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Sample records for precursor pathway engineering

  1. Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume.

    PubMed

    Li, Penghui; Dong, Qiang; Ge, Shujun; He, Xianzhi; Verdier, Jerome; Li, Dongqin; Zhao, Jian

    2016-07-01

    MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying its promotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR promotes PA production by a direct repression of biosynthesis of isoflavones, the major flavonoids in legume, and by redirecting immediate precursors, such as anthocyanidins, flux into PA pathway. Ectopic expression of MtPAR repressed isoflavonoid production by directly binding and suppressing isoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up-regulated PA-specific genes and decreased the anthocyanin levels without altering the expression of anthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. MtPAR complemented PA-deficient phenotype of Arabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showed the direct interactions between MtPAR, MtTT8 and MtWD40-1 proteins from Medicago truncatula and Glycine max, to form a ternary complex to trans-activate PA-specific ANR gene. Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and whole plants only promoted the production of small amount of PAs, which was significantly enhanced by co-expression of MtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect between MtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requires more anthocyanidin precursors. This study provides new insights into the role and mechanism of MtPAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathways for a specific promotion of PA production. Based on this, a strategy by combining MtPAR and MtLAP1 co-expression to effectively improve metabolic engineering performance of PA production in legume forage was developed. PMID:26806316

  2. Designed biosynthesis of 36-methyl-FK506 by polyketide precursor pathway engineering

    PubMed Central

    Lechner, Anna; Wilson, Micheal C.; Ban, Yeon Hee; Hwang, Jae-yeon; Yoon, Yeo Joon; Moore, Bradley S.

    2012-01-01

    The polyketide synthase (PKS) biosynthetic code has recently expanded to include a newly recognized group of extender unit substrates derived from α,β-unsaturated acyl-CoA molecules that deliver diverse side chain chemistry to polyketide backbones. Herein we report the identification of a three-gene operon responsible for the biosynthesis of the PKS building block isobutyrylmalonyl-CoA associated with the macrolide ansalactam A from the marine bacterium Streptomyces sp. CNH189. Using a synthetic biology approach, we engineered the production of unnatural 36-methyl-FK506 in Streptomyces sp. KCTC 11604BP by incorporating the branched extender unit into FK506 biosynthesis in place of its natural C-21 allyl side chain, which has been shown to be critical for FK506’s potent immunosuppressant and neurite outgrowth activities. PMID:23654255

  3. Precursors of Young Women's Family Formation Pathways

    ERIC Educational Resources Information Center

    Amato, Paul R.; Landale, Nancy S.; Havasevich-Brooks, Tara C.; Booth, Alan; Eggebeen, David J.; Schoen, Robert; McHale, Susan M.

    2008-01-01

    We used latent class analysis to create family formation pathways for women between the ages of 18 and 23. Input variables included cohabitation, marriage, parenthood, full-time employment, and attending school. Data (n = 2,290) came from Waves I and III of the National Longitudinal Study of Adolescent Health (Add Health). The analysis revealed…

  4. Protein design for pathway engineering

    SciTech Connect

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  5. Protein Design for Pathway Engineering

    PubMed Central

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  6. Pathways to adulthood and their precursors and outcomes.

    PubMed

    Skogbrott Birkeland, Marianne; Leversen, Ingrid; Torsheim, Torbjørn; Wold, Bente

    2014-02-01

    Norway has an extensive welfare system which may provide adolescents with many options and high levels of flexibility in terms of pathways to adulthood. This study aimed to describe Norwegian developmental pathways to adulthood, including changes in role statuses (such as living situations, education, work, marriage/cohabitation and parenthood) from 16 to 30 years of age, and their precursors and outcomes. Repeated measures latent class analysis of longitudinal data from 998 Norwegian individuals indicated three main pathways to adulthood among women and men. In both sexes, most individuals undertook a long period of education and postponed family formation. However, some individuals started working early, a group of women established families with partners and children early, and a group of men remained primarily single between 16 and 30 years of age. Furthermore, the results show that pathways to adulthood in Norway are surprisingly similar to pathways in other countries such as the US, UK and Finland. The results indicate that pathways to adulthood are influenced by social reproduction factors in a country with high levels of welfare benefits as well. In addition, the results suggest that pathways involving living with a partner and either higher education or work are associated with high life satisfaction at age 30. PMID:24236443

  7. Reverse Engineering Adverse Outcome Pathways

    SciTech Connect

    Perkins, Edward; Chipman, J.K.; Edwards, Stephen; Habib, Tanwir; Falciani, Francesco; Taylor, Ronald C.; Van Aggelen, Graham; Vulpe, Chris; Antczak, Philipp; Loguinov, Alexandre

    2011-01-30

    The toxicological effects of many stressors are mediated through unknown, or poorly characterized, mechanisms of action. We describe the application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, metabolic, signaling) to characterize adverse outcome pathways (AOPs) for chemicals that disrupt the hypothalamus-pituitary-gonadal endocrine axis in fathead minnows. Gene expression changes in fathead minnow ovaries in response to 7 different chemicals, over different times, doses, and in vivo versus in vitro conditions were captured in a large data set of 868 arrays. We examined potential AOPs of the antiandrogen flutamide using two mutual information theory methods, ARACNE and CLR to infer gene regulatory networks and potential adverse outcome pathways. Representative networks from these studies were used to predict a network path from stressor to adverse outcome as a candidate AOP. The relationship of individual chemicals to an adverse outcome can be determined by following perturbations through the network in response to chemical treatment leading to the nodes associated with the adverse outcome. Identification of candidate pathways allows for formation of testable hypotheses about key biologic processes, biomarkers or alternative endpoints, which could be used to monitor an adverse outcome pathway. Finally, we identify the unique challenges facing the application of this approach in ecotoxicology, and attempt to provide a road map for the utilization of these tools. Key Words: mechanism of action, toxicology, microarray, network inference

  8. Selective overproduction of the proteasome inhibitor salinosporamide A via precursor pathway regulation

    PubMed Central

    Lechner, Anna; Eustáquio, Alessandra S.; Gulder, Tobias A. M.; Hafner, Mathias; Moore, Bradley S.

