Xylose utilization in recombinant Zymomonas

DOEpatents

Kahsay, Robel Y; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

2013-01-07

Zymomonas expressing xylose isomerase from A. missouriensis was found to have improved xylose utilization, growth, and ethanol production when grown in media containing xylose. Xylose isomerases related to that of A. missouriensis were identified structurally through molecular phylogenetic and Profile Hidden Markov Model analyses, providing xylose isomerases that may be used to improve xylose utilization.

Effect of manganese ions on ethanol fermentation by xylose isomerase expressing Saccharomyces cerevisiae under acetic acid stress.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Lee, Sun-Mi

2016-12-01

The efficient fermentation of lignocellulosic hydrolysates in the presence of inhibitors is highly desirable for bioethanol production. Among the inhibitors, acetic acid released during the pretreatment of lignocellulose negatively affects the fermentation performance of biofuel producing organisms. In this study, we evaluated the inhibitory effects of acetic acid on glucose and xylose fermentation by a high performance engineered strain of xylose utilizing Saccharomyces cerevisiae, SXA-R2P-E, harboring a xylose isomerase based pathway. The presence of acetic acid severely decreased the xylose fermentation performance of this strain. However, the acetic acid stress was alleviated by metal ion supplementation resulting in a 52% increased ethanol production rate under 2g/L of acetic acid stress. This study shows the inhibitory effect of acetic acid on an engineered isomerase-based xylose utilizing strain and suggests a simple but effective method to improve the co-fermentation performance under acetic acid stress for efficient bioethanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

USDA-ARS?s Scientific Manuscript database

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans.

PubMed

Guo, Jian; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

2018-04-30

Aureobasidium pullulans is a yeast-like fungus that can ferment xylose to generate high-value-added products, such as pullulan, heavy oil, and melanin. The combinatorial expression of two xylose reductase (XR) genes and two xylitol dehydrogenase (XDH) genes from Spathaspora passalidarum and the heterologous expression of the Piromyces sp. xylose isomerase (XI) gene were induced in A. pullulans to increase the consumption capability of A. pullulans on xylose. The overexpression of XYL1.2 (encoding XR) and XYL2.2 (encoding XDH) was the most beneficial for xylose utilization, resulting in a 17.76% increase in consumed xylose compared with the parent strain, whereas the introduction of the Piromyces sp. XI pathway failed to enhance xylose utilization efficiency. Mutants with superior xylose fermentation performance exhibited increased intracellular reducing equivalents. The fermentation performance of all recombinant strains was not affected when glucose or sucrose was utilized as the carbon source. The strain with overexpression of XYL1.2 and XYL2.2 exhibited excellent fermentation performance with mimicked hydrolysate, and pullulan production increased by 97.72% compared with that of the parent strain. The present work indicates that the P4 mutant (using the XR/XDH pathway) with overexpressed XYL1.2 and XYL2.2 exhibited the best xylose fermentation performance. The P4 strain showed the highest intracellular reducing equivalents and XR and XDH activity, with consequently improved pullulan productivity and reduced melanin production. This valuable development in aerobic fermentation by the P4 strain may provide guidance for the biotransformation of xylose to high-value products by A. pullulans through genetic approach.

Zymomonas with improved xylose utilization

DOEpatents

Viitanen, Paul V [West Chester, PA; Tao, Luan [Havertown, PA; Zhang, Yuying [New Hope, PA; Caimi, Perry G [Kennett Square, PA; McCutchen, Carol M [Wilmington, DE; McCole, Laura [East Fallowfield, PA; Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Lakewood, CO; Franden, Mary Ann [Centennial, CO

2011-08-16

Strains of Zymomonas were engineered by introducing a chimeric xylose isomerase gene that contains a mutant promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene. The promoter directs increased expression of xylose isomerase, and when the strain is in addition engineered for expression of xylulokinase, transaldolase and transketolase, improved utilization of xylose is obtained.

SAXS-WAXS studies of the low-resolution structure in solution of xylose/glucose isomerase from Streptomyces rubiginosus

NASA Astrophysics Data System (ADS)

Kozak, Maciej; Taube, Michał

2009-10-01

The structure and conformation of molecule of xylose/glucose isomerase from Streptomyces rubiginosus in solution (at pH 6 and 7.6; with and without the substrate) has been studied by small- and wide-angle scattering of synchrotron radiation (SAXS-WAXS). On the basis of the SAXS-WAXS data, the low-resolution structure in solution has been reconstructed using ab inito methods. A comparison of the models of glucose isomerase shows only small differences between the model in solution and the crystal structure.

A Novel Technique that Enables Efficient Conduct of Simultaneous Isomerization and Fermentation (SIF) of Xylose

NASA Astrophysics Data System (ADS)

Rao, Kripa; Chelikani, Silpa; Relue, Patricia; Varanasi, Sasidhar

Of the sugars recovered from lignocellulose, D-glucose can be readily converted into ethanol by baker's or brewer's yeast (Saccharomyces cerevisiae). However, xylose that is obtained by the hydrolysis of the hemicellulosic portion is not fermentable by the same species of yeasts. Xylose fermentation by native yeasts can be achieved via isomerization of xylose to its ketose isomer, xylulose. Isomerization with exogenous xylose isomerase (XI) occurs optimally at a pH of 7-8, whereas subsequent fermentation of xylulose to ethanol occurs at a pH of 4-5. We present a novel scheme for efficient isomerization of xylose to xylulose at conditions suitable for the fermentation by using an immobilized enzyme system capable of sustaining two different pH microenvironments in a single vessel. The proof-of-concept of the two-enzyme pellet is presented, showing conversion of xylose to xylulose even when the immobilized enzyme pellets are suspended in a bulk solution whose pH is sub-optimal for XI activity. The co-immobilized enzyme pellets may prove extremely valuable in effectively conducting "simultaneous isomerization and fermentation" (SIF) of xylose. To help further shift the equilibrium in favor of xylulose formation, sodium tetraborate (borax) was added to the isomerization solution. Binding of tetrahydroxyborate ions to xylulose effectively reduces the concentration of xylulose and leads to increased xylose isomerization. The formation of tetrahydroxyborate ions and the enhancement in xylulose production resulting from the complexation was studied at two different bulk pH values. The addition of 0.05 M borax to the isomerization solution containing our co-immobilized enzyme pellets resulted in xylose to xylulose conversion as high as 86% under pH conditions that are suboptimal for XI activity. These initial findings, which can be optimized for industrial conditions, have significant potential for increasing the yield of ethanol from xylose in an SIF approach.

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.

Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

PubMed Central

Batt, C A; Jamieson, A C; Vandeyar, M A

1990-01-01

Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme. Images PMID:2405386

Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

PubMed

Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

2015-12-01

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains. © 2015 Society for Laboratory Automation and Screening.

Xylose utilization in recombinant zymomonas

DOEpatents

Caimi, Perry G; McCole, Laura; Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V

2014-03-25

Xylose-utilizing Zymomonas strains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.

PubMed

Demeke, Mekonnen M; Dietz, Heiko; Li, Yingying; Foulquié-Moreno, María R; Mutturi, Sarma; Deprez, Sylvie; Den Abt, Tom; Bonini, Beatriz M; Liden, Gunnar; Dumortier, Françoise; Verplaetse, Alex; Boles, Eckhard; Thevelein, Johan M

2013-06-21

The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. An industrial yeast strain for bioethanol production with

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

PubMed Central

2013-01-01

Background The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions An industrial yeast strain for

Pnp gene modification for improved xylose utilization in Zymomonas

DOEpatents

Caimi, Perry G G; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

2014-12-16

The endogenous pnp gene encoding polynucleotide phosphorylase in the Zymomonas genome was identified as a target for modification to provide improved xylose utilizing cells for ethanol production. The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification, and are not limited in xylose isomerase activity in the absence of the pnp modification.

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.

Locating active-site hydrogen atoms in d-xylose isomerase: Time-of-flight neutron diffraction

PubMed Central

Katz, Amy K.; Li, Xinmin; Carrell, H. L.; Hanson, B. Leif; Langan, Paul; Coates, Leighton; Schoenborn, Benno P.; Glusker, Jenny P.; Bunick, Gerard J.

2006-01-01

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of d-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 Å) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which d-xylose is converted to d-xylulose or d-glucose to d-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an −NH2-terminal group (rather than NH3+). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 Å) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms. PMID:16707576

Isolation, purification, and characterization of thermophilic T80 isoenzyme of xylose isomerase from the xerophyte Cereus pterogonus.

PubMed

Ravikumar, Sambandam; Shyamala, Sivalingam; Muthuraman, Pandurangan; Srikumar, Kotteazeth

2011-01-01

A thermostable isoenzyme (T(80)) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K(m) and V(max)) of the T(80) xylose isomerase isoenzyme were investigated. The molecular mass of the purified T(80) isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T(80) isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84 KJ mol(-1). The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T(80) isoenzymes yielded an emission peak with λ(em) at 320 nm and 340 nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn(2+) and Co(2+) associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production.

Recombinant lactobacillus for fermentation of xylose to lactic acid and lactate

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Franden, Mary Ann; Mc Millan, James D.; Finkelstein, Mark

1998-01-01

A recombinant Lactobacillus MONT4 is provided which has been genetically engineered with xylose isomerase and xylulokinase genes from Lactobacillus pentosus to impart to the Lactobacillus MONT4 the ability to ferment lignocellulosic biomass containing xylose to lactic acid.

Recombinant lactobacillus for fermentation of xylose to lactic acid and lactate

DOEpatents

Picataggio, S.K.; Zhang, M.; Franden, M.A.; McMillan, J.D.; Finkelstein, M.

1998-08-25

A recombinant Lactobacillus MONT4 is provided which has been genetically engineered with xylose isomerase and xylulokinase genes from Lactobacillus pentosus to impart to the Lactobacillus MONT4 the ability to ferment lignocellulosic biomass containing xylose to lactic acid. 4 figs.

Mutations in iron-sulfur cluster proteins that improve xylose utilization

DOEpatents

Froehlich, Allan; Henningsen, Brooks; Covalla, Sean; Zelle, Rintze M.

2018-03-20

There is provided an engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use thereof.

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

PubMed Central

Parreiras, Lucas S.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; Higbee, Alan J.; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B.; Bice, Benjamin D.; Bonfert, Brandi L.; Pinhancos, Rebeca C.; Balloon, Allison J.; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M.; Li, Haibo; Pohlmann, Edward L.; Serate, Jose; Withers, Sydnor T.; Simmons, Blake A.; Hodge, David B.; Westphall, Michael S.; Coon, Joshua J.; Dale, Bruce E.; Balan, Venkatesh; Keating, David H.; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P.; Sato, Trey K.

2014-01-01

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH. PMID:25222864

Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui†

PubMed Central

Johnsen, Ulrike; Schönheit, Peter

2004-01-01

During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui. PMID:15342590

Spectroscopic investigation of new water soluble Mn(II)(2) and Mg(II)(2) complexes for the substrate binding models of xylose/glucose isomerases.

PubMed

Patra, Ayan; Bera, Manindranath

2014-01-30

In methanol, the reaction of stoichiometric amounts of Mn(OAc)(2)·4H(2)O and the ligand H(3)hpnbpda [H(3)hpnbpda=N,N'-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N'-diacetic acid] in the presence of NaOH, afforded a new water soluble dinuclear manganese(II) complex, [Mn2(hpnbpda)(μ-OAc)] (1). Similarly, the reaction of Mg(OAc)(2)·4H(2)O and the ligand H3hpnbpda in the presence of NaOH, in methanol, yielded a new water soluble dinuclear magnesium(II) complex, [Mg2(hpnbpda)(μ-OAc)(H2O)2] (2). DFT calculations have been performed for the structural optimization of complexes 1 and 2. The DFT optimized structure of complex 1 shows that two manganese(II) centers are in a distorted square pyramidal geometry, whereas the DFT optimized structure of complex 2 reveals that two magnesium(II) centers adopt a six-coordinate distorted octahedral geometry. To understand the mode of substrate binding and the mechanistic details of the active site metals in xylose/glucose isomerases (XGI), we have investigated the binding interactions of biologically important monosaccharides d-glucose and d-xylose with complexes 1 and 2, in aqueous alkaline solution by a combined approach of FTIR, UV-vis, fluorescence, and (13)C NMR spectroscopic techniques. Fluorescence spectra show the binding-induced gradual decrease in emission of complexes 1 and 2 accompanied by a significant blue shift upon increasing the concentration of sugar substrates. The binding modes of d-glucose and d-xylose with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in (13)C NMR spectra for C1 and C2 carbon atoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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.

Combined enzyme mediated fermentation of cellulous and xylose to ethanol by Schizosaccharoyces pombe, cellulase, .beta.-glucosidase, and xylose isomerase

DOEpatents

Lastick, Stanley M.; Mohagheghi, Ali; Tucker, Melvin P.; Grohmann, Karel

1994-01-01

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35.degree. C. to about 40.degree. C. until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol.

Combined enzyme mediated fermentation of cellulose and xylose to ethanol by Schizosaccharomyces pombe, cellulase, [beta]-glucosidase, and xylose isomerase

DOEpatents

Lastick, S.M.; Mohagheghi, A.; Tucker, M.P.; Grohmann, K.

1994-12-13

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35 C to about 40 C until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol. 2 figures.

An allosteric disulfide bond is involved in enhanced activation of factor XI by protein disulfide isomerase.

PubMed

Zucker, M; Seligsohn, U; Yeheskel, A; Mor-Cohen, R

2016-11-01

Essentials Reduction of three disulfide bonds in factor (F) XI enhances chromogenic substrate cleavage. We measured FXI activity upon reduction and identified a bond involved in the enhanced activity. Reduction of FXI augments FIX cleavage, probably by faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide bond is responsible for FXI enhanced activation upon its reduction. Background Reduction of factor (F) XI by protein disulfide isomerase (PDI) has been shown to enhance the ability of FXI to cleave its chromogenic substrate. Three disulfide bonds in FXI (Cys118-Cys147, Cys362-Cys482, and Cys321-Cys321) are involved in this augmented activation. Objectives To characterize the mechanisms by which PDI enhances FXI activity. Methods FXI activity was measured following PDI reduction. Thiols that were exposed in FXI after PDI reduction were labeled with 3-(N-maleimidopropionyl)-biocytin (MPB) and detected with avidin. The rate of conversion of FXI to activated FXI (FXIa) following thrombin activation was assessed with western blotting. FXI molecules harboring mutations that disrupt the three disulfide bonds (C147S, C321S, and C482S) were expressed in cells. The antigenicity of secreted FXI was measured with ELISA, and its activity was assessed by the use of a chromogenic substrate. The effect of disulfide bond reduction was analyzed by the use of molecular dynamics. Results Reduction of FXI by PDI enhanced cleavage of both its chromogenic substrate, S2366, and its physiologic substrate, FIX, and resulted in opening of the Cys362-Cys482 bond. The rate of conversion of FXI to FXIa was increased following its reduction by PDI. C482S-FXI showed enhanced activity as compared with both wild-type FXI and C321S-FXI. MD showed that disruption of the Cys362-Cys482 bond leads to a broader thrombin-binding site in FXI. Conclusions Reduction of FXI by PDI enhances its ability to cleave FIX, probably by causing faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide

Native xylose-inducible promoter expands the genetic tools for the biomass-degrading, extremely thermophilic bacterium Caldicellulosiruptor bescii.

PubMed

Williams-Rhaesa, Amanda M; Awuku, Nanaakua K; Lipscomb, Gina L; Poole, Farris L; Rubinstein, Gabriel M; Conway, Jonathan M; Kelly, Robert M; Adams, Michael W W

2018-07-01

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.

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.

Molecular and industrial aspects of glucose isomerase.

PubMed Central

Bhosale, S H; Rao, M B; Deshpande, V V

1996-01-01

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the

Molecular and industrial aspects of glucose isomerase.

PubMed

Bhosale, S H; Rao, M B; Deshpande, V V

1996-06-01

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the

Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production

PubMed Central

2013-01-01

Background There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose. Results We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations. Conclusion We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates

Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production

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

Wang, Xi; Goh, Ee-Been; Beller, Harry R.

Previously, we developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our mostmore » efficient methyl ket one-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. This work demonstrated a strategy for engineering simultaneous utilization of C 6 and C 5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.« less

Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production

DOE PAGES

Wang, Xi; Goh, Ee-Been; Beller, Harry R.

2018-01-27

Previously, we developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our mostmore » efficient methyl ket one-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. This work demonstrated a strategy for engineering simultaneous utilization of C 6 and C 5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.« less

Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

PubMed Central

2011-01-01

Background Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem. Results We designed a novel strategy in which Bacillus subtilis and Candida maltosa were combined and used to convert xylose in this mother liquor to xylitol, a product of higher value. First, the xylose mother liquor was detoxified with the yeast C. maltosa to remove furfural and 5-hydromethylfurfural (HMF), which are inhibitors of B. subtilis growth. The glucose present in the mother liquor was also depleted by this yeast, which was an added advantage because glucose causes carbon catabolite repression in B. subtilis. This detoxification treatment resulted in an inhibitor-free mother liquor, and the C. maltosa cells could be reused as biocatalysts at a later stage to reduce xylose to xylitol. In the second step, a recombinant B. subtilis strain with a disrupted xylose isomerase gene was constructed. The detoxified xylose mother liquor was used as the medium for recombinant B. subtilis cultivation, and this led to L-arabinose depletion and xylose enrichment of the medium. In the third step, the xylose was further reduced to xylitol by C. maltosa cells, and crystallized xylitol was obtained from this yeast transformation medium. C. maltosa transformation of the xylose-enriched medium resulted in xylitol with 4.25 g L-1·h-1 volumetric productivity and 0.85 g xylitol/g xylose specific productivity. Conclusion In this study, we developed a biological method for the purification of xylose from xylose mother liquor and subsequent preparation of xylitol by C. maltosa-mediated biohydrogenation of xylose

D-xylose absorption

MedlinePlus

Xylose tolerance test; Diarrhea - xylose; Malnutrition - xylose; Sprue - xylose; Celiac - xylose ... test if you have: Persistent diarrhea Signs of malnutrition Unexplained weight loss This test is primarily used ...

xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari.

PubMed

Guamán, Linda P; Oliveira-Filho, Edmar R; Barba-Ostria, Carlos; Gomez, José G C; Taciro, Marilda K; da Silva, Luiziana Ferreira

2018-03-01

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (Y P3HB/Xil  = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

A Novel simultaneous-Saccharification-Fermentation Strategy for Efficient Co-fermentation of C5 and C6 Sugars Using Native, Non-GMO Yeasts

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

Varanasi, Sasidhar; Relue, Patricia

Economic bioethanol production is critically dependent upon the ability to convert both the hexose (C6) and pentose (C5) sugars resulting from cellulose and hemicellulose. C5 sugars are not readily fermentable by native Saccharomyces cerevisiae. Genetically Modified Organisms (GMOs) are designed to ferment xylose, but their stability, ethanol yield, environmental impact, and survival under conditions of industrial fermentation are unproven. In this project, we developed a novel approach for efficient fermentation of both C5 and C6 sugars using native S. Cerevisiae by exploiting its ability to produce ethanol from xylulose - the keto-isomer of xylose. While the isomerization of xylose tomore » xylulose can be accomplished via commercially (and cheaply) available Xylose Isomerase (XI) (Sweetzyme™), this conversion has an extremely unfavorable equilibrium (xylose:xylose is about 5:1). To address this, we developed two alternate strategies. In the first, the two enzymes XI and urease are coimmobilized on solid support particles to enable complete isomerization of xylose to xylulose under pH conditions suitable for fermentation, in a simultaneous-isomerization-fermentation (SIF) mode. The ability of our technology to conduct isomerization of xylose under pH conditions suitable for both saccharification and fermentation opens the possibility of SSF with native yeasts for the first time. Herein, we performed specific research tasks for implementation of our technology in several modes of operation, including simultaneous-isomerization-and-fermentation (SIF), simultaneous-saccharification-and-isomerization (SSI) followed by fermentation, and SSF mode with the biomass feedstock poplar. The projected economics of our process are very favorable in comparison to the costs associated with engineering, licensing and propagating GMOs. This novel fermentation technology is readily accessible to rural farming economies for implementation in cellulosic ethanol production

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.

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

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.

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism.

PubMed

Kim, Soo Rin; Lee, Ki-Sung; Choi, Jin-Ho; Ha, Suk-Jin; Kweon, Dae-Hyuk; Seo, Jin-Ho; Jin, Yong-Su

2010-11-01

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through

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

Mixing {Xi}--{Xi}' Effects and Static Properties of Heavy {Xi}'s

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

Aliev, T. M.; Ozpineci, A.; Zamiralov, V. S.

It is shown the importance of mixing of heavy baryons {Xi}--{Xi}' with the new quantum numbers for analysis of its characteristics. The quark model of Ono is used as an example. Masses of new baryons as well as mixing angles of the states {Xi}--{Xi}' are obtained. The same reasoning is shown to be valid for the interpolating currents of these baryons in the framework of the QCD sum rules.

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

PubMed

Lee, Sung-Haeng; Kodaki, Tsutomu; Park, Yong-Cheol; Seo, Jin-Ho

2012-04-30

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only. Copyright © 2011 Elsevier B.V. All rights reserved.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2012-01-01

Economically viable production of solvents through acetone butanol ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of fivemore » proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.« less

An innovative biocatalyst for production of ethanol from xylose in a continuous bioreactor.

PubMed

Silva, C R; Zangirolami, T C; Rodrigues, J P; Matugi, K; Giordano, R C; Giordano, R L C

2012-01-05

The use of the hemicellulose fraction of biomass may be important for the feasibility of the production of second generation bioethanol. Wild strains of Saccharomyces cerevisiae are widely used in industry for production of 1st generation ethanol, and the robustness of this yeast is an important advantage in large scale applications. Isomerization of xylose to xylulose is an essential step in this process. This reaction is catalyzed by glucose isomerase (GI). A new biocatalyst is presented here for the simultaneous isomerization and fermentation (SIF) of xylose. GI from Streptomyces rubiginosus was immobilized in chitosan, through crosslinking with glutaraldehyde, and the support containing the immobilized GI (IGI-Ch) was co-immobilized with S. cerevisiae, in calcium alginate gel. The immobilization experiments led to high immobilized protein loads (30-68 mg × g(support)(-1)), high yields (circa of 100%) and high recovered enzyme activity (>90%). The IGI-Ch derivative with maximum activity presented 1700 IU × g(catalyst)(-1), almost twice the activity of a commercial immobilized GI, GENSWEET(®) IGI-HF. At typical operational conditions for xylose SIF operation (pH 5, 30-35 °C, presence of nutrients and ethanol concentrations in the medium up to 70 L(-1)), both derivatives, IGI-Ch and GENSWEET(®) IGI-HF retained app. 90% of the initial activity after 120 h, while soluble GI was almost completely inactive at pH 5, 30 °C. The isomerization xylose/xylulose, catalyzed by IGI-Ch, reached the equilibrium in batch experiments after 4h, with 12,000 IU × L(-1) (7 g(der) × L(-1)), at pH 5 and 30 °C, in the presence of fermentation nutrients. After co-immobilization of IGI-Ch with yeast in alginate gel, this biocatalyst succeeded in producing 12 g × L(-1) of ethanol, 9.5 g × L(-1) of xylitol, 2.5 g × L(-1) of glycerol and 1.9 g × L(-1) of acetate after consumption of 50 g × L(-1) of xylose, in 48 h, using 32.5 × 10(3) IU × L(-1) and 20 g(yeast) × L(-1), at 35

Xylose fermentation to ethanol

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

McMillan, J.D.

1993-01-01

The past several years have seen tremendous progress in the understanding of xylose metabolism and in the identification, characterization, and development of strains with improved xylose fermentation characteristics. A survey of the numerous microorganisms capable of directly fermenting xylose to ethanol indicates that wild-type yeast and recombinant bacteria offer the best overall performance in terms of high yield, final ethanol concentration, and volumetric productivity. The best performing bacteria, yeast, and fungi can achieve yields greater than 0.4 g/g and final ethanol concentrations approaching 5%. Productivities remain low for most yeast and particularly for fungi, but volumetric productivities exceeding 1.0 g/L-hmore » have been reported for xylose-fermenting bacteria. In terms of wild-type microorganisms, strains of the yeast Pichia stipitis show the most promise in the short term for direct high-yield fermentation of xylose without byproduct formation. Of the recombinant xylose-fermenting microorganisms developed, recombinant E. coli ATTC 11303 (pLOI297) exhibits the most favorable performance characteristics reported to date.« less

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

PubMed Central

2012-01-01

Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes. PMID:22672737

21 CFR 862.1570 - Phosphohexose isomerase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Phosphohexose isomerase test system. 862.1570... Systems § 862.1570 Phosphohexose isomerase test system. (a) Identification. A phosphohexose isomerase test system is a device intended to measure the activity of the enzyme phosphohexose isomerase in serum...

21 CFR 862.1570 - Phosphohexose isomerase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Phosphohexose isomerase test system. 862.1570... Systems § 862.1570 Phosphohexose isomerase test system. (a) Identification. A phosphohexose isomerase test system is a device intended to measure the activity of the enzyme phosphohexose isomerase in serum...

21 CFR 862.1570 - Phosphohexose isomerase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Phosphohexose isomerase test system. 862.1570... Systems § 862.1570 Phosphohexose isomerase test system. (a) Identification. A phosphohexose isomerase test system is a device intended to measure the activity of the enzyme phosphohexose isomerase in serum...

21 CFR 862.1570 - Phosphohexose isomerase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Phosphohexose isomerase test system. 862.1570... Systems § 862.1570 Phosphohexose isomerase test system. (a) Identification. A phosphohexose isomerase test system is a device intended to measure the activity of the enzyme phosphohexose isomerase in serum...

Mass formulas for {Xi}{sub c} and {Xi}{sub b} baryons

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

Aliev, T. M.; Zamiralov, V. S.; Ozpineci, A.

The importance of taking into account the mixing of the heavy cascade baryons {Xi} and {Xi}' that have new quantum numbers in analyzing their properties is shown. The Ono quark model is considered by way of example. The masses of the new baryons and the {Xi}-{Xi}' mixing angles are obtained. The same approach is applied to the interpolating currents of these baryons within QCD sum rules.

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

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

Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min -1•mg -1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

DOE PAGES

Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; ...

2016-01-19

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min -1•mg -1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

Xylose fermentation to ethanol. A review

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

McMillan, J D

1993-01-01

The past several years have seen tremendous progress in the understanding of xylose metabolism and in the identification, characterization, and development of strains with improved xylose fermentation characteristics. A survey of the numerous microorganisms capable of directly fermenting xylose to ethanol indicates that wild-type yeast and recombinant bacteria offer the best overall performance in terms of high yield, final ethanol concentration, and volumetric productivity. The best performing bacteria, yeast, and fungi can achieve yields greater than 0.4 g/g and final ethanol concentrations approaching 5%. Productivities remain low for most yeast and particularly for fungi, but volumetric productivities exceeding 1.0 g/L-hmore » have been reported for xylose-fermenting bacteria. In terms of wild-type microorganisms, strains of the yeast Pichia stipitis show the most promise in the short term for direct high-yield fermentation of xylose without byproduct formation. Of the recombinant xylose-fermenting microorganisms developed, recombinant E. coli ATTC 11303 (pLOI297) exhibits the most favorable performance characteristics reported to date.« less

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

PubMed

Ribeiro, Lucas Ferreira; Tullman, Jennifer; Nicholes, Nathan; Silva, Sérgio Ruschi Bergamachi; Vieira, Davi Serradella; Ostermeier, Marc; Ward, Richard John

2016-01-01

Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a β-loop in the XynA domain results in an increased access to the

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... device is exempt from the premarket notification procedures in subpart E of part 807 subject to the...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... device is exempt from the premarket notification procedures in subpart E of part 807 subject to the...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... device is exempt from the premarket notification procedures in subpart E of part 807 subject to the...

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

PubMed Central

Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

2008-01-01

Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains

Engineering a synthetic anaerobic respiration for reduction of xylose to xylitol using NADH output of glucose catabolism by Escherichia coli AI21.

PubMed

Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde

2016-04-16

Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae.

PubMed

Hou, Jin; Vemuri, Goutham N; Bao, Xiaoming; Olsson, Lisbeth

2009-04-01

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO(2) to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.

Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

PubMed

Kim, Hee Su; Oh, Young Hoon; Jang, Young-Ah; Kang, Kyoung Hee; David, Yokimiko; Yu, Ju Hyun; Song, Bong Keun; Choi, Jong-il; Chang, Yong Keun; Joo, Jeong Chan; Park, Si Jae

2016-06-03

Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is

Production of Xylitol from D-Xylose by Overexpression of Xylose Reductase in Osmotolerant Yeast Candida glycerinogenes WL2002-5.

PubMed

Zhang, Cheng; Zong, Hong; Zhuge, Bin; Lu, Xinyao; Fang, Huiying; Zhuge, Jian

2015-07-01

Efficient bioconversion of D-xylose into various biochemicals is critical for the developing lignocelluloses application. In this study, we compared D-xylose utilization in Candida glycerinogenes WL2002-5 transformants expressing xylose reductase (XYL1) in D-xylose metabolism. C. glycerinogenes WL2002-5 expressing XYL1 from Schefferomyces stipitis can produce xylitol. Xylitol production by the recombinant strains was evaluated using a xylitol fermentation medium with glucose as a co-substrate. As glucose was found to be an insufficient co-substrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best co-substrate. The effects of glycerol on the xylitol production rate by a xylose reductase gene (XYL1)-overexpressed mutant of C. glycerinogenes WL2002-5 were investigated. The XYL1-overexpressed mutant produced xylitol from D-xylose using glycerol as a co-substrate for cell growth and NAD (P) H regeneration: 100 g/L D-xylose was completely converted into xylitol when at least 20 g/L glycerol was used as a co-substrate. XYL1 overexpressed mutant grown on glycerol as co-substrate accumulated 2.1-fold increased xylitol concentration over those cells grown on glucose as co-substrate. XYL1 overexpressed mutant produced xylitol with a volumetric productivity of 0.83 g/L/h, and a xylitol yield of 98 % xylose. Recombinant yeast strains obtained in this study are promising candidates for xylitol production. This is the first report of XYL1 gene overexpression of C. glycerinogenes WL2002-5 for enhancing the efficiency of xylitol production.

Microaerobic conversion of xylose to ethanol in recombinant Saccharomyces cerevisiae SX6(MUT) expressing cofactor-balanced xylose metabolic enzymes and deficient in ALD6.

PubMed

Jo, Sung-Eun; Seong, Yeong-Je; Lee, Hyun-Soo; Lee, Soo Min; Kim, Soo-Jung; Park, Kyungmoon; Park, Yong-Cheol

2016-06-10

Xylose is a major monosugar in cellulosic biomass and should be utilized for cost-effective ethanol production. In this study, xylose-converting ability of recombinant Saccharomyces cerevisiae SX6(MUT) expressing NADH-preferring xylose reductase mutant (R276H) and other xylose-metabolic enzymes, and deficient in aldehyde dehydrogenase 6 (Ald6p) were characterized at microaerobic conditions using various sugar mixtures. The reduction of air supply from 0.5vvm to 0.1vvm increased specific ethanol production rate by 75% and did not affect specific xylose consumption rate. In batch fermentations using various concentrations of xylose (50-104g/L), higher xylose concentration enhanced xylose consumption rate and ethanol productivity but reduced ethanol yield, owing to the accumulation of xylitol and glycerol from xylose. SX6(MUT) consumed monosugars in pitch pine hydrolysates and produced 23.1g/L ethanol from 58.7g/L sugars with 0.39g/g ethanol yield, which was 14% higher than the host strain of S. cerevisiae D452-2 without the xylose assimilating enzymes. In conclusion, S. cerevisiae SX6(MUT) was characterized to possess high xylose-consuming ability in microaerobic conditions and a potential for ethanol production from cellulosic biomass. Copyright © 2016 Elsevier B.V. All rights reserved.

Zymomonas with improved xylose utilization in stress conditions

DOEpatents

Caimi, Perry G; Emptage, Mark; Li, Xu; Viitanen, Paul V; Chou, Yat-Chen; Franden, Mary Ann; Zhang, Min

2013-06-18

Strains of xylose utilizing Zymomonas with improved xylose utilization and ethanol production during fermentation in stress conditions were obtained using an adaptation method. The adaptation involved continuously growing xylose utilizing Zymomonas in media containing high sugars, acetic acid, ammonia, and ethanol.

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing xylose catabolism genes for glucose and xylose co-utilization.

PubMed

Fu, Hongxin; Yang, Shang-Tian; Wang, Minqi; Wang, Jufang; Tang, I-Ching

2017-06-01

Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering yeasts for xylose metabolism

Treesearch

Thomas W. Jeffries

2006-01-01

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux...

Observation of the $$\\Xi_b^0$$ Baryon

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

Aaltonen, T.; /Helsinki Inst. of Phys.; Alvarez Gonzalez, B.

The observation of the bottom, strange baryon {Xi}{sup 0}{sub b} through the decay chain {Xi}{sup 0}{sub b} {yields} {Xi}{sup +}{sub c} {pi}{sup -}, where {Xi}{sup +}{sub c} {yields} {Xi}{sup -} {pi}{sup +} {pi}{sup +}, {Xi}{sup -} {yields} {Lambda} {yields} p {pi}{sup -}, is reported using data corresponding to an integrated luminosity of 4.2 ft{sup -1} from p{anti p} collisions at {radical}{ovr s} = 1.96 TeV recorded with the Collider Detector at Fermilab. A signal of 25.3{sup +5.6}{sub -5.4} candidates is observed whose probability of arising from a background fluctuation is 3.6 x 10{sup -12}, corresponding to 6.8 Gaussian standard deviations.more » The {Xi}{sup 0}{sub b} mass is measured to be 5787.8 {+-} 5.0(stat) {+-} 1.3(syst) MeV/c{sup 2}. In addition, the {Xi}{sup -}{sub b} is observed through the process {Xi}{sup -}{sub b} {yields} {Xi}{sup 0}{sub c} {pi}{sup -}, where {Xi}{sup 0}{sub c} {yields} {Xi}{sup -} {pi}{sup +}, {Xi}{sup -} {yields} {Lambda} {pi}{sup -}, and {Lambda} {yields} p {pi}{sup -}.« less

Structure of triosephosphate isomerase from Cryptosporidium parvum.

PubMed

Nguyen, Trang N; Abendroth, Jan; Leibly, David J; Le, Kristen P; Guo, Wenjin; Kelley, Angela; Stewart, Lance; Myler, Peter J; Van Voorhis, Wesley C

2011-09-01

Cryptosporidium parvum is one of several Cryptosporidium spp. that cause the parasitic infection cryptosporidiosis. Cryptosporidiosis is a diarrheal infection that is spread via the fecal-oral route and is commonly caused by contaminated drinking water. Triosephosphate isomerase is an enzyme that is ubiquitous to all organisms that perform glycolysis. Triosephosphate isomerase catalyzes the formation of glyceraldehyde 3-phosphate from dihydroxyacetone phosphate, which is a critical step to ensure the maximum ATP production per glucose molecule. In this paper, the 1.55 Å resolution crystal structure of the open-loop form of triosephosphate isomerase from C. parvum Iowa II is presented. An unidentified electron density was found in the active site.

