maintained in the cell. Possible terminal acceptors include O{sub 2}, CO{sub 2}, organic carbon, or various oxyanions. Cells possess regulatory mechanisms to balance the activity of the pathways which supply energy, such as photosynthesis, and those that consume energy, such as CO{sub 2} assimilation or N{sub 2} fixation. The major route for CO{sub 2} assimilation is the CBB reductive pentose phosphate pathway, whose key enzyme is ribulose 1,5-biphosphate carboxylase/oxygenase (RubisCO). In addition to providing virtually all cellular carbon during autotrophic metabolism, RubisCO-mediated CO{sub 2} assimilation is also very important for nonsulfur purple photosynthetic bacteria under photoheterotrophic growth conditions since CO{sub 2} becomes the major electron sink under these conditions. In this work, Ensemble Modeling (EM) was developed to examine the behavior of CBB-compromised RubisCO knockout mutant strains of the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides. Mathematical models of metabolism can be a great aid in studying the effects of large perturbations to the system, such as the inactivation of RubisCO. Due to the complex and highly-interconnected nature of these networks, it is not a trivial process to understand what the effect of perturbations to the metabolic network will be, or vice versa, what enzymatic perturbations are necessary to yield a desired effect. Flux distribution is controlled by multiple enzymes in the network, often indirectly linked to the pathways of interest. Further, depending on the state of the cell and the environmental conditions, the effect of a perturbation may center around how it effects the carbon flow in the network, the balancing of cofactors, or both. Thus, it is desirable to develop mathematical models to describe, understand, and predict network behavior. Through the development of such models, one may gain the ability to generate a set of testable hypotheses for system behavior.« less

The comprehensive profile of fermentation products during in situ CO2 recycling by Rubisco-based engineered Escherichia coli.

PubMed

Yang, Cheng-Han; Liu, En-Jung; Chen, Yi-Ling; Ou-Yang, Fan-Yu; Li, Si-Yu

2016-08-02

In our previous study, the feasibility of Rubisco-based engineered E. coli (that contains heterologous phosphoribulokinase (PrkA) and Rubisco) for in situ CO2 recycling during the fermentation of pentoses or hexoses was demonstrated. Nevertheless, it is perplexing to see that only roughly 70 % of the carbon fed to the bacterial culture could be accounted for in the standard metabolic products. This low carbon recovery during fermentation occurred even though CO2 emission was effectively reduced by Rubisco-based engineered pathway. In this study, the heterologous expression of form I Rubisco was found to enhance the accumulation of pyruvate in Escherichia coli MZLF [E. coli BL21(DE3) Δzwf, Δldh, Δfrd]. This may be attributed to the enhanced glycolytic reaction supported by the increased biomass and the ethanol/acetate ratio. Besides, it was found that the transcription of arcA (encodes the redox-dependent transcriptional activators ArcA that positively regulates the transcription of pyruvate formate-lyase) was down-regulated in the presence of Rubisco. The enhanced accumulation of pyruvate also occurs when PrkA is co-expressed with Rubisco in E. coli MZLF. Furthermore, E. coli containing Rubisco-based engineered pathway has a distinct profile of the fermentation products, indicating CO2 was converted into fermentation products. By analyzing the ratio of total C-2 (2-carbon fermentation products) to total C-1 (1-carbon fermentation product) of MZLFB (MZLF containing Rubisco-based engineered pathway), it is estimated that 9 % of carbon is directed into Rubisco-based engineered pathway. Here, we report for the first time the complete profile of fermentation products using E. coli MZLF and its derived strains. It has been shown that the expression of Rubisco alone in MZLF enhances the accumulation of pyruvate. By including the contribution of pyruvate accumulation, the perplexing problem of low carbon recovery during fermentation by E. coli containing Rubisco-based engineered pathway has been solved. 9 % of glucose consumption is directed from glycolysis to Rubisco-based engineered pathway in MZLFB. The principle characteristics of mixotroph MZLFB are the high bacterial growth and the low CO2 emission.

A metabolomics strategy to explore urinary biomarkers and metabolic pathways for assessment of interaction between Danhong injection and low-dose aspirin during their synergistic treatment.