    2011-01-01

    The chlorinated natural product salinosporamide A is a potent 20S proteasome inhibitor currently in clinical trials as an anticancer agent. To deepen our understanding of salinosporamide biosynthesis, we investigated the function of a LuxR-type pathway-specific regulatory gene, salR2, and observed a selective effect on the production of salinosporamide A over its less active aliphatic analogs. SalR2 was shown to specifically activate genes involved in the biosynthesis of the halogenated precursor chloroethylmalonyl-CoA, which is a dedicated precursor of salinosporamide A. Specifically, SalR2 activates transcription of two divergent operons – one of which contains the unique S-adenosyl-L-methionine-dependent chlorinase encoding gene salL. By applying this knowledge towards rational engineering, we were able to selectively double salinosporamide A production. This study exemplifies the specialized regulation of a polyketide precursor pathway and its application to the selective overproduction of a specific natural product congener. PMID:22195555

  9. Comparative Proteomics Analysis of Engineered Saccharomyces cerevisiae with Enhanced Biofuel Precursor Production

    PubMed Central

    Tang, Xiaoling; Feng, Huixing; Zhang, Jianhua; Chen, Wei Ning

    2013-01-01

    The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production. PMID:24376832

  10. Snail Coordinately Regulates Downstream Pathways to Control Multiple Aspects of Mammalian Neural Precursor Development

    PubMed Central

    Zander, Mark A.; Burns, Sarah E.; Yang, Guang; Kaplan, David R.

    2014-01-01

    The Snail transcription factor plays a key role in regulating diverse developmental processes but is not thought to play a role in mammalian neural precursors. Here, we have examined radial glial precursor cells of the embryonic murine cortex and demonstrate that Snail regulates their survival, self-renewal, and differentiation into intermediate progenitors and neurons via two distinct and separable target pathways. First, Snail promotes cell survival by antagonizing a p53-dependent death pathway because coincident p53 knockdown rescues survival deficits caused by Snail knockdown. Second, we show that the cell cycle phosphatase Cdc25b is regulated by Snail in radial precursors and that Cdc25b coexpression is sufficient to rescue the decreased radial precursor proliferation and differentiation observed upon Snail knockdown. Thus, Snail acts via p53 and Cdc25b to coordinately regulate multiple aspects of mammalian embryonic neural precursor biology. PMID:24719096

  11. Pathways to an Engineering Career

    ERIC Educational Resources Information Center

    Pearson, Willie, Jr.; Miller, Jon D.

    2012-01-01

    Utilizing data from the 20-year record of the Longitudinal Study of American Youth (LSAY), this analysis uses a set of variables to predict employment in engineering for a national sample of adults aged 34 to 37. The LSAY is one of the longest longitudinal studies of the impact of secondary education and postsecondary education conducted in the…

  12. Precursors and metabolic pathway for guaiacol production by Alicyclobacillus acidoterrestris.

    PubMed

    Cai, Rui; Yuan, Yahong; Wang, Zhouli; Guo, Chunfeng; Liu, Bin; Liu, Laping; Wang, Yutang; Yue, Tianli

    2015-12-01

    Alicyclobacillus acidoterrestris has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which was manifested by the production of guaiacol. Vanillic acid and vanillin have been accepted as the biochemical precursors of guaiacol in fruit juices. The purpose of this study was to try to find other precursors and elucidate details about the conversion of vanillic acid and vanillin to guaiacol by A. acidoterrestris. Four potential substrates including ferulic acid, catechol, phenylalanine and tyrosine were analyzed, but they could not be metabolized to guaiacol by all the thirty A. acidoterrestris strains tested. Resting cell studies and enzyme assays demonstrated that vanillin was reduced to vanillyl alcohol by NADPH-dependent vanillin reductase and oxidized to vanillic acid by NAD(P)(+)-dependent vanillin dehydrogenases in A. acidoterrestris DSM 3923. Vanillic acid underwent a nonoxidative decarboxylation to guaiacol. The reversible vanillic acid decarboxylase involved was oxygen insensitive and pyridine nucleotide-independent. PMID:26241489

  13. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis.

    PubMed

    Kuzuyama, Tomohisa; Seto, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  14. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

    PubMed Central

    KUZUYAMA, Tomohisa; SETO, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  15. Phosphoketolase pathway engineering for carbon-efficient biocatalysis.

    PubMed

    Henard, Calvin Andrew; Freed, Emily Frances; Guarnieri, Michael Thomas

    2015-12-01

    Recent advances in metabolic engineering have facilitated the development of microbial biocatalysts capable of producing an array of bio-products, ranging from fuels to drug molecules. These bio-products are commonly generated through an acetyl-CoA intermediate, which serves as a key precursor in the biological conversion of carbon substrates. Conventional biocatalytic upgrading strategies proceeding through this route are limited by low carbon efficiencies, in large part due to carbon losses associated with pyruvate decarboxylation to acetyl-CoA. Bypass of pyruvate decarboxylation offers a means to dramatically enhance carbon yields and, in turn, bioprocess economics. Herein, we discuss recent advances and prospects for employing the phosphoketolase pathway for direct biosynthesis of acetyl-CoA from carbon substrates, and phosphoketolase-based metabolic engineering strategies for carbon efficient biocatalysis. PMID:26360872

  16. Phosphoketolase Pathway Engineering for Carbon-Efficient Biocatalysis

    SciTech Connect

    Henard, Calvin Andrew; Freed, Emily Frances; Guarnieri, Michael Thomas

    2015-09-08

    Recent advances in metabolic engineering have facilitated the development of microbial biocatalysts capable of producing an array of bio-products, ranging from fuels to drug molecules. These bio-products are commonly generated through an acetyl-CoA intermediate, which serves as a key precursor in the biological conversion of carbon substrates. Moreover, conventional biocatalytic upgrading strategies proceeding through this route are limited by low carbon efficiencies, in large part due to carbon losses associated with pyruvate decarboxylation to acetyl-CoA. Bypass of pyruvate decarboxylation offers a means to dramatically enhance carbon yields and, in turn, bioprocess economics. Here, we discuss recent advances and prospects for employing the phosphoketolase pathway for direct biosynthesis of acetyl-CoA from carbon substrates, and phosphoketolase-based metabolic engineering strategies for carbon efficient biocatalysis.