Recombinant Zymomonas mobilis with improved xylose utilization

DOEpatents

Zhang, Min

2003-05-20

A strain derived from Zymomonas mobilis ATCC31821 or its derivative capable of producing ethanol upon fermentation of a carbohydrate medium containing xylose to provide enhanced xylose utilization and enhanced ethanol process yield, the strain or its derivative comprising exogenous genes encoding xylose isornerase, xylulokinase, transaldolase and transketolase, the genes are fused to at least one promotor recognized by Zymomonas which regulates the expression of at least one of the genes.

Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation

PubMed Central

Ha, Suk-Jin; Galazka, Jonathan M.; Rin Kim, Soo; Choi, Jin-Ho; Yang, Xiaomin; Seo, Jin-Ho; Louise Glass, N.; Cate, Jamie H. D.; Jin, Yong-Su

2011-01-01

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production. PMID:21187422

Xi0 and anti-Xi0 Polarization Measurements at 800-GeV/c

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

Abouzaid, E.; Alavi-Harati, A.; Alexopoulos, T.

The polarization of {Xi}{sup 0} and {bar {Xi}}{sup 0} hyperons produced by 800 GeV/c protons on a BeO target at a fixed targeting angle of 4.8 mrad is measured by the KTeV experiment at Fermilab. The result of 9.7% for {Xi}{sup 0} polarization shows no significant energy dependence when compared to a result obtained at 400 GeV/c production energy and at twice the targeting angle. The polarization of the {Xi}{sup 0} is measured for the first time and found to be consistent with zero. They also examine the dependence of polarization on production p{sub t}.

Alcoholic Fermentation of d-Xylose by Yeasts

PubMed Central

Toivola, Ansa; Yarrow, David; van den Bosch, Eduard; van Dijken, Johannes P.; Scheffers, W. Alexander

1984-01-01

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used. PMID:16346558

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

PubMed

Kwak, Suryang; Kim, Soo Rin; Xu, Haiqing; Zhang, Guo-Chang; Lane, Stephan; Kim, Heejin; Jin, Yong-Su

2017-11-01

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems

PubMed Central

Talts, Kristiina; Ilau, Birger; Ojangu, Eve-Ly; Tanner, Krista; Peremyslov, Valera V.; Dolja, Valerian V.; Truve, Erkki; Paves, Heiti

2016-01-01

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis. PMID:28066484

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

Escherichia coli arabinose isomerase and Staphylococcus aureus tagatose-6-phosphate isomerase: which is a better template for directed evolution of non-natural substrate isomerization?

PubMed

Kim, Hye Jung; Uhm, Tae Guk; Kim, Seong Bo; Kim, Pil

2010-06-01

Metallic and non-metallic isomerases can be used to produce commercially important monosaccharides. To determine which category of isomerase is more suitable as a template for directed evolution to improve enzymes for galactose isomerization, L-arabinose isomerase from Escherichia coli (ECAI; E.C. 5.3.1.4) and tagatose-6-phosphate isomerase from Staphylococcus aureus (SATI; E.C. 5.3.1.26) were chosen as models of a metallic and non-metallic isomerase, respectively. Random mutations were introduced into the genes encoding ECAI and SATI at the same rate, resulting in the generation of 515 mutants of each isomerase. The isomerization activity of each of the mutants toward a non-natural substrate (galactose) was then measured. With an average mutation rate of 0.2 mutations/kb, 47.5% of the mutated ECAIs showed an increase in activity compared with wild-type ECAI, and the remaining 52.5% showed a decrease in activity. Among the mutated SATIs, 58.6% showed an increase in activity, whereas 41.4% showed a decrease in activity. Mutant clones showing a significant change in relative activity were sequenced and specific increases in activity were measured. The maximum increase in activity achieved by mutation of ECAI was 130%, and that for SATI was 190%. Based on these results, the characteristics of the different isomerases are discussed in terms of their usefulness for directed evolution of non-natural substrate isomerization.

Enzymatic and Microbial Preparation of d-Xylulose from d-Xylose

PubMed Central

Chiang, Lin-Chang; Hsiao, Humg-Yu; Ueng, Pear P.; Tsao, George T.

1981-01-01

A high-d-xylulose mixture (d-xylose-d-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized d-xylose solution (d-xylose-d-xylulose = 77:23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize d-xylose in the mixture of d-xylose and d-xylulose. Chromatographically pure d-xylulose was thus obtained in 90% yield. A high-d-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiella pneumoniae, Candida utilis, or Mucor rouxii.d-Xylose and d-xylulose were simultaneously consumed. When borate was added to the mixture, a d-xylulose-borate complex was formed, and it could be used to protect d-xylulose from being utilized. PMID:16345816

Co-fermentation of glucose, xylose and/or cellobiose by yeast

DOEpatents

Jeffries, Thomas W.; Willis, Laura B.; Long, Tanya M.; Su, Yi-Kai

2013-09-10

Provided herein are methods of using yeast cells to produce ethanol by contacting a mixture comprising xylose with a Spathaspora yeast cell under conditions suitable to allow the yeast to ferment at least a portion of the xylose to ethanol. The methods allow for efficient ethanol production from hydrolysates derived from lignocellulosic material and sugar mixtures including at least xylose and glucose or xylose, glucose and cellobiose.

Exclusive photoproduction of the cascade (Xi) hyperon

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

John Price; Bernard Nefkens; Justin Ducote

2004-09-01

We report on the first measurement of exclusive {Xi}{sup -}(1321) hyperon photoproduction in {gamma}p {yields} K{sup +}K{sup +}{Xi}{sup -} for 3.2 < E{sub {gamma}} < 3.9 GeV. The final state is identified by the missing mass in p({gamma}, K{sup +}K{sup +})X measured with the CLAS detector at Jefferson Laboratory. We have detected a significant number of the ground-state {Xi}{sup -}(132)1/2{sup +}, and have estimated the total cross section for its production. We have also observed the first excited state {Xi}{sup -}(1530)3/2{sup +}. Photoproduction provides a copious source of {Xi}'s. We discuss the possibilities of a search for the recently proposedmore » {Xi}{sub 5}{sup --} and {Xi}{sub 5}{sup +} pentaquarks.« less

Xylose induces cellulase production in Thermoascus aurantiacus.

PubMed

Schuerg, Timo; Prahl, Jan-Philip; Gabriel, Raphael; Harth, Simon; Tachea, Firehiwot; Chen, Chyi-Shin; Miller, Matthew; Masson, Fabrice; He, Qian; Brown, Sarah; Mirshiaghi, Mona; Liang, Ling; Tom, Lauren M; Tanjore, Deepti; Sun, Ning; Pray, Todd R; Singer, Steven W

2017-01-01

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus . Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

Light {xi} hypernuclei in four-body cluster models

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

Hiyama, E.; Yamamoto, Y.; Motoba, T.

Detailed structure calculations in {sub {xi}{sup -}}{sup 12}Be, {sub {xi}{sup -}}{sup 5}H, {sub {xi}{sup -}}{sup 9}Li, {sub {xi}{sup -}}{sup 7}H, and {sub {xi}{sup -}}{sup 10}Li are performed within the framework of the microscopic two-, three-, and four-body cluster models using the Gaussian expansion method. We adopted effective {xi}N interactions derived from the Nijmegen interaction models, which give rise to substantially attractive {xi}-nucleus potentials in accordance with the experimental indications. {sub {xi}{sup -}}{sup 7}H and {sub {xi}{sup -}}{sup 10}Li are predicted to have bound states. we propose to observe the bound states in future (K{sup -},K{sup +}) experiments using {sup 7}Limore » and {sup 10}B targets in addition to the standard {sup 12}C target. The experimental confirmation of these states will provide information on the spin- and isospin-averaged {xi}N interaction.« less

Perfectly Cold Crystals: What Happens When They Are X-rayed?

NASA Technical Reports Server (NTRS)

vanderWoerd, Mark; Ferree, Darren S.; Snell, Edward H.

2003-01-01

For many macromolecular crystals the cryo-preservation of these crystals during X-ray data collection is of crucial importance, particularly at synchrotron facilities where the crystals rapidly receive a high dose of radiation. A practical variable to ensure adequate preservation is the variation of the cryo-protectant present when the crystal is preserved. Our initial approach to study X-ray diffraction data quality as a function of cryo-protectant present when preserving a xylose isomerase crystal shows that the data quality can be tremendously improved by recipe adjustment. Guided by crystal mosaicity estimates, we optimized crystal growth conditions to obtain cryo-preserved xylose isomerase crystals that withstand a very high dose of X-rays, with only the smallest amount of radiation damage at ultra-high resolution (1.2Angstroms). The rate at which damage occurs allowed us to collect a series of complete data sets, which show how the data degradation proceeds over time. We are here presenting data for the xylose isomerase crystallization recipe improvement and our interpretation of the crystal degradation process during X-ray data collection.

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

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.

Pilot-scale steam explosion for xylose production from oil palm empty fruit bunches and the use of xylose for ethanol production.

PubMed

Duangwang, Sairudee; Ruengpeerakul, Taweesak; Cheirsilp, Benjamas; Yamsaengsung, Ram; Sangwichien, Chayanoot

2016-03-01

Pilot-scale steam explosion equipments were designed and constructed, to experimentally solubilize xylose from oil palm empty fruit bunches (OPEFB) and also to enhance an enzyme accessibility of the residual cellulose pulp. The OPEFB was chemically pretreated prior to steam explosion at saturated steam (SS) and superheated steam (SHS) conditions. The acid pretreated OPEFB gave the highest xylose recovery of 87.58 ± 0.21 g/kg dried OPEFB in the liquid fraction after explosion at SHS condition. These conditions also gave the residual cellulose pulp with high enzymatic accessibility of 73.54 ± 0.41%, which is approximately threefold that of untreated OPEFB. This study has shown that the acid pretreatment prior to SHS explosion is an effective method to enhance both xylose extraction and enzyme accessibility of the exploded OPEFB. Moreover, the xylose solution obtained in this manner could directly be fermented by Candida shehatae TISTR 5843 giving high ethanol yield of 0.30 ± 0.08 g/g xylose. Copyright © 2015 Elsevier Ltd. All rights reserved.

Searches for the pentaquark states {xi}(1860){sup 0} and {xi}(1860){sup --} in neutron-carbon interactions via the EXCHARM experiment

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

Aleev, A. N.; Amaglobeli, N. S.; Balandin, V. P.

Results of searches for the pentaquark states {xi}(1860){sup 0} and {xi}(1860){sup --} in neutron-carbon interactions at an average neutron energy of about 51 GeV via the EXCHARM experiment are presented. No such states are found in the {xi}{sup -}{pi}{sup +} and {xi}{sup -}{pi}{sup -} effective-mass spectra examined in this study. Upper limits on the cross sections for {xi}(1860){sup 0} and {xi}(1860){sup --} production are estimated.

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2003-01-01

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes(XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into...

Engineering genome-reduced Bacillus subtilis for acetoin production from xylose.

PubMed

Yan, Panpan; Wu, Yuanqing; Yang, Li; Wang, Zhiwen; Chen, Tao

2018-02-01

To investigate the capacity of a genome-reduced Bacillus subtilis strain as chassis cell for acetoin production from xylose. To endow the genome-reduced Bacillus subtilis strain BSK814 with the ability to utilize xylose, we inserted a native xyl operon into its genome and deleted the araR gene. The resulting strain BSK814A2 produced 2.94 g acetoin/l from 10 g xylose/l, which was 39% higher than control strain BSK19A2. The deletion of the bdhA and acoA genes further improved xylose utilization efficiency and increased acetoin production to 3.71 g/l in BSK814A4. Finally, BSK814A4 produced up to 23.3 g acetoin/l from 50 g xylose/l, with a yield of 0.46 g/g xylose. Both the titer and yield were 39% higher than those of control strain BSK19A4. As a chassis cell, genome-reduced B. subtilis showed significantly improved capacity for the production of the overflow product acetoin from xylose compared with wild-type strain.

Observation of the {Xi}{sub b}{sup 0} Baryon

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

Aaltonen, T.; Brucken, E.; Devoto, F.

The observation of the bottom, strange baryon {Xi}{sub b}{sup 0} through the decay chain {Xi}{sub b}{sup 0}{yields}{Xi}{sub c}{sup +}{pi}{sup -}, where {Xi}{sub c}{sup +}{yields}{Xi}{sup -}{pi}{sup +}{pi}{sup +}, {Xi}{sup -}{yields}{Lambda}{pi}{sup -}, and {Lambda}{yields}p{pi}{sup -}, is reported by using data corresponding to an integrated luminosity of 4.2 fb{sup -1} from pp collisions at {radical}(s)=1.96 TeV recorded with the Collider Detector at Fermilab. A signal of 25.3{sub -5.4}{sup +5.6} candidates is observed whose probability of arising from a background fluctuation is 3.6x10{sup -12}, corresponding to 6.8 Gaussian standard deviations. The {Xi}{sub b}{sup 0} mass is measured to be 5787.8{+-}5.0(stat){+-}1.3(syst) MeV/c{sup 2}. In addition,more » the {Xi}{sub b}{sup -} baryon is observed through the process {Xi}{sub b}{sup -}{yields}{Xi}{sub c}{sup 0}{pi}{sup -}, where {Xi}{sub c}{sup 0}{yields}{Xi}{sup -}{pi}{sup +}, {Xi}{sup -}{yields}{Lambda}{pi}{sup -}, and {Lambda}{yields}p{pi}{sup -}.« less

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion.

PubMed

Hector, Ronald E; Dien, Bruce S; Cotta, Michael A; Qureshi, Nasib

2011-09-01

Saccharomyces' physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04-0.17 h(-1). Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17-0.31 g/l/h, with ethanol yields 0.18-0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13-17% more ethanol than the parental control strains because of their ability to ferment xylose.

RNAi assisted genome evolution unveils yeast mutants with improved xylose utilization.

PubMed

HamediRad, Mohammad; Lian, Jiazhang; Li, Hejun; Zhao, Huimin

2018-06-01

Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8. Followed by screening in media containing xylose as the sole carbon source, yeast mutants with 29% faster xylose utilization, and 45% higher ethanol productivity were obtained relative to the parent strain. Two known and two new effector genes were identified in these mutant strains. Notably, down-regulation of CDC11, an essential gene, resulted in faster xylose utilization, and this gene target cannot be identified in genetic knock-out screens. © 2018 Wiley Periodicals, Inc.

Molecular cloning and biochemical characterization of rabbit factor XI.

PubMed Central

Sinha, Dipali; Marcinkiewicz, Mariola; Gailani, David; Walsh, Peter N

2002-01-01

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160 kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80 kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process. PMID:12084014

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2004-01-01

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast raditionally...

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.

Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production

DOEpatents

Viitanen, Paul V.; Chou, Yat-Chen; McCutchen, Carol M.; Zhang, Min

2010-06-22

A strain of xylose-utilizing Zymomonas was engineered with a genetic modification to the glucose-fructose oxidoreductase gene resulting in reduced expression of GFOR enzyme activity. The engineered strain exhibits reduced production of xylitol, a detrimental by-product of xylose metabolism. It also consumes more xylose and produces more ethanol during mixed sugar fermentation under process-relevant conditions.

Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response

Treesearch

Yong-Su Jin; Jose M. Laplaza; Thomas W. Jeffries

2004-01-01

Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for D-xylose utilization through the heterologous expression of genes for aldose reductase ( XYL1), xylitol dehydrogenase (XYL2), and D-xylulokinase ( XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1, XYL2,...

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

PubMed

Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

2017-02-01

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation.

PubMed

Nambu-Nishida, Yumiko; Sakihama, Yuri; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

2018-01-01

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, P TDH3 , P FBA1 , and P TDH1 were favorable for high expression, and P SED1 , P HXT7 , P PDC1 , P TEF1 , P TPI1 , and P PGK1 were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. P TEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. P ZWF1 and P SOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. P ALD3 and P TKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation. Copyright © 2017 The Society for Biotechnology, Japan. Published by 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...

Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis

Treesearch

Thomas W. Jeffries; Igor V. Grigroriev; Jane Grimwood; Jose M. Laplaza; Andrea Aerts; Asaf Salamov; Jeremy Schmutz; Erika Lindquist; Paramvir Dehal; Harris Shapiro; Yong-Su Jin; Volkmar Passoth; Paul M. Richardson

2007-01-01

Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose...

Active-site-directed irreversible inhibitors of isopentenyl diphosphate isomerase

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

Muhlbacher, M.

1987-01-01

Seven analogues of isopentenyl diphosphate, containing fluorine, epoxy, or ammonium functionalities were found to irreversibly inhibit isopentenyl diphosphate:dimethylallyl diphosphate isomerase isolated from the mold Claviceps purpurea. The mechanism of their inhibition of isomerase was studied. Syntheses of 3-(fluoromethyl)-3-buten-1-yl diphosphate, 2-dimethylamino-1-ethyl diphosphate, 3,4-epoxy-3-methyl-1-butyl diphosphate, 3,4,-epoxy-1-butyl diphosphate, and 2,3-epoxy-3-methyl-1-butyl diphosphate were developed and carried out in high overall yield affording 100 mg quantities of the triammonium diphosphate salts. Radiolabeled materials of these analogues with {sup 3}H, {sup 14}C, and {sup 32}P at appropriate positions were also prepared. Inactivation kinetics, substrate protection studies, and labeling experiments demonstrated that the analogues interact stoichiometrically withmore » the active-site of isomerase. Radioactive enzyme-inactivator complexes were isolated, that are stable to extended dialysis and chaotropic reagents. The complexes resulting from inactivation of the enzyme by 3-(fluoromethyl)-3-buten-1-yl diphosphate and 3,4-epoxy-3-methyl-1-butyl diphosphate are stable to ion exchange chromatography and gel electrophoresis. Stoichiometric fluoride ion release occurs during inactivation of isomerase with 3-(fluoromethyl)-3-buten-1-yl diphosphate. The complexes are not stable to high concentrations of mixtures of 2-mercaptoethanol-sodium dodecyl sulfate. The radiolabeled 2-dimethylamino-1-ethyl diphosphate isomerase complex loses radioactivity almost instantaneously when treated with base. Partial fragmentation of the inactivator molecule was observed.« less

Conversion of xylose to ethanol under aerobic conditions by Candida tropicalis

Treesearch

T. W. Jeffries

1981-01-01

Candida tropicalis converts xylose to ethanol under aerobic, but not anaerobic, conditions. Ethanol production lags behind growth and is accelerated by increased aeration. Adding xylose to active cultures stimulates ethanol production as does serial subculture in a medium containing xylose as a sole carbon source.

Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi.

PubMed

Ostoa-Saloma, P; Garza-Ramos, G; Ramírez, J; Becker, I; Berzunza, M; Landa, A; Gómez-Puyou, A; Tuena de Gómez-Puyou, M; Pérez-Montfort, R

1997-03-15

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to

Mammalian peptide isomerase: platypus-type activity is present in mouse heart.

PubMed

Koh, Jennifer M S; Chow, Stephanie J P; Crossett, Ben; Kuchel, Philip W

2010-06-01

Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2014-01-07

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Xylose induces cellulase production in Thermoascus aurantiacus

DOE PAGES

Schuerg, Timo; Prahl, Jan -Philip; Gabriel, Raphael; ...

2017-11-15

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted tomore » produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. CXylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.« less

Xylose induces cellulase production in Thermoascus aurantiacus

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

Schuerg, Timo; Prahl, Jan -Philip; Gabriel, Raphael

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted tomore » produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. CXylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.« less

Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

PubMed

Zhang, Biao; Zhang, Jia; Wang, Dongmei; Han, Ruixiang; Ding, Rui; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

2016-09-01

Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob. Copyright © 2016 Elsevier Ltd. All rights reserved.

Measurement of the Spin of the $$\\Xi(1530)$$ Resonance

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

Aubert, B.; Bona, M.; Karyotakis, Y.

The properties of the {Xi}(1530) resonance are investigated in the {Lambda}{sub c}{sup +} {yields} {Xi}{sup -}{pi}{sup +}K{sup +} decay process. The data sample was collected with the BABAR detector at the SLAC PEP-II asymmetric-energy e{sup +}e{sup -} collider operating at center of mass energies 10.58 and 10.54 GeV. The corresponding integrated luminosity is approximately 230 fb{sup -1}. The spin of the {Xi}(1530) is established to be 3/2. The existence of an S-wave amplitude in the {Xi}{sup -}{pi}{sup +} system is inferred, and its interference with the {Xi}(1530)0 amplitude provides the first clear demonstration of the Breit-Wigner phase motion expected formore » the {Xi}(1530). The P{sub 1}(cos {theta}{sub {Xi}{sup -}}) Legendre polynomial moment indicates the presence of a significant S-wave amplitude for {Xi}{sup -}{pi}{sup +} mass values above 1.6 GeV/c{sup 2}, and a dip in the mass distribution at approximately 1.7 GeV/c{sup 2} is interpreted as due to coherent addition of a {Xi}(1690){sup 0} contribution to this amplitude. This would imply J{sup P} = 1/2{sup -} for the {Xi}(1690). Attempts at fitting the {Xi}(1530){sup 0} lineshape yield unsatisfactory results, and this failure is attributed to interference effects associated with the amplitudes describing the K{sup +}{pi}{sup +} and/or {Xi}{sup -}K{sup +} systems.« less

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2013-05-14

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2017-09-12

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2011-05-17

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2016-08-09

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

First measurements of J/{psi} decays into {sigma}{sup +}{sigma}{sup -} and {xi}{sup 0}{xi}{sup 0}

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

Ablikim, M.; Bai, J. Z.; Bai, Y.

Based on 58x10{sup 6} J/{psi} events collected with the BESII detector at the Beijing Electron-Positron Collider, the baryon pair processes J/{psi}{yields}{sigma}{sup +}{sigma}{sup -} and J/{psi}{yields}{xi}{sup 0}{xi}{sup 0} are observed for the first time. The branching fractions are measured to be B(J/{psi}{yields}{sigma}{sup +}{sigma}{sup -})=(1.50{+-}0.10{+-}0.22)x10{sup -3} and B(J/{psi}{yields}{xi}{sup 0}{xi}{sup 0})=(1.20{+-}0.12{+-}0.21)x10{sup -3}, where the first errors are statistical and the second ones are systematic.

Measurement of the spin of the {xi}(1530) resonance

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

Aubert, B.; Bona, M.; Karyotakis, Y.

The properties of the {xi}(1530) resonance are investigated in the {lambda}{sub c}{sup +}{yields}{xi}{sup -}{pi}{sup +}K{sup +} decay process. The data sample was collected with the BABAR detector at the SLAC PEP-II asymmetric-energy e{sup +}e{sup -} collider operating at center-of-mass energies 10.58 and 10.54 GeV. The corresponding integrated luminosity is approximately 230 fb{sup -1}. The spin of the {xi}(1530) is established to be 3/2. The existence of an S-wave amplitude in the {xi}{sup -}{pi}{sup +} system is inferred, and its interference with the {xi}(1530){sup 0} amplitude provides the first clear demonstration of the Breit-Wigner phase motion expected for the {xi}(1530). Themore » P{sub 1}(cos{theta}{sub {xi}}{sub {sup -}}) Legendre polynomial moment indicates the presence of a significant S-wave amplitude for {xi}{sup -}{pi}{sup +} mass values above 1.6 GeV/c{sup 2}, and a dip in the mass distribution at approximately 1.7 GeV/c{sup 2} is interpreted as due to the coherent addition of a {xi}(1690){sup 0} contribution to this amplitude. This would imply J{sup P}=1/2{sup -} for the {xi}(1690). Attempts at fitting the {xi}(1530){sup 0} line shape yield unsatisfactory results, and this failure is attributed to interference effects associated with the amplitudes describing the K{sup +}{pi}{sup +} and/or {xi}{sup -}K{sup +} systems.« less

Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

PubMed

Li, Yun-Cheng; Mitsumasu, Kanako; Gou, Zi-Xi; Gou, Min; Tang, Yue-Qin; Li, Guo-Ying; Wu, Xiao-Lei; Akamatsu, Takashi; Taguchi, Hisataka; Kida, Kenji

2016-02-01

Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol.

An in vivo, label-free quick assay for xylose transport in Escherichia coli.

PubMed

Chen, Tingjian; Zhang, Jingqing; Liang, Ling; Yang, Rong; Lin, Zhanglin

2009-07-01

Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-beta-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms.

Search for CP Violation in Hyperon Decay: $$\\Xi^- / \\bar{\\Xi}^+$$ and $$\\Lambda / \\bar{\\Lambda}$$ (in French)

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

Leros, Nicolas

2001-06-01

The HyperCP(EB71) experiment, performed at the Fermi National Accelerator Laboratory in the United States, provides a primary search for direct OP violation in the decays ofmore » $$\\Xi^-/\\bar{\\Xi}^+$$ and $$\\Lambda/ \\bar{\\Lambda}$$ hyperons....« less

Cofermentation of Glucose, Xylose, and Cellobiose by the Beetle-Associated Yeast Spathaspora passalidarum

PubMed Central

Long, Tanya M.; Su, Yi-Kai; Headman, Jennifer; Higbee, Alan; Willis, Laura B.

2012-01-01

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts. PMID:22636012

Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

PubMed

Jin, Li-Qun; Xu, Qi; Liu, Zhi-Qiang; Jia, Dong-Xu; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2017-09-01

Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min -1 . The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

NASA Technical Reports Server (NTRS)

Shing, Y. W.; Akagi, J. M.; Himes, R. H.

1972-01-01

It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

Group 16SrXI phytoplasma strains, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D, are associated with sugar cane white leaf.

PubMed

Zhang, Rong-Yue; Li, Wen-Feng; Huang, Ying-Kun; Wang, Xiao-Yan; Shan, Hong-Li; Luo, Zhi-Ming; Yin, Jiong

2016-01-01

Sugar cane white leaf (SCWL) is a serious disease caused by phytoplasmas. In this study, we performed nested PCR with phytoplasma universal primer pairs (P1/P7 and R16F2n/R16R2) for the 16S rRNA gene to detect SCWL phytoplasmas in 31 SCWL samples collected from Baoshan and Lincang, Yunnan, China. We cloned and sequenced the nested PCR products, revealing that the 16S rRNA gene sequences from 31 SCWL samples were all 1247 bp in length and shared more than 99 % nucleotide sequence similarity with the 16S rRNA gene sequences of SCWL phytoplasmas from various countries. Based on the reported 16S rRNA gene sequence data from SCWL isolates of various countries, we conducted phylogenetic and virtual RFLP analysis. In the resulting phylogenetic tree, all SCWL isolates clustered into two branches, with the Lincang and Baoshan SCWL phytoplasma isolates belonging to different branches. The virtual RFLP patterns show that phytoplasmas of the Lincang branch belong to subgroup 16SrXI-B. However, the virtual RFLP patterns revealed by HaeIII digestion of phytoplasmas of the Baoshan branch differed from those of subgroup 16SrXI-B. According to the results of phylogenetic and virtual RFLP analysis, we propose that the phytoplasmas of the Baoshan branch represent a new subgroup, 16SrXI-D. These findings suggest that SCWL is caused by phytoplasmas from group 16SrXI, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D.

Genomic sequence of the xylose fermenting, insect-inhabitingyeast, Pichia stipitis

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

Jeffries, Thomas W.; Grigoriev, Igor; Grimwood, Jane

2007-06-25

Xylose is a major constituent of angiosperm lignocellulose,so its fermentation is important for bioconversion to fuels andchemicals. Pichia stipitis is the best-studied native xylose fermentingyeast. Genes from P. stipitis have been used to engineer xylosemetabolism in Saccharomycescerevisiae, and the regulation of the P.stipitis genome offers insights into the mechanisms of xylose metabolismin yeasts. We have sequenced, assembled and finished the genome ofP.stipitis. As such, it is one of only a handful of completely finishedeukaryotic organisms undergoing analysis and manual curation. Thesequence has revealed aspects of genome organization, numerous genes forbiocoversion, preliminary insights into regulation of central metabolicpathways, numerous examples ofmore » co-localized genes with related functions,and evidence of how P. stipitis manages to achieve redox balance whilegrowing on xylose under microaerobic conditions.« less

First Observation of the Cabibbo-suppressed Decays Xi+(c) ---> Sigma+ pi- pi+ and Xi+(c) ---> Sigma- pi+ pi+ and Measurement of their Branching Ratios

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

Vazquez-Jauregui, E.; /San Luis Potosi U.; Engelfried, J.

The authors report the first observation of two Cabibbo-suppressed decay modes, {Xi}{sub c}{sup +} {yields} {Sigma}{sup +}{pi}{sup -}{pi}{sup +} and {Xi}{sub c}{sup +} {yields} {Sigma}{sup -} {pi}{sup +}{pi}{sup +}. They observe 56 {+-} 13 over a background of 21, and 23 {+-} 7 over a background of 12 events, respectively, for the signals. The data were accumulated using the SELEX spectrometer during the 1996-1997 fixed target run at Fermilab, chiefly from a 600 GeV/c {Sigma}{sup -} beam. The branching ratios of the decays relative to the Cabibbo-favored {Xi}{sub c}{sup +} {yields} {Xi}{sup -}{pi}{sup +}{pi}{sup +} are measured to be B({Xi}{submore » c}{sup +} {yields} {Sigma}{sup +}{pi}{sup -}{pi}{sup +})/B({xi}{sub c}{sup +} {yields} {Xi}{sup -} {pi}{sup +}{pi}{sup +}) = 0.50 {+-} 0.20, and B({Xi}{sub c}{sup +} {yields} {Sigma}{sup -}{pi}{sup +}{pi}{sup +})/B({Xi}{sub c}{sup +} {yields} {Xi}{sup -}{pi}{sup +}{pi}{sup +}) = 0.23 {+-} 0.11, respectively. They also report branching ratios for the same decay modes of the {Lambda}{sub c}{sup +} relative to {Lambda}{sub c}{sup +} {yields} pK{sup -}{pi}{sup +}.« less

Light-cone distribution amplitudes of {xi} and their applications

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

Liu Yonglu; Huang Mingqiu

We present the light-cone distribution amplitudes of the {xi} baryons up to twist six on the basis of QCD conformal partial wave expansion to the leading order conformal spin accuracy. The nonperturbative parameters relevant to the DAs are determined in the framework of the QCD sum rule. The light-cone QCD sum rule approach is used to investigate both the electromagnetic form factors of {xi} and the exclusive semileptonic decay of {xi}{sub c} as applications. Our estimations on the magnetic moments are {mu}{sub {xi}{sup 0}}=-(1.92{+-}0.34){mu}{sub N} and {mu}{sub {xi}{sup -}}=-(1.19{+-}0.03){mu}{sub N}. The decay width of the process {xi}{sub c}{yields}{xi}e{sup +}{nu}{sub e}more » is evaluated to be {gamma}=8.73x10{sup -14} GeV, which is in accordance with the experimental measurements and other theoretical approaches.« less

Genetics Home Reference: triosephosphate isomerase deficiency

MedlinePlus

... more common in particular ethnic groups? Genetic Changes Mutations in the TPI1 gene cause triosephosphate isomerase deficiency . ... down to produce energy for cells. TPI1 gene mutations lead to the production of unstable enzymes or ...

Pulsed addition of HMF and furfural to batch-grown xylose-utilizing Saccharomyces cerevisiae results in different physiological responses in glucose and xylose consumption phase

PubMed Central

2013-01-01

Background Pretreatment of lignocellulosic biomass generates a number of undesired degradation products that can inhibit microbial metabolism. Two of these compounds, the furan aldehydes 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), have been shown to be an impediment for viable ethanol production. In the present study, HMF and furfural were pulse-added during either the glucose or the xylose consumption phase in order to dissect the effects of these inhibitors on energy state, redox metabolism, and gene expression of xylose-consuming Saccharomyces cerevisiae. Results Pulsed addition of 3.9 g L-1 HMF and 1.2 g L-1 furfural during either the glucose or the xylose consumption phase resulted in distinct physiological responses. Addition of furan aldehydes in the glucose consumption phase was followed by a decrease in the specific growth rate and the glycerol yield, whereas the acetate yield increased 7.3-fold, suggesting that NAD(P)H for furan aldehyde conversion was generated by acetate synthesis. No change in the intracellular levels of NAD(P)H was observed 1 hour after pulsing, whereas the intracellular concentration of ATP increased by 58%. An investigation of the response at transcriptional level revealed changes known to be correlated with perturbations in the specific growth rate, such as protein and nucleotide biosynthesis. Addition of furan aldehydes during the xylose consumption phase brought about an increase in the glycerol and acetate yields, whereas the xylitol yield was severely reduced. The intracellular concentrations of NADH and NADPH decreased by 58 and 85%, respectively, hence suggesting that HMF and furfural drained the cells of reducing power. The intracellular concentration of ATP was reduced by 42% 1 hour after pulsing of inhibitors, suggesting that energy-requiring repair or maintenance processes were activated. Transcriptome profiling showed that NADPH-requiring processes such as amino acid biosynthesis and sulfate and

Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.

PubMed

Puchner, Claudia; Eixelsberger, Thomas; Nidetzky, Bernd; Brecker, Lothar

2017-01-02

Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via 1 H and 13 C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala 79 . In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site. Copyright © 2016. Published by Elsevier Ltd.

Efficient xylose fermentation by the brown rot fungus Neolentinus lepideus.

PubMed

Okamoto, Kenji; Kanawaku, Ryuichi; Masumoto, Masaru; Yanase, Hideshi

2012-02-10

The efficient production of bioethanol on an industrial scale requires the use of renewable lignocellulosic biomass as a starting material. A limiting factor in developing efficient processes is identifying microorganisms that are able to effectively ferment xylose, the major pentose sugar found in hemicellulose, and break down carbohydrate polymers without pre-treatment steps. Here, a basidiomycete brown rot fungus was isolated as a new biocatalyst with unprecedented fermentability, as it was capable of converting not only the 6-carbon sugars constituting cellulose, but also the major 5-carbon sugar xylose in hemicelluloses, to ethanol. The fungus was identified as Neolentinus lepideus and was capable of assimilating and fermenting xylose to ethanol in yields of 0.30, 0.33, and 0.34 g of ethanol per g of xylose consumed under aerobic, oxygen-limited, and anaerobic conditions, respectively. A small amount of xylitol was detected as the major by-product of xylose metabolism. N. lepideus produced ethanol from glucose, mannose, galactose, cellobiose, maltose, and lactose with yields ranging from 0.34 to 0.38 g ethanol per g sugar consumed, and also exhibited relatively favorable conversion of non-pretreated starch, xylan, and wheat bran. These results suggest that N. lepideus is a promising candidate for cost-effective and environmentally friendly ethanol production from lignocellulosic biomass. To our knowledge, this is the first report on efficient ethanol fermentation from various carbohydrates, including xylose, by a naturally occurring brown rot fungus. Copyright © 2011 Elsevier Inc. All rights reserved.

Ethanol production in fermentation of mixed sugars containing xylose

DOEpatents

Viitanen, Paul V [West Chester, PA; Mc Cutchen, Carol M [Wilmington, DE; Li,; Xu, [Newark, DE; Emptage, Mark [Wilmington, DE; Caimi, Perry G [Kennett Square, PA; Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Lakewood, CO; Franden, Mary Ann [Centennial, CO

2009-12-08

Xylose-utilizing Z. mobilis strains were found to have improved ethanol production when grown in medium containing mixed sugars including xylose if sorbitol or mannitol was included in the medium. The effect was seen in concentrations of mixed sugars where no growth lag period occurs, as well as in higher sugars concentrations.