PubMed

Li, Jianping; Guo, Jianming; Shang, Erxin; Zhu, Zhenhua; Zhu, Kevin Yue; Li, Shujiao; Zhao, Buchang; Jia, Lifu; Zhao, Jing; Tang, Zhishu; Duan, Jinao

2016-07-15

The drug combination of Danhong injection (DHI) and low-dose aspirin (ASA) was frequently applied for the treatment of cardiovascular and cerebrovascular diseases. Due to the drug interactions, a lot of potential benefits and risks might exist side by side in the course of combination therapy. However, there had been no studies of interaction between DHI and ASA. Metabolomics was a powerful tool to explore endogenous biomarkers and metabolic pathways. In present study, metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS) coupled with multivariate statistical analysis was performed to provide insight into understanding the interaction between DHI and low-dose ASA. Eleven potential biomarkers of three types were identified and seven metabolic pathways were constructed. The results showed that the interaction between DHI and low-dose ASA during synergistic treatment indeed affected some key endogenous biomarkers and metabolic pathways, which could not happen when DHI or low-dose ASA was used alone. The quality and quantity of endogenous metabolite were both influenced by interaction between DHI and low-dose ASA. In details, the amount of flavin mononucleotide, L-2, 4-diaminobutyric acid (DABA) and 4-aminohippuric acid were significantly increased. On the contrary, the amount of 3-methyluridine, 4, 6-dihydroxyquinoline, cortolone-3-glucuronide, and serotonin were significantly decreased. Furthermore, O-phosphotyrosine, 3-methyl-2-butenal, indoxyl sulfate and dolichyl diphosphate were disappeared in urine. As to metabolic pathways, riboflavin metabolism, pentose and glucuronate interconversions, and tryptophan metabolism were all significantly influenced. The emerging alterations of biomarkers and metabolic pathways were associated with a lot of drugs and diseases based on literature researches, which might influence the co-administration of other drugs or the treatments of relevant diseases. Our paper presented some hints to uncover the mechanism of interaction between DHI and low-dose ASA, which would provide some references for application of DHI and low-dose ASA combination. Copyright © 2015 Elsevier B.V. All rights reserved.

Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells.

PubMed

Driscoll, Timothy P; Verhoeve, Victoria I; Guillotte, Mark L; Lehman, Stephanie S; Rennoll, Sherri A; Beier-Sexton, Magda; Rahman, M Sayeedur; Azad, Abdu F; Gillespie, Joseph J

2017-09-26

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia ( Alphaproteobacteria ; Rickettsiaceae ). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia , arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail. Copyright © 2017 Driscoll et al.

Charting the Emergence of Corporate Procurement of Utility-Scale PV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heeter, Jenny S.; Cook, Jeffrey J.; Bird, Lori A.

Through July 2017, corporate customers contracted for more than 2,300 MW of utility-scale solar. This paper examines the benefits, challenges, and outlooks for large-scale off-site solar purchasing through four pathways: PPAs, retail choice, utility partnerships (green tariffs and bilateral contracts with utilities), and by becoming a licensed wholesale seller of electricity. Each pathway differs based on where in the United States it is available, the value provided to a corporate off-taker, and the ease of implementation. The paper concludes with a discussion of future pathway comparison, noting that to deploy more corporate off-site solar, new procurement pathways are needed.

C5 glycolipids of heterocystous cyanobacteria track symbiont abundance in the diatom Hemiaulus hauckii across the tropical North Atlantic

NASA Astrophysics Data System (ADS)

Bale, Nicole J.; Villareal, Tracy A.; Hopmans, Ellen C.; Brussaard, Corina P. D.; Besseling, Marc; Dorhout, Denise; Sinninghe Damsté, Jaap S.; Schouten, Stefan

2018-03-01

Diatom-diazotroph associations (DDAs) include marine heterocystous cyanobacteria found as exosymbionts and endosymbionts in multiple diatom species. Heterocysts are the site of N2 fixation and have thickened cell walls containing unique heterocyst glycolipids which maintain a low oxygen environment within the heterocyst. The endosymbiotic cyanobacterium Richelia intracellularis found in species of the diatom genus Hemiaulus and Rhizosolenia makes heterocyst glycolipids (HGs) which are composed of C30 and C32 diols and triols with pentose (C5) moieties that are distinct from limnetic cyanobacterial HGs with predominantly hexose (C6) moieties. Here we applied a method for analysis of intact polar lipids to the study of HGs in suspended particulate matter (SPM) and surface sediment from across the tropical North Atlantic. The study focused on the Amazon plume region, where DDAs are documented to form extensive surface blooms, in order to examine the utility of C5 HGs as markers for DDAs as well as their transportation to underlying sediments. C30 and C32 triols with C5 pentose moieties were detected in both marine SPM and surface sediments. We found a significant correlation between the water column concentration of these long-chain C5 HGs and DDA symbiont counts. In particular, the concentrations of both the C5 HGs (1-(O-ribose)-3,27,29-triacontanetriol (C5 HG30 triol) and 1-(O-ribose)-3,29,31-dotriacontanetriol (C5 HG32 triol)) in SPM exhibited a significant correlation with the number of Hemiaulus hauckii symbionts. This result strengthens the idea that long-chain C5 HGs can be applied as biomarkers for marine endosymbiotic heterocystous cyanobacteria. The presence of the same C5 HGs in surface sediment provides evidence that they are effectively transported to the sediment and hence have potential as biomarkers for studies of the contribution of DDAs to the paleo-marine N cycle.