  17. Pyrolysis Pathways of Sulfonated Polyethylene, an Alternative Carbon Fiber Precursor

    SciTech Connect

    Younker, Jarod M; Saito, Tomonori; Hunt, Marcus A; Beste, Ariana; Naskar, Amit K

    2013-01-01

    Sulfonated polyethylene is an emerging precursor for the production of carbon fibers. Pyrolysis of sulfonated polyethylene was characterized by thermogravimetric analysis (TGA). n-heptane-4-sulfonic acid (H4S) was selected as a model compound for the study of sulfonated polyethylene. Density functional theory and conventional transition state theory were used to determine the rate constants of pyrolysis for H4S from 300-1000 K. Multiple reaction channels from two different mechanisms were explored: 1) internal five-centered elimination (Ei 5) and 2) radical chain reaction. The pyrolysis of H4S was simulated with kinetic Monte Carlo (kMC) to obtain TGA plots that compared favorably to experiment. We observed that at tem- peratures < 550 K, the radical mechanism was dominant and yielded the trans-alkene, whereas cis-alkene was formed at higher temperatures from the internal elimination. The maximum rates of % mass loss became independent of initial OH radical concentration at 440-480 K. Experimentally, the maximum % mass loss occurred from 440-460 K (heating rate dependent). Activation energies derived from the kMC-simulated TGAs of H4S (26-29 kcal/mol) agreed with experiment for sulfonated polyethylene ( 31 kcal/mol). The simulations revealed that in this region, decomposition of radical HOSO2 became competitive to H abstraction by HOSO2, making OH the carrying radical for the reaction chain. The maximum rate of % mass loss for internal elimination was observed at temperatures > 600 K. Low-scale carbonization utilizes temperatures < 620 K; thus, internal elimination will not be competitive. Ei5 elimination has been studied for sulfoxides and sulfones, but this represents the first study of internal elimination in sulfonic acids. Nonlinear Arrhenius plots were found for all bimolecular reactions. The most significant nonlinear behavior was observed for reactions where the barrier was small. For reactions with low activation barriers, nonlinearity was traced to

  18. General low-temperature reaction pathway from precursors to monomers before nucleation of compound semiconductor nanocrystals.

    PubMed

    Yu, Kui; Liu, Xiangyang; Qi, Ting; Yang, Huaqing; Whitfield, Dennis M; Y Chen, Queena; Huisman, Erik J C; Hu, Changwei

    2016-01-01

    Little is known about the molecular pathway to monomers of semiconductor nanocrystals. Here we report a general reaction pathway, which is based on hydrogen-mediated ligand loss for the precursor conversion to 'monomers' at low temperature before nucleation. We apply (31)P nuclear magnetic resonance spectroscopy to monitor the key phosphorous-containing products that evolve from MXn+E=PPh2H+HY mixtures, where MXn, E=PPh2H, and HY are metal precursors, chalcogenide precursors, and additives, respectively. Surprisingly, the phosphorous-containing products detected can be categorized into two groups, Ph2P-Y and Ph2P(E)-Y. On the basis of our experimental and theoretical results, we propose two competing pathways to the formation of M2En monomers, each of which is accompanied by one of the two products. Our study unravels the pathway of precursor evolution into M2En monomers, the stoichiometry of which directly correlates with the atomic composition of the final compound nanocrystals. PMID:27531507

  19. General low-temperature reaction pathway from precursors to monomers before nucleation of compound semiconductor nanocrystals

    PubMed Central

    Yu, Kui; Liu, Xiangyang; Qi, Ting; Yang, Huaqing; Whitfield, Dennis M.; Y. Chen, Queena; Huisman, Erik J. C.; Hu, Changwei

    2016-01-01

    Little is known about the molecular pathway to monomers of semiconductor nanocrystals. Here we report a general reaction pathway, which is based on hydrogen-mediated ligand loss for the precursor conversion to ‘monomers' at low temperature before nucleation. We apply 31P nuclear magnetic resonance spectroscopy to monitor the key phosphorous-containing products that evolve from MXn+E=PPh2H+HY mixtures, where MXn, E=PPh2H, and HY are metal precursors, chalcogenide precursors, and additives, respectively. Surprisingly, the phosphorous-containing products detected can be categorized into two groups, Ph2P–Y and Ph2P(E)–Y. On the basis of our experimental and theoretical results, we propose two competing pathways to the formation of M2En monomers, each of which is accompanied by one of the two products. Our study unravels the pathway of precursor evolution into M2En monomers, the stoichiometry of which directly correlates with the atomic composition of the final compound nanocrystals. PMID:27531507

  20. Engineering the MEP pathway enhanced ajmalicine biosynthesis.

    PubMed

    Chang, Kai; Qiu, Fei; Chen, Min; Zeng, Lingjiang; Liu, Xiaoqiang; Yang, Chunxian; Lan, Xiaozhong; Wang, Qiang; Liao, Zhihua

    2014-01-01

    The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR-overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co-overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes. PMID:24237015

  1. Construction and engineering of large biochemical pathways via DNA assembler

    PubMed Central

    Shao, Zengyi; Zhao, Huimin

    2015-01-01

    Summary DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated. PMID:23996442

  2. Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies

    PubMed Central

    Schmid, Jochen; Sieber, Volker; Rehm, Bernd

    2015-01-01

    Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications. PMID:26074894

  3. Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

    PubMed

    Ro, Dae-Kyun; Paradise, Eric M; Ouellet, Mario; Fisher, Karl J; Newman, Karyn L; Ndungu, John M; Ho, Kimberly A; Eachus, Rachel A; Ham, Timothy S; Kirby, James; Chang, Michelle C Y; Withers, Sydnor T; Shiba, Yoichiro; Sarpong, Richmond; Keasling, Jay D

    2006-04-13

    Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices. PMID:16612385

  4. Engineering the Microstructure of Solution Precursor Plasma-Sprayed Coatings

    NASA Astrophysics Data System (ADS)

    Bertolissi, G.; Chazelas, C.; Bolelli, G.; Lusvarghi, L.; Vardelle, M.; Vardelle, A.