Production and Decay of {xi}{sub c}{sup 0} at BABAR

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

Aubert, B.; Barate, R.; Boutigny, D.

Using 116.1 fb{sup -1} of data collected by the BABAR detector, we present an analysis of {xi}{sub c}{sup 0} production in B decays and from the cc continuum, with the {xi}{sub c}{sup 0} decaying into {omega}{sup -}K{sup +} and {xi}{sup -}{pi}{sup +} final states. We measure the ratio of branching fractions B({xi}{sub c}{sup 0}{yields}{omega}{sup -}K{sup +})/B({xi}{sub c}{sup 0}{yields}{xi}{sup -}{pi}{sup +}) to be 0.294{+-}0.018{+-}0.016, where the first uncertainty is statistical and the second is systematic. The {xi}{sub c}{sup 0} momentum spectrum is measured on and 40 MeV below the {upsilon}(4S) resonance. From these spectra the branching fraction product B(B{yields}{xi}{sub c}{sup 0}X)xB({xi}{submore » c}{sup 0}{yields}{xi}{sup -}{pi}{sup +}) is measured to be (2.11{+-}0.19{+-}0.25)x10{sup -4}, and the cross-section product {sigma}(e{sup +}e{sup -}{yields}{xi}{sub c}{sup 0}X)xB({xi}{sub c}{sup 0}{yields}{xi}{sup -}{pi}{sup +}) from the continuum is measured to be (388{+-}39{+-}41) fb at a center-of-mass energy of 10.58 GeV.« less

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.

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

PubMed

Watanabe, Seiya; Abu Saleh, Ahmed; Pack, Seung Pil; Annaluru, Narayana; Kodaki, Tsutomu; Makino, Keisuke

2007-09-01

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

Ethanol production using xylitol synthesis mutant of xylose-utilizing zymomonas

DOEpatents

Viitanen, Paul V.; McCutchen, Carol M.; Emptage, Mark; Caimi, Perry G.; Zhang, Min; Chou, Yat-Chen

2010-06-22

Production of ethanol using a strain of xylose-utilizing Zymomonas with a genetic modification of the glucose-fructose oxidoreductase gene was found to be improved due to greatly reduced production of xylitol, a detrimental by-product of xylose metabolism synthesized during fermentation.

Increased xylose affinity of Hxt2 through gene shuffling of hexose transporters in Saccharomyces cerevisiae.

PubMed

Nijland, J G; Shin, H Y; de Waal, P P; Klaassen, P; Driessen, A J M

2018-02-01

Optimizing D-xylose transport in Saccharomyces cerevisiae is essential for efficient bioethanol production from cellulosic materials. We have used a gene shuffling approach of hexose (Hxt) transporters in order to increase the affinity for D-xylose. Various libraries were transformed to a hexose transporter deletion strain, and shuffled genes were selected via growth on low concentrations of D-xylose. This screening yielded two homologous fusion proteins (fusions 9,4 and 9,6), both consisting of the major central part of Hxt2 and various smaller parts of other Hxt proteins. Both chimeric proteins showed the same increase in D-xylose affinity (8·1 ± 3·0 mmol l -1 ) compared with Hxt2 (23·7 ± 2·1 mmol l -1 ). The increased D-xylose affinity could be related to the C terminus, more specifically to a cysteine to proline mutation at position 505 in Hxt2. The Hxt2 C505P mutation increased the affinity for D-xylose for Hxt2, thus providing a way to increase D-xylose transport flux at low D-xylose concentration. The gene shuffling protocol using the highly homologues hexose transporters family provides a powerful tool to enhance the D-xylose affinity of Hxt transporters in S. cerevisiae, thus providing a means to increase the D-xylose uptake flux at low D-xylose concentrations. © 2017 The Society for Applied Microbiology.

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

USDA-ARS?s Scientific Manuscript database

Saccharomyces physiology and fermentation related properties vary broadly among industrial strains. In this study, six industrial strains of varied genetic background were engineered to ferment xylose. Aerobic growth rates on xylose were 0.040 h**-1 to 0.167 h**-1. Fermentation of xylose, glucose/xy...

Utilization of xylose as a carbon source for mixotrophic growth of Scenedesmus obliquus.

PubMed

Yang, Suling; Liu, Guijun; Meng, Youting; Wang, Ping; Zhou, Sijing; Shang, Hongzhong

2014-11-01

Mixotrophic cultivation is one potential mode for microalgae production, and an economically acceptable and environmentally sustainable organic carbon source is essential. The potential use of xylose for culturing Scenedesmus obliquus in a mixotrophic mode and physiological features of xylose-grown S. obliquus were studied. S. obliquus had a certain xylose tolerance, and was capable of utilizing xylose for growth. At a xylose concentration of 4gL(-1), the maximal cell density was 2.2gL(-1), being 2.9-fold of that under photoautotrophic condition and arriving to the level of mixotrophic growth using 4gL(-1) glucose. No changes in cellular morphology of the cells grown with or without xylose were detected. Fluorescence emission from photosystem II (PS II) relative to photosystem I (PS I) was decreased in mixotrophic cells, implying that the PSII activity was decreased. The biomass lipid content was enhanced and carbohydrate concentration was decreased, in relation to photoautotrophic controls. Copyright © 2014 Elsevier Ltd. All rights reserved.

Xylose-fermenting Pichia stipitis by genome shuffling for improved ethanol production.

PubMed

Shi, Jun; Zhang, Min; Zhang, Libin; Wang, Pin; Jiang, Li; Deng, Huiping

2014-03-01

Xylose fermentation is necessary for the bioconversion of lignocellulose to ethanol as fuel, but wild-type Saccharomyces cerevisiae strains cannot fully metabolize xylose. Several efforts have been made to obtain microbial strains with enhanced xylose fermentation. However, xylose fermentation remains a serious challenge because of the complexity of lignocellulosic biomass hydrolysates. Genome shuffling has been widely used for the rapid improvement of industrially important microbial strains. After two rounds of genome shuffling, a genetically stable, high-ethanol-producing strain was obtained. Designated as TJ2-3, this strain could ferment xylose and produce 1.5 times more ethanol than wild-type Pichia stipitis after fermentation for 96 h. The acridine orange and propidium iodide uptake assays showed that the maintenance of yeast cell membrane integrity is important for ethanol fermentation. This study highlights the importance of genome shuffling in P. stipitis as an effective method for enhancing the productivity of industrial strains. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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

Fermentation of xylose into ethanol by a new fungus strain Pestalotiopsis sp. XE-1.

PubMed

Pang, Zong-wen; Liang, Jing-juan; Huang, Ri-bo

2011-08-01

A new fungus, Pestalotiopsis sp. XE-1, which produced ethanol from xylose with yield of 0.47 g ethanol/g of consumed xylose was isolated. It also produced ethanol from arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, and sucrose with yields of 0.38, 0.47, 0.45, 0.46, 0.31, 0.25, 0.31, and 0.34 g ethanol/g of sugar consumed, respectively. It produced maximum ethanol from xylose at pH 6.5, 30°C under a semi-aerobic condition. Acetic acid produced in xylose fermenting process inhibited ethanol production of XE-1. The ethanol yield in the pH-uncontrolled batch fermentation was about 27% lower than that in the pH-controlled one. The ethanol tolerance of XE-1 was higher than most xylose-fermenting, ethanol-producing microbes, but lower than Saccharomyces cerevisiae and Hansenula polymorpha. XE-1 showed tolerance to high concentration of xylose, and was able to grow and produce ethanol even when it was cultivated in 97.71 g/l xylose.

Design of Xylose-Based Semisynthetic Polyurethane Tissue Adhesives with Enhanced Bioactivity Properties.

PubMed

Balcioglu, Sevgi; Parlakpinar, Hakan; Vardi, Nigar; Denkbas, Emir Baki; Karaaslan, Merve Goksin; Gulgen, Selam; Taslidere, Elif; Koytepe, Suleyman; Ates, Burhan

2016-02-01

Developing biocompatible tissue adhesives with high adhesion properties is a highly desired goal of the tissue engineering due to adverse effects of the sutures. Therefore, our work involves synthesis, characterization, adhesion properties, protein adsorption, in vitro biodegradation, in vitro and in vivo biocompatibility properties of xylose-based semisynthetic polyurethane (NPU-PEG-X) bioadhesives. Xylose-based semisynthetic polyurethanes were developed by the reaction among 4,4'-methylenebis(cyclohexyl isocyanate) (MCI), xylose and polyethylene glycol 200 (PEG). Synthesized polyurethanes (PUs) showed good thermal stability and high adhesion strength. The highest values in adhesion strength were measured as 415.0 ± 48.8 and 94.0 ± 2.8 kPa for aluminum substrate and muscle tissue in 15% xylose containing PUs (NPU-PEG-X-15%), respectively. The biodegradation of NPU-PEG-X-15% was also determined as 19.96 ± 1.04% after 8 weeks of incubation. Relative cell viability of xylose containing PU was above 86%. Moreover, 10% xylose containing NPU-PEG-X (NPU-PEG-X-10%) sample has favorable tissue response, and inflammatory reaction between 1 and 6 weeks implantation period. With high adhesiveness and biocompatibility properties, NPU-PEG-X can be used in the medical field as supporting materials for preventing the fluid leakage after abdominal surgery or wound closure.

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

PubMed

Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

2017-03-01

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Xi-cam: Flexible High Throughput Data Processing for GISAXS

NASA Astrophysics Data System (ADS)

Pandolfi, Ronald; Kumar, Dinesh; Venkatakrishnan, Singanallur; Sarje, Abinav; Krishnan, Hari; Pellouchoud, Lenson; Ren, Fang; Fournier, Amanda; Jiang, Zhang; Tassone, Christopher; Mehta, Apurva; Sethian, James; Hexemer, Alexander

With increasing capabilities and data demand for GISAXS beamlines, supporting software is under development to handle larger data rates, volumes, and processing needs. We aim to provide a flexible and extensible approach to GISAXS data treatment as a solution to these rising needs. Xi-cam is the CAMERA platform for data management, analysis, and visualization. The core of Xi-cam is an extensible plugin-based GUI platform which provides users an interactive interface to processing algorithms. Plugins are available for SAXS/GISAXS data and data series visualization, as well as forward modeling and simulation through HipGISAXS. With Xi-cam's advanced mode, data processing steps are designed as a graph-based workflow, which can be executed locally or remotely. Remote execution utilizes HPC or de-localized resources, allowing for effective reduction of high-throughput data. Xi-cam is open-source and cross-platform. The processing algorithms in Xi-cam include parallel cpu and gpu processing optimizations, also taking advantage of external processing packages such as pyFAI. Xi-cam is available for download online.

Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose.

PubMed

Panagiotou, Gianni; Christakopoulos, Paul; Grotkjaer, Thomas; Olsson, Lisbeth

2006-09-01

Dissimilatory nitrate reduction metabolism, of the natural xylose-fermenting fungus Fusarium oxysporum, was used as a strategy to achieve anaerobic growth and ethanol production from xylose. Beneficial alterations of the redox fluxes and thereby of the xylose metabolism were obtained by taking advantage of the regeneration of the cofactor NAD(+) during the denitrification process. In batch cultivations, nitrate sustained growth under anaerobic conditions (1.21 g L(-1) biomass) and simultaneously a maximum yield of 0.55 moles of ethanol per mole of xylose was achieved, whereas substitution of nitrate with ammonium limited the growth significantly (0.15 g L(-1) biomass). Using nitrate, the maximum acetate yield was 0.21 moles per mole of xylose and no xylitol excretion was observed. Furthermore, the network structure in the central carbon metabolism of F. oxysporum was characterized in steady state. F. oxysporum grew anaerobically on [1-(13)C] labelled glucose and unlabelled xylose in chemostat cultivation with nitrate as nitrogen source. The use of labelled substrate allowed the precise determination of the glucose and xylose contribution to the carbon fluxes in the central metabolism of this poorly described microorganism. It was demonstrated that dissimilatory nitrate reduction allows F. oxysporum to exhibit typical respiratory metabolic behaviour with a highly active TCA cycle and a large demand for NADPH.

Transposon mutagenesis to improve the growth of recombinant Saccharomyces cerevisiae on D-xylose

Treesearch

Haiying Ni; Jose M. Laplaza; Thomas W. Jeffries

2007-01-01

Saccharomyces cerevisiae L2612 transformed with genes for xylose reductase and xylitol dehydrogenase (XYL1 and XYL2) grows well on glucose but very poorly on D-xylose. When a gene for D-xylulokinase (XYL3 or XKS1) is overexpressed, growth on glucose is unaffected, but growth on xylose is blocked. Spontaneous or chemically induced mutants of this engineered yeast that...

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production.

PubMed

Yuvadetkun, Prawphan; Leksawasdi, Noppol; Boonmee, Mallika

2017-03-16

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8 g/L in xylose and 52.6 g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4 g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40 g/L of ethanol and ethanol production capacity of the yeast was 52 g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170 g/L sugar concentrations.

Absence of FKBP10 in Recessive Type XI Osteogenesis Imperfecta Leads to Diminished Collagen Cross-Linking and Reduced Collagen Deposition in Extracellular Matrix

PubMed Central

Barnes, Aileen M.; Cabral, Wayne A.; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L.; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C.

2012-01-01

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. PMID:22718341

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

PubMed

Turner, Timothy L; Zhang, Guo-Chang; Kim, Soo Rin; Subramaniam, Vijay; Steffen, David; Skory, Christopher D; Jang, Ji Yeon; Yu, Byung Jo; Jin, Yong-Su

2015-10-01

Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.

Production of xylitol from D-xylose by Debaryomyces hansenii

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

Dominguez, J.M.; Gong, Cheng S.; Tsao, G.T.

1997-12-31

Xylitol, a naturally occurring five-carbon sugar alcohol, can be produced from D-xylose through microbial hydrogenation. Xylitol has found increasing use in the food industries, especially in confectionary. It is the only so-called {open_quotes}second-generation polyol sweeteners{close_quotes} that is allowed to have the specific health claims in some world markets. In this study, the effect of cell density on the xylitol production by the yeast Debaryomyces hansenii NRRL Y-7426 from D-xylose under microaerobic conditions was examined. The rate of xylitol production increased with increasing yeast cell density to 3 g/L. Beyond this amount there was no increase in the xylitol production withmore » increasing cell density. The optimal pH range for xylitol production was between 4.5 and 5.5. The optimal temperature was between 28 and 37{degrees}C, and the optimal shaking speed was 300 rpm. The rate of xylitol production increased linearly with increasing initial xylose concentration. A high concentration of xylose (279 g/L) was converted rapidly and efficiently to produce xylitol with a product concentration of 221 g/L was reached after 48 h of incubation under optimum conditions. 18 refs., 5 figs.« less

Xi-CAM v1.2.3

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

PANDOLFI, RONALD; KUMAR, DINESH; VENKATAKRISHNAN, SINGANALLUR

Xi-CAM aims to provide a community driven platform for multimodal analysis in synchrotron science. The platform core provides a robust plugin infrastructure for extensibility, allowing continuing development to simply add further functionality. Current modules include tools for characterization with (GI)SAXS, Tomography, and XAS. This will continue to serve as a development base as algorithms for multimodal analysis develop. Seamless remote data access, visualization and analysis are key elements of Xi-CAM, and will become critical to synchrotron data infrastructure as expectations for future data volume and acquisition rates rise with continuously increasing throughputs. The highly interactive design elements of Xi-cam willmore » similarly support a generation of users which depend on immediate data quality feedback during high-throughput or burst acquisition modes.« less

Molecular mechanism of environmental d-xylose perception by a XylFII-LytS complex in bacteria.

PubMed

Li, Jianxu; Wang, Chengyuan; Yang, Gaohua; Sun, Zhe; Guo, Hui; Shao, Kai; Gu, Yang; Jiang, Weihong; Zhang, Peng

2017-08-01

d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. In bacteria, the first step for d-xylose metabolism is signal perception at the membrane. We previously identified a three-component system in Firmicutes bacteria comprising a membrane-associated sensor protein (XylFII), a transmembrane histidine kinase (LytS) for periplasmic d-xylose sensing, and a cytoplasmic response regulator (YesN) that activates the transcription of the target ABC transporter xylFGH genes to promote the uptake of d-xylose. The molecular mechanism underlying signal perception and integration of these processes remains elusive, however. Here we purified the N-terminal periplasmic domain of LytS (LytSN) in a complex with XylFII and determined the conformational structures of the complex in its d-xylose-free and d-xylose-bound forms. LytSN contains a four-helix bundle, and XylFII contains two Rossmann fold-like globular domains with a xylose-binding cleft between them. In the absence of d-xylose, LytSN and XylFII formed a heterodimer. Specific binding of d-xylose to the cleft of XylFII induced a large conformational change that closed the cleft and brought the globular domains closer together. This conformational change led to the formation of an active XylFII-LytSN heterotetramer. Mutations at the d-xylose binding site and the heterotetramer interface diminished heterotetramer formation and impaired the d-xylose-sensing function of XylFII-LytS. Based on these data, we propose a working model of XylFII-LytS that provides a molecular basis for d-xylose utilization and metabolic modification in bacteria.

Characterization of equine GST A3-3 as a steroid isomerase.

PubMed

Lindström, Helena; Peer, Shawna M; Ing, Nancy H; Mannervik, Bengt

2018-04-01

Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ 5 -androstene-3,17-dione to Δ 4 -androstene-3,17-dione and of Δ 5 -pregnene-3,20-dione to Δ 4 -pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse (Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones. Copyright © 2017 Elsevier Ltd. All rights reserved.

Absence of Diauxie during Simultaneous Utilization of Glucose and Xylose by Sulfolobus acidocaldarius▿ †

PubMed Central

Joshua, Chijioke J.; Dahl, Robert; Benke, Peter I.; Keasling, Jay D.

2011-01-01

Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h−1 and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h−1) as the sole carbon source than on an equal amount of glucose (0.022 h−1). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h−1 to 0.085 h−1 as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated. PMID:21239580

Direct Observation of the Strange b Baryon {xi}{sub b}{sup -}

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

Abazov, V. M.; Alexeev, G. D.; Kalinin, A. M.

We report the first direct observation of the strange b baryon {xi}{sub b}{sup -}({xi}{sub b}{sup +}). We reconstruct the decay {xi}{sub b}{sup -}{yields}J/{psi}{xi}{sup -}, with J/{psi}{yields}{mu}{sup +}{mu}{sup -}, and {xi}{sup -}{yields}{lambda}{pi}{sup -}{yields}p{pi}{sup -}{pi}{sup -} in pp collisions at {radical}(s)=1.96 TeV. Using 1.3 fb{sup -1} of data collected by the D0 detector, we observe 15.2{+-}4.4(stat){sub -0.4}{sup +1.9}(syst) {xi}{sub b}{sup -} candidates at a mass of 5.774{+-}0.011(stat){+-}0.015(syst) GeV. The significance of the observed signal is 5.5{sigma}, equivalent to a probability of 3.3x10{sup -8} of it arising from a background fluctuation. Normalizing to the decay {lambda}{sub b}{yields}J/{psi}{lambda}, we measure the relative rate ({sigma}({xi}{submore » b}{sup -})xB({xi}{sub b}{sup -}{yields}J/{psi}{xi}{sup -})/{sigma}({lambda}{sub b})xB({lambda}{sub b}{yields}J/{psi}{lambda}))=0.28{+-}0.09(stat){sub -0.08}{sup +0.09}(syst)« less

Simultaneous glucose and xylose uptake by an acetone/butanol/ethanol producing laboratory Clostridium beijerinckii strain SE-2.

PubMed

Zhang, Jie; Zhu, Wen; Xu, Haipeng; Li, Yan; Hua, Dongliang; Jin, Fuqiang; Gao, Mintian; Zhang, Xiaodong

2016-04-01

Most butanol-producing strains of Clostridium prefer glucose over xylose, leading to a slower butanol production from lignocellulose hydrolysates. It is therefore beneficial to find and use a strain that can simultaneously use both glucose and xylose. Clostridium beijerinckii SE-2 strain assimilated glucose and xylose simultaneously and produced ABE (acetone/butanol/ethanol). The classic diauxic growth behavior was not seen. Similar rates of sugar consumption (4.44 mM glucose h(-1) and 6.66 mM xylose h(-1)) were observed suggesting this strain could use either glucose or xylose as the substrate and it has a similar capability to degrade these two sugars. With different initial glucose:xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. ABE production profiles were similar on different substrates. Transcriptional studies on the effect of glucose and xylose supplementation, however, suggests a clear glucose inhibition on xylose metabolism-related genes is still present.

Optimized Production of Xylitol from Xylose Using a Hyper-Acidophilic Candida tropicalis.

PubMed

Tamburini, Elena; Costa, Stefania; Marchetti, Maria Gabriella; Pedrini, Paola

2015-08-19

The yeast Candida tropicalis DSM 7524 produces xylitol, a natural, low-calorie sweetener, by fermentation of xylose. In order to increase xylitol production rate during the submerged fermentation process, some parameters-substrate (xylose) concentration, pH, aeration rate, temperature and fermentation strategy-have been optimized. The maximum xylitol yield reached at 60-80 g/L initial xylose concentration, pH 5.5 at 37 °C was 83.66% (w/w) on consumed xylose in microaerophilic conditions (kLa = 2·h(-1)). Scaling up on 3 L fermenter, with a fed-batch strategy, the best xylitol yield was 86.84% (w/w), against a 90% of theoretical yield. The hyper-acidophilic behaviour of C. tropicalis makes this strain particularly promising for industrial application, due to the possibility to work in non-sterile conditions.

Co-immobilization of glucose oxidase and xylose dehydrogenase displayed whole cell on multiwalled carbon nanotube nanocomposite films modified electrode for simultaneous voltammetric detection of D-glucose and D-xylose.

PubMed

Li, Liang; Liang, Bo; Li, Feng; Shi, Jianguo; Mascini, Marco; Lang, Qiaolin; Liu, Aihua

2013-04-15

In this paper, we first report the construction of Nafion/glucose oxidase (GOD)/xylose dehydrogenase displayed bacteria (XDH-bacteria)/multiwalled carbon nanotubes (MWNTs) modified electrode for simultaneous voltammetric determination of D-glucose and D-xylose. The optimal conditions for the immobilized enzymes were established. Both enzymes retained their good stability and activities. In the mixture solution of D-glucose and D-xylose containing coenzyme NAD⁺ (the oxidized form of nicotinamide adenine dinucleotide), the Nafion/GOD/XDH-bacteria/MWNTs modified electrode exhibited quasi-reversible oxidation-reduction peak at -0.5 V (vs. saturated calomel electrode, SCE) originating from the catalytic oxidation of D-glucose, and oxidation peak at +0.55 V(vs. SCE) responding to the oxidation of NADH (the reduced form of nicotinamide adenine dinucleotide) by the carbon nanotubes, where NADH is the resultant product of coenzyme NAD⁺ involved in the catalysis of D-xylose by XDH-displayed bacteria. For the proposed biosensor, cathodic peak current at -0.5 V was linear with the concentration of D-glucose within the range of 0.25-6 mM with a low detection limit of 0.1 mM D-glucose (S/N=3), and the anodic peak current at +0.55 V was linear with the concentration of d-xylose in the range of 0.25∼4 mM with a low detection limit of 0.1 mM D-xylose (S/N=3). Further, D-xylose and D-glucose did not interfere with each other. 300-fold excess saccharides including D-maltose, D-galactose, D-mannose, D-sucrose, D-fructose, D-cellobiose, and 60-fold excess L-arabinose, and common interfering substances (100-fold excess ascorbic acid, dopamine, uric acid) as well as 300-fold excess D-xylitol did not affect the detection of D-glucose and D-xylose (both 1 mM). Therefore, the proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which holds great potential in real applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Solubility of glucose isomerase in ammonium sulphate solutions

NASA Astrophysics Data System (ADS)

Chayen, N.; Akins, J.; Campbell-Smith, S.; Blow, D. M.

1988-07-01

In order to quantify protein crystallization techniques, a method for measuring protein solubility in high salt concentration has been developed. It is based on a sensitive protein concentration assay, using binding to Coomassie blue dye. The protein concentration in a supernatant from which glucose isomerase is crystallising has been studied as a function of time. Equilibrium is established in 3-5 weeks, and the protein concentration remaining in solution is defined as the solubility of the protein. The solubility of glucose isomerase has been determined as a function of ammonium sulphate concentration; its variation with pH in 1.50M ammonium sulphate has also been studied. A remarkable dependence on pH over the range of 5.5 to 6.5 has been observed.

Xi-cam: a versatile interface for data visualization and analysis

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

Pandolfi, Ronald J.; Allan, Daniel B.; Arenholz, Elke

Xi-cam is an extensible platform for data management, analysis and visualization.Xi-camaims to provide a flexible and extensible approach to synchrotron data treatment as a solution to rising demands for high-volume/high-throughput processing pipelines. The core ofXi-camis an extensible plugin-based graphical user interface platform which provides users with an interactive interface to processing algorithms. Plugins are available for SAXS/WAXS/GISAXS/GIWAXS, tomography and NEXAFS data. WithXi-cam's `advanced' mode, data processing steps are designed as a graph-based workflow, which can be executed live, locally or remotely. Remote execution utilizes high-performance computing or de-localized resources, allowing for the effective reduction of high-throughput data.Xi-cam's plugin-based architecture targetsmore » cross-facility and cross-technique collaborative development, in support of multi-modal analysis.Xi-camis open-source and cross-platform, and available for download on GitHub.« less

Xi-cam: a versatile interface for data visualization and analysis

DOE PAGES

Pandolfi, Ronald J.; Allan, Daniel B.; Arenholz, Elke; ...

2018-05-31

Xi-cam is an extensible platform for data management, analysis and visualization.Xi-camaims to provide a flexible and extensible approach to synchrotron data treatment as a solution to rising demands for high-volume/high-throughput processing pipelines. The core ofXi-camis an extensible plugin-based graphical user interface platform which provides users with an interactive interface to processing algorithms. Plugins are available for SAXS/WAXS/GISAXS/GIWAXS, tomography and NEXAFS data. WithXi-cam's `advanced' mode, data processing steps are designed as a graph-based workflow, which can be executed live, locally or remotely. Remote execution utilizes high-performance computing or de-localized resources, allowing for the effective reduction of high-throughput data.Xi-cam's plugin-based architecture targetsmore » cross-facility and cross-technique collaborative development, in support of multi-modal analysis.Xi-camis open-source and cross-platform, and available for download on GitHub.« less

Protein disulfide isomerase a multifunctional protein with multiple physiological roles

NASA Astrophysics Data System (ADS)

Ali Khan, Hyder; Mutus, Bulent

2014-08-01

Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as well as its physiological and pathophysiological functions related to redox regulation and protein folding.

Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase.

PubMed

Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen

2013-11-01

Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

Furfural and glucose can enhance conversion of xylose to xylitol by Candida magnoliae TISTR 5663.

PubMed

Wannawilai, Siwaporn; Lee, Wen-Chien; Chisti, Yusuf; Sirisansaneeyakul, Sarote

2017-01-10

Xylitol production from xylose by the yeast Candida magnoliae TISTR 5663 was enhanced by supplementing the fermentation medium with furfural (300mg/L) and glucose (3g/L with an initial mass ratio of glucose to xylose of 1:10) together under oxygen limiting conditions. In the presence of furfural and glucose, the final concentration of xylitol was unaffected relative to control cultures but the xylitol yield on xylose increased by about 5%. Supplementation of the culture medium with glucose alone at an initial concentration of 3g/L, stimulated the volumetric and specific rates of xylose consumption and the rate of xylitol production from xylose. In a culture medium containing 30g/L xylose, 300mg/L furfural and 3g/L glucose, the volumetric production rate of xylitol was 1.04g/L h and the specific production rate was 0.169g/g h. In the absence of furfural and glucose, the volumetric production rate of xylitol was ∼35% lower and the specific production rate was nearly 30% lower. In view of these results, xylose-containing lignocellulosic hydrolysates contaminated with furfural can be effectively used for producing xylitol by fermentation so long as the glucose-to-xylose mass ratio in the hydrolysate does not exceed 1:10 and the furfural concentration is ≤300mg/L. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetics Home Reference: glucose phosphate isomerase deficiency

MedlinePlus

... patient homozygous for the L487F mutation in the human GPI gene. Int J Hematol. 2012 Aug;96(2):263- ... PubMed Xu W, Beutler E. The characterization of gene mutations for human glucose phosphate isomerase deficiency associated with chronic hemolytic ...

Optimized Production of Xylitol from Xylose Using a Hyper-Acidophilic Candida tropicalis

PubMed Central

Tamburini, Elena; Costa, Stefania; Marchetti, Maria Gabriella; Pedrini, Paola

2015-01-01

The yeast Candida tropicalis DSM 7524 produces xylitol, a natural, low-calorie sweetener, by fermentation of xylose. In order to increase xylitol production rate during the submerged fermentation process, some parameters-substrate (xylose) concentration, pH, aeration rate, temperature and fermentation strategy-have been optimized. The maximum xylitol yield reached at 60–80 g/L initial xylose concentration, pH 5.5 at 37 °C was 83.66% (w/w) on consumed xylose in microaerophilic conditions (kLa = 2·h−1). Scaling up on 3 L fermenter, with a fed-batch strategy, the best xylitol yield was 86.84% (w/w), against a 90% of theoretical yield. The hyper-acidophilic behaviour of C. tropicalis makes this strain particularly promising for industrial application, due to the possibility to work in non-sterile conditions. PMID:26295411

Improved Ethanol Production from Xylose by Candida shehatae Induced by Dielectric Barrier Discharge Air Plasma

NASA Astrophysics Data System (ADS)

Chen, Huixia; Xiu, Zhilong; Bai, Fengwu

2014-06-01

Xylose fermentation is essential for ethanol production from lignocellulosic biomass. Exposure of the xylose-fermenting yeast Candida shehatae (C. shehatae) CICC1766 to atmospheric pressure dielectric barrier discharge (DBD) air plasma yields a clone (designated as C81015) with stability, which exhibits a higher ethanol fermentation rate from xylose, giving a maximal enhancement in ethanol production of 36.2% compared to the control (untreated). However, the biomass production of C81015 is lower than that of the control. Analysis of the NADH (nicotinamide adenine dinucleotide)- and NADPH (nicotinamide adenine dinucleotide phosphate)-linked xylose reductases and NAD+-linked xylitol dehydrogenase indicates that their activities are enhanced by 34.1%, 61.5% and 66.3%, respectively, suggesting that the activities of these three enzymes are responsible for improving ethanol fermentation in C81015 with xylose as a substrate. The results of this study show that DBD air plasma could serve as a novel and effective means of generating microbial strains that can better use xylose for ethanol fermentation.

Metabolism of Linoleic Acid by Barley Lipoxygenase and Hydroperoxide Isomerase 1

PubMed Central

Lulai, Edward C.; Baker, Charles W.; Zimmerman, Don C.

1981-01-01

The oxidation of linoleic acid in incubation mixtures containing extracts of barley lipoxygenase and hydroperoxide isomerase, and the production of these enzymes in quiescent and germinated barley, were investigated. The ratio of 9-hydroperoxylinoleic acid to 13-hydroperoxylinoleic acid was higher for incubation mixtures containing extracts of quiescent barley than for mixtures containing extracts of germinated barley; production of 13-hydroperoxylinoleic acid from germinated barley exceeded that of quiescent barley. Hydroperoxy metabolites of linoleic acid were converted to 9-hydroxy-10-oxo-cis-12-octadecenoic acid, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, and small amounts of 11-hydroxy-12,13-epoxy-cis-9-octadecenoic acid and 11-hydroxy-9,10-epoxy-cis-13-octadecenoic acid whether quiescent or germinated barley was the enzyme source; a fifth product, 13-hydroxy-12-oxo-cis-9-octadecenoic acid was formed only when germinated barley was the enzyme source. Lipoxygenase was readily extracted by buffer, but hydroperoxide isomerase was bound in a catalytically active state to the insoluble barley grist and was efficiently extracted only when Triton X-100 was included in the extraction buffer. Hydroperoxide isomerase was localized in the embryo of quiescent barley, but it was present in the embryo, acrospire, and in small but concentrated amounts in the rootlet of germinating barley. The levels of both lipoxygenase and hydroperoxide isomerase increased through the thirteenth day of germination. Images PMID:16662032

Coproduction of xylose, lignosulfonate and ethanol from wheat straw.

PubMed

Zhu, Shengdong; Huang, Wangxiang; Huang, Wenjing; Wang, Ke; Chen, Qiming; Wu, Yuanxin

2015-06-01

A novel integrated process to coproduce xylose, lignosulfonate and ethanol from wheat straw was investigated. Firstly, wheat straw was treated by dilute sulfuric acid and xylose was recovered from its hydrolyzate. Its optimal conditions were 1.0wt% sulfuric acid, 10% (w/v) wheat straw loading, 100°C, and 2h. Then the acid treated wheat straw was treated by sulfomethylation reagent and its hydrolyzate containing lignosulfonate was directly recovered. Its optimal conditions were 150°C, 15% (w/v) acid treated wheat straw loading, and 5h. Finally, the two-step treated wheat straw was converted to ethanol through enzymatic hydrolysis and microbial fermentation. Under optimal conditions, 1kg wheat straw could produce 0.225kg xylose with 95% purity, 4.16kg hydrolyzate of sulfomethylation treatment containing 5.5% lignosulfonate, 0.183kg ethanol and 0.05kg lignin residue. Compared to present technology, this process is a potential economically profitable wheat straw biorefinery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Search for the lepton-number-violating decay Xi(-)-->pmu(-)mu(-).

PubMed

Rajaram, D; Burnstein, R A; Chakravorty, A; Chan, A; Chen, Y C; Choong, W S; Clark, K; Dukes, E C; Durandet, C; Felix, J; Gidal, G; Gu, P; Gustafson, H R; Ho, C; Holmstrom, T; Huang, M; James, C; Jenkins, C M; Kaplan, D M; Lederman, L M; Leros, N; Longo, M J; Lopez, F; Lu, L C; Luebke, W; Luk, K B; Nelson, K S; Park, H K; Perroud, J-P; Rubin, H A; Teng, P K; Volk, J; White, C G; White, S L; Zyla, P

2005-05-13

A sensitive search for the lepton-number-violating decay Xi(-)-->pmu(-)mu(-) has been performed using a sample of approximately 10(9) Xi(-) hyperons produced in 800 GeV/c p-Cu collisions. We obtain B(Xi(-)-->pmu(-)mu(-))<4.0x10(-8) at 90% confidence, improving on the best previous limit by 4 orders of magnitude.

A Burkholderia sacchari cell factory: production of poly-3-hydroxybutyrate, xylitol and xylonic acid from xylose-rich sugar mixtures.