Beta-hydroxybutyrate increases reactive oxygen species in late but not in early postimplantation embryonic cells in vitro.

PubMed

Forsberg, H; Eriksson, U J; Melefors, O; Welsh, N

1998-02-01

Embryonic dysmorphogenesis has been blocked by antioxidant treatment in vivo and in vitro, suggesting that embryonic excess of reactive oxygen species (ROS) has a role in the teratogenic process of diabetic pregnancy. We report that the basal levels of ROS in dispersed rat embryonic cells in vitro, as determined by fluorescence of dichlorofluorescein (DCF), were not different in cells from control and diabetic pregnancy at day 10 or 12. Beta-hydroxybutyrate (beta-HB) and succinic acid monomethyl ester both augmented DCF fluorescence in cells from day 12 embryos of normal and diabetic rats but not from day 10 embryos. Cells of day 10 and day 12 embryos from normal and diabetic rats responded to increasing glucose concentrations with a dosage-dependent alleviation of DCF fluorescence. Day 10 embryonic cells exhibited high glucose utilization rates and high pentose phosphate shunt rates, but low mitochondrial oxidation rates. Moreover, in vitro culture of embryos between gestational days 9 and 10 in the presence of 20% oxygen induced an increased and glucose-sensitive oxidation of glucose compared with embryos not cultured in vitro. At gestation day 12, however, pentose phosphate shunt rates showed a decrease, whereas the mitochondrial beta-HB oxidation rates were increased compared with those at gestation day 10. This was paralleled by a lower expression of glucose 6-phosphate dehydrogenase- and phosphofructokinase-mRNA levels at day 12 than at day 10. On the other hand, H-ferritin mRNA expression at day 12 was high compared with day 10. None of the mRNA species investigated were affected by the diabetic state of the mother. It was concluded that beta-HB-induced stimulation of mitochondrial oxidative events may lead to the generation of ROS at gestational day 12, but probably not at day 10, when only a minute amount of mitochondrial activity occurs. Thus our results do not support the notion of diabetes-induced mitochondrial oxidative stress before the development of a placental supply of oxygen.

Pathways for Off-site Corporate PV Procurement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heeter, Jenny S

Through July 2017, corporate customers contracted for more than 2,300 MW of utility-scale solar. This paper examines the benefits, challenges, and outlooks for large-scale off-site solar purchasing through four pathways: power purchase agreements, retail choice, utility partnerships (green tariffs and bilateral contracts with utilities), and by becoming a licensed wholesale seller of electricity. Each pathway differs based on where in the United States it is available, the value provided to a corporate off-taker, and the ease of implementation. The paper concludes with a discussion of future pathway comparison, noting that to deploy more corporate off-site solar, new procurement pathways aremore » needed.« less

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

PubMed

Carta, M C; Mattana, A; Camici, M; Allegrini, S; Tozzi, M G; Sgarrella, F

2001-10-03

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Cd²⁺-induced alteration of the global proteome of human skin fibroblast cells.

PubMed

Prins, John M; Fu, Lijuan; Guo, Lei; Wang, Yinsheng

2014-03-07

Cadmium (Cd(2+)) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd(2+) on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd(2+), few have been targeted toward investigating the ability of Cd(2+) to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd(2+) exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC-MS/MS, to assess the Cd(2+)-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd(2+) exposure. Our results unveiled that Cd(2+) treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd(2+) treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd(2+) exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd(2+)-induced toxicity in human cells.

Autophagy inhibitor chloroquine increases sensitivity to cisplatin in QBC939 cholangiocarcinoma cells by mitochondrial ROS.