    2012-12-01

    This study examines the fundamental reactions that occur in-flight during the solution precursor plasma spraying (SPPS) of solutions containing Zr- and Y-based salts in water or ethanol solvent. The effect of plasma jet composition (pure Ar, Ar-H2 and Ar-He-H2 mixtures) on the mechanical break-up and thermal treatment of the solution, mechanically injected in the form of a liquid stream, was investigated. Observation of the size evolution of the solution droplets in the plasma flow by means of a laser shadowgraphy technique, showed that droplet break-up was more effective and solvent evaporation was faster when the ethanol-based solution was injected into binary or ternary plasma gas mixtures. In contrast with water-based solutions, residual liquid droplets were always detected at the substrate location. The morphology and structure of the material deposited onto stainless steel substrates during single-scan experiments were characterised by SEM, XRD and micro-Raman spectroscopy and were shown to be closely related to in-flight droplet behaviour. In-flight pyrolysis and melting of the precursor led to well-flattened splats, whereas residual liquid droplets at the substrate location turned into non pyrolysed inclusions. The latter, although subsequently pyrolysed by the plasma heat during the deposition of entire coatings, resulted in porous "sponge-like" structures in the deposit.

  5. Pathway engineering strategies for production of beneficial carotenoids in microbial hosts.

    PubMed

    Ye, Victor M; Bhatia, Sujata K

    2012-08-01

    Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet. PMID:22488437

  6. Effect of Na-doped Mo on Selenization Pathways for CuGa/In Metallic Precursors

    SciTech Connect

    Krishnan, Rangarajan; Tong, Gabriel; Kim, Woo Kyoung; Payzant, E Andrew; Adelhelm, Christoph; Franzke, Enrico; Winkler, Jörg; Anderson, Timothy J

    2013-01-01

    Reaction pathways were followed for selenization of CuGa/In precursor structures using in-situ high temperature X-ray diffraction (HTXRD). Precursor films were deposited on Na-free and Na-doped Mo (3 and 5 at %)/Na-free glass. The precursor film was constituted with CuIn, In, Cu9Ga4, Cu3Ga, Cu16In9 and Mo. HTXRD measurements during temperature ramp selenization showed CIS formation occurs first, followed by CGS formation, and then mixing on the group III sub-lattice to form CIGS. CIGS formation was observed to be complete at ~450 C for samples deposited on 5 at % Na-doped Mo substrates. MoSe2 formation was evidenced after the CIGS synthesis reaction was complete. The Ga distribution in the annealed CIGS was determined by Rietveld refinement. Isothermal reaction studies were conducted for CIGS (112) formation in the temperature range 260-320 C to estimate the rate constants.

  7. Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast.

    PubMed

    Qu, Yang; Easson, Michael L A E; Froese, Jordan; Simionescu, Razvan; Hudlicky, Tomas; De Luca, Vincenzo

    2015-05-12

    Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine. PMID:25918424

  8. Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast

    PubMed Central

    Qu, Yang; Easson, Michael L. A. E.; Froese, Jordan; Simionescu, Razvan; Hudlicky, Tomas; De Luca, Vincenzo

    2015-01-01

    Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine. PMID:25918424

  9. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

    PubMed Central

    2013-01-01

    Background Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial

  10. A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes.

    PubMed

    Andersen, Gorm; Björnberg, Olof; Polakova, Silvia; Pynyaha, Yuriy; Rasmussen, Anna; Møller, Kasper; Hofer, Anders; Moritz, Thomas; Sandrini, Michael Paolo Bastner; Merico, Anna-Maria; Compagno, Concetta; Akerlund, Hans-Erik; Gojković, Zoran; Piskur, Jure

    2008-07-18

    Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1-6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date. PMID:18550080

  11. Enhancing the Community College Pathway to Engineering Careers

    ERIC Educational Resources Information Center

    Mattis, Mary C., Ed.; Sislin, John, Ed.

    2005-01-01

    Community colleges play an important role in starting students on the road to engineering careers, but students often face obstacles in transferring to four-year educational institutions to continue their education. "Enhancing the Community College Pathway to Engineering Careers," a new book from the National Academy of Engineering and the…

  12. Metabolite Valves: Dynamic Control of Metabolic Flux for Pathway Engineering

    NASA Astrophysics Data System (ADS)

    Prather, Kristala

    2015-03-01

    Microbial strains have been successfully engineered to produce a wide variety of chemical compounds, several of which have been commercialized. As new products are targeted for biological synthesis, yield is frequently considered a primary driver towards determining feasibility. Theoretical yields can be calculated, establishing an upper limit on the potential conversion of starting substrates to target compounds. Such yields typically ignore loss of substrate to byproducts, with the assumption that competing reactions can be eliminated, usually by deleting the genes encoding the corresponding enzymes. However, when an enzyme encodes an essential gene, especially one involved in primary metabolism, deletion is not a viable option. Reducing gene expression in a static fashion is possible, but this solution ignores the metabolic demand needed for synthesis of the enzymes required for the desired pathway. We have developed Metabolite valves to address this challenge. The valves are designed to allow high flux through the essential enzyme during an initial period where growth is favored. Following an external perturbation, enzyme activity is then reduced, enabling a higher precursor pool to be diverted towards the pathway of interest. We have designed valves with control at both the transcriptional and post-translational levels. In both cases, key enzymes in glucose metabolism are regulated, and two different compounds are targeted for heterologous production. We have measured increased concentrations of intracellular metabolites once the valve is closed, and have demonstrated that these increased pools lead to increased product yields. These metabolite valves should prove broadly useful for dynamic control of metabolic flux, resulting in improvements in product yields.

  13. Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.

    PubMed

    Xu, Wei; Weissmiller, April M; White, Joseph A; Fang, Fang; Wang, Xinyi; Wu, Yiwen; Pearn, Matthew L; Zhao, Xiaobei; Sawa, Mariko; Chen, Shengdi; Gunawardena, Shermali; Ding, Jianqing; Mobley, William C; Wu, Chengbiao

    2016-05-01

    The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its β-C-terminal fragment (β-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and β-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. β-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or β-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS. PMID:27064279

  14. On the possible involvement of bovine serum albumin precursor in lipofection pathway.

    PubMed

    Mukherjee, Anubhab; Bhattacharyya, Jayanta; Chaudhuri, Arabinda

    2014-03-01

    Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10 percent (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10 percent FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway. PMID:24499789

  15. Amyloid precursor protein–mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration

    PubMed Central

    Xu, Wei; Weissmiller, April M.; White, Joseph A.; Fang, Fang; Wang, Xinyi; Wu, Yiwen; Pearn, Matthew L.; Zhao, Xiaobei; Chen, Shengdi; Gunawardena, Shermali; Ding, Jianqing; Mobley, William C.