PubMed

Raposo, Rodrigo S; de Almeida, M Catarina M D; de Oliveira, M da Conceição M A; da Fonseca, M Manuela; Cesário, M Teresa

2017-01-25

Efficient production of poly-3-hydroxybutyrate (P(3HB)) based on glucose-xylose mixtures simulating different types of lignocellulosic hydrolysate (LCH) was addressed using Burkholderia sacchari, a wild strain capable of metabolizing both sugars and producing P(3HB). Carbon catabolite repression was avoided by maintaining glucose concentration below 10g/L. Xylose concentrations above 30g/L were inhibitory for growth and production. In fed-batch cultivations, pulse size and feed addition rate were controlled in order to reach high productivities and efficient sugar consumptions. High xylose uptake and P(3HB) productivity were attained with glucose-rich mixtures (glucose/xylose ratio in the feed=1.5w/w) using high feeding rates, while with xylose-richer feeds (glucose/xylose=0.8w/w), a lower feeding rate is a robust strategy to avoid xylose build-up in the medium. Xylitol production was observed with xylose concentrations in the medium above 30-40g/L. With sugar mixtures featuring even lower glucose/xylose ratios, i.e. xylose-richer feeds (glucose/xylose=0.5), xylonic acid (a second byproduct) was produced. This is the first report of the ability of Burkholderia sacchari to produce both xylitol and xylonic acid. Copyright © 2016 Elsevier B.V. All rights reserved.

Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis

PubMed Central

Liu, Lixia; Zhang, Lei; Tang, Wei; Gu, Yang; Hua, Qiang; Yang, Sheng; Jiang, Weihong

2012-01-01

Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-13C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter−1. To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation. PMID:22865845

Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.

PubMed

Barnes, Aileen M; Cabral, Wayne A; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C

2012-11-01

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. Published 2012 Wiley Periodicals, Inc.*This article is a US Government work and, as such, is in the public domain of the United States of America.

Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

PubMed

Wang, R K; Zhan, S F; Zhao, T J; Zhou, X L; Wang, C E

2015-03-20

Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae.

PubMed

Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki

2009-04-01

In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates.

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

PubMed

Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

2006-07-01

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

Transcriptional activator Cat8 is involved in regulation of xylose alcoholic fermentation in the thermotolerant yeast Ogataea (Hansenula) polymorpha.

PubMed

Ruchala, Justyna; Kurylenko, Olena O; Soontorngun, Nitnipa; Dmytruk, Kostyantyn V; Sibirny, Andriy A

2017-02-28

Efficient xylose alcoholic fermentation is one of the key to a successful lignocellulosic ethanol production. However, regulation of this process in the native xylose-fermenting yeasts is poorly understood. In this work, we paid attention to the transcriptional factor Cat8 and its possible role in xylose alcoholic fermentation in Ogataea (Hansenula) polymorpha. In Saccharomyces cerevisiae, organism, which does not metabolize xylose, gene CAT8 encodes a Zn-cluster transcriptional activator necessary for expression of genes involved in gluconeogenesis, respiration, glyoxylic cycle and ethanol utilization. Xylose is a carbon source that could be fermented to ethanol and simultaneously could be used in gluconeogenesis for hexose synthesis. This potentially suggests involvement of CAT8 in xylose metabolism. Here, the role of CAT8 homolog in the natural xylose-fermenting thermotolerant yeast O. polymorpha was characterized. The CAT8 ortholog was identified in O. polymorpha genome and deleted both in the wild-type strain and in advanced ethanol producer from xylose. Constructed cat8Δ strain isolated from wild strain showed diminished growth on glycerol, ethanol and xylose as well as diminished respiration on the last substrate. At the same time, cat8Δ mutant isolated from the best available O. polymorpha ethanol producer showed only visible defect in growth on ethanol. CAT8 deletant was characterized by activated transcription of genes XYL3, DAS1 and RPE1 and slight increase in the activity of several enzymes involved in xylose metabolism and alcoholic fermentation. Ethanol production from xylose in cat8Δ mutants in the background of wild-type strain and the best available ethanol producer from xylose increased for 50 and 30%, respectively. The maximal titer of ethanol during xylose fermentation was 12.5 g ethanol/L at 45 °C. Deletion of CAT8 did not change ethanol production from glucose. Gene CAT8 was also overexpressed under control of the strong constitutive

Lactic acid production from xylose by Geobacillus stearothermophilus strain 15

NASA Astrophysics Data System (ADS)

Kunasundari, B.; Naresh, S.; Chu, J. E.

2017-09-01

Lactic acid is an important compound with a wide range of industrial applications. The present study tested the efficiency of xylose, as a sole carbon source to be converted to lactic acid by Geobacillus stearothermophilus strain 15. To the best of our knowledge, limited information is available on the directed fermentation of xylose to lactic acid by this bacterium. The effects of different parameters such as temperature, pH, incubation time, agitation speed, concentrations of nitrogen and carbon sources on the lactic acid production were investigated statistically. It was found that the bacterium exhibited poor assimilation of xylose to lactic acid. Temperature, agitation rate and incubation time were determined to improve the lactic acid production slightly. The highest lactic acid yield obtained was 8.9% at 45°C, 300 RPM, 96 h, pH of 6.0 with carbon and nitrogen source concentrations were fixed at 5% w/v.

Study of excited charm-strange baryons with evidence for new baryons {xi}{sub c}(3055){sup +} and {xi}{sub c}(3123){sup +}

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

Aubert, B.; Bona, M.; Boutigny, D.

We present a study of excited charm-strange baryon states produced in e{sup +}e{sup -} annihilations at or near a center-of-mass energy of 10.58 GeV, in a data sample with an integrated luminosity of 384 fb{sup -1} recorded with the BABAR detector at the PEP-II e{sup +}e{sup -} storage rings at the Stanford Linear Accelerator Center. We study strong decays of charm-strange baryons to {lambda}{sub c}{sup +}K{sub S}{sup 0}, {lambda}{sub c}{sup +}K{sup -}, {lambda}{sub c}{sup +}K{sup -}{pi}{sup +}, {lambda}{sub c}{sup +}K{sub S}{sup 0}{pi}{sup -}, {lambda}{sub c}{sup +}K{sub S}{sup 0}{pi}{sup -}{pi}{sup +}, and {lambda}{sub c}{sup +}K{sup -}{pi}{sup +}{pi}{sup -}. This study confirmsmore » the existence of the states {xi}{sub c}(2980){sup +}, {xi}{sub c}(3077){sup +}, and {xi}{sub c}(3077){sup 0}, with a more accurate determination of the {xi}{sub c}(2980){sup +} mass and width. We also present evidence for two new states, {xi}{sub c}(3055){sup +} and {xi}{sub c}(3123){sup +}, decaying through the intermediate-resonant modes {sigma}{sub c}(2455){sup ++}K{sup -} and {sigma}{sub c}(2520){sup ++}K{sup -}, respectively. For each of these baryons, we measure the yield in each final state, determine the statistical significance, and calculate the product of the production cross section and branching fractions. We also measure the masses and widths of these excited charm-strange baryons.« less

Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from pseudomonas sp. strain E-3

PubMed

Okuyama; Ueno; Enari; Morita; Kusano

1998-01-01

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 &mgr;mol h-1 (mg protein)-1 and a Km of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7-8 and 30 degrees C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as alpha-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1, 10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed.

A viable method and configuration for fermenting biomass sugars to ethanol using native Saccharomyces cerevisiae.

PubMed

Yuan, Dawei; Rao, Kripa; Varanasi, Sasidhar; Relue, Patricia

2012-08-01

A system that incorporates a packed bed reactor for isomerization of xylose and a hollow fiber membrane fermentor (HFMF) for sugar fermentation by yeast was developed for facile recovery of the xylose isomerase enzyme pellets and reuse of the cartridge loaded with yeast. Fermentation of pre-isomerized poplar hydrolysate produced using ionic liquid pretreatment in HFMF resulted in ethanol yields equivalent to that of model sugar mixtures of xylose and glucose. By recirculating model sugar mixtures containing partially isomerized xylose through the packed bed and the HFMF connected in series, 39 g/l ethanol was produced within 10h with 86.4% xylose utilization. The modular nature of this configuration has the potential for easy scale-up of the simultaneous isomerization and fermentation process without significant capital costs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation and Characterization of the cis-trans-Unsaturated Fatty Acid Isomerase of Pseudomonas oleovorans GPo12

PubMed Central

Pedrotta, Valerian; Witholt, Bernard

1999-01-01

Pseudomonas oleovorans contains an isomerase which catalyzes the cis-trans conversion of the abundant unsaturated membrane fatty acids 9-cis-hexadecenoic acid (palmitoleic acid) and 11-cis-octadecenoic acid (vaccenic acid). We purified the isomerase from the periplasmic fraction of Pseudomonas oleovorans. The molecular mass of the enzyme was estimated to be 80 kDa under denaturing conditions and 70 kDa under native conditions, suggesting a monomeric structure of the active enzyme. N-terminal sequencing showed that the isomerase derives from a precursor with a signal sequence which is cleaved from the primary translation product in accord with the periplasmic localization of the enzyme. The purified isomerase acted only on free unsaturated fatty acids and not on esterified fatty acids. In contrast to the in vivo cis-trans conversion of lipids, this in vitro isomerization of free fatty acids did not require the addition of organic solvents. Pure phospholipids, even in the presence of organic solvents, could not serve as substrate for the isomerase. However, when crude membranes from Pseudomonas or Escherichia coli cells were used as phospholipid sources, a cis-trans isomerization was detectable which occurred only in the presence of organic solvents. These results indicate that isolated membranes from Pseudomonas or E. coli cells must contain factors which, activated by the addition of organic solvents, enable and control the cis-trans conversion of unsaturated acyl chains of membrane phospholipids by the periplasmic isomerase. PMID:10322030

Fermentation of xylose to ethanol by genetically modified enteric bacteria

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

Tolan, J.S.

1987-01-01

This thesis describes the fermentation of D-xylose by wild type and recombinant Klebsiella planticola ATCC 33531 and Erwinia chrysanthemi B374. The recombinant strains bear multi-copy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene in K. planticola markedly increased the yield of ethanol, up to 1.3 mole/mole xylose, or 25.1 g/L. Concurrently, there were significant decreases in the yields of formation acetate, lactate, and butanediol. Transconjugant Klebsiella grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even inmore » the absence of selective pressure by antibiotics to maintain the plasmid. The cells produced 31.6 g/L ethanol from 79.6 g/L of a D-glucose-D-xylose-L-arabinose mixture designed to simulate hydrolyzed hemicellulose. The physiology of the wild type K. planticola is described in more detail than in the original report of its isolation. E. chrysanthemi PDC transconjugants also produced ethanol in high yield (up to 1.45 mole/mole xylose). However, transconjugant E. chrysanthemi grew only 1/4 as rapidly as the wild type and tolerated only 2% ethanol. The plasmid PZM15 apparently exhibits pleiotropic effects when inserted into K. planticola and into E. chrysanthemi.« less

Deep eutectic solvent and inorganic salt pretreatment of lignocellulosic biomass for improving xylose recovery.

PubMed

Loow, Yu-Loong; Wu, Ta Yeong; Yang, Ge Hoa; Ang, Lin Yang; New, Eng Kein; Siow, Lee Fong; Md Jahim, Jamaliah; Mohammad, Abdul Wahab; Teoh, Wen Hui

2018-02-01

Deep eutectic solvents (DESs) have received considerable attention in recent years due to their low cost, low toxicity, and biodegradable properties. In this study, a sequential pretreatment comprising of a DES (choline chloride:urea in a ratio of 1:2) and divalent inorganic salt (CuCl 2 ) was evaluated, with the aim of recovering xylose from oil palm fronds (OPF). At a solid-to-liquid ratio of 1:10 (w/v), DES alone was ineffective in promoting xylose extraction from OPF. However, a combination of DES (120°C, 4h) and 0.4mol/L of CuCl 2 (120°C, 30min) resulted in a pretreatment hydrolysate containing 14.76g/L of xylose, remarkably yielding 25% more xylose than the CuCl 2 -only pretreatment (11.87g/L). Characterization studies such as FE-SEM, BET, XRD, and FTIR confirmed the delignification of OPF when DES was implemented. Thus, the use of this integrated pretreatment system enabled xylose recoveries which were comparable with other traditional pretreatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

PubMed

Matsushika, Akinori; Hoshino, Tamotsu

2015-12-01

The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

Enhanced Furfural Yields from Xylose Dehydration in the gamma-Valerolactone/Water Solvent System at Elevated Temperatures.

PubMed

Sener, Canan; Motagamwala, Ali Hussain; Alonso, David Martin; Dumesic, James

2018-05-18

High yields of furfural (>90%) were achieved from xylose dehydration in a sustainable solvent system composed of -valerolactone (GVL), a biomass derived solvent, and water. It is identified that high reaction temperatures (e.g., 498 K) are required to achieve high furfural yield. Additionally, it is shown that the furfural yield at these temperatures is independent of the initial xylose concentration, and high furfural yield is obtained for industrially relevant xylose concentrations (10 wt%). A reaction kinetics model is developed to describe the experimental data obtained with solvent system composed of 80 wt% GVL and 20 wt% water across the range of reaction conditions studied (473 - 523 K, 1-10 mM acid catalyst, 66 - 660 mM xylose concentration). The kinetic model demonstrates that furfural loss due to bimolecular condensation of xylose and furfural is minimized at elevated temperature, whereas carbon loss due to xylose degradation increases with increasing temperature. Accordingly, the optimal temperature range for xylose dehydration to furfural in the GVL/H2O solvent system is identified to be from 480 to 500 K. Under these reaction conditions, furfural yield of 93% is achieved at 97% xylan conversion from lignocellulosic biomass (maple wood). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production.

PubMed

Chaves, Julie E; Romero, Paloma Rueda; Kirst, Henning; Melis, Anastasios

2016-12-01

Heterologous production of isoprene (C 5 H 8 ) hydrocarbons in cyanobacteria, emanating from sunlight, CO 2 , and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.

Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.

PubMed

Reznicek, O; Facey, S J; de Waal, P P; Teunissen, A W R H; de Bont, J A M; Nijland, J G; Driessen, A J M; Hauer, B

2015-07-01

Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass. © 2015 The Society for Applied Microbiology.

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

PubMed

Kim, P; Yoon, S H; Roh, H J; Choi, J H

2001-01-01

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose.

PubMed

Nichols, Nancy N; Saha, Badal C

2016-05-01

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical-chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5-hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild-type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606-612, 2016. © 2016 American Institute of Chemical Engineers.

Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

PubMed

Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

2016-07-01

Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

Bacterial L-arabinose isomerases: industrial application for D-tagatose production.

PubMed

Boudebbouze, Samira; Maguin, Emmanuelle; Rhimi, Moez

2011-12-01

D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.

Observation of B{sup +}{yields}{xi}{sub c}{sup 0}{lambda}{sub c}{sup +} and evidence for B{sup 0}{yields}{xi}{sub c}{sup -}{lambda}{sub c}{sup +}

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

Chistov, R.; Aushev, T.; Balagura, V.

We report the first observation of the decay B{sup +}{yields}{xi}{sub c}{sup 0}{lambda}{sub c}{sup +} with a significance of 8.7{sigma} and evidence for the decay B{sup 0}{yields}{xi}{sub c}{sup -}{lambda}{sub c}{sup +} with a significance of 3.8{sigma}. The product B(B{sup +}{yields}{xi}{sub c}{sup 0}{lambda}{sub c}{sup +})xB({xi}{sub c}{sup 0}{yields}{xi}{sup +}{pi}{sup -}) is measured to be (4.8{sub -0.9}{sup +1.0}{+-}1.1{+-}1.2)x10{sup -5}, and B(B{sup 0}{yields}{xi}{sub c}{sup -}{lambda}{sub c}{sup +})xB({xi}{sub c}{sup -}{yields}{xi}{sup +}{pi}{sup -}{pi}{sup -}) is measured to be (9.3{sub -2.8}{sup +3.7}{+-}1.9{+-}2.4)x10{sup -5}. The errors are statistical, systematic and the error of the {lambda}{sub c}{sup +}{yields}pK{sup -}{pi}{sup +} branching fraction, respectively. The decay B{sup +}{yields}{xi}{sub c}{sup 0}{lambda}{sub c}{supmore » +} is the first example of a two-body exclusive B{sup +} decay into two charmed baryons. The data used for this analysis was accumulated at the {upsilon}(4S) resonance, using the Belle detector at the e{sup +}e{sup -} asymmetric-energy collider KEKB. The integrated luminosity of the data sample is equal to 357 fb{sup -1}, corresponding to 386x10{sup 6} BB pairs.« less

Methods of measuring Protein Disulfide Isomerase activity: a critical overview

NASA Astrophysics Data System (ADS)

Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

2014-09-01

Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase.

PubMed

Mishra, Abha; Debnath Das, Meera

2002-01-01

pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60 degrees C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.

MuPeXI: prediction of neo-epitopes from tumor sequencing data.

PubMed

Bjerregaard, Anne-Mette; Nielsen, Morten; Hadrup, Sine Reker; Szallasi, Zoltan; Eklund, Aron Charles

2017-09-01

Personalization of immunotherapies such as cancer vaccines and adoptive T cell therapy depends on identification of patient-specific neo-epitopes that can be specifically targeted. MuPeXI, the mutant peptide extractor and informer, is a program to identify tumor-specific peptides and assess their potential to be neo-epitopes. The program input is a file with somatic mutation calls, a list of HLA types, and optionally a gene expression profile. The output is a table with all tumor-specific peptides derived from nucleotide substitutions, insertions, and deletions, along with comprehensive annotation, including HLA binding and similarity to normal peptides. The peptides are sorted according to a priority score which is intended to roughly predict immunogenicity. We applied MuPeXI to three tumors for which predicted MHC-binding peptides had been screened for T cell reactivity, and found that MuPeXI was able to prioritize immunogenic peptides with an area under the curve of 0.63. Compared to other available tools, MuPeXI provides more information and is easier to use. MuPeXI is available as stand-alone software and as a web server at http://www.cbs.dtu.dk/services/MuPeXI .

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state.

PubMed

Hoshida, Hisashi; Kidera, Kenta; Takishita, Ryuta; Fujioka, Nobuhisa; Fukagawa, Taiki; Akada, Rinji

2018-06-01

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K. marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Directed evolution reveals unexpected epistatic interactions that alter metabolic regulation and enable anaerobic xylose use by Saccharomyces cerevisiae

DOE PAGES

Sato, Trey K.; Tremaine, Mary; Parreiras, Lucas S.; ...

2016-10-14

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactionsmore » among genes encoding a xylose reductase ( GRE3), a component of MAP Kinase (MAPK) signaling ( HOG1), a regulator of Protein Kinase A (PKA) signaling ( IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis ( ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Lastly, our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.« less

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

PubMed

Sato, Trey K; Tremaine, Mary; Parreiras, Lucas S; Hebert, Alexander S; Myers, Kevin S; Higbee, Alan J; Sardi, Maria; McIlwain, Sean J; Ong, Irene M; Breuer, Rebecca J; Avanasi Narasimhan, Ragothaman; McGee, Mick A; Dickinson, Quinn; La Reau, Alex; Xie, Dan; Tian, Mingyuan; Reed, Jennifer L; Zhang, Yaoping; Coon, Joshua J; Hittinger, Chris Todd; Gasch, Audrey P; Landick, Robert

2016-10-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae

PubMed Central

Tremaine, Mary; Hebert, Alexander S.; Myers, Kevin S.; Sardi, Maria; Dickinson, Quinn; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert

2016-01-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. PMID:27741250

21 CFR 862.1570 - Phosphohexose isomerase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Phosphohexose isomerase test system. 862.1570 Section 862.1570 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...

Diversity and Fermentation Products of Xylose-Utilizing Yeasts Isolated from Buffalo Feces in Thailand

PubMed Central

Lorliam, Wanlapa; Akaracharanya, Ancharida; Suzuki, Motofumi; Ohkuma, Moriya; Tanasupawat, Somboon

2013-01-01

Twenty-eight xylose-utilizing yeast strains were isolated by enrichment culture from 11 samples of feces from the rectum of Murrah buffalo and Swamp buffalo in Thailand. On the basis of their morphological and biochemical characteristics, including sequence analysis of the D1/D2 region of the large-subunit ribosomal RNA gene (LSU rDNA), they were identified as Candida tropicalis (designated as Group I, 11 isolates), Candida parasilosis (Group II, 2 isolates), Candida mengyuniae (Group III, 2 isolates), Sporopachydermia lactativora (Group IV, 2 isolates), Geotrichum sp. (Group V, 5 isolates) and Trichosporon asahii (Group VI, 6 isolates). All isolates utilized xylose as the sole carbon source but 27 isolates could ferment xylose to ethanol (0.006–0.602 g L−1) and 21 isolates could ferment xylose to xylitol (0.19–22.84 g L−1). Candida tropicalis isolates produced the highest yield of xylitol (74.80%). Their ability to convert xylose to xylitol and ethanol ranged from 15.06 g L−1 to 22.84 g L−1 xylitol and 0.110 g L−1 to 0.602 g L−1 ethanol, respectively. PMID:24005843

Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

PubMed

Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

2014-12-01

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

The Influence of Sugar Cane Bagasse Type and Its Particle Size on Xylose Production and Xylose-to-Xylitol Bioconversion with the Yeast Debaryomyces hansenii.

PubMed

Aghcheh, Razieh Karimi; Bonakdarpour, Babak; Ashtiani, Farzin Zokaee

2016-11-01

In the present study, the effect of the type of sugar cane bagasse (non-depithed or depithed) and its particle size on the production of xylose and its subsequent fermentation to xylitol by Debaryomyces hansenii CBS767 was investigated using a full factorial experimental design. It was found that the particle size range and whether bagasse was depithed or not had a significant effect on the concentration and yield of xylose in the resulting hemicellulose hydrolysate. Depithed bagasse resulted in higher xylose concentrations compared to non-depithed bagasse. The corresponding detoxified hemicellulose hydrolysates were used as fermentation media for the production of xylitol. The hemicellulose hydrolysate prepared from depithed bagasse also yielded meaningfully higher xylitol fermentation rates compared to non-depithed bagasse. However, in the case of non-depithed bagasse, the hemicellulose hydrolysate prepared from larger particle size range resulted in higher xylitol fermentation rates, whereas the effect in the case of non-depithed bagasse was not pronounced. Therefore, depithing of bagasse is an advantageous pretreatment when it is to be employed in bioconversion processes.

Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose

PubMed Central

2014-01-01

Background There has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis. Results In MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose. Conclusions Our transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

PubMed Central

Wei, Na; Xu, Haiqing; Kim, Soo Rin

2013-01-01

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD+/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation. PMID:23475614

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

PubMed

Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

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

USDA-ARS?s Scientific Manuscript database

Background: The limited xylose utilizing ability of native Saccharomyces cerevisiae has been a major obstacle for efficient cellulosic ethanol production from lignocellulosic materials. Haploid laboratory strains of S. cerevisiae are commonly used for genetic engineering to enable its xylose utiliza...

Fermentation of D-xylose and L-arabinose to ethanol by Erwinia chrysanthemi

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

Tolan, J.S.; Finn, R.K.

1987-09-01

Erwinia spp. are gram-negative facultative anaerobes within the family Enterobacteriacae which possess several desirable traits for the conversion of pentose sugars to ethanol, such as the ability to ferment a broad range of carbohydrates and the ease with which they can be genetically modified. Twenty-eight strains of Erwinia carotovora and E. chrysanthemi were screened for the ability to ferment D-xylose to ethanol. E. chrysanthemi B374 was chosen for further study on the basis of its superior (4%) ethanol tolerance. They have characterized the fermentation of D-xylose and L-arabinose by the wild type and mutants which bear plasmids containing the pyruvatemore » decarboxylase gene from Zymomonas mobilis. Expression of the gene markedly increased the yields of ethanol (from 0.7 up to 1.45 mol/mol of xylose) and decreased the yields of formate, acetate, and lactate. However, the cells with pyruvate decarboxylase grew only one-fourth as fast as the wild type and tolerated only 2% ethanol. Alcohol tolerance was stimulated by the addition of yeast extract to the growth medium. Xylose catabolism was characterized by a high saturation constant K/sub s/ (4.5 mM).« less

Aristolochic acid-related nephropathy associated with the popular Chinese herb Xi Xin.

PubMed

Yang, Huang-Yu; Lin, J-L; Chen, Kuan-Hsing; Yu, Chun-Chen; Hsu, Po-Yaur; Lin, Chun-Liang

2006-01-01

Chinese herbs nephropathy is known as a subacute interstitial nephritis attributed to aristolochic acid. This work describes the case of a 49-year-old male who displayed subacute renal failure induced by ingestion of herbal powder containing Xi Xin, which includes aristolochic acid. Since Xi Xin is a common ingredient in traditional formulae, care needs to be taken in the future to identify the aristolochic acid concentration of different components of Xi Xin. Xi Xin containing aristolochic acid should be forbidden for use in remedies in order to prevent the harmful effects of aristolochic acid.

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

Introduction of unnatural amino acids into chalcone isomerase.

PubMed

Bednar, R A; McCaffrey, C; Shan, K

1991-01-01

The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denatured and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cysteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cysteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2',4',4-trihydroxychalcone, 2',4',6',4-tetrahydroxychalcone, or 2'-hydroxy-4-methoxychalcone as substrates. The competitive inhibition constant for 4',4-dihydroxychalcone, the substrate inhibition constant for 2',4',4-trihydroxychalcone, and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4',4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.

Cofermentation of Glucose, Xylose, and Cellobiose by the Beetle-Associated Yeast Spathaspora passalidarum

Treesearch

Tanya M. Long; Yi-Kai Su; Jennifer Headman; Alan Higbee; Laura B. Willis; Thomas W. Jeffries

2012-01-01

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most...

Donor motivation in Xi'an, China: comparison with Canadian donors.

PubMed

O'Brien, S F; Shao, Z-J; Osmond, L; Yi, Q-L; Li, C-Y; An, Q-X

2013-04-01

In China, paid donation is prohibited by law. There is little literature assessing donor motivation in China, and comparison with western countries such as Canada is important in understanding the application of Western literature. We compared motivational factors in donors from the city of Xi'an, China, with Canadian donors matched for age, sex and donation status. A total of 218 donors in Xi'an completed an interview about motivation as did 218 Canadian donors matched for age, sex and donation status. Frequencies and percentages of responses to questions were tabulated and compared using the Chi-squared test. Donors in Xi'an and Canada felt a personal responsibility to donate blood (81·2% vs. 78·0%, P = 0·2057), but Xi'an donors were more likely to consider blood donation a social responsibility (81·7% vs. 45·2%, P < 0·0001). Xi'an donors more often believed that society views donation as a normal activity (98·6% vs. 48·4%, P < 0·0001) and that the social atmosphere promotes donation (90·3% vs. 53·5%, P < 0·0001) and saw greater health benefit (52·3% vs. 12·5%, P < 0·0001). Most Xi'an donors believed in balance between their life force (Qi) and blood (86·7% vs. 49·8%, P < 0·0001) but did not believe blood lost from donating would affect this (0·5% vs. 3·8%, P = 0·01). While traditional Chinese beliefs may not be seen as a barrier among people in Xi'an who donate blood, blood donation is seen differently than by Canadian donors. There is a need for more research specific to China to tailor recruitment strategies. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

Metabolic Engineering of Escherichia coli K12 for Homofermentative Production of L-Lactate from Xylose.

PubMed

Jiang, Ting; Zhang, Chen; He, Qin; Zheng, Zhaojuan; Ouyang, Jia

2018-02-01

The efficient utilization of xylose is regarded as a technical barrier to the commercial production of bulk chemicals from biomass. Due to the desirable mechanical properties of polylactic acid (PLA) depending on the isomeric composition of lactate, biotechnological production of lactate with high optical pure has been increasingly focused in recent years. The main objective of this work was to construct an engineered Escherichia coli for the optically pure L-lactate production from xylose. Six chromosomal deletions (pflB, ldhA, ackA, pta, frdA, adhE) and a chromosomal integration of L-lactate dehydrogenase-encoding gene (ldhL) from Bacillus coagulans was involved in construction of E. coli KSJ316. The recombinant strain could produce L-lactate from xylose resulting in a yield of 0.91 g/g xylose. The chemical purity of L-lactate was 95.52%, and the optical purity was greater than 99%. Moreover, three strategies, including overexpression of L-lactate dehydrogenase, intensification of xylose catabolism, and addition of additives to medium, were designed to enhance the production. The results showed that they could increase the concentration of L-lactate by 32.90, 20.13, and 233.88% relative to the control, respectively. This was the first report that adding formate not only could increase the xylose utilization but also led to the fewer by-product levels.

Separating xylose from glucose using spiral wound nanofiltration membrane: Effect of cross-flow parameters on sugar rejection

NASA Astrophysics Data System (ADS)

Roli, N. F. M.; Yussof, H. W.; Seman, M. N. A.; Saufi, S. M.; Mohammad, A. W.

2016-11-01

A solution model consisted of two different monosaccharides namely xylose and glucose were separated using a pilot scale spiral wound cross-flow system. This system was equipped by a commercial spiral wound nanofiltration (NF) membrane, Desal-5 DK, having a molecular weight cut off (MWCO) of 150-300 g mol-1. The aim of this present work is to investigate the effect of the cross-flow parameters: the trans-membrane pressure (TMP) and the feed concentration (C0) on the xylose separation from glucose. The filtration experiments were carried out in total reflux mode with different feed concentration of 2, 5, and 10 g/L at different TMP of 5,8 and 10 bar. The performances of the NF membrane were evaluated by measuring the permeate flux and sugar rejection for each experiment. All the samples were quantified using a high performance liquid chromatography equipped by a fractive index detector. The experimental results indicated an increase in pressure from 5 to 10 bar which was a notable increase to the permeate fluxes from 2.66 × 10-3 to 4.14 × 10-3L m-2s-1. Meanwhile, an increase in the C0 increases the xylose rejection. At TMP of 10 bar and C0 of 5 g/L, the observed xylose rejection and glucose rejection were measured at 67.19% and 91.82%, respectively. The lower rejection in xylose than glucose suggested that larger glucose molecule were not able to easily pass through the membrane compared to the smaller xylose molecule. The results of this phenomena proved that NF with spiral wound configuration has the potential to separate xylose from glucose, which is valuable to the purification of xylose in xylose production as an alternative to chromatographic processes.

Enhancement of xylose utilization from corn stover by a recombinant bacterium for ethanol production

USDA-ARS?s Scientific Manuscript database

Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia ...

A synthetic hybrid promoter for xylose-regulated control of gene expression in Saccharomyces yeasts

USDA-ARS?s Scientific Manuscript database

Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DN...

The effect of initial cell concentration on xylose fermentation by Pichia stipitis

Treesearch

Frank K. Agbogbo; Guillermo Coward-Kelly; Mads Torry-Smith; Kevin Wenger; Thomas W. Jeffries

2007-01-01

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was...

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.

xiSPEC: web-based visualization, analysis and sharing of proteomics data.

PubMed

Kolbowski, Lars; Combe, Colin; Rappsilber, Juri

2018-05-08

We present xiSPEC, a standard compliant, next-generation web-based spectrum viewer for visualizing, analyzing and sharing mass spectrometry data. Peptide-spectrum matches from standard proteomics and cross-linking experiments are supported. xiSPEC is to date the only browser-based tool supporting the standardized file formats mzML and mzIdentML defined by the proteomics standards initiative. Users can either upload data directly or select files from the PRIDE data repository as input. xiSPEC allows users to save and share their datasets publicly or password protected for providing access to collaborators or readers and reviewers of manuscripts. The identification table features advanced interaction controls and spectra are presented in three interconnected views: (i) annotated mass spectrum, (ii) peptide sequence fragmentation key and (iii) quality control error plots of matched fragments. Highlighting or selecting data points in any view is represented in all other views. Views are interactive scalable vector graphic elements, which can be exported, e.g. for use in publication. xiSPEC allows for re-annotation of spectra for easy hypothesis testing by modifying input data. xiSPEC is freely accessible at http://spectrumviewer.org and the source code is openly available on https://github.com/Rappsilber-Laboratory/xiSPEC.

Search for Exotic Baryons in 800 GeV pp{yields}p{xi}{sup {+-}}{pi}{sup {+-}}X Reactions

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

Christian, D.C.; Gottschalk, E.E.; Gutierrez, G.

We report the results of a high-statistics, sensitive search for narrow baryon resonances decaying to {xi}{sup -}{pi}{sup -}, {xi}{sup -}{pi}{sup +}, {xi}{sup +}{pi}{sup -}, and {xi}{sup +}{pi}{sup +}. The only resonances observed are the well known {xi}{sup 0}(1530) and {xi}{sup 0}(1530). No evidence is found for the states near 1862 MeV, previously reported by NA49 [Phys. Rev. Lett. 92, 042003 (2003)]. At the 95% confidence level, we find the upper limit for the production of a Gaussian enhancement with {sigma}=7.6 MeV in the {xi}{sup -}{pi}{sup -} effective mass spectrum to be 0.3% of the number of observed {xi}{sup 0}(1530){yields}{xi}{sup -}{pi}{supmore » +}. We find similarly restrictive upper limits for an enhancement at 1862 MeV in the {xi}{sup -}{pi}{sup +}, {xi}{sup +}{pi}{sup -}, and {xi}{sup +}{pi}{sup +} mass spectra.« less

The Da Vinci Xi and robotic radical prostatectomy-an evolution in learning and technique.

PubMed

Goonewardene, S S; Cahill, D

2017-06-01

The da Vinci Xi robot has been introduced as the successor to the Si platform. The promise of the Xi is to open the door to new surgical procedures. For robotic-assisted radical prostatectomy (RARP)/pelvic surgery, the potential is better vision and longer instruments. How has the Xi impacted on operative and pathological parameters as indicators of surgical performance? This is a comparison of an initial series of 42 RARPs with the Xi system in 2015 with a series using the Si system immediately before Xi uptake in the same calendar year, and an Si series by the same surgeon synchronously as the Xi series using operative time, blood loss, and positive margins as surrogates of surgical performance. Subjectively and objectively, there is a learning curve to Xi uptake in longer operative times but no impact on T2 positive margins which are the most reflective single measure of RARP outcomes. Subjectively, the vision of the Xi is inferior to the Si system, and the integrated diathermy system and automated setup are quirky. All require experience to overcome. There is a learning curve to progress from the Si to Xi da Vinci surgical platforms, but this does not negatively impact the outcome.

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: Construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

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

Street, I.P.; Poulter, C.D.

1990-08-14

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinantmore » IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with (4-{sup 3}H)FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.« less

Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

PubMed

Salusjärvi, Laura; Kaunisto, Sanna; Holmström, Sami; Vehkomäki, Maija-Leena; Koivuranta, Kari; Pitkänen, Juha-Pekka; Ruohonen, Laura

2013-12-01

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

Interaction of p53 with prolyl isomerases: Healthy and unhealthy relationships.

PubMed

Mantovani, Fiamma; Zannini, Alessandro; Rustighi, Alessandra; Del Sal, Giannino

2015-10-01

The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic Analysis of the Secretory Response of Aspergillus niger to D-Maltose and D-Xylose

PubMed Central

Ferreira de Oliveira, José Miguel P.; van Passel, Mark W. J.; Schaap, Peter J.; de Graaff, Leo H.

2011-01-01

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on d-sorbitol, small amounts of d-maltose or d-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by d-maltose or d-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on d-maltose and β-xylosidase D on d-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra d-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of d-xylose or d-maltose. Furthermore, d-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers d-maltose and d-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by d-maltose or d-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for d-xylose induction, d-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation. PMID:21698107

Proteomic analysis of the secretory response of Aspergillus niger to D-maltose and D-xylose.