PubMed

Qu, Xianzhi; Sheng, Jiyao; Shen, Luyan; Su, Jing; Xu, Yunjie; Xie, Qi; Wu, Yao; Zhang, Xuewen; Sun, Liankun

2017-01-01

The tumor cells have some metabolic characteristics of the original tissues, and the metabolism of the tumor cells is closely related to autophagy. However, the mechanism of autophagy and metabolism in chemotherapeutic drug resistance is still poorly understood. In this study, we investigated the role and mechanism of autophagy and glucose metabolism in chemotherapeutic drug resistance by using cholangiocarcinoma QBC939 cells with primary cisplatin resistance and hepatocellular carcinoma HepG2 cells. We found that QBC939 cells with cisplatin resistance had a higher capacity for glucose uptake, consumption, and lactic acid generation, and higher activity of the pentose phosphate pathway compared with HepG2 cells, and the activity of PPP was further increased after cisplatin treatment in QBC939 cells. It is suggested that there are some differences in the metabolism of glucose in hepatocellular carcinoma and cholangiocarcinoma cells, and the activation of PPP pathway may be related to the drug resistance. Through the detection of autophagy substrates p62 and LC3, found that QBC939 cells have a higher flow of autophagy, autophagy inhibitor chloroquine can significantly increase the sensitivity of cisplatin in cholangiocarcinoma cells compared with hepatocellular carcinoma HepG2 cells. The mechanism may be related to the inhibition of QBC939 cells with higher activity of the PPP, the key enzyme G6PDH, which reduces the antioxidant capacity of cells and increases intracellular ROS, especially mitochondrial ROS. Therefore, we hypothesized that autophagy and the oxidative stress resistance mediated by glucose metabolism may be one of the causes of cisplatin resistance in cholangiocarcinoma cells. It is suggested that according to the metabolism characteristics of tumor cells, inhibition of autophagy lysosome pathway with chloroquine may be a new route for therapeutic agents against cholangiocarcinoma.

Identification and Metabolite Profiling of Chemical Activators of Lipid Accumulation in Green Algae.

PubMed

Wase, Nishikant; Tu, Boqiang; Allen, James W; Black, Paul N; DiRusso, Concetta C

2017-08-01

Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii , and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity, and 15 were selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using gas chromatography-mass spectrometry quantification of total fatty acids, and targeted TAG and galactolipid measurements were performed using liquid chromatography-multiple reaction monitoring/mass spectrometry. These results demonstrated that TAG accumulation does not necessarily proceed at the expense of galactolipid. Untargeted metabolite profiling provided important insights into pathway shifts due to five different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle, and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds also was demonstrated in three other algal species. These lipid-inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation. © 2017 American Society of Plant Biologists. All Rights Reserved.

Oil biosynthesis and transcriptome profiles in developing endosperm and oil characteristic analyses in Paeonia ostii var. lishizhenii.

PubMed

Xiu, Yu; Wu, Guodong; Tang, Wensi; Peng, Zhengfeng; Bu, Xiangpan; Chao, Longjun; Yin, Xue; Xiong, Jiannan; Zhang, Haiwu; Zhao, Xiaoqing; Ding, Jing; Ma, Lvyi; Wang, Huafang; van Staden, Johannes

2018-06-04

Paeonia ostii var. lishizhenii, a well-known medicinal and horticultural plant, is indigenous to China. Recent studies have shown that its seed has a high oil content, and it was approved as a novel resource of edible oil with a high level of α-linolenic acid by the Chinese Government. This study measured the seed oil contents and fatty acid components of P. ostii var. lishizhenii and six other peonies, P. suffruticosa, P. ludlowii, P. decomposita, P. rockii, and P. lactiflora Pall. 'Heze' and 'Gansu'. The results show that P. ostii var. lishizhenii exhibits the average oil characteristics of tested peonies, with an oil content of 21.3%, α-linolenic acid 43.8%, and unsaturated fatty acids around 92.1%. Hygiene indicators for the seven peony seed oils met the Chinese national food standards. P. ostii var. lishizhenii seeds were used to analyze transcriptome gene regulation networks on endosperm development and oil biosynthesis. In total, 124,117 transcripts were obtained from six endosperm developing stages (S0-S5). The significant changes in differential expression genes (DEGs) clarify three peony endosperm developmental phases: the endosperm cell mitotic phase (S0-S1), the TAG biosynthesis phase (S1-S4), and the mature phase (S5). The DEGs in plant hormone signal transduction, DNA replication, cell division, differentiation, transcription factors, and seed dormancy pathways regulate the endosperm development process. Another 199 functional DEGs participate in glycolysis, pentose phosphate pathway, citrate cycle, FA biosynthesis, TAG assembly, and other pathways. A key transcription factor (WRI1) and some important target genes (ACCase, FATA, LPCAT, FADs, and DGAT etc.) were found in the comprehensive genetic networks of oil biosynthesis. Copyright © 2018 Elsevier GmbH. All rights reserved.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes*

PubMed Central

Nam, Young-Woo; Nihira, Takanori; Arakawa, Takatoshi; Saito, Yuka; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

2015-01-01

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes. PMID:26041776

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616