    2016-01-01

    The endosome/lysosome pathway is disrupted early in the course of both Alzheimer’s disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its β-C-terminal fragment (β-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and β-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. β-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or β-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS. PMID:27064279

  16. TAPP1 inhibits the differentiation of oligodendrocyte precursor cells via suppressing the Mek/Erk pathway.

    PubMed

    Chen, Yidan; Mei, Ruyi; Teng, Peng; Yang, Aifen; Hu, Xuemei; Zhang, Zunyi; Qiu, Mengsheng; Zhao, Xiaofeng

    2015-10-01

    Oligodendrocytes (OLs) are glial cells that form myelin sheaths around axons in the central nervous system (CNS). Loss of the myelin sheath in demyelinating and neurodegenerative diseases can lead to severe impairment of movement. Understanding the extracellular signals and intracellular factors that regulate OL differentiation and myelination during development can help to develop novel strategies for enhancing myelin repair in neurological disorders. Here, we report that TAPP1 was selectively expressed in differentiating OL precursor cells (OPCs). TAPP1 knockdown promoted OL differentiation and myelin gene expression in culture. Conversely, over-expression of TAPP1 in immature OPCs suppressed their differentiation. Moreover, TAPP1 inhibition in OPCs altered the expression of Erk1/2 but not AKT. Taken together, our results identify TAPP1 as an important negative regulator of OPC differentiation through the Mek/Erk signaling pathway. PMID:26242484

  17. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  18. Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Sesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial applications. Here, we construct an efficient Saccharomyces cerevisiae cell factory for α-santalene production. Results A multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product. To do so, genetic modifications were introduced acting to optimize the farnesyl diphosphate branch point, modulate the mevalonate pathway, modify the ammonium assimilation pathway and enhance the activity of a transcriptional activator. The approach employed resulted in an overall α-santalene yield of a 0.0052 Cmmol (Cmmol glucose)-1 corresponding to a 4-fold improvement over the reference strain. This strategy, combined with a specifically developed continuous fermentation process, led to a final α-santalene productivity of 0.036 Cmmol (g biomass)-1 h-1. Conclusions The results reported in this work illustrate how the combination of a metabolic engineering strategy with fermentation technology optimization can be used to obtain significant amounts of the high-value sesquiterpene α-santalene. This represents a starting point toward the construction of a yeast “sesquiterpene factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods. PMID:22938570

  19. Reverse Engineering Adverse Outcome Pathways in Ecotoxicology

    EPA Science Inventory

    The toxicological effects of many stressors are mediated through unknown, or incompletely characterized, mechanisms of action. We describe the application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, meabolic, signaling) t...

  20. Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

    PubMed Central

    Sengupta, Sudeshna; Goonewardena, Lakshani; Juturu, Veeresh

    2015-01-01

    cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroFFBR, aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars. PMID:26362984

  1. Metabolic engineering of a novel muconic acid biosynthesis pathway via 4-hydroxybenzoic acid in Escherichia coli.

    PubMed

    Sengupta, Sudeshna; Jonnalagadda, Sudhakar; Goonewardena, Lakshani; Juturu, Veeresh

    2015-12-01

    cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroF(FBR), aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars. PMID:26362984

  2. Bioremediation: environmental clean-up through pathway engineering.

    PubMed

    Singh, Shailendra; Kang, Seung Hyun; Mulchandani, Ashok; Chen, Wilfred

    2008-10-01

    Given the immense risk posed by widespread environmental pollution by inorganic and organic chemicals, novel methods of decontamination and clean-up are required. Owing to the relatively high cost and the non-specificity of conventional techniques, bioremediation is a promising alternative technology for pollutant clean-up. Advances in bioremediation harness molecular, genetic, microbiology, and protein engineering tools and rely on identification of novel metal-sequestering peptides, rational and irrational pathway engineering, and enzyme design. Recent advances have been made for enhanced inorganic chemical remediation and organic chemical degradation using various pathway-engineering approaches and these are discussed in this review. PMID:18760355

  3. [Engineering MEP pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control].

    PubMed

    Wang, Jianfeng; Xiong, Zhiqiang; Zhang, Siliang; Wang, Yong

    2014-01-01

    The pathway of 2-methyl-D-erythritol-4-phosphate (MEP) is the exclusive isoprenoid precursor biosynthetic pathway in Escherichia coli, with a higher theoretical yield than mevalonate (MVA) pathway. However, due to lack of information about the regulation of MEP pathway, only engineering MEP pathway in E. coli achieved limited improvement of heterologous isoprenoid production. We used exogenous MEP pathway genes to improve MEP pathway in E. coli and optimized the glucose feeding to release the potential of MEP pathway. The results demonstrate that co-expression of dxs2 from Streptomyces avermitilis and idi from Bacillus subtilis can increase amorphadiene production with 12.2-fold compared with the wild-type strain in shake flask fermentation. Then we established a high-cell density fermentation process for the engineered strain, and found that the phase from 24 to 72 h is important for product biosynthesis. The optimization of glucose feeding rate during 24 to 72 h significantly improved product accumulation, which was improved from 2.5 to 4.85 g/L, within the same process time. Considering the attenuation of strain metabolism after 72 h, this study further modulated the glucose feeding rate during exponential phase to control strain growth and the amorphadiene yield eventually reached to 6.1 g/L. These results provided useful information to develop engineered E. coli for isoprenoid production through MEP pathway engineering. PMID:24818480

  4. Effect of temperature on the reaction pathway of calcium carbonate formation via precursor phases

    NASA Astrophysics Data System (ADS)