PubMed

de Oliveira, José Miguel P Ferreira; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2011-01-01

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and β-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Co-Utilization of Glucose and Xylose for Enhanced Lignocellulosic Ethanol Production with Reverse Membrane Bioreactors

PubMed Central

Ishola, Mofoluwake M.; Ylitervo, Päivi; Taherzadeh, Mohammad J.

2015-01-01

Integrated permeate channel (IPC) flat sheet membranes were examined for use as a reverse membrane bioreactor (rMBR) for lignocellulosic ethanol production. The fermenting organism, Saccharomyces cerevisiae (T0936), a genetically-modified strain with the ability to ferment xylose, was used inside the rMBR. The rMBR was evaluated for simultaneous glucose and xylose utilization as well as in situ detoxification of furfural and hydroxylmethyl furfural (HMF). The synthetic medium was investigated, after which the pretreated wheat straw was used as a xylose-rich lignocellulosic substrate. The IPC membrane panels were successfully used as the rMBR during the batch fermentations, which lasted for up to eight days without fouling. With the rMBR, complete glucose and xylose utilization, resulting in 86% of the theoretical ethanol yield, was observed with the synthetic medium. Its application with the pretreated wheat straw resulted in complete glucose consumption and 87% xylose utilization; a final ethanol concentration of 30.3 g/L was obtained, which corresponds to 83% of the theoretical yield. Moreover, complete in situ detoxification of furfural and HMF was obtained within 36 h and 60 h, respectively, with the rMBR. The use of the rMBR is a promising technology for large-scale lignocellulosic ethanol production, since it facilitates the co-utilization of glucose and xylose; moreover, the technology would also allow the reuse of the yeast for several batches. PMID:26633530

Homo-D-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum.

PubMed

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

2011-10-01

In order to achieve efficient D-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-D-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of D-lactic acid of 99.5%. Finally, we successfully demonstrated homo-D-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-D-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

Xylose fermentation to ethanol by new Galactomyces geotrichum and Candida akabanensis strains.

PubMed

Valinhas, Raquel V; Pantoja, Lílian A; Maia, Ana Carolina F; Miguel, Maria Gabriela C P; Vanzela, Ana Paula F C; Nelson, David L; Santos, Alexandre S

2018-01-01

The conversion of pentoses into ethanol remains a challenge and could increase the supply of second-generation biofuels. This study sought to isolate naturally occurring yeasts from plant biomass and determine their capabilities for transforming xylose into ethanol. Three yeast strains with the ability to ferment xylose were isolated from pepper, tomato and sugarcane bagasse. The strains selected were characterized by morphological and auxanographic assays, and they were identified by homology analysis of 5.8 S and 26 S ribosomal RNA gene sequences. The identities of two lineages of microrganism were associated with Galactomyces geotrichum , and the other was associated with Candida akabanensis . Fermentative processes were conducted with liquid media containing only xylose as the carbon source. Y P/S values for the production of ethanol ranging between 0.29 and 0.35 g g -1 were observed under non-optimized conditions.

Structural analysis of N-linked carbohydrate chains of funnel web spider (Agelenopsis aperta) venom peptide isomerase.

PubMed

Shikata, Y; Ohe, H; Mano, N; Kuwada, M; Asakawa, N

1998-06-01

The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose alpha 1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

PubMed

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

Da Vinci Xi Robot–Assisted Penetrating Keratoplasty

PubMed Central

Chammas, Jimmy; Sauer, Arnaud; Pizzuto, Joëlle; Pouthier, Fabienne; Gaucher, David; Marescaux, Jacques; Mutter, Didier; Bourcier, Tristan

2017-01-01

Purpose This study aims (1) to investigate the feasibility of robot-assisted penetrating keratoplasty (PK) using the new Da Vinci Xi Surgical System and (2) to report what we believe to be the first use of this system in experimental eye surgery. Methods Robot-assisted PK procedures were performed on human corneal transplants using the Da Vinci Xi Surgical System. After an 8-mm corneal trephination, four interrupted sutures and one 10.0 monofilament running suture were made. For each procedure, duration and successful completion of the surgery as well as any unexpected events were assessed. The depth of the corneal sutures was checked postoperatively using spectral-domain optical coherence tomography (SD-OCT). Results Robot-assisted PK was successfully performed on 12 corneas. The Da Vinci Xi Surgical System provided the necessary dexterity to perform the different steps of surgery. The mean duration of the procedures was 43.4 ± 8.9 minutes (range: 28.5–61.1 minutes). There were no unexpected intraoperative events. SD-OCT confirmed that the sutures were placed at the appropriate depth. Conclusions We confirm the feasibility of robot-assisted PK with the new Da Vinci Surgical System and report the first use of the Xi model in experimental eye surgery. Operative time of robot-assisted PK surgery is now close to that of conventional manual surgery due to both improvement of the optical system and the presence of microsurgical instruments. Translational Relevance Experimentations will allow the advantages of robot-assisted microsurgery to be identified while underlining the improvements and innovations necessary for clinical use. PMID:28660096

Da Vinci Xi Robot-Assisted Penetrating Keratoplasty.

PubMed

Chammas, Jimmy; Sauer, Arnaud; Pizzuto, Joëlle; Pouthier, Fabienne; Gaucher, David; Marescaux, Jacques; Mutter, Didier; Bourcier, Tristan

2017-06-01

This study aims (1) to investigate the feasibility of robot-assisted penetrating keratoplasty (PK) using the new Da Vinci Xi Surgical System and (2) to report what we believe to be the first use of this system in experimental eye surgery. Robot-assisted PK procedures were performed on human corneal transplants using the Da Vinci Xi Surgical System. After an 8-mm corneal trephination, four interrupted sutures and one 10.0 monofilament running suture were made. For each procedure, duration and successful completion of the surgery as well as any unexpected events were assessed. The depth of the corneal sutures was checked postoperatively using spectral-domain optical coherence tomography (SD-OCT). Robot-assisted PK was successfully performed on 12 corneas. The Da Vinci Xi Surgical System provided the necessary dexterity to perform the different steps of surgery. The mean duration of the procedures was 43.4 ± 8.9 minutes (range: 28.5-61.1 minutes). There were no unexpected intraoperative events. SD-OCT confirmed that the sutures were placed at the appropriate depth. We confirm the feasibility of robot-assisted PK with the new Da Vinci Surgical System and report the first use of the Xi model in experimental eye surgery. Operative time of robot-assisted PK surgery is now close to that of conventional manual surgery due to both improvement of the optical system and the presence of microsurgical instruments. Experimentations will allow the advantages of robot-assisted microsurgery to be identified while underlining the improvements and innovations necessary for clinical use.

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2011-01-01

Background: Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetonebutanol- ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylosemore » substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion: Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.« less

Study of the Rare Hyperon Decay $${\\boldmath \\Omega^\\mp \\to \\Xi^\\mp \\: \\pi^+\\pi^-}$$

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

Kamaev, O.; Solomey, N.; Burnstein, R.A.

The authors report a new measurement of the decay {Omega}{sup -} {yields} {Xi}{sup -} {pi}{sup +}{pi}{sup -} with 76 events and a first observation of the decay {bar {Omega}}{sup +} {yields} {bar {Xi}}{sup +} {pi}{sup +}{pi}{sup -} with 24 events, yielding a combined branching ratio (3.74{sub -0.56}{sup +0.67}) x 10{sup -4}. This represents a factor 25 increase in statistics over the best previous measurement. No evidence is seen for CP violation, with {Beta}({Omega}{sup -} {yields} {Xi}{sup -} {pi}{sup +}{pi}{sup -}) = 4.04{sub -0.71}{sup +0.83} x 10{sup -4} and {Beta}({bar {Omega}}{sup +} {yields} {bar {Xi}}{sup +} {pi}{sup +}{pi}{sup -}) = 3.15{submore » -0.89}{sup +1.12} x 10{sup -4}. Contrary to theoretical expectation, they see little evidence for the decays {Omega}{sup -} {yields} {Xi}*{sub 1530}{sup 0} {pi}{sup -} and {bar {Omega}}{sup +} {yields} {bar {Xi}}*{sub 1530}{sup 0} {pi}{sup +} and place a 90% C.L. upper limit on the combined branching ratio {Beta}({Omega}{sup -}({bar {Omega}}{sup +}) {yields} {Xi}*{sub 1530}{sup 0} ({bar {Xi}}*{sub 1530}{sup 0}){pi}{sup {-+}}) < 7.0 x 10{sup -5}.« less

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.

Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B.

PubMed

Zhao, Jinfang; Xu, Liyuan; Wang, Yongze; Zhao, Xiao; Wang, Jinhua; Garza, Erin; Manow, Ryan; Zhou, Shengde

2013-06-07

Polylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing "white pollution". However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass. In this study, an ethanologenic Escherichia coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA), was reengineered for homofermentative production of L-lactic acid from xylose (1.2 mole xylose = > 2 mole L-lactic acid), by deleting the alcohol dehydrogenase gene (adhE) and integrating the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici. The resulting strain, WL203, was metabolically evolved further through serial transfers in screw-cap tubes containing xylose, resulting in the strain WL204 with improved anaerobic cell growth. When tested in 70 g L-1 xylose fermentation (complex medium), WL204 produced 62 g L-1 L-lactic acid, with a maximum production rate of 1.631 g L-1 h-1 and a yield of 97% based on xylose metabolized. HPLC analysis using a chiral column showed that an L-lactic acid optical purity of 99.5% was achieved by WL204. These results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates, which contain a

Small intestinal malabsorption in chronic alcoholism: a retrospective study of alcoholic patients by the ¹⁴C-D-xylose breath test.

PubMed

Hope, Håvar; Skar, Viggo; Sandstad, Olav; Husebye, Einar; Medhus, Asle W

2012-04-01

The ¹⁴C-D-xylose breath test was used at Ullevål University Hospital in the period from 1986 TO 1995 for malabsorption testing. The objective of this retrospective study was to reveal whether patients with chronic alcoholism may have intestinal malabsorption. The consecutive ¹⁴C-D-xylose breath test database was reviewed and patients with the diagnosis of chronic alcoholism were identified. ¹⁴C-D-xylose breath test results of the alcoholic patients were compared with the results of untreated celiac patients and patient and healthy controls. In the ¹⁴C-D-xylose breath test, ¹⁴C-D-xylose was dissolved in water and given orally after overnight fast. Breath samples were taken at 30-min intervals for 210 min, and ¹⁴CO₂ : ¹²CO₂ ratios were calculated for each time point, presenting a time curve for ¹⁴C-D-xylose absorption. Urine was collected after 210 min and the fraction of the total d-xylose passed was calculated (U%). ¹⁴CO₂ in breath and ¹⁴C-D-xylose in urine were analyzed using liquid scintillation. Both breath and urine analysis revealed a pattern of malabsorption in alcoholics comparable with untreated celiac patients, with significantly reduced absorption of d-xylose compared with patient and healthy controls. Alcoholic patients have a significantly reduced ¹⁴C-D-xylose absorption, comparable with untreated celiac patients. This indicates a reduced intestinal function in chronic alcoholism.

Rapid increase of ozone concentrations in Xi'an, China: Anthropogenically or naturally?

NASA Astrophysics Data System (ADS)

Wu, J.; Li, G.; Junji, C.

2017-12-01

The air quality in the Guanzhong basin, China has deteriorated recently caused by growing industries, city expansions, and increasing transportation activity. We report here a substantial increasing trend of ozone (O3) concentrations in Xi'an, the largest city of the basin, and the average observed O3 concentration in the afternoon during summertime has increased by 39% from 2013 to 2016. There are two main possible reasons for the rapid O3 increase. Motor vehicle has been reported to increase by 35% in Xi'an, which enhances the O3 precursors emissions to facilitate the O3 formation. In addition, the surface solar radiation at the meteorological site in Xi'an has been observed to intensify by 30%, which increases the photolysis rates to expedite the O3 production. A persistent high O3 episode from 16 to 22 June 2016 in Xi'an has been simulated using the WRF-CHEM model to evaluate the contribution of the transportation emission and solar radiation enhancement on the O3 trend. The model generally performs reasonably well in simulating the temporal variation and spatial distribution of near-surface O3 and NO2 concentrations against measurements in Xi'an. Sensitivity studies have revealed that the enhancement of transportation emissions and the solar radiation explains about 70% of the O3 trend from 2013 to 2016. Considering that large amounts of biogenic emissions are released over the Qinling Mountains on the south of Xi'an, which can be delivered to Xi'an under favorable meteorological conditions, enhancing O3 formation. Therefore, future studies need to be performed to evaluate impacts of the solar radiation enhancement on the biogenic emissions and further the O3 formation in Xi'an.

Oxidative production of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth by Gluconobacter oxydans.

PubMed

Zhang, Hongsen; Han, Xushen; Wei, Chengxiang; Bao, Jie

2017-01-01

An oxidative production process of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth was designed, experimentally investigated, and evaluated. Dry dilute acid pretreated and biodetoxified corn stover was simultaneously saccharified and fermented into 59.80g/L of ethanol (no xylose utilization). 65.39g/L of xylose was obtained in the distillation stillage without any concentrating step after ethanol was distillated. Then the xylose was completely converted into 66.42g/L of xylonic acid by Gluconobacter oxydans. The rigorous Aspen Plus modeling shows that the wastewater generation and energy consumption was significantly reduced comparing to the previous xylonic acid production process using xylose in pretreatment liquid. This study provided a practical process option for xylonic acid production from lignocellulose feedstock with significant reduction of wastewater and energy consumption. Copyright © 2016 Elsevier Ltd. All rights reserved.

Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

PubMed Central

Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.

2015-01-01

BACKGROUND Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that specifically reduces factor XI levels. We compared the efficacy and safety of FXI-ASO with those of enoxaparin in patients undergoing total knee arthroplasty. METHODS In this open-label, parallel-group study, we randomly assigned 300 patients who were undergoing elective primary unilateral total knee arthroplasty to receive one of two doses of FXI-ASO (200 mg or 300 mg) or 40 mg of enoxaparin once daily. The primary efficacy outcome was the incidence of venous thromboembolism (assessed by mandatory bilateral venography or report of symptomatic events). The principal safety outcome was major or clinically relevant nonmajor bleeding. RESULTS Around the time of surgery, the mean (±SE) factor XI levels were 0.38±0.01 units per milliliter in the 200-mg FXI-ASO group, 0.20±0.01 units per milliliter in the 300-mg FXI-ASO group, and 0.93±0.02 units per milliliter in the enoxaparin group. The primary efficacy outcome occurred in 36 of 134 patients (27%) who received the 200-mg dose of FXI-ASO and in 3 of 71 patients (4%) who received the 300-mg dose of FXI-ASO, as compared with 21 of 69 patients (30%) who received enoxaparin. The 200-mg regimen was noninferior, and the 300-mg regimen was superior, to enoxaparin (P<0.001). Bleeding occurred in 3%, 3%, and 8% of the patients in the three study groups, respectively. CONCLUSIONS This study showed that factor XI contributes to postoperative venous thromboembolism; reducing factor XI levels in patients undergoing elective primary unilateral total knee arthroplasty was an effective method for its prevention and appeared to be safe with respect to the risk of bleeding. (Funded by Isis Pharmaceuticals; FXI-ASO TKA ClinicalTrials.gov number

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

PubMed Central

Meinander, N Q; Hahn-Hägerdal, B

1997-01-01

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess. PMID:9143128

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.

Improving Xylose Utilization of Saccharomyces cerevisiae by Expressing the MIG1 Mutant from the Self-Flocculating Yeast SPSC01.

PubMed

Xu, Jian-Ren; Zhao, Xin-Qing; Liu, Chen-Guang; Bai, Feng-Wu

2018-01-01

The major carbohydrate components of lignocellulosic biomass are cellulose and hemicelluloses. Saccharomyces cerevisiae cannot efficiently utilize xylose derived upon the hydrolysis of hemicelluloses. Although engineering the yeast with xylose metabolic pathway has been intensively studied, challenges are still ahead for developing robust strains for lignocellulosic bioethanol production. The main objective of this study was to reveal the role of the MIG1 mutant isolated from the self-flocculating S. cerevisiae SPSC01 in xylose utilization, glucose repression and ethanol fermentation by S. cerevisiae. The MIG1 mutant was amplified from S. cerevisiae SPSC01 by PCR and MIG1- overexpression-cassette was transformed into S. cerevisiae S288c and xylose-metabolizing strain YB-2625-T through homologous recombination. Yeast growth was measured by colony assay on plates with or without xylose supplementation. Then xylose utilization and ethanol production were further evaluated through flask fermentation when mixed sugars of glucose and xylose at 3:1 and 2:1, respectively, were supplied. Fermentation products were detected by HPLC, and activities of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) were also measured. The transcription of genes regulated by the expression of the MIG1 mutant was analyzed by RTqPCR. Evolutionary relationship of various MIG1s was developed by gene sequencing and sequence alignment. No difference was observed for S288c growing with xylose when it was engineered with the overexpression or deletion of its native MIG1, but its growth was enhanced when overexpressing the MIG1 mutant from SPSC01. The submerged culture of YB-2625-T MIG1-SPSC engineered with xylose-metabolic pathway and the MIG1 mutant indicated that xylitol accumulation was decreased, and consequently, more biomass was accumulated. Furthermore, improved activities of the key enzymes such as XR, XDH and XK were detected in YB-2625-T MIG1-SPSC. Evolutionary

Decay widths of ground-state and excited {Xi}{sub b} baryons in a nonrelativistic quark model

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

Limphirat, Ayut; Thailand Center of Excellence in Physics; Department of Applied Physics, Faculty of Sciences and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima 30000

Decay processes of ground and excited bottom baryons are studied in the {sup 3}P{sub 0} nonrelativistic quark model with all model parameters fixed in the sector of light quarks. Using as an input the recent mass of {Xi}{sub b} and the theoretical masses of {Xi}{sub b}{sup *} and {Xi}{sub b}{sup '}, narrow decay widths are predicted for the ground-state bottom baryons {Xi}{sub b}{sup *} and {Xi}{sub b}{sup '}. The work predicts large decay widths, about 100 MeV for the {rho}-type orbital excitation states of {Xi}{sub b}.

Dehydration of xylose to furfural over MCM-41-supported niobium-oxide catalysts.

PubMed

García-Sancho, Cristina; Sádaba, Irantzu; Moreno-Tost, Ramón; Mérida-Robles, Josefa; Santamaría-González, José; López-Granados, Manuel; Maireles-Torres, Pedro

2013-04-01

A series of silica-based MCM-41-supported niobium-oxide catalysts are prepared, characterized by using XRD, N2 adsorption-desorption, X-ray photoelectron spectroscopy, Raman spectroscopy, and pyridine adsorption coupled to FTIR spectroscopy, and tested for the dehydration of D-xylose to furfural. Under the operating conditions used all materials are active in the dehydration of xylose to furfural (excluding the MCM-41 silica support). The xylose conversion increases with increasing Nb2 O5 content. At a loading of 16 wt % Nb2 O5 , 74.5 % conversion and a furfural yield of 36.5 % is achieved at 170 °C, after 180 min reaction time. Moreover, xylose conversion and furfural yield increase with the reaction time and temperature, attaining 82.8 and 46.2 %, respectively, at 190 °C and after 100 min reaction time. Notably, the presence of NaCl in the reaction medium further increases the furfural yield (59.9 % at 170 °C after 180 min reaction time). Moreover, catalyst reutilization is demonstrated by performing at least three runs with no loss of catalytic activity and without the requirement for an intermediate regeneration step. No significant niobium leaching is observed, and a relationship between the structure of the catalyst and the activity is proposed. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of canola meal tannins on the intestinal absorption capacity of broilers using a D-xylose test.

PubMed

Mansoori, B; Rogiewicz, A; Slominski, B A

2015-12-01

In three D-xylose absorption experiments, the effect of 1% HCl/methanol, 70% methanol or 70% acetone extracts of canola meal (CM) or 70% acetone extract of soybean meal (SBM) containing polyphenols, phenolic acids, tannins and phytic acid on intestinal absorption capacity of broilers was determined. In Exp. 1, the experimental groups received orally D-xylose solution alone or with methanol/HCl, methanol or acetone extracts of CM. In Exp. 2, the experimental groups received D-xylose alone or with acetone extracts of CM or SBM. In Exp. 3, the experimental groups received D-xylose plus sucrose solution or D-xylose plus acetone extracts of CM or SBM. In Exps. 2 and 3, the CM extracts contained 2.7 and 2.6, 2.4 and 2.3, 3.2 and 3.2, and 2.4 and 2.2 times higher polyphenols, phenolic acids, tannins and condensed tannins than the corresponding SBM extracts respectively. Blood samples were collected in 40-min intervals, and plasma D-xylose was measured. Compared to the Control, plasma D-xylose in Exp. 1 was lower (p < 0.001) by 81, 69 and 73% at 40-min, by 41, 44 and 37% at 80-min and by 22, 31, and 23% at 120-min post-ingestion of the HCl/methanol, methanol and acetone extracts respectively. In both Exps. 2 and 3, plasma D-xylose level was lower (p < 0.001) in groups dosed with CM extract or SBM extract at each time of blood collection, when compared to the respective Control group. However, in Exp. 3, birds dosed with SBM extract had higher plasma D-xylose than CM extract-dosed birds by 28, 8 and 21% at 40, 80 and 120 min respectively (p < 0.01). In conclusion, although CM extract caused a lower absorption of D-xylose, based on 5 to 10% of CM inclusion levels in practical broiler rations, the soluble bioactive components of CM will likely have minor impact on the absorption capacity of the chicken intestine. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

Efficient non-sterilized fermentation of biomass-derived xylose to lactic acid by a thermotolerant Bacillus coagulans NL01.

PubMed

Ouyang, Jia; Cai, Cong; Chen, Hai; Jiang, Ting; Zheng, Zhaojuan

2012-12-01

Xylose is the major pentose and the second most abundant sugar in lignocellulosic feedstock. Its efficient utilization is regarded as a technical barrier to the commercial production of bulk chemicals from lignocellulosic biomass. This work aimed at evaluating the lactic acid production from the biomass-derived xylose using non-sterilized fermentation by Bacillus coagulans NL01. A maximum lactic acid concentration of about 75 g/L was achieved from xylose of 100 g/L after 72 h batch fermentation. Acetic acid and levulinic acid were identified as important inhibitors in xylose fermentation, which markedly reduced lactic acid productivity at 15 and 1.0 g/L, respectively. But low concentrations of formic acid (<2 g/L) exerted a stimulating effect on the lactic acid production. When prehydrolysate containing total 25.45 g/L monosaccharide was fermented with B. coagulans NL01, the same preference for glucose, xylose, and arabinose was observed and18.2 g/L lactic acid was obtained after 48 h fermentation. These results proved that B. coagulans NL01 was potentially well-suited for producing lactic acid from underutilized xylose-rich prehydrolysates.

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

Inhibitor tolerance of a recombinant flocculating industrial Saccharomyces cerevisiae strain during glucose and xylose co-fermentation.

PubMed

Li, Yun-Cheng; Gou, Zi-Xi; Zhang, Ying; Xia, Zi-Yuan; Tang, Yue-Qin; Kida, Kenji

Lignocellulose-derived inhibitors have negative effects on the ethanol fermentation capacity of Saccharomyces cerevisiae. In this study, the effects of eight typical inhibitors, including weak acids, furans, and phenols, on glucose and xylose co-fermentation of the recombinant xylose-fermenting flocculating industrial S. cerevisiae strain NAPX37 were evaluated by batch fermentation. Inhibition on glucose fermentation, not that on xylose fermentation, correlated with delayed cell growth. The weak acids and the phenols showed additive effects. The effect of inhibitors on glucose fermentation was as follows (from strongest to weakest): vanillin>phenol>syringaldehyde>5-HMF>furfural>levulinic acid>acetic acid>formic acid. The effect of inhibitors on xylose fermentation was as follows (from strongest to weakest): phenol>vanillin>syringaldehyde>furfural>5-HMF>formic acid>levulinic acid>acetic acid. The NAPX37 strain showed substantial tolerance to typical inhibitors and showed good fermentation characteristics, when a medium with inhibitor cocktail or rape straw hydrolysate was used. This research provides important clues for inhibitors tolerance of recombinant industrial xylose-fermenting S. cerevisiae. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors

PubMed Central

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-01-01

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy. DOI: http://dx.doi.org/10.7554/eLife.14709.001 PMID:27304073

Acid-catalysed xylose dehydration into furfural in the presence of kraft lignin.

PubMed

Lamminpää, Kaisa; Ahola, Juha; Tanskanen, Juha

2015-02-01

In this study, the effects of kraft lignin (Indulin AT) on acid-catalysed xylose dehydration into furfural were studied in formic and sulphuric acids. The study was done using D-optimal design. Three variables in both acids were included in the design: time (20-80 min), temperature (160-180°C) and initial lignin concentration (0-20 g/l). The dependent variables were xylose conversion, furfural yield, furfural selectivity and pH change. The results showed that the xylose conversion and furfural yield decreased in sulphuric acid, while in formic acid the changes were minor. Additionally, it was showed that lignin has an acid-neutralising capacity, and the added lignin increased the pH of reactant solutions in both acids. The pH rise was considerably lower in formic acid than in sulphuric acid. However, the higher pH did not explain all the changes in conversion and yield, and thus lignin evidently inhibits the formation of furfural. Copyright © 2014 Elsevier Ltd. All rights reserved.

The growth and lipid accumulation of Scenedesmus quadricauda during batch mixotrophic/heterotrophic cultivation using xylose as a carbon source.

PubMed

Song, Mingming; Pei, Haiyan

2018-05-10

To overcome the bottlenecks of high cost and low production yields that restrict the commercial production of microalgae biodiesel, the use of xylose was evaluate by Scenedesmus quadricauda FACHB-1297, which was shown to be capable of mixotrophic and heterotrophic growth and lipid production on xylose, rich in the waste streams from pulp and paper industry, with increases in lipid productivities of 35.8-fold (mixotrophic) and 9.2-fold (heterotrophic) in comparison to photoautotrophic lipid yields. Five doses of xylose were tested to determine the effects and mechanisms of the carbon source on microalgae in mixotrophic mode. At the optimal xylose dosage of 4 g/L, the highest lipid content (38.61%) and productivity (139.55 mg/L/d) were achieved besides maximum biomass productivity (361.4 mg/L/d), nutrient removal efficiency of 68.4% (nitrogen), 97.2% (phosphorus) and 35.2% (xylose). Those indicated that S. quadricauda FACHB-1297 was suitable for further development of using xylose from certain waste streams for biofuel production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Xylose and cellulose fractionation from corncob with three different strategies and separate fermentation of them to bioethanol.

PubMed

Chen, Yefu; Dong, Boyu; Qin, Weijun; Xiao, Dongguang

2010-09-01

To the aim of efficient utilization of both of xylose and cellulose, a laboratory xylose/cellulose fractionation and separate fermentation (XCFSF) bioethanol process was performed. Three xylose/cellulose fractionation strategies: (A) dilute sulfur acid hydrolysis and detoxification, (B) lime pretreatment and xylanase hydrolysis, (C) bio-treatment with Phanerochaete chrysosporium and xylanase hydrolysis were applied to corn cobs. As a result, the maximum xylose yields obtained from A, B and C fractionation methods were 78.47%, 57.84% and 42.54%, respectively, and 96.81%, 92.14% and 80.34% of cellulose were preserved in the corresponding solid residues. The xylose dissolved in acid and enzymatic hydrolysates was fermented to ethanol by Candida shahatae and the cellulose remaining in solid residues was converted to ethanol by simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae. Finally, for A, B, C fractionation methods, 70.40%, 52.87%, 39.22% of hemicellulose and 89.77%, 84.30%, 71.90% of cellulose in corn cobs was converted to ethanol, respectively. Copyright 2010 Elsevier Ltd. All rights reserved.

Engineered yeast with a CO2-fixation pathway to improve the bio-ethanol production from xylose-mixed sugars.

PubMed

Li, Yun-Jie; Wang, Miao-Miao; Chen, Ya-Wei; Wang, Meng; Fan, Li-Hai; Tan, Tian-Wei

2017-03-06

Bio-ethanol production from lignocellulosic raw materials could serve as a sustainable potential for improving the supply of liquid fuels in face of the food-to-fuel competition and the growing energy demand. Xylose is the second abundant sugar of lignocelluloses hydrolysates, but its commercial-scale conversion to ethanol by fermentation is challenged by incomplete and inefficient utilization of xylose. Here, we use a coupled strategy of simultaneous maltose utilization and in-situ carbon dioxide (CO 2 ) fixation to achieve efficient xylose fermentation by the engineered Saccharomyces cerevisiae. Our results showed that the introduction of CO 2 as electron acceptor for nicotinamide adenine dinucleotide (NADH) oxidation increased the total ethanol productivity and yield at the expense of simultaneous maltose and xylose utilization. Our achievements present an innovative strategy using CO 2 to drive and redistribute the central pathways of xylose to desirable products and demonstrate a possible breakthrough in product yield of sugars.

The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

PubMed

Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

2002-07-16

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

KINETICS OF GROWTH AND ETHANOL PRODUCTION ON DIFFERENT CARBON SUBSTRATES USING GENETICALLY ENGINEERED XYLOSE-FERMENTING YEAST

EPA Science Inventory

Saccharomyces cerevisiae 424A (LNH-ST) strain was used for fermentation of glucose and xylose. Growth kinetics and ethanol productivity were calculated for batch fermentation on media containing different combinations of glucose and xylose to give a final sugar concentra...

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.

75 FR 8920 - Grant of Authority for Subzone Status; Danisco USA, Inc., Sweeteners Division (Xylitol, Xylose...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-26

... Status; Danisco USA, Inc., Sweeteners Division (Xylitol, Xylose, Galactose and Mannose); Thomson, IL...., Sweeteners Division, located in Thomson, Illinois, (FTZ Docket 4-2009, filed 2/4/2009); Whereas, notice... xylitol, xylose, galactose and mannose at the facility of Danisco USA, Inc., Sweeteners Division, located...

Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

PubMed

Cadete, Raquel M; Melo, Monaliza A; Dussán, Kelly J; Rodrigues, Rita C L B; Silva, Silvio S; Zilli, Jerri E; Vital, Marcos J S; Gomes, Fátima C O; Lachance, Marc-André; Rosa, Carlos A

2012-01-01

This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L · h to 0.75 g/L · h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L · h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose.

PubMed

Fu, Hongxin; Yu, Le; Lin, Meng; Wang, Jufang; Xiu, Zhilong; Yang, Shang-Tian

2017-03-01

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8g/L vs. 19.4g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28g/L·h vs. 0.16g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53g/L·h vs. 0.26g/L·h) and yield (0.32g/g vs. 0.28g/g). When the initial total sugar concentration was ~120g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4g/L, yield of 0.43g/g sugar consumed, productivity of 0.87g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Ultrabass Sounds of the Giant Star xi Hya

NASA Astrophysics Data System (ADS)

2002-05-01

First Observations of Solar-type Oscillations in a Star Very Different from the Sun Summary About 30 years ago, astronomers realised that the Sun resonates like a giant musical instrument with well-defined periods (frequencies). It forms a sort of large, spherical organ pipe. The energy that excites these sound waves comes from the turbulent region just below the Sun's visible surface. Observations of the solar sound waves (known as " helioseismology ") have resulted in enormous progress in the exploration of the interior of the Sun, otherwise hidden from view. As is the case on Earth, seismic techniques can be applied and the detailed interpretation of the observed oscillation periods has provided quite accurate information about the structure and motions inside the Sun, our central star. It has now also become possible to apply this technique to some solar-type stars. The first observations concerned the northern star eta Bootis (cf. ESO PR 16/94 ). Last year, extensive and much more accurate observations with the 1.2-m Swiss telescope at the ESO La Silla Observatory proved that Alpha Centauri , a solar "twin", behaves very much like the Sun (cf. ESO PR 15/01 ), and that some of the periods are quite similar to those in the Sun. These new observational data were of a superb quality, and that study marked a true break-through in the new research field of " asteroseismology " (seismology of the stars) for solar-type stars. But what about other types of stars, for instance those that are much larger than the Sun? Based on an extremely intensive observing project with the same telescope, an international group of astronomers [1] has found that the giant star xi Hya ("xi" is the small greek letter [2]; "Hya" is an abbreviation of "Hydrae") behaves like a giant sub-ultra-bass instrument . This star is located in the constellation Hydra (the Water-Monster) at a distance of 130 light-years, it has a radius about 10 times that of the Sun and its luminosity is about 60

21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

Code of Federal Regulations, 2014 CFR

2014-04-01

... isomerase enzyme preparations are used in the production of high fructose corn syrup described in § 184.1866... defined in § 170.3(o)(9) of this chapter, to convert glucose to fructose. (2) The ingredient is used in high fructose corn syrup, at levels not to exceed current good manufacturing practice. [48 FR 5720, Feb...

Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain.

PubMed

Ko, Ja Kyong; Jung, Je Hyeong; Altpeter, Fredy; Kannan, Baskaran; Kim, Ha Eun; Kim, Kyoung Heon; Alper, Hal S; Um, Youngsoon; Lee, Sun-Mi

2018-05-01

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Separate hydrolysis and co-fermentation for improved xylose utilization in integrated ethanol production from wheat meal and wheat straw

PubMed Central

2012-01-01

Background The commercialization of second-generation bioethanol has not been realized due to several factors, including poor biomass utilization and high production cost. It is generally accepted that the most important parameters in reducing the production cost are the ethanol yield and the ethanol concentration in the fermentation broth. Agricultural residues contain large amounts of hemicellulose, and the utilization of xylose is thus a plausible way to improve the concentration and yield of ethanol during fermentation. Most naturally occurring ethanol-fermenting microorganisms do not utilize xylose, but a genetically modified yeast strain, TMB3400, has the ability to co-ferment glucose and xylose. However, the xylose uptake rate is only enhanced when the glucose concentration is low. Results Separate hydrolysis and co-fermentation of steam-pretreated wheat straw (SPWS) combined with wheat-starch hydrolysate feed was performed in two separate processes. The average yield of ethanol and the xylose consumption reached 86% and 69%, respectively, when the hydrolysate of the enzymatically hydrolyzed (18.5% WIS) unwashed SPWS solid fraction and wheat-starch hydrolysate were fed to the fermentor after 1 h of fermentation of the SPWS liquid fraction. In the other configuration, fermentation of the SPWS hydrolysate (7.0% WIS), resulted in an average ethanol yield of 93% from fermentation based on glucose and xylose and complete xylose consumption when wheat-starch hydrolysate was included in the feed. Increased initial cell density in the fermentation (from 5 to 20 g/L) did not increase the ethanol yield, but improved and accelerated xylose consumption in both cases. Conclusions Higher ethanol yield has been achieved in co-fermentation of xylose and glucose in SPWS hydrolysate when wheat-starch hydrolysate was used as feed, then in co-fermentation of the liquid fraction of SPWS fed with the mixed hydrolysates. Integration of first-generation and second

Purification of a d-Mannose Isomerase from Mycobacterium smegmatis1

PubMed Central

Hey-Ferguson, Ann; Elbein, Alan D.