    Purgstaller, Bettina; Mavromatis, Vasileios; Konrad, Florian; Dietzel, Martin

    2016-04-01

    It has been earlier postulated that some biogenic and sedimentary calcium carbonate (CaCO3) minerals (e.g. calcite and aragonite) are secondary in origin and have originally formed via a metastable calcium carbonate precursor phase (e.g. amorphous CaCO3, [1-2]). Such formation pathways are likely affected by various physicochemical parameters including aqueous Mg and temperature. In an effort to improve our understanding on the formation mechanism of CaCO3 minerals, precipitation experiments were carried out by the addition of a 0.6 M (Ca,Mg)Cl2 solution at distinct Mg/Ca ratios (1/4 and 1/8) into a 1 M NaHCO3 solution under constant pH conditions(8.3 ±0.1). The formation of CaCO3 was systematically examined as a function of temperature (6, 12, 18 and 25 ±0.3° C). During the experimental runs mineral precipitation was monitored by in situ Raman spectroscopy as well as by continuous sampling and analyzing of precipitates and reactive solutions. The results revealed two pathways of CaCO3 formation depending on the initial Mg/Ca ratio and temperature: (i) In experiments with a Mg/Ca ratio of 1/4 at ≤ 12° C as well as in experiments with a Mg/Ca ratio of 1/8 at ≤ 18° C, ikaite (CaCO3 6H2O) acts as a precursor phase for aragonite formation. (ii) In contrast higher temperatures induced the formation of Mg-rich amorphous CaCO3 (Mg-ACC) which was subsequently transformed to Mg-rich calcite. In situ Raman spectra showed that the transformation of Mg-ACC to Mg-calcite occurs at a higher rate (˜ 8 min) compared to that of ikaite to aragonite (> 2 h). Thus, the formation of aragonite rather than of Mg-calcite occurs due to the slower release of Ca2+and CO32‑ ions into the Mg-rich reactive solution during retarded ikaite dissolution. This behavior is generally consistent with the observation that calcite precipitation is inhibited at elevated aqueous Mg/Ca ratios. [1] Addadi L., Raz S. and Weiner S. (2003) Advanced Materials 15, 959-970. [2] Rodriguez-Blanco J. D

  5. Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of coenzyme A.

    PubMed

    Rothmann, Michael; Kang, MinJin; Villa, Reymundo; Ntai, Ioanna; La Clair, James J; Kelleher, Neil L; Chapman, Eli; Burkart, Michael D

    2013-04-24

    Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues. PMID:23550886

  6. Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of Coenzyme A

    PubMed Central

    Rothmann, Michael; Kang, MinJin; Villa, Reymundo; Ntai, Ioanna; La Clair, James J.; Kelleher, Neil L.; Chapman, Eli; Burkart, Michael D.

    2013-01-01

    Pantetheine, and its corresponding disulfide pantethine, play a key role in metabolism as a building block of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and non-ribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we demonstrate that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one carbon deletion from pantetheine, can rescue a mutant E. coli strain MG1655CΔpanC lacking a functional pantothenate synthetase. Using mass spectral methods, we demonstrate that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogs. PMID:23550886

  7. Engineered biofiltration for the removal of disinfection by-product precursors and genotoxicity.

    PubMed

    McKie, Michael J; Taylor-Edmonds, Liz; Andrews, Susan A; Andrews, Robert C

    2015-09-15

    Disinfection by-products (DBPs) are formed when naturally occurring organic matter reacts with chlorine used in drinking water treatment, and DBPs formed in chlorinated drinking water samples have been shown to cause a genotoxic response. The objective of the current study was to further understand the principles of biofiltration and the resulting impacts on the formation of DBPs and genotoxicity. Pilot-scale systems were utilized to assess the performance of engineered biofilters enhanced with hydrogen peroxide, in-line coagulants, and nutrients when compared to passively operated biofilters and conventional treatment (coagulation, flocculation, sedimentation, non-biological filtration). Organic fractionation was completed using liquid chromatography-organic carbon detection (LC-OCD). Water samples were chlorinated after collection and examined for the removal of trihalomethane (THM), haloacetic acid (HAA), and adsorbable organic halide (AOX) precursors. Additionally, the formation potential of two halogenated furanones, 3-chloro-4(dichloromethyl)-2(5H)-furanone (MX) and mucochloric acid (MCA), and genotoxicity was determined. Biofiltration was shown to preferentially remove more DBP precursors than dissolved organic carbon (DOC). Formation potential of the unregulated DBPs, including MX and MCA, and genotoxic response was shown to be correlated to THM formation. These results infer that monitoring for THMs and HAAs provide insight to the formation of more mutagenic DBPs such as halogenated furanones, and that biofiltration may preferentially remove precursors to DBPs at a rate exceeding the removal of DOC. PMID:26065391

  8. Engineering the lactococcal mevalonate pathway for increased sesquiterpene production.

    PubMed

    Song, Adelene A; Abdullah, Janna Ong; Abdullah, Mohd P; Shafee, Norazizah; Othman, Roohaida; Noor, Normah Mohd; Rahim, Raha A

    2014-06-01

    Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced. PMID:24828482

  9. Cofactor Engineering for Enhancing the Flux of Metabolic Pathways

    PubMed Central

    Akhtar, M. Kalim; Jones, Patrik R.

    2014-01-01

    The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering. PMID:25221776

  10. Natural Variation in Epigenetic Pathways Affects the Specification of Female Gamete Precursors in Arabidopsis[OPEN

    PubMed Central

    Rodríguez-Leal, Daniel; León-Martínez, Gloria; Abad-Vivero, Ursula; Vielle-Calzada, Jean-Philippe

    2015-01-01

    In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants. PMID:25829442

  11. Natural variation in epigenetic pathways affects the specification of female gamete precursors in Arabidopsis.

    PubMed

    Rodríguez-Leal, Daniel; León-Martínez, Gloria; Abad-Vivero, Ursula; Vielle-Calzada, Jean-Philippe

    2015-04-01

    In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants. PMID:25829442

  12. Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus.

    PubMed

    Jiang, Lingjuan; Wei, Junhong; Li, Lei; Niu, Guoqing; Tan, Huarong

    2013-12-01

    Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus . It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics. PMID:24121866

  13. Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli.

    PubMed

    Mirza, Nadia; Crocoll, Christoph; Erik Olsen, Carl; Ann Halkier, Barbara

    2016-05-01

    The methionine-derived glucosinolate glucoraphanin is associated with the health-promoting properties of broccoli. This has developed a strong interest in producing this compound in high amounts from a microbial source. Glucoraphanin synthesis starts with a five-gene chain elongation pathway that converts methionine to dihomo-methionine, which is subsequently converted to glucoraphanin by the seven-gene glucosinolate core structure pathway. As dihomo-methionine is the precursor amino acid for glucoraphanin production, a first challenge is to establish an expression system for production of dihomo-methionine. In planta, the methionine chain elongation enzymes are physically separated within the cell with the first enzyme in the cytosol while the rest are located in the chloroplast. A de-compartmentalization approach was applied to produce dihomo-methionine by expression of the respective plant genes in Escherichia coli cytosol. Introduction of two plasmids encoding the methionine chain elongation pathway into E. coli resulted in production of 25mgL(-1) of dihomo-methionine. In addition to chain-elongated methionine products, side-products from chain elongation of leucine were produced. Methionine supplementation enhanced dihomo-methionine production to 57mgL(-1), while keeping a steady level of the chain-elongated leucine products. Engineering of the de-compartmentalized pathway of dihomo-methionine in E. coli cytosol provides an important first step for microbial production of the health-promoting glucoraphanin. PMID:26410451

  14. An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors

    PubMed Central

    Mihardja, Shirley S.; Liszewski, Walter; Erle, David J.; Lee, Randall J.; Bernstein, Harold S.