1970-01-01

An enzyme, d-mannose ketol isomerase, catalyzing the isomerization of d-mannose and d-fructose was purified approximately 60-fold from cells of Mycobacterium smegmatis grown on mannose as the sole carbon source. This enzyme was shown to catalyze the conversion of d-mannose and d-lyxose to ketoses. The ketose produced from mannose was identified as fructose by chemical and chromatographic methods. The reaction was shown to be reversible, the equilibrium ratio of fructose to mannose being approximately 65 to 35. The pH optimum was about 7.5, and the Km for mannose was estimated to be 7 × 10−3m. Mannose isomerase activity was greatest in cells grown on mannose, whereas cells grown on fructose had about 30% as much activity. Very low levels of activity were detected in cells grown on other substrates. There was an immediate increase in enzyme activity on transfer of cells from nutrient broth to a mannose mineral salts medium. PMID:5438047

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

PubMed

Latimer, Luke N; Dueber, John E

2017-06-01

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-xylose

Treesearch

Jennifer Van Vleet; Thomas W. Jeffries; Lisbeth Olsson

2008-01-01

Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled...

Genetically engineered Escherichia coli FBR5: Part II. Ethanol production from xylose and simultaneous product recovery

USDA-ARS?s Scientific Manuscript database

In these studies concentrated xylose solution was fermented to ethanol employing Escherichia coli FBR5 which can ferment both lignocellulosic sugars (hexoses and pentoses). E. coli FBR5 can produce 40-50 gL-1 ethanol from 100 gL-1 xylose in batch reactors. Increasing sugar concentration beyond this...

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.

Hadronic production of the doubly heavy baryon {Xi}{sub bc} at the LHC

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

Zhang Jiawei; Wu Xinggang; Zhong Tao

We investigate the hadronic production of the doubly heavy baryon {Xi}{sub bc} at the Large Hadron Collider (LHC), where contributions from the four (bc)-diquark states (bc){sub 3,6}[{sup 1}S{sub 0}] and (bc){sub 3,6}[{sup 3}S{sub 1}] have been taken into consideration. Numerical results show that under the condition of p{sub T}>4 GeV and |y|<1.5, sizable {Xi}{sub bc} events about 1.7x10{sup 7} and 3.5x10{sup 9} per year can be produced for the center-of-mass energy {radical}(S)=7 TeV and {radical}(S)=14 TeV, respectively. For experimental usage, the total and the interested differential cross sections are estimated under some typical p{sub T} and y cuts for themore » LHC detectors CMS, ATLAS, and LHCb. The main uncertainties are discussed and a comparative study on the hadronic production of {Xi}{sub cc}, {Xi}{sub bc}, and {Xi}{sub bb} at the LHC are also presented.« less

Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

PubMed

Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

2016-08-26

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. Copyright © 2016. Published by Elsevier Inc.

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.

Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Jewett, Kathy; Sandwick, Roger K.

2011-01-01

The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

A selective and sensitive D-xylose electrochemical biosensor based on xylose dehydrogenase displayed on the surface of bacteria and multi-walled carbon nanotubes modified electrode.

PubMed

Li, Liang; Liang, Bo; Shi, Jianguo; Li, Feng; Mascini, Marco; Liu, Aihua

2012-03-15

A novel Nafion/bacteria-displaying xylose dehydrogenase (XDH)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode was fabricated and applied for the sensitive and selective determination of d-xylose (INS 967), where the XDH-displayed bacteria (XDH-bacteria) was prepared using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. The XDH-displayed bacteria can be used directly, eliminating further enzyme-extraction and purification, thus greatly improved the stability of the enzyme. The optimal conditions for the construction of biosensor were established: homogeneous Nafion-MWNTs composite dispersion (10 μL) was cast onto the inverted glassy carbon electrode, followed by casting 10-μL of XDH-bacteria aqueous solution to stand overnight to dry, then a 5-μL of Nafion solution (0.05 wt%) is syringed to the electrode surface. The bacteria-displaying XDH could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD(+) in 0.1M PBS buffer (pH7.4), where NAD(+) (nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around 0.50 V (vs. Ag/AgCl). In contrast, the bacteria-XDH-only modified electrode showed oxidation peak at higher potential of 0.7 V and less sensitivity. Therefore, the electrode/MWNTs/bacteria-XDH/Nafion exhibited good analytical performance such as long-term stability, a wide dynamic range of 0.6-100 μM and a low detection limit of 0.5 μM D-xylose (S/N=3). No interference was observed in the presence of 300-fold excess of other saccharides including D-glucose, D-fructose, D-maltose, D-galactose, D-mannose, D-sucrose, and D-cellbiose as well as 60-fold excess of L-arabinose. The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds

Alcoholic fermentation of d-xylose by yeasts. [Brettanomyces naardenensis; Candida shehatae; Candida tenuis; Pachysolen tannaphilus, Pichia segobiensis; Pichia stipitis

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

Toivola, A.; Yarrow, D.; van den Bosch, E.

1984-06-01

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of D-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment D-cellobiose revealed that only Candida tenuis CBS 4435 was a goodmore » fermenter of both xylose and cellobiose under the test conditions used.« less

A Dependence Study of $$\\Xi^{*0}$$ and $$\\bar{\\Xi}^{*0}$$ in 250 GeV/c $$\\pi^-$$. $K^-$ -nucleon Interactions (in Portuguese)

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

Seixas de Rezende, Fabio Antonio; /Rio de Janeiro, CBPF

A direct measurement of the mass number (A) dependence of the production of the hyperon {Xi}*{sup 0} and its opposite {bar {Xi}}*{sup 0} in {pi}{sup -}, K{sup -} beam-nucleon interactions at 250 GeV/c is reported. The data derive from the experiment E769 at Fermilab. The results were obtained for different targets: Be, Al, Cu and W. It was observed the data are found to be well described by the parametrization {sigma}{sub A} = {sigma}{sub 0}A{sup {alpha}}, {alpha} being calculated for different beams. The results obtained are compared with those results of E769 experiment. The results shown here are preliminary.

Enzymatic Characterization of AMP Phosphorylase and Ribose-1,5-Bisphosphate Isomerase Functioning in an Archaeal AMP Metabolic Pathway

PubMed Central

Aono, Riku; Sato, Takaaki; Yano, Ayumu; Yoshida, Shosuke; Nishitani, Yuichi; Miki, Kunio; Imanaka, Tadayuki

2012-01-01

AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis. PMID:23065974

Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite, Reticulitermes chinensis

PubMed Central

Xie, Rongrong; Zhou, Feng; Huang, Miao

2017-01-01

The effective fermentation of xylose remains an intractable challenge in bioethanol industry. The relevant xylanase enzyme is also in a high demand from industry for several biotechnological applications that inevitably in recent times led to many efforts for screening some novel microorganisms for better xylanase production and fermentation performance. Recently, it seems that wood-feeding termites can truly be considered as highly efficient natural bioreactors. The highly specialized gut systems of such insects are not yet fully realized, particularly, in xylose fermentation and xylanase production to advance industrial bioethanol technology as well as industrial applications of xylanases. A total of 92 strains from 18 yeast species were successfully isolated and identified from the gut of wood-feeding termite, Reticulitermes chinensis. Of these yeasts and strains, seven were identified for new species: Candida gotoi, Candida pseudorhagii, Hamamotoa lignophila, Meyerozyma guilliermondii, Sugiyamaella sp.1, Sugiyamaella sp. 2, and Sugiyamaella sp.3. Based on the phylogenetic and phenotypic characterization, the type strain of C. pseudorhagii sp. nov., which was originally designated strain SSA-1542T, was the most frequently occurred yeast from termite gut samples, showed the highly xylanolytic activity as well as D-xylose fermentation. The highest xylanase activity was recorded as 1.73 and 0.98 U/mL with xylan or D-xylose substrate, respectively, from SSA-1542T. Among xylanase-producing yeasts, four novel species were identified as D-xylose-fermenting yeasts, where the yeast, C. pseudorhagii SSA-1542T, showed the highest ethanol yield (0.31 g/g), ethanol productivity (0.31 g/L·h), and its fermentation efficiency (60.7%) in 48 h. Clearly, the symbiotic yeasts isolated from termite guts have demonstrated a competitive capability to produce xylanase and ferment xylose, suggesting that the wood-feeding termite gut is a promising reservoir for novel xylanases

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

PubMed

Xiong, Wei; Reyes, Luis H; Michener, William E; Maness, Pin-Ching; Chou, Katherine J

2018-03-15

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals. © 2018 Wiley Periodicals, Inc.

Multiquadrant robotic colorectal surgery: the da Vinci Xi vs Si comparison.

PubMed

Protyniak, Bogdan; Jorden, Jeffrey; Farmer, Russell

2018-03-01

The newly introduced da Vinci Xi Surgical System hopes to address the shortcomings of its predecessor, specifically robotic arm restrictions and difficulty working in multiple quadrants. We compare the two robot platforms in multiquadrant surgery at a major colorectal referral center. Forty-four patients in the da Vinci Si group and 26 patients in the Xi group underwent sigmoidectomy or low anterior resection between 2014 and 2016. Patient demographics, operative variables, and postoperative outcomes were compared using descriptive statistics. Both groups were similar in age, sex, BMI, pelvic surgeries, and ASA class. Splenic flexure was mobilized in more (p = 0.045) da Vinci Xi cases compared to da Vinci Si both for sigmoidectomy (50 vs 15.4%) and low anterior resection (60 vs 29%). There was no significant difference in operative time (219.9 vs 224.7 min; p = 0.640), blood loss (170.0 vs 188.1 mL; p = 0.289), length of stay (5.7 vs 6 days; p = 0.851), or overall complications (26.9 vs 22.7%; p = 0.692) between the da Vinci Xi and Si groups, respectively. Single-dock multiquadrant robotic surgery, measured by splenic flexure mobilization with concomitant pelvic dissection, was more frequently performed using the da Vinci Xi platform with no increase in operative time, bleeding, or postoperative complications. The new platform provides surgeons an easier alternative to the da Vinci Si dual docking or combined robotic/laparoscopic multiquadrant surgery.

Improved ethanol production from xylose in the presence of acetic acid by the overexpression of the HAA1 gene in Saccharomyces cerevisiae.

PubMed

Sakihama, Yuri; Hasunuma, Tomohisa; Kondo, Akihiko

2015-03-01

The hydrolysis of lignocellulosic biomass liberates sugars, primarily glucose and xylose, which are subsequently converted to ethanol by microbial fermentation. The rapid and efficient fermentation of xylose by recombinant Saccharomyces cerevisiae strains is limited by weak acids generated during biomass pretreatment processes. In particular, acetic acid negatively affects cell growth, xylose fermentation rate, and ethanol production. The ability of S. cerevisiae to efficiently utilize xylose in the presence of acetic acid is an essential requirement for the cost-effective production of ethanol from lignocellulosic hydrolysates. Here, an acetic acid-responsive transcriptional activator, HAA1, was overexpressed in a recombinant xylose-fermenting S. cerevisiae strain to yield BY4741X/HAA1. This strain exhibited improved cell growth and ethanol production from xylose under aerobic and oxygen limited conditions, respectively, in the presence of acetic acid. The HAA1p regulon enhanced transcript levels in BY4741X/HAA1. The disruption of PHO13, a p-nitrophenylphosphatase gene, in BY4741X/HAA1 led to further improvement in both yeast growth and the ability to ferment xylose, indicating that HAA1 overexpression and PHO13 deletion act by different mechanisms to enhance ethanol production. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity.

PubMed

Perry, J E; Ishii-Ohba, H; Stalvey, J R

1991-06-01

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

Time-based comparative transcriptomics in engineered xylose-utilizing Saccharomyces cerevisiae identifies temperature-responsive genes during ethanol production.

PubMed

Ismail, Ku Syahidah Ku; Sakamoto, Takatoshi; Hasunuma, Tomohisa; Kondo, Akihiko

2013-09-01

Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 °C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 °C) and control temperature (30 °C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell's mechanism to protect itself from heat degradation.

Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari.

PubMed

Guamán, Linda P; Barba-Ostria, Carlos; Zhang, Fuzhong; Oliveira-Filho, Edmar R; Gomez, José Gregório C; Silva, Luiziana F

2018-05-15

Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors. First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB). These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.

Study of the strong {sigma}{sub c}{yields}{lambda}{sub c}{pi},{sigma}{sub c}*{yields}{lambda}{sub c}{pi} and {xi}{sub c}*{yields}{xi}{sub c}{pi} decays in a nonrelativistic quark model

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

Albertus, C.; Nieves, J.; Hernandez, E.

We present results for the strong widths corresponding to the {sigma}{sub c}{yields}{lambda}{sub c}{pi}, {sigma}{sub c}*{yields}{lambda}{sub c}{pi} and {xi}{sub c}*{yields}{xi}{sub c}{pi} decays. The calculations have been done in a nonrelativistic constituent quark model with wave functions that take advantage of the constraints imposed by heavy quark symmetry. Partial conservation of axial current hypothesis allows us to determine the strong vertices from an analysis of the axial current matrix elements. Our results {gamma}({sigma}{sub c}{sup ++}{yields}{lambda}{sub c}{sup +}{pi}{sup +})=2.41{+-}0.07{+-}0.02 MeV, {gamma}({sigma}{sub c}{sup +}{yields}{lambda}{sub c}{sup +}{pi}{sup 0})=2.79{+-}0.08{+-}0.02 MeV, {gamma}({sigma}{sub c}{sup 0}{yields}{lambda}{sub c}{sup +}{pi}{sup -})=2.37{+-}0.07{+-}0.02 MeV, {gamma}({sigma}{sub c}*{sup ++}{yields}{lambda}{sub c}{sup +}{pi}{sup +})=17.52{+-}0.74{+-}0.12 MeV, {gamma}({sigma}{sub c}*{supmore » +}{yields}{lambda}{sub c}{sup +}{pi}{sup 0})=17.31{+-}0.73{+-}0.12 MeV, {gamma}({sigma}{sub c}*{sup 0}{yields}{lambda}{sub c}{sup +}{pi}{sup -})=16.90{+-}0.71{+-}0.12 MeV, {gamma}({xi}{sub c}*{sup +}{yields}{xi}{sub c}{sup 0}{pi}{sup +}+{xi}{sub c}{sup +}{pi}{sup 0})=3.18{+-}0.10{+-}0.01 MeV, and {gamma}({xi}{sub c}*{sup 0}{yields}{xi}{sub c}{sup +}{pi}{sup -}+{xi}{sub c}{sup 0}{pi}{sup 0})=3.03{+-}0.10{+-}0.01 MeV are in good agreement with experimental determinations.« less

Factor XI and XII as antithrombotic targets.

PubMed

Müller, Felicitas; Gailani, David; Renné, Thomas

2011-09-01

Arterial and venous thrombosis are major causes of morbidity and mortality, and the incidence of thromboembolic diseases increases as a population ages. Thrombi are formed by activated platelets and fibrin. The latter is a product of the plasma coagulation system. Currently available anticoagulants such as heparins, vitamin K antagonists and inhibitors of thrombin or factor Xa target enzymes of the coagulation cascade that are critical for fibrin formation. However, fibrin is also necessary for terminating blood loss at sites of vascular injury. As a result, anticoagulants currently in clinical use increase the risk of bleeding, partially offsetting the benefits of reduced thrombosis. This review focuses on new targets for anticoagulation that are associated with minimal or no therapy-associated increased bleeding. Data from experimental models using mice and clinical studies of patients with hereditary deficiencies of coagulation factors XI or XII have shown that both of these clotting factors are important for thrombosis, while having minor or no apparent roles in processes that terminate blood loss (hemostasis). Hereditary deficiency of factor XII (Hageman factor) or factor XI, plasma proteases that initiate the intrinsic pathway of coagulation, impairs thrombus formation and provides protection from vascular occlusive events, while having a minimal impact on hemostasis. As the factor XII-factor XI pathway contributes to thrombus formation to a greater extent than to normal hemostasis, pharmacological inhibition of these coagulation factors may offer the exciting possibility of anticoagulation therapies with minimal or no bleeding risk.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates

USDA-ARS?s Scientific Manuscript database

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes needed for xylose-fermentation integrated into its genome was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than their parental strain (p < 0.05) and abl...

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.

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes*

PubMed Central

Kaulfürst-Soboll, Heidi; Rips, Stephan; Koiwa, Hisashi; Kajiura, Hiroyuki; Fujiyama, Kazuhito; von Schaewen, Antje

2011-01-01

Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins. PMID:21478158

Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain

PubMed Central

2014-01-01

Background The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. Results In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. Conclusions In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain.

PubMed

Signori, Lorenzo; Passolunghi, Simone; Ruohonen, Laura; Porro, Danilo; Branduardi, Paola

2014-04-08

The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the

Detection of a compact companion of the mild barium star Xi-1 Ceti

NASA Technical Reports Server (NTRS)

Bohm-Vitense, E.; Johnson, H. R.

1985-01-01

In the present paper, the observation of a white dwarf companion of the mild Ba star Xi-1 Ceti (= 65 Ceti = HR 649 = HD 13611) is reported, taking into account also the properties of the mild Ba star and of its companion. The UV spectrum of Xi-1 Ceti is discussed along with an interpretation of this spectrum. Attention is given to the effective temperature of the companion, the absorption bands in the spectrum, the radius and mass of the Xi-1 Ceti companion, and questions regarding the obscuration of the companion by the atmosphere of the Ba star. It is found that the overall energy distribution of the Xi-1 Ceti companion can best be matched with a 14,000 K DA white dwarf of log g = 8 or less. However, the absolute intensity is too small and would require a radius too small and a mass too large for such a gravity.

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.

Identification of triosephosphate isomerase as a novel allergen in octopus fangsiao

USDA-ARS?s Scientific Manuscript database

A 28 kDa-protein was purified from octopus (Octopus fangsiao) and identified to be triosephosphate isomerase (TIM). The purified TIM is a glycoprotein with 1.7% carbohydrates and the isoelectric point is 7.6. TIM aggregated after heating above 45 °C, and the secondary structure was altered in extre...

The Obama - Xi Accord: A Need for Further Action

NASA Astrophysics Data System (ADS)

Tribett, W. R.; Hope, A. P.; Canty, T. P.; Salawitch, R. J.

2015-12-01

Presidents Barrack Obama of the United States and Jinping Xi of China recently announced a bilateral framework to reduce the total carbon emissions of their respective countries. The U.S. agreed to reduce annual carbon emissions such that by 2025, emissions would be 27% below 2005 levels. China agreed to achieve peak carbon emissions around 2030 coupled with a best effort to peak early. Here we analyze the implications of the Obama-Xi accord for total global carbon emissions (GCE) out to year 2060, using projections of population, economic growth, and carbon intensity for the rest of the world as well as various assumptions regarding how emissions from the U.S. and China will evolve after the timeframe of the Obama-Xi accord. Our GCE projections will be compared to those of the four Representative Concentration Pathway (RCP) emission scenarios used in the IPCC Fifth Assessment Report (AR5). The Obama-Xi accord is shown to be a meaningful first step: if followed, the actual GCE will likely fall below RCP 8.5 between now and 2060. The U.S., China, and rest of the world presently emit 4.5, 2.0, and 1.1 tonne of carbon per person per year (tpy), respectively. We show that if the world's nations adopt a strategy of "Contraction and Convergence", such that per capita emission for each country reaches 1.0 tpy by 2060, actual GCE will approach that of RCP 4.5 by year 2060. Such action may be needed to reduce the risk of the most dire global warming forecasts within IPCC AR5.

Improved bioethanol production using fusants of Saccharomyces cerevisiae and xylose-fermenting yeasts.

PubMed

Kumari, Rajni; Pramanik, K

2012-06-01

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose-xylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8 ± 0.31 g L(-1)), ethanol productivity (1.06 g L(-1) h(-1)) and ethanol yield (0.458 g g(-1)) by fermentation of glucose-xylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08 ± 0.142 g L(-1), ethanol yield of 0.44 g g(-1), productivity of 0.45 g L(-1) h(-1) and biomass yield of 0.40 g g(-1).

2017 Scientific Sessions Sol Sherry Distinguished Lecture in Thrombosis: Factor XI as a Target for New Anticoagulants.

PubMed

Weitz, Jeffrey I; Fredenburgh, James C

2018-02-01

The goal of anticoagulant therapy is to attenuate thrombosis without compromising hemostasis. Although the direct oral anticoagulants are associated with less intracranial hemorrhage than vitamin K antagonists, bleeding remains their major side effect. Factor XI has emerged as a promising target for anticoagulants that may be safer than those currently available. The focus on factor XI stems from epidemiological evidence of its role in thrombosis, the observation of attenuated thrombosis in factor XI-deficient mice, identification of novel activators, and the fact that factor XI deficiency is associated with only a mild bleeding diathesis. Proof-of-concept comes from the demonstration that compared with enoxaparin, factor XI knockdown reduces venous thromboembolism without increasing bleeding after elective knee arthroplasty. This article rationalizes the selection of factor XI as a target for new anticoagulants, reviews the agents under development, and outlines a potential path forward for their development. © 2017 American Heart Association, Inc.

Level structure and production cross section of {sub {Xi}}{sup 12} Be studied with coupled-channels antisymmetrized molecular dynamics

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

Matsumiya, H.; Tsubakihara, K.; Kimura, M.

A theoretical framework of coupled-channels antisymmetrized molecular dynamics that describes the multistrangeness system with mixing between different baryon species is developed and applied to {sub {Lambda}}{sup 12}C and {sub {Xi}}{sup 12}Be. By introducing a minor modification to the YN G-matrix interaction derived from the Nijmegen model-D, the low-lying level structure and production cross section of {sub {Lambda}}{sup 12}C are reasonably described. It is found that the low-lying states of {sub {Xi}}{sup 12}Be are dominated by the {sup 11}B {circle_times} {Xi}{sup -} channel and their order strongly depends on {Xi}N effective interactions used in the calculation. The calculated peak position ofmore » the production cross section depends on the {Xi}N effective interaction and the magnitude of spin-flip and non-spin-flip cross sections of K{sup -}p{yields}K{sup +}{Xi}{sup -} elemental processes. We suggest that the {sup 12}C(K{sup -},K{sup +}){sub {Xi}}{sup 12}Be reaction possibly provides us information about the {Xi}N interaction.« less

Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

Treesearch

Gionata Scalcinati; Jose´ Manuel Otero; Jennifer R.H. Van Vleet; Thomas W. Jeffries; Lisbeth Olsson; Jens Nielsen

2012-01-01

Industrial biotechnology aims to develop robust microbial cell factories, such as , to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research...

Analysis of metabolisms and transports of xylitol using xylose- and xylitol-assimilating Saccharomyces cerevisiae.

PubMed

Tani, Tatsunori; Taguchi, Hisataka; Akamatsu, Takashi

2017-05-01

To clarify the relationship between NAD(P) + /NAD(P)H redox balances and the metabolisms of xylose or xylitol as carbon sources, we analyzed aerobic and anaerobic batch cultures of recombinant Saccharomyces cerevisiae in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C. The TDH3p-GAL2 or gal80Δ strain completely consumed the xylose within 24 h and aerobically consumed 92-100% of the xylitol within 96 h, but anaerobically consumed only 20% of the xylitol within 96 h. Cells of both strains grew well in aerobic culture. The addition of acetaldehyde (an effective oxidizer of NADH) increased the xylitol consumption by the anaerobically cultured strain. These results indicate that in anaerobic culture, NAD + generated in the NAD(P)H-dependent xylose reductase reaction was likely needed in the NAD + -dependent xylitol dehydrogenase reaction, whereas in aerobic culture, the NAD + generated by oxidation of NADH in the mitochondria is required in the xylitol dehydrogenase reaction. The role of Gal2 and Fps1 in importing xylitol into the cytosol and exporting it from the cells was analyzed by examining the xylitol consumption in aerobic culture and the export of xylitol metabolized from xylose in anaerobic culture, respectively. The xylitol consumptions of gal80Δ gal2Δ and gal80Δ gal2Δ fps1Δ strains were reduced by 81% and 88% respectively, relative to the gal80Δ strain. The maximum xylitol concentration accumulated by the gal80Δ, gal80Δ gal2Δ, and gal80Δ gal2Δ fps1Δ strains was 7.25 g/L, 5.30 g/L, and 4.27 g/L respectively, indicating that Gal2 and Fps1 transport xylitol both inward and outward. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

42 CFR 476.86 - Correlation of Title XI functions with Title XVIII functions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 4 2011-10-01 2011-10-01 false Correlation of Title XI functions with Title XVIII functions. 476.86 Section 476.86 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF...) Qio Review Functions § 476.86 Correlation of Title XI functions with Title XVIII functions. (a...

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...

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

PubMed

Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

2012-12-01

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Evaluation of fermentation kinetics of acid-treated corn cob hydrolysate for xylose fermentation in the presence of acetic acid by Pichia stipitis.

PubMed

Kashid, Mohan; Ghosalkar, Anand

2017-08-01

The efficient utilization of lignocellulosic biomass for ethanol production depends on the fermentability of the biomass hydrolysate obtained after pretreatment. In this work we evaluated the kinetics of ethanol production from xylose using Pichia stipitis in acid-treated corn cob hydrolysate. Acetic acid is one of the main inhibitors in corn cob hydrolysate that negatively impacts kinetics of xylose fermentation by P. stipitis. Unstructured kinetic model has been formulated that describes cell mass growth and ethanol production as a function of xylose, oxygen, ethanol, and acetic acid concentration. Kinetic parameters were estimated under different operating conditions affecting xylose fermentation. This is the first report on kinetics of xylose fermentation by P. stipitis which includes inhibition of acetic acid on growth and product formation. In the presence of acetic acid in the hydrolysate, the model accurately predicted reduction in maximum specific growth rate (from 0.23 to 0.15 h -1 ) and increase in ethanol yield per unit biomass (from 3 to 6.2 gg -1 ), which was also observed during experimental trials. Presence of acetic acid in the fermentation led to significant reduction in the cell growth rate, reduction in xylose consumption and ethanol production rate. The developed model accurately described physiological state of P. stipitis during corn cob hydrolysate fermentation. Proposed model can be used to predict the influence of xylose, ethanol, oxygen, and acetic acid concentration on cell growth and ethanol productivity in industrial fermentation.

Hydrothermal conversion of xylose, glucose, and cellulose under the catalysis of transition metal sulfates.

PubMed

Cao, Xuefei; Peng, Xinwen; Sun, Shaoni; Zhong, Linxin; Chen, Wei; Wang, Sha; Sun, Run-Cang

2015-03-15

Hydrothermal conversion (HTC) is an important thermochemical process to upgrade low-cost biomass into valuable chemicals or fuels. As compared with non-catalytic HTC, catalytic HTC shows high energy efficiency on biomass upgradation. In this work, the catalytic performances of various transition metal sulfates (Mn(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), and Zn(2+)) in the HTCs of xylose, glucose, and cellulose under different conditions were explored. Among these catalysts, Zn(2+) and Ni(2+) showed obvious effects on the conversions of xylose, glucose, and cellulose into lactic acid, while Cu(2+) and Fe(3+), which could significantly accelerate the hydrolysis of cellulose into glucose at 200°C, displayed high efficiency on converting glucose and cellulose into levulinic acid and formic acid at high temperature. Additionally, significant positive correlative relationships among xylose, glucose, and cellulose degradations were observed. This study is helpful for screening appropriate catalysts for biomass upgradation through catalytic HTC of monosaccharide. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced L-lactic acid production from biomass-derived xylose by a mutant Bacillus coagulans.

PubMed

Zheng, Zhaojuan; Cai, Cong; Jiang, Ting; Zhao, Mingyue; Ouyang, Jia

2014-08-01

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient L-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for L-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett-Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l(-1)  L-lactic acid was obtained from 100 g l(-1) xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l(-1)  L-lactic acid was obtained in 36 h and the yield was 83.09 %.

Low acid hydrothermal fractionation of Giant Miscanthus for production of xylose-rich hydrolysate and furfural.

PubMed

Kim, Tae Hyun; Ryu, Hyun Jin; Oh, Kyeong Keun

2016-10-01

Low acid hydrothermal (LAH) fractionation was developed for the effective recovery of hemicellulosic sugar (mainly xylose) from Miscanthus sacchariflorus Goedae-Uksae 1 (M. GU-1). The xylose yield was maximized at 74.75% when the M. GU-1 was fractionated at 180°C and 0.3wt.% of sulfuric acid for 10min. At this condition, the hemicellulose (mainly xylan) degradation was 86.41%. The difference between xylan degradation and xylose recovery yield, i.e., xylan loss, was 11.66%, as indicated by the formation of decomposed products. The furfural, the value added biochemical product, was also obtained by 0.42g/L at this condition, which was 53.82% of furfural production yield based on the xylan loss. After then, the furfural production continued to increase to a maximum concentration of 1.87g/L, at which point the xylan loss corresponded to 25.87%. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Effect of Initial Cell Concentration on Xylose Fermentation by Pichia stipitis

NASA Astrophysics Data System (ADS)

Agbogbo, Frank K.; Coward-Kelly, Guillermo; Torry-Smith, Mads; Wenger, Kevin; Jeffries, Thomas W.

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R 2 of 0.99.

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

PubMed

Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

2008-11-01

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

Da Vinci Xi and Si platforms have equivalent perioperative outcomes during robot-assisted partial nephrectomy: preliminary experience.

PubMed

Abdel Raheem, Ali; Sheikh, Abulhasan; Kim, Dae Keun; Alatawi, Atalla; Alabdulaali, Ibrahim; Han, Woong Kyu; Choi, Young Deuk; Rha, Koon Ho

2017-03-01

The aims of this study were to compare the perioperative outcomes of da Vinci Xi to Si during robotic-assisted partial nephrectomy (RAPN) and to discuss the feasibility of our novel port placement scheme for the da Vinci Xi platform, to overcome the existing kinetic and technical difficulties we faced with the linear port placement in patients with a small body habitus. A retrospective data analysis of patients who underwent RPN using da Vinci Xi (n = 18) was carried out. The outcomes of the Xi group were compared with the Si group (n = 18) selected using a case-matched methodology. For da Vinci Xi, we applied the universal linear port placement in 12 patients and our modified port placement in the remaining 6 patients. The Xi group had a shorter mean docking time of 17.8 ± 2.6 min compared to the Si group of 20.5 ± 2.1 min (p = 0.002); otherwise, no significant difference was present with regard to the remaining perioperative variables (p > 0.05). The modified Xi port placement had a shorter mean console time of 70.8 ± 9.7 min compared to the universal linear port placement of 89.3 ± 17.2 min (p = 0.03). Moreover, it provided a broader field of vision with excellent robotic arms movement, minimizing collisions and allowing an easier and comfortable surgical assist. Da Vinci Xi appears to be feasible and safe during RPN with similar outcomes to Si. The novel Xi port placement makes surgery easier in patients with low BMI.

Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

PubMed

Currin, Andrew; Dunstan, Mark S; Johannissen, Linus O; Hollywood, Katherine A; Vinaixa, Maria; Jervis, Adrian J; Swainston, Neil; Rattray, Nicholas J W; Gardiner, John M; Kell, Douglas B; Takano, Eriko; Toogood, Helen S; Scrutton, Nigel S

2018-03-02

The realization of a synthetic biology approach to microbial (1 R ,2 S ,5 R )-( - )-menthol ( 1 ) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing ( R )-(+)-pulegone (( R )- 2 ) from (+)- cis -isopulegone ( 3 ). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3 . This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

DOE PAGES

Xiong, Wei; Reyes, Luis H.; Michener, William E.; ...

2018-04-10

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration ofmore » xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.« less

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

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

Xiong, Wei; Reyes, Luis H.; Michener, William E.

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration ofmore » xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.« less

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review.

PubMed

Chen, Yanli

2011-05-01

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49-0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.

Optimization of CDT-1 and XYL1 Expression for Balanced Co-Production of Ethanol and Xylitol from Cellobiose and Xylose by Engineered Saccharomyces cerevisiae

PubMed Central

Zha, Jian; Li, Bing-Zhi; Shen, Ming-Hua; Hu, Meng-Long; Song, Hao; Yuan, Ying-Jin

2013-01-01

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular β-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g). PMID:23844185

Factor XI and Contact Activation as Targets for Antithrombotic Therapy

PubMed Central

Gailani, David; Bane, Charles E.; Gruber, Andras

2015-01-01

Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

OMEGA{sup -}, XI*{sup -}, SIGMA*{sup -}, and DELTA{sup -} decuplet baryon magnetic moments

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

Slaughter, Milton Dean

The properties of the ground-state U spin =(3/2) baryon decuplet magnetic moments DELTA{sup -}, XI*{sup -}, SIGMA*{sup -}, and OMEGA{sup -} and their ground-state spin-(1/2) cousins p, n, LAMBDA, SIGMA{sup +}, SIGMA{sup 0}, SIGMA{sup -}, XI{sup +}, and XI{sup -} have been studied for many years with a modicum of success. The magnetic moments of many are yet to be determined. Of the decuplet baryons, only the magnetic moment of the OMEGA{sup -} has been accurately determined. We calculate the magnetic moments of the physical decuplet U spin =(3/2) quartet members without ascribing any specific form to their quark structuremore » or intraquark interactions.« less

Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production.

PubMed

Demeke, Mekonnen M; Dumortier, Françoise; Li, Yingying; Broeckx, Tom; Foulquié-Moreno, María R; Thevelein, Johan M

2013-08-26

In addition to efficient pentose utilization, high inhibitor tolerance is a key trait required in any organism used for economically viable industrial bioethanol production with lignocellulose biomass. Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiae strains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates. The aim of the present work was to combine high xylose fermentation activity and high inhibitor tolerance in a single industrial yeast strain. We have screened 580 yeast strains for high inhibitor tolerance using undetoxified acid-pretreated spruce hydrolysate and identified a triploid industrial baker's yeast strain as having the highest inhibitor tolerance. From this strain, a mating competent diploid segregant with even higher inhibitor tolerance was obtained. It was crossed with the recently developed D-xylose fermenting diploid industrial strain GS1.11-26, with the Ethanol Red genetic background. Screening of 819 diploid segregants from the tetraploid hybrid resulted in two strains, GSF335 and GSF767, combining high inhibitor tolerance and efficient xylose fermentation. In a parallel approach, meiotic recombination of GS1.11-26 with a haploid segregant of Ethanol Red and screening of 104 segregants resulted in a similar inhibitor tolerant diploid strain, GSE16. The three superior strains exhibited significantly improved tolerance to inhibitors in spruce hydrolysate, higher glucose consumption rates, higher aerobic growth rates and higher maximal ethanol accumulation capacity in very-high gravity fermentation, compared to GS1.11-26. In complex medium, the D-xylose utilization rate by the three superior strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that of GS1.11-26 (1.10 g/g DW/h). On the other hand, in batch fermentation of undetoxified acid-pretreated spruce hydrolysate, the

Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production

PubMed Central

2013-01-01

Background In addition to efficient pentose utilization, high inhibitor tolerance is a key trait required in any organism used for economically viable industrial bioethanol production with lignocellulose biomass. Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiae strains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates. The aim of the present work was to combine high xylose fermentation activity and high inhibitor tolerance in a single industrial yeast strain. Results We have screened 580 yeast strains for high inhibitor tolerance using undetoxified acid-pretreated spruce hydrolysate and identified a triploid industrial baker’s yeast strain as having the highest inhibitor tolerance. From this strain, a mating competent diploid segregant with even higher inhibitor tolerance was obtained. It was crossed with the recently developed D-xylose fermenting diploid industrial strain GS1.11-26, with the Ethanol Red genetic background. Screening of 819 diploid segregants from the tetraploid hybrid resulted in two strains, GSF335 and GSF767, combining high inhibitor tolerance and efficient xylose fermentation. In a parallel approach, meiotic recombination of GS1.11-26 with a haploid segregant of Ethanol Red and screening of 104 segregants resulted in a similar inhibitor tolerant diploid strain, GSE16. The three superior strains exhibited significantly improved tolerance to inhibitors in spruce hydrolysate, higher glucose consumption rates, higher aerobic growth rates and higher maximal ethanol accumulation capacity in very-high gravity fermentation, compared to GS1.11-26. In complex medium, the D-xylose utilization rate by the three superior strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that of GS1.11-26 (1.10 g/g DW/h). On the other hand, in batch fermentation of undetoxified acid-pretreated spruce

Nutritional and metabolic implications of replacing cornstarch with D-xylose in broiler chickens fed corn and soybean meal-based diet.

PubMed

Regassa, A; Kiarie, E; Sands, J S; Walsh, M C; Kim, W K; Nyachoti, C M

2017-02-01

Effects of substituting cornstarch with D-xylose on growth performance, nutrients digestibility, serum metabolites, and expression of select hepatic genes involved in glucose and lipid metabolism were investigated in broiler chickens. A total of 360 one-day-old male Ross chicks were fed 3 diets (n = 24; 5 chicks/cage) for 21 days. A control corn-soybean meal-based diet with 25% cornstarch was formulated to meet specifications. Two additional diets were formulated by substituting cornstarch with 5 or 15% D-xylose w/w. Growth performance and digestibility by index method were determined in 12 replicate cages. Birds in these replicates had free access to feed and water, the BW and feed intake (FI) were monitored weekly and the excreta samples were collected on d 18 to 20. The other 12 replicates were used for blood and liver sampling by serial slaughter. On d 18, baseline (t0) birds were sampled following a 12 h overnight fasting and birds allowed 30 min access to the feed; samples were subsequently taken at 60, 120, 180, 240, and 300 min post feeding. Serum metabolites (glucose, xylose, and insulin) were assayed at all time points, whereas expression of hepatic transcripts was evaluated at zero, 180 and 300 min. Xylose linearly reduced (P < 0.05) FI, BWG, gross energy digestibility, and feed conversion ratio (FCR) but increased (P < 0.05) serum xylose level. Serum glucose and insulin levels were higher (P < 0.05) in the post-fed state compared with baseline, irrespective of treatments. There was an interaction (P < 0.05) between diet and sampling time on the expression of hepatic genes. At t0, xylose linearly increased (P < 0.05) the expression of pyruvate carboxylase, Acetyl Co-A acethyltransferase 2 (ACAT2), and glucose transporter 2. Xylose linearly reduced (P < 0.05) the expression of ACAT2 at 300 min post feeding. In conclusion, 5% or more xylose reduced growth performance and utilization of nutrients linked to hepatic enzymes and transcription

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

PubMed Central

Lee, Jung-Kul; Pan, Cheol-Ho

2013-01-01

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

Electrochemistry for the Generation of Renewable Chemicals: One-Pot Electrochemical Deoxygenation of Xylose to δ-Valerolactone.

PubMed

James, Olusola O; Sauter, Waldemer; Schröder, Uwe

2017-05-09

In this study, the electrochemical conversion of xylose to δ-valerolactone via carbonyl intermediates is demonstrated. The conversion was achieved in aqueous media and at ambient conditions. This study also demonstrates that the feedstock for production of renewable chemicals and biofuels through electrochemistry can be extended to primary carbohydrate molecules. This is the first report on a one-pot electrochemical deoxygenation of xylose to δ-valerolactone. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

PubMed

Chen, Yingying; Wu, Ying; Zhu, Baotong; Zhang, Guanyu; Wei, Na

2018-01-01

Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is

Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose.

PubMed

Ammar, Ehab M; Wang, Xiaoyi; Rao, Christopher V

2018-01-12

Catabolite repression refers to the process where the metabolism of one sugar represses the genes involved in metabolizing another sugar. While glucose provides the canonical example, many other sugars are also known to induce catabolite repression. However, less is known about the mechanism for catabolite repression by these non-glucose sugars. In this work, we investigated the mechanism of catabolite repression in the bacterium Escherichia coli during growth on lactose, L-arabinose, and D-xylose. The metabolism of these sugars is regulated in a hierarchical manner, where lactose is the preferred sugar, followed by L-arabinose, and then D-xylose. Previously, the preferential utilization of L-arabinose over D-xylose was found to result from transcriptional crosstalk. However, others have proposed that cAMP governs the hierarchical regulation of many non-glucose sugars. We investigated whether lactose-induced repression of L-arabinose and D-xylose gene expression is due to transcriptional crosstalk or cAMP. Our results demonstrate that it is due to cAMP and not transcriptional crosstalk. In addition, we found that repression is reciprocal, where both L-arabinose and D-xylose also repress the lactose gene expression, albeit to a lesser extent and also through a mechanism involving cAMP. Collectively, the results further our understanding of metabolism during growth on multiple sugars.

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

PubMed Central

Riggs, Daniel L.; Roberts, Patricia J.; Chirillo, Samantha C.; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F.

2003-01-01

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. PMID:12606580

Wild-type isopropylmalate isomerase in Salmonella typhimurium is composed of two different subunits.

PubMed Central

Fultz, P N; Kemper, J

1981-01-01

The isopropylmalate isomerase in Salmonella typhimurium is the second enzyme specific for leucine biosynthesis. It is a complex enzyme composed of two subunits which are coded for by two genes of the leucine operon, leuC and leuD. The two polypeptides have been shown to copurify through successive ammonium sulfate fractionations and have been identified on sodium dodecyl sulfate-polyacrylamide gels as having molecular weights of 51,000 (leuC gene product) and 23,500 (leuD gene product). They have also been shown to be fairly stable, since in vitro complementation of cell-free extracts of leuC and leuD mutant strains was demonstrated, with only a 40% loss of activity 16 h after preparation of the extracts. The native isopropylmalate isomerase was shown to have a Km for its substrate alpha-isopropylmalate of 3 x 10(-4)M. Images PMID:7026530

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium.

PubMed

Cordova, Lauren T; Long, Christopher P; Venkataramanan, Keerthi P; Antoniewicz, Maciek R

2015-11-01

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h(-1) on glucose and 1.52 h(-1) on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio's RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed

Chen, H; Juchau, M R

1997-11-01

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs.

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

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

Asther, M.; Khan, A.W.

1984-01-01

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobiamore » converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.« less

Adaptation of the xylose fermenting yeast Saccharomyces cerevisiae F12 for improving ethanol production in different fed-batch SSF processes.

PubMed

Tomás-Pejó, E; Ballesteros, M; Oliva, J M; Olsson, L

2010-11-01

An efficient fermenting microorganism for bioethanol production from lignocellulose is highly tolerant to the inhibitors released during pretreatment and is able to ferment efficiently both glucose and xylose. In this study, directed evolution was employed to improve the xylose fermenting Saccharomyces cerevisiae F12 strain for bioethanol production at high substrate loading. Adapted and parental strains were compared with respect to xylose consumption and ethanol production. Adaptation led to an evolved strain more tolerant to the toxic compounds present in the medium. When using concentrated prehydrolysate from steam-pretreated wheat straw with high inhibitor concentration, an improvement of 65 and 20% in xylose consumption and final ethanol concentration, respectively, were achieved using the adapted strain. To address the need of high substrate loadings, fed-batch SSF experiments were performed and an ethanol concentration as high as 27.4 g/l (61% of the theoretical) was obtained with 11.25% (w/w) of water insoluble solids (WIS).

Synthetic Consortium of Escherichia coli for n-Butanol Production by Fermentation of the Glucose-Xylose Mixture.

PubMed

Saini, Mukesh; Lin, Li-Jen; Chiang, Chung-Jen; Chao, Yun-Peng

2017-11-22

The microbial production of n-butanol using glucose and xylose, the major components of plant biomass, can provide a sustainable and renewable fuel as crude oil replacement. However, Escherichia coli prefers glucose to xylose as programmed by carbohydrate catabolite repression (CCR). In this study, a synthetic consortium consisting of two strains was developed by transforming the CCR-insensitive strain into a glucose-selective strain and a xylose-selective strain. Furthermore, the dual culture was reshaped by distribution of the synthetic pathway of n-butanol into two strains. Consequently, the co-culture system enabled effective co-utilization of both sugars and production of 5.2 g/L n-butanol at 30 h. The result leads to the conversion yield and productivity accounting for 63% of the theoretical yield and 0.17 g L -1 h -1 , respectively. Overall, the technology platform as proposed is useful for production of other value-added chemicals, which require complicated pathways for their synthesis by microbial fermentation of a sugar mixture.

Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2. Results With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50

Study on collaborative operation in Xi'an international inland port and airport

NASA Astrophysics Data System (ADS)

Jia, Guoling

2017-10-01

Xi 'an international inland port and airport are the important fulcrums for Shaanxi province to implement the strategy of "One Belt One Road" and to develop its export-oriented economy. Based on the general development situation of Xi 'an international inland port and airport and analyzing their similarities and differences, the external cause and internal cause of synergy are discussed. The contents of synergy from the strategy level, tactics level and business level are explained respectively.

Dehydration of D-xylose to furfural using acid-functionalized MWCNTs catalysts

NASA Astrophysics Data System (ADS)

Termvidchakorn, Chompoopitch; Itthibenchapong, Vorranutch; Songtawee, Siripit; Chamnankid, Busaya; Namuangruk, Supawadee; Faungnawakij, Kajornsak; Charinpanitkul, Tawatchai; Khunchit, Radchadaporn; Hansupaluk, Nanthiya; Sano, Noriaki; Hinode, Hirofumi

2017-09-01

Acid-functionalized multi-wall carbon nanotubes (MWCNTs) catalysts were prepared by a wet chemical sonication with various acid solutions, i.e. H2SO4, H3PO4, HNO3, and HCl. Sulfonic groups and carboxyl groups were detected on MWCNTs with H2SO4 treatment (s-MWCNTs), while only carboxyl groups were presented from other acid treatments. The catalytic dehydration of D-xylose into furfural was evaluated using a batch reactor at 170 °C for 3 h under N2 pressure of 15 bar. The highest furfural selectivity was achieved around 57% by s-MWCNTs catalyst, suggesting a positive role of the sulfonic functionalized groups. The effect of Co species was related to their Lewis acid property resulting in the enhancement of xylose conversion with low selectivity to furfural product. Invited talk at 5th Thailand International Nanotechnology Conference (Nano Thailand-2016), 27-29 November 2016, Nakhon Ratchasima, Thailand.

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed Central

Chen, H; Juchau, M R

1997-01-01

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs. PMID:9581548

Educational Pico-Satellite Project CUBESAT - University of Tokyo's CUBESAT XI and its Operation Plan

NASA Astrophysics Data System (ADS)

Tsuda, Y.; Sako, N.; Eishima, T.; Ito, T.; Arikawa, Y.; Miyamura, N.

2002-01-01

University of Tokyo ISSL (Intelligent Space Systems Laboratory) has been developing a pico-satellite called "CubeSat" as an international joint program. In CubeSat project, 10cm cubic satellites have been developed by several universities and launched to the low-earth orbit altogether by Russian rocket "Dnepr". ISSL has developed "XI" series ([sai]: X-factor Investigator) satellites, and the flight model is already fabricated and ready for delivery. The mission of XI satellite is the on-orbit technology demonstration of the ultra-small satellite bus system with an extensive use of commercial-off-the-shelf components. XI transmits the Morse beacon and FM packet telemetry which provides the health data of the satellite. Additionally, XI has a CMOS camera which provides 15,000 pixels panchromatic images as an advanced mission. Ground operation is one of the key issues for CubeSats. Now we are promoting international ground station network in which several universities' ground stations connected by internet collaboratively operate university-built small satellites, which enlarges the link opportunity. Collaboration with amateur HAM engineers is also indispensable for search for the satellite or get beacon signal to estimate the satellite orbit. We are now developing operation concept based on these ideas. As the launch is scheduled in this fall, the operation plan will be fixed at the time of this conference. In this presentation the final design of ISSL's CubeSat XI and operation plan will be presented.

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

PubMed

Jørgensen, F; Hansen, O C; Stougaard, P

2004-06-01

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

Early clinical experience with the da Vinci Xi Surgical System in general surgery.

PubMed

Hagen, Monika E; Jung, Minoa K; Ris, Frederic; Fakhro, Jassim; Buchs, Nicolas C; Buehler, Leo; Morel, Philippe

2017-09-01

The da Vinci Xi Surgical System (Intuitive Surgical Inc., Sunnyvale, CA, USA) has been released in 2014 to facilitate minimally invasive surgery. Novel features are targeted towards facilitating complex multi-quadrant procedures, but data is scarce so far. Perioperative data of patients who underwent robotic general surgery with the da Vinci Xi system within the first 6 month after installation were collected and analyzed. The gastric bypass procedures performed with the da Vinci Xi Surgical System were compared to an equal amount of the last procedures with the da Vinci Si Surgical System. Thirty-one foregut (28 Roux-en-Y gastric bypasses), 6 colorectal procedures and 1 revisional biliary procedure were performed. The mean operating room (OR) time was 221.8 (±69.0) minutes for gastric bypasses and 306.5 (±48.8) for colorectal procedures with mean docking time of 9.4 (±3.8) minutes. The gastric bypass procedure was transitioned from a hybrid to a fully robotic approach. In comparison to the last 28 gastric bypass procedures performed with the da Vinci Si Surgical System, the OR time was comparable (226.9 versus 230.6 min, p = 0.8094), but the docking time significantly longer with the da Vinci Xi Surgical System (8.5 versus 6.1 min, p = 0.0415). All colorectal procedures were performed with a single robotic docking. No intraoperative and two postoperative complications occurred. The da Vinci Xi might facilitate single-setups of totally robotic gastric bypass and colorectal surgeries. However, further comparable research is needed to clearly determine the significance of this latest version of the da Vinci Surgical System.

Harnessing genetic diversity in Saccharomyces cerevisiae for fermentation of xylose in hydrolysates of alkaline hydrogen peroxide-pretreated biomass.

PubMed

Sato, Trey K; Liu, Tongjun; Parreiras, Lucas S; Williams, Daniel L; Wohlbach, Dana J; Bice, Benjamin D; Ong, Irene M; Breuer, Rebecca J; Qin, Li; Busalacchi, Donald; Deshpande, Shweta; Daum, Chris; Gasch, Audrey P; Hodge, David B

2014-01-01

The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na(+), acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification

PubMed Central

2011-01-01

Background L-arabinose is an important intermediate for anti-virus drug synthesis and has also been used in food additives for diets-controlling in recent years. Commercial production of L-arabinose is a complex progress consisting of acid hydrolysis of gum arabic, followed by multiple procedures of purification, thus making high production cost. Therefore, there is a biotechnological and commercial interest in the development of new cost-effective and high-performance methods for obtaining high purity grade L-arabinose. Results An alternative, economical method for purifying L-arabinose from xylose mother liquor was developed in this study. After screening 306 yeast strains, a strain of Pichia anomala Y161 was selected as it could effectively metabolize other sugars but not L-arabinose. Fermentation in a medium containing xylose mother liquor permitted enrichment of L-arabinose by a significant depletion of other sugars. Biochemical analysis of this yeast strain confirmed that its poor capacity for utilizing L-arabinose was due to low activities of the enzymes required for the metabolism of this sugar. Response surface methodology was employed for optimization the fermentation conditions in shake flask cultures. The optimum conditions were: 75 h fermentation time, at 32.5°C, in a medium containing 21% (v/v) xylose mother liquor. Under these conditions, the highest purity of L-arabinose reached was 86.1% of total sugar, facilitating recovery of white crystalline L-arabinose from the fermentation medium by simple methods. Conclusion Yeast-mediated biopurification provides a dynamic method to prepare high purity of L-arabinose from the feedstock solution xylose mother liqour, with cost-effective and high-performance properties. PMID:21649890

Use of a mobile tower-based robot--The initial Xi robot experience in surgical oncology.

PubMed

Yuh, Bertram; Yu, Xian; Raytis, John; Lew, Michael; Fong, Yuman; Lau, Clayton

2016-01-01

The da Vinci Xi platform provides expanded movement of the arms relative to the base, theoretically allowing increased versatility in complex multi-field or multi-quadrant surgery. We describe the initial Xi experience in oncologic surgery at a tertiary cancer center. One hundred thirty unique robot-assisted procedures were performed using the Xi between 2014 and 2015, 112 of which were oncology surgeries. For procedures involving multiple quadrants, the robot was re-targeted. Complications were assessed according to Martin criteria and the Clavien-Dindo classification up to 90 days after operation. Thirteen different operations were performed in five oncology subspecialties (urology, gynecology, thoracic, hepatobiliary, and gastrointestinal surgery). Median operative times ranged from 183 min for nephroureterectomy to 543 min for esophagogastrectomy. Median estimated blood loss did not exceed 200 ml for any of the categorized procedures . No patients were transfused intraoperatively and no positioning injuries occurred. Conversions to open operation occurred in three cases (2.7%), though not related to complications or technical considerations. Overall complication rate was 26% with major complication rate of 4%. Readmissions were necessary in 11 (10%) patients. The da Vinci Xi can be safely assimilated into a surgical oncology program. The Xi offers versatility to various oncologic procedures with satisfactory complication and readmission rates. © 2015 Wiley Periodicals, Inc.

Catalytic conversion of xylose and corn stalk into furfural over carbon solid acid catalyst in γ-valerolactone.

PubMed

Zhang, Tingwei; Li, Wenzhi; Xu, Zhiping; Liu, Qiyu; Ma, Qiaozhi; Jameel, Hasan; Chang, Hou-min; Ma, Longlong

2016-06-01

A novel carbon solid acid catalyst was synthesized by the sulfonation of carbonaceous material which was prepared by carbonization of sucrose using 4-BDS as a sulfonating agent. TEM, N2 adsorption-desorption, elemental analysis, XPS and FT-IR were used to characterize the catalyst. Then, the catalyst was applied for the conversion of xylose and corn stalk into furfural in GVL. The influence of the reaction time, temperature and dosage of catalyst on xylose dehydration were also investigated. The Brønsted acid catalyst exhibited high activity in the dehydration of xylose, with a high furfural yield of 78.5% at 170°C in 30min. What's more, a 60.6% furfural yield from corn stalk was achieved in 100min at 200°C. The recyclability of the sulfonated carbon catalyst was perfect, and it could be reused for 5times without the loss of furfural yields. Copyright © 2016 Elsevier Ltd. All rights reserved.

Highly efficient production of L-lactic acid from xylose by newly isolated Bacillus coagulans C106.

PubMed

Ye, Lidan; Zhou, Xingding; Hudari, Mohammad Sufian Bin; Li, Zhi; Wu, Jin Chuan

2013-03-01

Cost-effective production of optically pure lactic acid from lignocellulose sugars is commercially attractive but challenging. Bacillus coagulans C106 was isolated from environment and used to produce l-lactic acid from xylose at 50°C and pH 6.0 in mineral salts medium containing 1-2% (w/v) of yeast extract without sterilizing the medium before fermentation. In batch fermentation with 85g/L of xylose, lactic acid titer and productivity reached 83.6g/L and 7.5g/Lh, respectively. When fed-batch (120+80+60g/L) fermentation was applied, they reached 215.7g/L and 4.0g/Lh, respectively. In both cases, the lactic acid yield and optical purity reached 95% and 99.6%, respectively. The lactic acid titer and productivity on xylose are the highest among those ever reported. Ca(OH)2 was found to be a better neutralizing agent than NaOH in terms of its giving higher lactic acid titer (1.2-fold) and productivity (1.8-fold) under the same conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

PubMed Central

2011-01-01

Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase

A novel cleaning process for industrial production of xylose in pilot scale from corncob by using screw-steam-explosive extruder.

PubMed

Zhang, Hong-Jia; Fan, Xiao-Guang; Qiu, Xue-Liang; Zhang, Qiu-Xiang; Wang, Wen-Ya; Li, Shuang-Xi; Deng, Li-Hong; Koffas, Mattheos A G; Wei, Dong-Sheng; Yuan, Qi-Peng

2014-12-01

Steam explosion is the most promising technology to replace conventional acid hydrolysis of lignocellulose for biomass pretreatment. In this paper, a new screw-steam-explosive extruder was designed and explored for xylose production and lignocellulose biorefinery at the pilot scale. We investigated the effect of different chemicals on xylose yield in the screw-steam-explosive extrusion process, and the xylose production process was optimized as followings: After pre-impregnation with sulfuric acid at 80 °C for 3 h, corncob was treated at 1.55 MPa with 9 mg sulfuric acid/g dry corncob (DC) for 5.5 min, followed by countercurrent extraction (3 recycles), decoloration (activated carbon dosage 0.07 g/g sugar, 75 °C for 40 min), and ion exchange (2 batches). Using this process, 3.575 kg of crystal xylose was produced from 22 kg corncob, almost 90 % of hemicellulose was released as monomeric sugar, and only a small amount of by-products was released (formic acid, acetic acid, fural, 5-hydroxymethylfurfural, and phenolic compounds were 0.17, 1.14, 0.53, 0.19, and 1.75 g/100 g DC, respectively). All results indicated that the screw-steam-explosive extrusion provides a more effective way to convert hemicellulose into xylose and could be an alternative method to traditional sulfuric acid hydrolysis process for lignocellulose biorefinery.

Cristobalite X-I: A bridge between low and high density silica polymorphs

NASA Astrophysics Data System (ADS)

Shelton, H.; Tiange, B.; Zurek, E.; Smith, J.; Dera, P.

2017-12-01

SiO2 is one of the most common compounds found on Earth. Despite its chemical simplicity, and because of its crystal chemical characteristics, SiO2 exhibits a complex phase diagram. SiO2 has a wide variety of thermodynamically stable crystalline phases, as well as numerous metastable crystalline and amorphous polymorphs. Many of the phase transition sequences that produce metastable phases of SiO2 are strongly path-dependent, where the rate of change controls the transition just as much as the final conditions. The elusive metastable polymorphs of SiO2 may provide a better understanding of the factors controlling its densification. On compression of α-cristobalite (the high temperature tetrahedral phase of SiO2) to pressures above 12 GPa, a new polymorph known as cristobalite X-I forms. Existence of cristobalite X-I has been known for several decades, however, consensus regarding the exact atomic arrangement has not yet been reached. The X-I phase constitutes an important step in the silica densification process, separating low-density tetrahedral framework structures from high-density octahedral polymorphs. It is unique in being the only non-quenchable high-density SiO2 phase, which reverts back to the tetrahedral low-density form on decompression at ambient temperature. Our new single crystal synchrotron X-ray diffraction experiments, with quasihydrostatic neon as the pressure medium, revealed the structure of this enigmatic phase to consist of octahedral silicate chains with 4-60°-2 zigzag chain geometry. This geometry has not been considered before, but is closely related to post-quartz, stishovite and seifertite. Density functional theory calculations support this observation, confirming the dynamic stability of the X-I arrangement and reasonably reproducing the pressure at which the transformation takes place. The enthalpy of cristobalite X-I is higher than stishovite and seifertite, but it is favored as a high-pressure successor of cristobalite due to a

Robotic da Vinci Xi-assisted nipple-sparing mastectomy: First clinical report.

PubMed

Sarfati, Benjamin; Honart, Jean-Francois; Leymarie, Nicolas; Rimareix, Francoise; Al Khashnam, Heba; Kolb, Frederic

2018-05-01

Nipple-sparing mastectomy (NSM) is increasingly popular for the treatment of selected breast cancers and prophylactic mastectomy. Surgical scarring and esthetic outcomes are important patient-related cosmetic considerations. Today, the concept of minimally invasive surgery has become popular, especially using robotic surgery. The authors report the first case of NSM using the latest version of the da Vinci Xi surgical system (Xi). The final incision used to remove the entire mammary gland was located behind the axillary line. In this position, hidden by the arm of the patient, the incision was not visible and was compatible with immediate breast reconstruction. © 2017 Wiley Periodicals, Inc.

Thermochemical hydrolysis of macroalgae Ulva for biorefinery: Taguchi robust design method

NASA Astrophysics Data System (ADS)

Jiang, Rui; Linzon, Yoav; Vitkin, Edward; Yakhini, Zohar; Chudnovsky, Alexandra; Golberg, Alexander

2016-06-01

Understanding the impact of all process parameters on the efficiency of biomass hydrolysis and on the final yield of products is critical to biorefinery design. Using Taguchi orthogonal arrays experimental design and Partial Least Square Regression, we investigated the impact of change and the comparative significance of thermochemical process temperature, treatment time, %Acid and %Solid load on carbohydrates release from green macroalgae from Ulva genus, a promising biorefinery feedstock. The average density of hydrolysate was determined using a new microelectromechanical optical resonator mass sensor. In addition, using Flux Balance Analysis techniques, we compared the potential fermentation yields of these hydrolysate products using metabolic models of Escherichia coli, Saccharomyces cerevisiae wild type, Saccharomyces cerevisiae RN1016 with xylose isomerase and Clostridium acetobutylicum. We found that %Acid plays the most significant role and treatment time the least significant role in affecting the monosaccharaides released from Ulva biomass. We also found that within the tested range of parameters, hydrolysis with 121 °C, 30 min 2% Acid, 15% Solids could lead to the highest yields of conversion: 54.134-57.500 gr ethanol kg-1 Ulva dry weight by S. cerevisiae RN1016 with xylose isomerase. Our results support optimized marine algae utilization process design and will enable smart energy harvesting by thermochemical hydrolysis.

Genetic engineering and improvement of a Zymomonas mobilis for arabinose utilization and its performance on pretreated corn stover hydrolyzate

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

Chou, Yat -Chen; Linger, Jeffrey; Yang, Shihui

In this paper, a glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizingmore » arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. Finally, we speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.« less

Genetic engineering and improvement of a Zymomonas mobilis for arabinose utilization and its performance on pretreated corn stover hydrolyzate

DOE PAGES

Chou, Yat -Chen; Linger, Jeffrey; Yang, Shihui; ...

2015-04-28

In this paper, a glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizingmore » arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. Finally, we speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.« less

Optimised formation of blue Maillard reaction products of xylose and glycine model systems and associated antioxidant activity.

PubMed

Yin, Zi; Sun, Qian; Zhang, Xi; Jing, Hao

2014-05-01

A blue colour can be formed in the xylose (Xyl) and glycine (Gly) Maillard reaction (MR) model system. However, there are fewer studies on the reaction conditions for the blue Maillard reaction products (MRPs). The objective of this study is to investigate characteristic colour formation and antioxidant activities in four different MR model systems and to determine the optimum reaction conditions for the blue colour formation in a Xyl-Gly MR model system, using the random centroid optimisation program. The blue colour with an absorbance peak at 630 nm appeared before browning in the Xyl-Gly MR model system, while no blue colour formation but only browning was observed in the xylose-alanine, xylose-aspartic acid and glucose-glycine MR model systems. The Xyl-Gly MR model system also showed higher antioxidant activity than the other three model systems. The optimum conditions for blue colour formation were as follows: xylose and glycine ratio 1:0.16 (M:M), 0.20 mol L⁻¹ NaHCO₃, 406.1 mL L⁻¹ ethanol, initial pH 8.63, 33.7°C for 22.06 h, which gave a much brighter blue colour and a higher peak at 630 nm. A characteristic blue colour could be formed in the Xyl-Gly MR model system and the optimum conditions for the blue colour formation were proposed and confirmed. © 2013 Society of Chemical Industry.

Discovery and Biochemical Characterization of the UDP-Xylose Biosynthesis Pathway in Sphaerobacter thermophilus.

PubMed

Gu, Bin; Laborda, Pedro; Wei, Shuang; Duan, Xu-Chu; Song, Hui-Bo; Liu, Li; Voglmeir, Josef

2016-01-01

The biosynthesis of UDP-xylose requires the stepwise oxidation/ decarboxylation of UDP-glucose, which is catalyzed by the enzymes UDPglucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS). UDPxylose biosynthesis is ubiquitous in animals and plants. However, only a few UGD and UXS isoforms of bacterial origin have thus far been biochemically characterized. Sphaerobacter thermophilus DSM 20745 is a bacterium isolated from heated sewage sludge, and therefore can be a valuable source of thermostable enzymes of biotechnological interest. However, no biochemical characterizations of any S. thermophilus enzymes have yet been reported. Herein, we describe the cloning and characterization of putative UGD (StUGD) and UXS (StUXS) isoforms from this organism. HPLC- and plate reader-based activity tests of the recombinantly expressed StUGD and StUXS showed that they are indeed active enzymes. Both StUGD and StUXS showed a temperature optimum of 70°C, and a reasonable thermal stability up to 60°C. No metal ions were required for enzymatic activities. StUGD had a higher pH optimum than StUXS. The simple purification procedures and the thermotolerance of StUGD and StUXS make them valuable biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose at elevated temperatures. The biosynthetic potential of StUGD was further exemplified in a coupled enzymatic reaction with an UDP-glucuronosyltransferase, allowing the glucuronylation of the natural model substrate bilirubin.

Distribution and source analysis of aluminum in rivers near Xi'an City, China.

PubMed

Wang, Dongqi; He, Yanling; Liang, Jidong; Liu, Pei; Zhuang, Pengyu

2013-02-01

To study the status and source of aluminum (Al) contamination, a total of 21 sampling sites along six rivers near Xi'an City (Shaanxi province, China) were investigated during 2008-2010. The results indicated that the average concentration of total Al (Al(t)) in the six rivers increased by 1.6 times from 2008 to 2010. The spatial distribution of Al(t) concentrations in the rivers near Xi'an City was significantly different, ranged from 367 μg/L (Bahe River) to 1,978 μg/L (Taiping River). The Al(t) concentration was highest near an industrial area for pulp and paper-making (2,773 μg/L), where the Al level greatly exceeded the water quality criteria of both the USA (Criterion Continuous Concentration, 87 μg/L) and Canada (100 μg/L). The average concentration of inorganic monometric aluminum (Al(im)) was 72 μg/L which would pose threats to fishes and other aquatic lives in the rivers. The concentrations of exchangeable Al (Al(ex)) in the sediment of the Taiping River sampled were relatively high, making it to be an alternative explanation of increasing Al concentrations in the rivers near Xi'an City. Furthermore, an increasing Al level has been detected in the upstream watershed near Xi'an City in recent years, which might indicate another notable pollution source of Al.

Monitoring on Xi'an ground fissures deformation with TerraSAR-X data

USGS Publications Warehouse

Zhao, C.; Zhang, Q.; Zhu, W.; Lu, Z.

2012-01-01

Owing to the fine resolution of TerraSAR-X data provided since 2007, this paper applied 6 TerraSAR data (strip mode) during 3rd Dec. 2009 to 23rd Mar. 2010 to detect and monitor the active fissures over Xi'an region. Three themes have been designed for high precision detection and monitoring of Xi'an-Chang'an fissures, as small baseline subsets (SBAS) to test the atmospheric effects of differential interferograms pair stepwise, 2-pass differential interferogram with very short baseline perpendicular to generate the whole deformation map with 44 days interval, and finally, corner reflector (CR) technique was used to closely monitor the relative deformation time series between two CRs settled crossing two ground fissures. Results showed that TerraSAR data are a good choice for small-scale ground fissures detection and monitoring, while special considerations should be taken for their great temporal and baseline decorrelation. Secondly, ground fissures in Xi'an were mostly detected at the joint section of stable and deformable regions. Lastly, CR-InSAR had potential ability to monitor relative deformation crossing fissures with millimeter precision.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels.

PubMed

Li, Xin; Yu, Vivian Yaci; Lin, Yuping; Chomvong, Kulika; Estrela, Raíssa; Park, Annsea; Liang, Julie M; Znameroski, Elizabeth A; Feehan, Joanna; Kim, Soo Rin; Jin, Yong-Su; Glass, N Louise; Cate, Jamie H D

2015-02-03

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels

PubMed Central

Li, Xin; Yu, Vivian Yaci; Lin, Yuping; Chomvong, Kulika; Estrela, Raíssa; Park, Annsea; Liang, Julie M; Znameroski, Elizabeth A; Feehan, Joanna; Kim, Soo Rin; Jin, Yong-Su; Glass, N Louise; Cate, Jamie HD

2015-01-01

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production. DOI: http://dx.doi.org/10.7554/eLife.05896.001 PMID:25647728

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

PubMed Central

2011-01-01

Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we reported the purification and the

Xylose-rich polysaccharides from the primary walls of embryogenic cell line of Pinus caribaea.

PubMed

Mollard, A; Domon, J M; David, H; Joseleau, J P

1997-08-01

Embryogenic cell lines of Pinus caribaea were isolated from somatic embryogenesis from zygotic embryos. Previous studies showed that the proteins and glycoproteins were characteristic of the embryogenic state. In the present work we were seeking typical feature in the polysaccharide from the cell walls of embryogenic calli at nine days of culture. Sequential extraction with water, ammonium oxalate, dimethyl sulfoxide, sodium borohydride and 4.3 M potassium hydroxide revealed that the extracted polysaccharides contained high proportions of arabinose and significant amounts of xylose. Fractionation of the hydrosoluble polymers on DEAE cellulose afforded a xylose-rich fraction (80% xylose, 24% glucose and lower properties of fucose and mannose). Methylation analysis and 13C-NMR spectra showed that the glycan backbone consisted of beta 1 --> 4 linked xylosyl residues Similar study of the fractions extracted respectively with DMSO and 4.3 M KOH showed the presence of polydisperse glycoxylans but excluded the presence of xyloglucan in significant amount. This could be a characteristic feature of embryogenic cells walls of Pinus caribaea or could be typical of cells grown as calluses. In the various fractions obtained from DEAE cellulose chromatography of the alkaline extract the infrequent occurrence of fucoxylans beside an arabinogalactan showed again the unusual nature of the cell wall polymers of this embryogenic lines, which seems to differ greatly from those found in the primary wall of cells from suspension cultures.

Xylose utilizing Zymomonas mobilis with improved ethanol production in biomass hydrolysate medium

DOEpatents

Caimi, Perry G; Hitz, William D; Viitanen, Paul V; Stieglitz, Barry

2013-10-29

Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

Xylose utilizing zymomonas mobilis with improved ethanol production in biomass hydrolysate medium

DOEpatents

Caimi, Perry G; Hitz, William D; Stieglitz, Barry; Viitanen, Paul V

2013-07-02

Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

The active centre of triose phosphate isomerase

PubMed Central

Burton, Pamela M.; Waley, S. G.

1966-01-01

The molecular weight and amino acid composition of triose phosphate isomerase have been determined. The molecular weight (43000) is lower and the molecular activity (500000) higher than those of most other glycolytic enzymes. Reaction with iodoacetate (studied with radioactive reagent) takes place in two phases: in the first phase, at pH6·3, cysteine and methionine groups react and enzymic activity is unimpaired; in the second phase, histidine reacts and enzymic activity is lost. Photo-oxidation leads to inactivation, with loss of cysteine, of histidine and of tryptophan, but little loss of tyrosine. The mechanism postulated for the action of the enzyme demands the intervention of a group functioning as a base, and the results obtained are consistent with histidine's being the basic group in the active centre. PMID:5969283

[HIV/STD prevalence and related behaviors among male STD clinic attendees in Xi'an and Xianyang cities, Shaanxi province].