    2011-01-01

    Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease. PMID:21245908

  15. Enzyme engineering in the context of novel pathways and products.

    PubMed

    Otte, Konrad B; Hauer, Bernhard

    2015-12-01

    In recent years, enzyme engineering was used to fine-tune a diverse set of proteins to realize new biosynthetic pathways and gain access to novel products. However, enzymes in nature do not always meet the required demands in terms of activity, selectivity and stability. In these cases enzyme engineering has been used to improve the enzyme properties, which facilitated the development of tailor-made functional biocatalysts, even beyond their natural capabilities. Examples can be found in the three main areas of chemical biotechnology: single-step biocatalysis, metabolic engineering and enzymatic cascades. In this review we highlight recently published work in all of these three fields and emphasize the main trends and differences. PMID:25585003

  16. Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry, free-energy conservation and redox-cofactor balancing.

    PubMed

    van Rossum, Harmen M; Kozak, Barbara U; Pronk, Jack T; van Maris, Antonius J A

    2016-07-01

    Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories. PMID:27016336

  17. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    DOE PAGESBeta

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-12-21

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through themore » systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Lastly, our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.« less

  18. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    SciTech Connect

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-12-21

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Lastly, our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.

  19. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    PubMed Central

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-01-01

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries. PMID:26686655

  20. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-12-01

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.

  1. Optimization of Engineered Production of the Glucoraphanin Precursor Dihomomethionine in Nicotiana benthamiana.

    PubMed

    Crocoll, Christoph; Mirza, Nadia; Reichelt, Michael; Gershenzon, Jonathan; Halkier, Barbara Ann

    2016-01-01

    Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we obtained fivefold to eightfold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: (1) coexpression of the large subunit (LSU1) of the heterodimeric isopropylmalate isomerase and (2) coexpression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomomethionine (DHM) levels to a maximum of 432 nmol g(-1) fresh weight that is equivalent to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host. PMID:26909347

  2. Optimization of Engineered Production of the Glucoraphanin Precursor Dihomomethionine in Nicotiana benthamiana

    PubMed Central

    Crocoll, Christoph; Mirza, Nadia; Reichelt, Michael; Gershenzon, Jonathan; Halkier, Barbara Ann

    2016-01-01

    Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we obtained fivefold to eightfold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: (1) coexpression of the large subunit (LSU1) of the heterodimeric isopropylmalate isomerase and (2) coexpression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomomethionine (DHM) levels to a maximum of 432 nmol g−1 fresh weight that is equivalent to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host. PMID:26909347

  3. An engineered eukaryotic protein glycosylation pathway in Escherichia coli.

    PubMed

    Valderrama-Rincon, Juan D; Fisher, Adam C; Merritt, Judith H; Fan, Yao-Yun; Reading, Craig A; Chhiba, Krishan; Heiss, Christian; Azadi, Parastoo; Aebi, Markus; DeLisa, Matthew P

    2012-05-01

    We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins. PMID:22446837

  4. Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

    PubMed Central

    Ben Aissa, Manel; April, Marie-Claude; Bergeron, Lucien-Junior; Perreault, Jean-Pierre; Levesque, Georges

    2012-01-01

    Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD. PMID:22482079

  5. Genetically engineered mouse models of human B-cell precursor leukemias

    PubMed Central

    Hauer, Julia; Borkhardt, Arndt; Sánchez-García, Isidro; Cobaleda, César

    2014-01-01

    B-cell precursor acute lymphoblastic leukemias (pB-ALLs) are the most frequent type of malignancies of the childhood, and also affect an important proportion of adult patients. In spite of their apparent homogeneity, pB-ALL comprises a group of diseases very different both clinically and pathologically, and with very diverse outcomes as a consequence of their biology, and underlying molecular alterations. Their understanding (as a prerequisite for their cure) will require a sustained multidisciplinary effort from professionals coming from many different fields. Among all the available tools for pB-ALL research, the use of animal models stands, as of today, as the most powerful approach, not only for the understanding of the origin and evolution of the disease, but also for the development of new therapies. In this review we go over the most relevant (historically, technically or biologically) genetically engineered mouse models (GEMMs) of human pB-ALLs that have been generated over the last 20 years. Our final aim is to outline the most relevant guidelines that should be followed to generate an “ideal” animal model that could become a standard for the study of human pB-ALL leukemia, and which could be shared among research groups and drug development companies in order to unify criteria for studies like drug testing, analysis of the influence of environmental risk factors, or studying the role of both low-penetrance mutations and cancer susceptibility alterations. PMID:25486471

  6. Engineering an isoprenoid pathway in Escherichia coli for production of 2-methyl-3-buten-2-ol: a potential biofuel.

    PubMed

    Gupta, Dinesh; Summers, Michael L; Basu, Chhandak

    2014-06-01

    2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcohol produced by several pine species that have the potential to be used as biofuel. MBO has a high energy content making it superior to ethanol in terms of energy output, and due to its volatility and lower solubility in water, MBO is easier to recover than ethanol. Pine's MBO synthase enzyme utilizes the intermediate dimethylallyl pyrophosphate (DMAPP) produced by the methyl-erythritol-4-phosphate isoprenoid pathway for the production of MBO. In this study, we performed metabolic engineering of Escherichia coli to express an alternate mevalonate dependent pathway for production of DMAPP, along with a codon optimized Pinus sabiniana MBO synthase gene. This heterologous expressed pathway carried out the conversion of an acetyl CoA precursor to DMAPP leading to production of MBO. PMID:24271564

  7. Exploring the Engineering Student Experience: Findings from the Academic Pathways of People Learning Engineering Survey (APPLES). TR-10-01

    ERIC Educational Resources Information Center

    Sheppard, Sheri; Gilmartin, Shannon; Chen, Helen L.; Donaldson, Krista; Lichtenstein, Gary; Eris, Ozgur; Lande, Micah; Toye, George

    2010-01-01

    This report is based on data from the Academic Pathways of People Learning Engineering Survey (APPLES), administered to engineering students at 21 U.S. engineering colleges and schools in the spring of 2008. The first comprehensive set of analyses completed on the APPLES dataset presented here looks at how engineering students experience their…

  8. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids.