PubMed

Hu, T; Dong, L F; Ding, Z W; Jia, H; Li, X; Zhang, J S; Song, Y L; Chang, W H

2017-12-10

Objective: To compare the prevalence of HIV/STD and related health care seeking behaviors among male STD clinic attendees between Xi'an and Xianyang cities. Methods: During June and July 2016, 206 male STD clinic attendees were studied in Xi'an city, with another 221 male STD clinic attendees in Xianyang city. Cross-sectional questionnaire survey was used to collect attendees'behavioral information. Blood samples were collected via HIV/HCV/Syphilis testing. Results: The prevalence rate of HIV infection was 2.4% (5/206) in Xi'an and 0.9% (2/221) in Xianyang, with no statistical significant difference between the two cities. The prevalence rate of syphilis was 4.9% (10/206) in Xi'an, which was significantly lower than 13.6% (30/221) in Xianyang. The proportion of respondents, diagnosed with other sexually transmitted diseases, in Xi'an was higher than that of Xianyang. The proportions of commercial heterosexual sex and sex with temporary sexual partners in the past 3 months were 18.0% (37/206) and 15.5% (32/206) in Xi'an, lower than 46.6% (103/221) and 15.8% (35/221) in Xianyang ( χ (2)=39.70, Р <0.01; χ (2)=0.01, P =0.93) . The proportions of condom use with commercial sex workers or temporary sexual partners in the past 3 months among Xi'an were 37.8% (14/37) and 6.3% (2/32) , lower than 93.1% (95/102) and 57.1% (20/35) in Xianyang ( χ (2)=49.06, Р <0.01; χ (2)=19.63, Р <0.01) . Conclusion: Differences were noticed between Xi'an and Xianyang city in terms of STD and HIV prevalences, behaviors related to commercial sex and use of condoms among the male STD clinic attendees that calling for targeted actions in control of high risk behavlors in both HIV/AIDS and STDs transmission.

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

PubMed

Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

2015-10-01

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Copyright © 2015 John Wiley & Sons, Ltd.

The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate.

PubMed

Lu, Xin; Sun, Jibin; Nimtz, Manfred; Wissing, Josef; Zeng, An-Ping; Rinas, Ursula

2010-04-20

The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most

The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate

PubMed Central

2010-01-01

Background The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. Results The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was

4-O-beta-D-galactopyranosyl-D-xylose: a new synthesis and application to the evaluation of intestinal lactase.

PubMed

Rivera-Sagredo, A; Fernández-Mayoralas, A; Jiménez-Barbero, J; Martín-Lomas, M; Villanueva, D; Aragón, J J

1992-04-10

4-O-beta-D-Galactopyranosyl-D-xylose (2) was prepared from benzyl 2,3-O-isopropylidene-beta-D-xylopyranoside by glycosylation with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide and subsequent deprotection. Compound 2 was hydrolyzed in vitro by intestinal lactase; the Vmax was 25% of that with lactose and the Km was 370mM (cf. 27mM for lactose). Oral administration of 2 suckling rats led to urinary excretion of D-xylose which could be estimated colorimetrically.

Comparison of Gavage, Water Bottle, and a High-Moisture Diet Bolus as Dosing Methods for Quantitative D-xylose Administration to B6D2F1 (Mus musculus) Mice

NASA Technical Reports Server (NTRS)

Zimmer, J. Paul; Lewis, Sherry M.; Moyer, Jerry L.

1993-01-01

Gavage, water bottle, and diet incorporation are 3 dosing methods used orally to administer test compounds to rodents. These 3 methods were compared in mice to determine which represented the most quantitative delivery system. For dietary incorporation, a high-moisture bolus form of NIH-31 rodent meal was developed using hydroxypropyl methylcellulose as an autoclave-stable binding agent. A high-moisture bolus were selected to increase the acceptability of the dosed diet and to promote quantitative consumption through reduced wastage. The test compound used was D-xylose, a pentose sugar that may be quantitatively detected, colorimetrically, in urine following oral dosing. Six male and 6 female B6D2FI mice were placed in metabolism cages and dosed with a known quantity of D-xylose by each of the 3 methods. Urine was collected before and after each method of administration and analysed for total D-xylose; the per cent recovery was based upon the amount of D-xylose consumed. Quantitative consumption was apparently greatest for water bottle dosing with an average recovery of 56.0% of the original D-xylose dose. High-moisture bolus incorporation ranked second with 50.0% D-xylose recovery, and gavage was third with 41.0% D-xylose recovery.

Dynamic flux balance modeling of microbial co-cultures for efficient batch fermentation of glucose and xylose mixtures.

PubMed

Hanly, Timothy J; Henson, Michael A

2011-02-01

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture. © 2010 Wiley Periodicals, Inc.

Operative technique and early experience for robotic-assisted laparoscopic nephroureterectomy (RALNU) using da Vinci Xi.

PubMed

Darwiche, Fadi; Swain, Sanjaya; Kallingal, George; Punnen, Sanoj; Manoharan, Murugesan; Parekh, Dipen J; Gonzalgo, Mark L

2015-01-01

Robotic-assisted laparoscopic nephroureterectomy (RALNU) has been previously utilized for management of upper tract urothelial carcinoma. The da Vinci Xi surgical system was released in April of 2014. We describe our operative technique and early experience for RALNU using the da Vinci Xi system highlighting unique features of this surgical platform. A total of 10 patients with a diagnosis of upper tract urothelial carcinoma underwent RALNU using the da Vinci Xi system between April and November of 2014. A novel, oblique "in line" robotic trocar configuration was utilized to access the upper abdomen (nephrectomy portion) and pelvis (bladder cuff excision) without undocking. The port hopping feature of da Vinci Xi was utilized to facilitate optimal, multi-quadrant visualization during RALNU. Robotic-assisted laparoscopic nephroureterectomy was successfully completed without open conversion in all 10 patients. Mean operative time was 184 min (range 140-300 min), mean estimated blood loss was 121 cc (range 60-300 cc), and mean hospital stay was 2.4 days. Final pathology demonstrated high grade urothelial carcinoma in all patients. Surgical margins were negative in all patients. No intra-operative complications were encountered. One patient developed a pulmonary embolus after being discharged. No patients required a blood transfusion. Mean patient follow-up was 130 days (range 15-210 days). The use of da Vinci Xi with a novel, oblique "in line" port configuration and camera port hopping technique allows for an efficient and reproducible method for RALNU without the need for repositioning the patient or the robot during surgery.

25 CFR 36.31 - Standard XI-Student promotion requirements.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 25 Indians 1 2012-04-01 2011-04-01 true Standard XI-Student promotion requirements. 36.31 Section 36.31 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR EDUCATION MINIMUM ACADEMIC STANDARDS FOR THE BASIC EDUCATION OF INDIAN CHILDREN AND NATIONAL CRITERIA FOR DORMITORY SITUATIONS Student...

25 CFR 36.31 - Standard XI-Student promotion requirements.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 25 Indians 1 2011-04-01 2011-04-01 false Standard XI-Student promotion requirements. 36.31 Section 36.31 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR EDUCATION MINIMUM ACADEMIC STANDARDS FOR THE BASIC EDUCATION OF INDIAN CHILDREN AND NATIONAL CRITERIA FOR DORMITORY SITUATIONS Student...

Cloning and characterization of XiR1, a locus responsible for dagger nematode resistance in grape.

PubMed

Hwang, Chin-Feng; Xu, Kenong; Hu, Rong; Zhou, Rita; Riaz, Summaira; Walker, M Andrew

2010-08-01

The dagger nematode, Xiphinema index, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) x V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks.

Cloning and characterization of XiR1, a locus responsible for dagger nematode resistance in grape

PubMed Central

Hwang, Chin-Feng; Xu, Kenong; Hu, Rong; Zhou, Rita; Riaz, Summaira

2010-01-01

The dagger nematode, Xiphinemaindex, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) × V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks. PMID:20490447

D-Xylose as a sugar complement regulates blood glucose levels by suppressing phosphoenolpyruvate carboxylase (PEPCK) in streptozotocin-nicotinamide-induced diabetic rats and by enhancing glucose uptake in vitro

PubMed Central

Kim, Eunju; Kim, Yoo-Sun; Kim, Kyung-Mi; Jung, Sangwon; Yoo, Sang-Ho

2016-01-01

BACKGROUND/OBJECTIVES Type 2 diabetes (T2D) is more frequently diagnosed and is characterized by hyperglycemia and insulin resistance. D-Xylose, a sucrase inhibitor, may be useful as a functional sugar complement to inhibit increases in blood glucose levels. The objective of this study was to investigate the anti-diabetic effects of D-xylose both in vitro and stretpozotocin (STZ)-nicotinamide (NA)-induced models in vivo. MATERIALS/METHODS Wistar rats were divided into the following groups: (i) normal control; (ii) diabetic control; (iii) diabetic rats supplemented with a diet where 5% of the total sucrose content in the diet was replaced with D-xylose; and (iv) diabetic rats supplemented with a diet where 10% of the total sucrose content in the diet was replaced with D-xylose. These groups were maintained for two weeks. The effects of D-xylose on blood glucose levels were examined using oral glucose tolerance test, insulin secretion assays, histology of liver and pancreas tissues, and analysis of phosphoenolpyruvate carboxylase (PEPCK) expression in liver tissues of a STZ-NA-induced experimental rat model. Levels of glucose uptake and insulin secretion by differentiated C2C12 muscle cells and INS-1 pancreatic β-cells were analyzed. RESULTS In vivo, D-xylose supplementation significantly reduced fasting serum glucose levels (P < 0.05), it slightly reduced the area under the glucose curve, and increased insulin levels compared to the diabetic controls. D-Xylose supplementation enhanced the regeneration of pancreas tissue and improved the arrangement of hepatocytes compared to the diabetic controls. Lower levels of PEPCK were detected in the liver tissues of D-xylose-supplemented rats (P < 0.05). In vitro, both 2-NBDG uptake by C2C12 cells and insulin secretion by INS-1 cells were increased with D-xylose supplementation in a dose-dependent manner compared to treatment with glucose alone. CONCLUSIONS In this study, D-xylose exerted anti-diabetic effects in vivo by

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.

Effect of salts on the Co-fermentation of glucose and xylose by a genetically engineered strain of Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background A challenge currently facing the cellulosic biofuel industry is the efficient fermentation of both C5 and C6 sugars in the presence of inhibitors. To overcome this challenge, microorganisms that are capable of mixed-sugar fermentation need to be further developed for increased inhibitor tolerance. However, this requires an understanding of the physiological impact of inhibitors on the microorganism. This paper investigates the effect of salts on Saccharomyces cerevisiae 424A(LNH-ST), a yeast strain capable of effectively co-fermenting glucose and xylose. Results In this study, we show that salts can be significant inhibitors of S. cerevisiae. All 6 pairs of anions (chloride and sulfate) and cations (sodium, potassium, and ammonium) tested resulted in reduced cell growth rate, glucose consumption rate, and ethanol production rate. In addition, the data showed that the xylose consumption is more strongly affected by salts than glucose consumption at all concentrations. At a NaCl concentration of 0.5M, the xylose consumption rate was reduced by 64.5% compared to the control. A metabolomics study found a shift in metabolism to increased glycerol production during xylose fermentation when salt was present, which was confirmed by an increase in extracellular glycerol titers by 4 fold. There were significant differences between the different cations. The salts with potassium cations were the least inhibitory. Surprisingly, although salts of sulfate produced twice the concentration of cations as compared to salts of chloride, the degree of inhibition was the same with one exception. Potassium salts of sulfate were less inhibitory than potassium paired with chloride, suggesting that chloride is more inhibitory than sulfate. Conclusions When developing microorganisms and processes for cellulosic ethanol production, it is important to consider salt concentrations as it has a significant negative impact on yeast performance, especially with regards to xylose

Biochemical properties of L-arabinose isomerase from Clostridium hylemonae to produce D-tagatose as a functional sweetener.

PubMed

Nguyen, Tien-Kieu; Hong, Moon-Gi; Chang, Pahn-Shick; Lee, Byung-Hoo; Yoo, Sang-Ho

2018-01-01

d-Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA, encoding l-arabinose isomerase (l-AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l-AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7-7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-1) of l-AI from C. hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d-tagatose using the C. hylemonae l-arabinose isomerase at 60°C reached approximately 46% from 10 mM of d-galactose after 2 h. From these results, it is suggested that the l-arabinose isomerase from C. hylemonae could be utilized as a potential enzyme for d-tagatose production due to its high conversion yield at an industrially competitive temperature.

Biochemical properties of L-arabinose isomerase from Clostridium hylemonae to produce D-tagatose as a functional sweetener

PubMed Central

Nguyen, Tien-Kieu; Hong, Moon-Gi; Chang, Pahn-Shick; Lee, Byung-Hoo

2018-01-01

d-Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA, encoding l-arabinose isomerase (l-AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l-AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7–7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-1) of l-AI from C. hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d-tagatose using the C. hylemonae l-arabinose isomerase at 60°C reached approximately 46% from 10 mM of d-galactose after 2 h. From these results, it is suggested that the l-arabinose isomerase from C. hylemonae could be utilized as a potential enzyme for d-tagatose production due to its high conversion yield at an industrially competitive temperature. PMID:29684065

Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

PubMed

Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

2009-05-01

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

PubMed

Pratter, S M; Eixelsberger, T; Nidetzky, B

2015-12-01

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ground Subsidence Monitoring with MT-InSAR and Mechanism Inversion Over Xi'an, China

NASA Astrophysics Data System (ADS)

Peng, M. M.; Zhao, C. Y.; Zhang, Q.; Zhang, J.; Liu, Y. Y.

2018-04-01

The ancient Xi'an, China, has been suffering severe land subsidence and ground fissure hazards since the 1960s, which has affected the safety of Subways. Multi-sensor SAR data are conducted to monitor the latest complex ground deformation and its influence on subway line No.3 over Xi'an. Annual deformation rates have been retrieved to reveal the spatiotemporal evolution of ground subsidence in Xi'an city from 2013 to 2017. Meanwhile, the correlation between land subsidence and ground fissures are analyzed by retrieving the deformation differences in both sides of the fissures. Besides, the deformation along subway line No. 3 is analyzed, and the fast deformation section is quantitatively studied. Finally, a flat lying sill model with distributed contractions is implemented to model the InSAR deformation over YHZ subsidence center, which manifests that the ground deformation is mainly caused by groundwater withdrawal.

Triosephosphate isomerase and filamin C share common epitopes as novel allergens of Procambarus clarkii

USDA-ARS?s Scientific Manuscript database

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity...

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

PubMed

Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

2015-04-16

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation

PubMed Central

Marsolier, J.; Perichon, M.; DeBarry, JD.; Villoutreix, BO.; Chluba, J.; Lopez, T.; Garrido, C.; Zhou, XZ.; Lu, KP.; Fritsch, L.; Ait-Si-Ali, S.; Mhadhbi, M; Medjkane, S.; Weitzman, JB.

2014-01-01

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack the genetic and epigenetic machinery to change phenotypic states. Amongst the Apicomplexa phylum of obligate intracellular parasites which cause veterinary and human diseases, Theileria is the only genus which transforms its mammalian host cells1. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-12. The transformed phenotypes are reversed by treatment with the theilericidal drug Buparvaquone3. We used comparative genomics to identify a homologue of the Peptidyl Prolyl Isomerase Pin1 (designated TaPin1) in T. annulata which is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPin1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7 leading to its degradation and subsequent stabilization of c-Jun which promotes transformation. We performed in vitro analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPin1 is directly inhibited by the anti-parasite drug Buparvaquone (and other known Pin1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerisation is thus a conserved mechanism which is important in cancer and is used by Theileria parasites to manipulate host oncogenic signaling. PMID:25624101

Inhibiting prolyl isomerase activity by hybrid organic-inorganic molecules containing rhodium(II) fragments.

PubMed

Coughlin, Jane M; Kundu, Rituparna; Cooper, Julian C; Ball, Zachary T

2014-11-15

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site. Copyright © 2014 Elsevier Ltd. All rights reserved.

Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co-utilization in Escherichia coli.

PubMed

Groot, Joost; Cepress-Mclean, Sidney C; Robbins-Pianka, Adam; Knight, Rob; Gill, Ryan T

2017-04-01

Engineering the simultaneous consumption of glucose and xylose sugars is critical to enable the sustainable production of biofuels from lignocellulosic biomass. In most major industrial microorganisms glucose completely inhibits the uptake of xylose, limiting efficient sugar mixture conversion. In E. coli removal of the major glucose transporter PTS allows for glucose and xylose co-consumption but only after prolonged adaptation, which is an effective process but hard to control and prone to co-evolving undesired traits. Here we synthetically engineer mutants to target sugar co-consumption properties; we subject a PTS - mutant to a short adaptive step and subsequently either delete or overexpress key genes previously suggested to affect sugar consumption. Screening the co-consumption properties of these mutants individually is very laborious. We show we can evaluate sugar co-consumption properties in parallel by culturing the mutants in selection and applying a novel approach that computes mutant growth rates in selection using chromosomal barcode counts obtained from Next-Generation Sequencing. We validate this multiplex growth rate phenotyping approach with individual mutant pure cultures, identify new instances of mutants cross-feeding on metabolic byproducts, and, importantly, find that the rates of glucose and xylose co-consumption can be tuned by altering glucokinase expression in our PTS - background. Biotechnol. Bioeng. 2017;114: 885-893. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Heavy metals pollution and analysis of seasonal variation runoff in Xi'an].

PubMed

Yuan, Hong-Lin; Li, Xing-Yu; Wang, Xiao-Chang

2014-11-01

In order to explore heavy metals pollution situation,changes in characteristics, the correlation between each heavy mental and pollution source analysis of Xi'an various regions in different season in one year. This study collected several samples of Xi'an rainfall typical urban trunk roads throughout the year in 2013 and used inductively coupled plasma mass spectrometry (ICP-MS) to determine the level of Fe, Mn, Pb, Zn, Al, Cd of the samples, then, analyzed the seasonal change of heavy mental. Studies have shown that: the heavy metal of Xi'an road runoff pollutes seriously, the concentration of Fe over three times of the national standard and maintain the higher levels throughout the year, meanwhile the concentration with the intensity of human activities increases. The concentration of Mn and Zn in one year show a trends: winter > autumn > summer> spring. Pb concentration increases with the increase in traffic volume, while showing: winter > spring > summer > autumn. Factor analysis shows: Fe and Al was affected by the same sources-natural sources; Zn, Cd affected by anthropogenic sources of large; Mn, Pb affected by the larger traffic sources.

25 CFR 36.31 - Standard XI-Student promotion requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 25 Indians 1 2014-04-01 2014-04-01 false Standard XI-Student promotion requirements. 36.31 Section 36.31 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR EDUCATION MINIMUM ACADEMIC... programs, in a minimum of 160 instructional days per academic term or 80 instructional days per semester...

25 CFR 36.31 - Standard XI-Student promotion requirements.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 25 Indians 1 2013-04-01 2013-04-01 false Standard XI-Student promotion requirements. 36.31 Section 36.31 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR EDUCATION MINIMUM ACADEMIC... programs, in a minimum of 160 instructional days per academic term or 80 instructional days per semester...

Research on Xi Jinping's Thought of Ecological Civilization and Environment Sustainable Development

NASA Astrophysics Data System (ADS)

Xiang-chao, Pan

2018-05-01

Since the reform and opening up, China’s sustained and rapid economic development, but the environment problem increasingly is prominent in our country. It has seriously affected the sustainability of economic development in China. Environment overall situation is not optimistic, and environmental management is imperative. Since the 18th national congress of the Communist Party of China (CPC), Xi Jin-ping has put forward the thought of building a beautiful China with ecological civilization and realizing the sustainable development of economic construction and environmental protection. Sticking to Xi's Thought of Ecological Civilization is a fundamental guarantee for the sustainable development of environment and building a new era of ecological civilization.

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2016-03-01

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

Transoral robotic surgery for the base of tongue squamous cell carcinoma: a preliminary comparison between da Vinci Xi and Si.

PubMed

Alessandrini, Marco; Pavone, Isabella; Micarelli, Alessandro; Caporale, Claudio

2017-09-13

Considering the emerging advantages related to da Vinci Xi robotic platform, the aim of this study is to compare for the first time the operative outcomes of this tool to the previous da Vinci Si during transoral robotic surgery (TORS), both performed for squamous cell carcinomas (SCC) of the base of tongue (BOT). Intra- and peri-operative outcomes of eight patients with early stage (T1-T2) of the BOT carcinoma and undergoing TORS by means of the da Vinci Xi robotic platform (Xi-TORS) are compared with the da Vinci Si group ones (Si-TORS). With respect to Si-TORS group, Xi-TORS group demonstrated a significantly shorter overall operative time, console time, and intraoperative blood loss, as well as peri-operative pain intensity and length of mean hospital stays and nasogastric tube positioning. Considering recent advantages offered by surgical robotic techniques, the da Vinci Xi Surgical System preliminary outcomes could suggest its possible future routine implementation in BOT squamous cell carcinoma procedures.

Quantitative investigations of xylose and arabinose substituents in hydroxypropylated and hydroxyvinylethylated arabinoxylans.

PubMed

Lorenz, Dominic; Knöpfle, Anna; Akil, Youssef; Saake, Bodo

2017-11-01

The chemical structures obtained by the modification of arabinoxylans with the cyclic carbonates propylene carbonate (PC) and 4-vinyl-1,3-dioxolan-2-one (VEC) with varying degrees of substitution were investigated. Therefore, a new analytical method was developed that is based on a microwave-assisted hydrolysis of the polysaccharides with trifluoroacetic acid and the reductive amination with 2-aminobenzoic acid. The peak assignment was achieved by HPLC-MS and the carbohydrate derivatives were quantified by HPLC-fluorescence. The obtained maximum molar substitution of PC-derivatized xylan (X HP ) was 1.8; the molar substitution of VEC-derivatized xylan (X HVE ) was 2.3. Investigations of xylose and arabinose based mono- and disubstituted derivatives revealed a preferred reaction of the cyclic carbonates with arabinose. Conversion rates were up to 2.4 times higher for monosubstitution and up to 3.0 times for disubstitution compared to xylose. Furthermore, the reaction with VEC was preferred due to higher reactivity of the newly introduced side chains. Copyright © 2017 Elsevier Ltd. All rights reserved.

An artificial transport metabolon facilitates improved substrate utilization in yeast.

PubMed

Thomik, Thomas; Wittig, Ilka; Choe, Jun-Yong; Boles, Eckhard; Oreb, Mislav

2017-11-01

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes

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

Liavonchanka, Alena; Hornung, Ellen; Feussner, Ivo

2006-02-01

Low iodide concentrations were sufficient to allow SAD and SIRAS phasing of cubic crystals of a novel fatty acid isomerase using Cu Kα radiation. The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy- or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellow-coloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2{submore » 1}3, with a unit-cell parameter of 160.4 Å, and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å, β = 115.8°. Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å. Initial phases were obtained by SIRAS from in-house data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M.« less

40 CFR Appendix Xi to Part 86 - Sampling Plans for Selective Enforcement Auditing of Light-Duty Vehicles

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 19 2010-07-01 2010-07-01 false Sampling Plans for Selective Enforcement Auditing of Light-Duty Vehicles XI Appendix XI to Part 86 Protection of Environment ENVIRONMENTAL... Enforcement Auditing of Light-Duty Vehicles 40% AQL Table 1—Sampling Plan Code Letter Annual sales of...

Genes related to xylose fermentation and methods of using same for enhanced biofuel production

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2015-09-29

The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

Genes related to xylose fermentation and methods of using same for enhanced biofuel production

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2016-11-29

The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

Genes related to xylose fermentation and methods of using same for enhanced biofuel production

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2014-08-05

The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

Improvement of ACE inhibitory activity of casein hydrolysate by Maillard reaction with xylose.

PubMed

Hong, Xu; Meng, Jun; Lu, Rong-Rong

2015-01-01

The Maillard reaction is widely used to improve the functional properties or biological activities of food. The purpose of this study was to investigate the effect of the Maillard reaction on angiotensin I converting enzyme (ACE) inhibitory activity in a casein hydrolysate-xylose system. Two-step hydrolysis was used to prepare casein ACE inhibitory peptides. Maillard reaction products (MRPs) were prepared by heating hydrolyzed casein with xylose at pH 8.0, 110 °C for up to 16 h. The results showed that the content of free amino group decreased (P < 0.05); however, browning intensity and absorbance at 294 nm increased because of the Maillard reaction (P < 0.05). The ACE inhibitory activity improved greatly within 2 h (from 63.48% to 90.23%), which was mainly due to carbonyl ammonia condensation reaction in the MRPs. The study shows that the Maillard reaction under appropriate conditions can improve the ACE inhibitory activity of casein hydrolysate effectively. © 2014 Society of Chemical Industry.

25 CFR 36.31 - Standard XI-Student promotion requirements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 25 Indians 1 2010-04-01 2010-04-01 false Standard XI-Student promotion requirements. 36.31 Section... Instructional Evaluation § 36.31 Standard XI—Student promotion requirements. Each school shall establish and implement a promotion policy which shall be submitted to and approved by the local school board and Agency...

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.

Does transition from the da Vinci Si to Xi robotic platform impact single-docking technique for robot-assisted laparoscopic nephroureterectomy?

PubMed

Patel, Manish N; Aboumohamed, Ahmed; Hemal, Ashok

2015-12-01

To describe our robot-assisted nephroureterectomy (RNU) technique for benign indications and RNU with en bloc excision of bladder cuff (BCE) and lymphadenectomy (LND) for malignant indications using the da Vinci Si and da Vinci Xi robotic platform, with its pros and cons. The port placement described for Si can be used for standard and S robotic systems. This is the first report in the literature on the use of the da Vinci Xi robotic platform for RNU. After a substantial experience of RNU using different da Vinci robots from the standard to the Si platform in a single-docking fashion for benign and malignant conditions, we started using the newly released da Vinci Xi robot since 2014. The most important differences are in port placement and effective use of the features of da Vinci Xi robot while performing simultaneous upper and lower tract surgery. Patient positioning, port placement, step-by-step technique of single docking RNU-LND-BCE using the da Vinci Si and da Vinci Xi robot are shown in an accompanying video with the goal that centres using either robotic system benefit from the hints and tips. The first segment of video describes RNU-LND-BCE using the da Vinci Si followed by the da Vinci Xi to highlight differences. There was no need for patient repositioning or robot re-docking with the new da Vinci Xi robotic platform. We have experience of using different robotic systems for single docking RNU in 70 cases for benign (15) and malignant (55) conditions. The da Vinci Xi robotic platform helps operating room personnel in its easy movement, allows easier patient side-docking with the help of its boom feature, in addition to easy and swift movements of the robotic arms. The patient clearance feature can be used to avoid collision with the robotic arms or the patient's body. In patients with challenging body habitus and in situations where bladder cuff management is difficult, modifications can be made through reassigning the camera to a different port with

Ethanol fermentation by xylose-assimilating Saccharomyces cerevisiae using sugars in a rice straw liquid hydrolysate concentrated by nanofiltration.

PubMed

Sasaki, Kengo; Sasaki, Daisuke; Sakihama, Yuri; Teramura, Hiroshi; Yamada, Ryosuke; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

2013-11-01

Concentrating sugars using membrane separation, followed by ethanol fermentation by recombinant xylose-assimilating Saccharomyces cerevisiae, is an attractive technology. Three nanofiltration membranes (NTR-729HF, NTR-7250, and ESNA3) were effective in concentrating glucose, fructose, and sucrose from dilute molasses solution and no permeation of sucrose. The separation factors of acetate, formate, furfural, and 5-hydroxymethyl furfural, which were produced by dilute acid pretreatment of rice straw, over glucose after passage through these three membranes were 3.37-11.22, 4.71-20.27, 4.32-16.45, and 4.05-16.84, respectively, at pH 5.0, an applied pressure of 1.5 or 2.0 MPa, and 25 °C. The separation factors of these fermentation inhibitors over xylose were infinite, as there was no permeation of xylose. Ethanol production from approximately two-times concentrated liquid hydrolysate using recombinant S. cerevisiae was double (5.34-6.44 g L(-1)) that compared with fermentation of liquid hydrolysate before membrane separation (2.75 g L(-1)). Copyright © 2013 Elsevier Ltd. All rights reserved.

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

PubMed

Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

2000-02-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

PubMed Central

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

PubMed

Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

2018-06-16

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

PubMed

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-04-03

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

PubMed Central

Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

2017-01-01

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

Disulfide isomerase-like protein AtPDIL1–2 is a good candidate for trichlorophenol phytodetoxification

PubMed Central

Peng, Ri-He; Qiu, Jin; Tian, Yong-Sheng; Gao, Jian-jie; Han, Hong-juan; Fu, Xiao-Yan; Zhu, Bo; Xu, Jing; Wang, Bo; Li, Zhen-jun; Wang, Li-juan; Yao, Quan-Hong

2017-01-01

Trichlorophenol (TCP) is a widely used and persistent environmentally toxic compound that poses a carcinogenic risk to humans. Phytoremediation is a proficient cleanup technology for organic pollutants. In this study, we found that the disulfide isomerase-like protein AtPDIL1–2 in plants is a good candidate for enhancing 2,4,6-TCP phytoremediation. The expression of AtPDIL1-2 in Arabidopsis was induced by 2,4,6-TCP. The heterologously expressed AtPDIL1-2 in Escherichia coli exhibited both oxidase and isomerase activities as protein disulfide isomerase and improved bacteria tolerance to 2,4,6-TCP. Further research revealed that transgenic tobacco overexpressing AtPDIL1-2 was more tolerant to high concentrations of 2,4,6-TCP and removed the toxic compound at far greater rates than the control plants. To elucidate the mechanism of action of AtPDIL1-2, we investigated the chemical interaction of AtPDIL1-2 with 2,4,6-TCP for the first time. HPLC analysis implied that AtPDIL1-2 exerts a TCP-binding activity. A suitable configuration of AtPDIL1-2-TCP binding was obtained by molecular docking studies using the AutoDock program. It predicted that the TCP binding site is located in the b-b′ domain of AtPDIL1-2 and that His254 of the protein is critical for the binding interaction. These findings imply that AtPDIL1-2 can be used for TCP detoxification by the way of overexpression in plants. PMID:28059139

Improvement on D-xylose to Xylitol Biotransformation by Candida guilliermondii Using Cells Permeabilized with Triton X-100 and Selected Process Conditions.

PubMed

Cortez, Daniela Vieira; Mussatto, Solange I; Roberto, Inês Conceição

2016-11-01

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by D-xylose and MgCl 2 ·6H 2 O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % D-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the D-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction.

PubMed

Ferreira de Oliveira, José Miguel P; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2010-07-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.

Presence of glucose, xylose, and glycerol fermenting bacteria in the deep biosphere of the former Homestake gold mine, South Dakota

PubMed Central

Rastogi, Gurdeep; Gurram, Raghu N.; Bhalla, Aditya; Gonzalez, Ramon; Bischoff, Kenneth M.; Hughes, Stephen R.; Kumar, Sudhir; Sani, Rajesh K.

2012-01-01

Eight fermentative bacterial strains were isolated from mixed enrichment cultures of a composite soil sample collected at 1.34 km depth from the former Homestake gold mine in Lead, SD, USA. Phylogenetic analysis of their 16S rRNA gene sequences revealed that these isolates were affiliated with the phylum Firmicutes belonging to genera Bacillus and Clostridium. Batch fermentation studies demonstrated that isolates had the ability to ferment glucose, xylose, or glycerol to industrially valuable products such as ethanol and 1,3-propanediol (PDO). Ethanol was detected as the major fermentation end product in glucose-fermenting cultures at pH 10 with yields of 0.205–0.304 g of ethanol/g of glucose. While a xylose-fermenting strain yielded 0.189 g of ethanol/g of xylose and 0.585 g of acetic acid/g of xylose at the end of fermentation. At pH 7, glycerol-fermenting isolates produced PDO (0.323–0.458 g of PDO/g of glycerol) and ethanol (0.284–0.350 g of ethanol/g of glycerol) as major end products while acetic acid and succinic acid were identified as minor by-products in fermentation broths. These results suggest that the deep biosphere of the former Homestake gold mine harbors bacterial strains which could be used in bio-based production of ethanol and PDO. PMID:23919089

Robotic resections in hepatobiliary oncology - initial experience with Xi da Vinci system in India.

PubMed

Chandarana, M; Patkar, S; Tamhankar, A; Garg, S; Bhandare, M; Goel, M

2017-01-01

Minimal invasive surgery has proven its advantages over open surgeries in the perioperative period. Food and Drug Administration approved da Vinci robot in 2000. The latest version, da Vinci Xi system has a mobile tower-based robot with several modifications to improve the functionality, versatility, and operative ease. None of the centers have reported exclusively on hepatobiliary oncology using the da Vinci Xi system. We report our initial experience. To study the feasibility, advantages, and discuss the operative technique of da Vinci Xi system in hepatobiliary oncology. Data were analyzed retrospectively from a prospectively maintained database from June 2015 to October 2016. Twenty-five patients with suspected or proven hepatobiliary malignancies were operated. Total robotic technique using da Vinci Xi system was used. Demographic details and perioperative outcomes were noted. Of the 25 surgeries, 14 patients had a suspected gallbladder malignancy, 11 patients had primary or metastatic liver tumor. Median age was 53 years. The average duration of surgery was 225 min with a median blood loss 150 ml. The median postoperative stay was 4 days. The median nodal yield for radical cholecystectomy was seven. Five patients required conversion. Two of these developed postoperative morbidity. Robotic surgery for hepatobiliary oncology is feasible and can be performed safely in experienced hands. Increasing experience in this field may equal or even prove advantageous over conventional or laparoscopic approach in future. A cautious approach with judicious patient selection is the key to establishing robotic surgery as a standard surgical approach.

The implementation of high fermentative 2,3-butanediol production from xylose by simultaneous additions of yeast extract, Na2EDTA, and acetic acid.

PubMed

Wang, Xiao-Xiong; Hu, Hong-Ying; Liu, De-Hua; Song, Yuan-Quan

2016-01-25

The effective use of xylose may significantly enhance the feasibility of using lignocellulosic hydrolysate to produce 2,3-butanediol (2,3-BD). Previous difficulties in 2,3-BD production include that the high-concentration xylose cannot be converted completely and the fermentation rate is slow. This study investigated the effects of yeast extract, ethylenediaminetetraacetic acid disodium salt (Na2EDTA), and acetic acid on 2,3-BD production from xylose. The central composite design approach was used to optimize the concentrations of these components. It was found that simultaneous addition of yeast extract, Na2EDTA, and acetic acid could significantly improve 2,3-BD production. The optimal concentrations of yeast extract, Na2EDTA, and acetic acid were 35.2, 1.2, and 4.5 g/L, respectively. The 2,3-BD concentration in the optimized medium reached 39.7 g/L after 48 hours of shake flask fermentation, the highest value ever reported in such a short period. The xylose utilization ratio and the 2,3-BD concentration increased to 99.0% and 42.7 g/L, respectively, after 48 hours of stirred batch fermentation. Furthermore, the 2,3-BD yield was 0.475 g/g, 95.0% of the theoretical maximum value. As the major components of lignocellulosic hydrolysate are glucose, xylose, and acetic acid, the results of this study indicate the possibility of directly using the hydrolysate to effectively produce 2,3-BD. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

PubMed Central

Lopez-Zavala, Alonso A.; Carrasco-Miranda, Jesus S.; Ramirez-Aguirre, Claudia D.; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G.; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S.; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

2016-01-01

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation. PMID:27614148