    PubMed

    Kishimoto, Shinji; Sato, Michio; Tsunematsu, Yuta; Watanabe, Kenji

    2016-01-01

    Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details. PMID:27548127

  9. Career Pursuit Pathways among Emerging Adult Men and Women: Psychosocial Correlates and Precursors

    ERIC Educational Resources Information Center

    Shulman, Shmuel; Barr, Tamuz; Livneh, Yaara; Nurmi, Jari-Erik; Vasalampi, Kati; Pratt, Michael

    2015-01-01

    The present study examined career pursuit pathways in 100 Israeli emerging adults (54 men) who were followed from age 22 to 29. Employing a semi-structured interview at the age of 29, participants were asked about current work and educational status, work and educational goals and status changes in recent years, and to reflect on the meaning of…

  10. Murine Muscle Engineered from Dermal Precursors: An In Vitro Model for Skeletal Muscle Generation, Degeneration, and Fatty Infiltration

    PubMed Central

    García-Parra, Patricia; Naldaiz-Gastesi, Neia; Maroto, Marcos; Padín, Juan Fernando; Goicoechea, María; Aiastui, Ana; Fernández-Morales, José Carlos; García-Belda, Paula; Lacalle, Jaione; Álava, Jose Iñaki; García-Verdugo, José Manuel; García, Antonio G.

    2014-01-01

    Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5–7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na+- and Ca2+-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca2+ transients; fiber contractions coupled to these Ca2+ transients suggest that Ca2+ entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca2+ transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration. PMID:23631552

  11. Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin

    PubMed Central

    Nabhan, Joseph F.; Gooch, Renea L.; Piatnitski Chekler, Eugene L.; Pierce, Betsy; Bulawa, Christine E.

    2015-01-01

    Friedreich’s Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein. PMID:26671574

  12. Stepwise modular pathway engineering of Escherichia coli for efficient one-step production of (2S)-pinocembrin.

    PubMed

    Wu, Junjun; Zhang, Xia; Dong, Mingsheng; Zhou, Jingwen

    2016-08-10

    Developing efficient microbial processes to produce flavonoids has been a metabolic engineering goal over the past decade due to their important functions. Previously, the de novo production of the main flavonoid precursor (2S)-pinocembrin was achieved. However, low productivity and two separate fermentation steps made it inappropriate for industrial scale low-cost production. Here, a stepwise modular engineering approach was introduced to systematically identify and eliminate metabolic pathway bottlenecks. The overall pathway was firstly divided into four modules and analysis then revealed that efficient conversion of l-phenylalanine to (2S)-pinocembrin is the major limiting factor. Therefore, the pathway from l-phenylalanine to (2S)-pinocembrin was re-cast into three modules to alleviate this bottleneck by modifying both the gene copy number and promoter strength. Furthermore, the expression of the rate-limiting enzyme PAL was found to be correlated with 5' region of mRNA structure. The efficiency of the synthetic pathway was then improved by customizing the PAL expression level based on modification of the mRNA secondary structure. Fed-batch cultivation of engineered strains in a 3-L fermentor resulted in a final (2S)-pinocembrin production of 432.4mg/L. The results presented here pave the way for the development of an economical and simple process for microbial production of flavonoids. PMID:27297547

  13. Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli.

    PubMed

    Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2014-05-01

    cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered strain is able to produce 1.2g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of up to 1.5g/L MA within 48h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites. PMID:24583236

  14. Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane.

    PubMed

    Kind, Stefanie; Jeong, Weol Kyu; Schröder, Hartwig; Wittmann, Christoph

    2010-07-01

    In the present work the Gram-positive bacterium Corynebacterium glutamicum was engineered into an efficient, tailor-made production strain for diaminopentane (cadaverine), a highly attractive building block for bio-based polyamides. The engineering comprised expression of lysine decarboxylase (ldcC) from Escherichia coli, catalyzing the conversion of lysine into diaminopentane, and systems-wide metabolic engineering of central supporting pathways. Substantially re-designing the metabolism yielded superior strains with desirable properties such as (i) the release from unwanted feedback regulation at the level of aspartokinase and pyruvate carboxylase by introducing the point mutations lysC311 and pycA458, (ii) an optimized supply of the key precursor oxaloacetate by amplifying the anaplerotic enzyme, pyruvate carboxylase, and deleting phosphoenolpyruvate carboxykinase which otherwise removes oxaloacetate, (iii) enhanced biosynthetic flux via combined amplification of aspartokinase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase, and (iv) attenuated flux into the threonine pathway competing with production by the leaky mutation hom59 in the homoserine dehydrogenase gene. Lysine decarboxylase proved to be a bottleneck for efficient production, since its in vitro activity and in vivo flux were closely correlated. To achieve an optimal strain having only stable genomic modifications, the combination of the strong constitutive C. glutamicum tuf promoter and optimized codon usage allowed efficient genome-based ldcC expression and resulted in a high diaminopentane yield of 200 mmol mol(-1). By supplementing the medium with 1 mgL(-1) pyridoxal, the cofactor of lysine decarboxylase, the yield was increased to 300 mmol mol(-1). In the production strain obtained, lysine secretion was almost completely abolished. Metabolic analysis, however, revealed substantial formation of an as yet unknown by-product. It was identified as an

  15. Characterization and engineering of the ethylmalonyl-CoA pathway towards the improved heterologous production of polyketides in Streptomyces venezuelae.

    PubMed

    Jung, Won Seok; Kim, Eunji; Yoo, Young Ji; Ban, Yeon Hee; Kim, Eun Ji; Yoon, Yeo Joon

    2014-04-01

    Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B₁₂-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (∼10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria. PMID:24413979

  16. HEB-deficient T-cell precursors lose T-cell potential and adopt an alternative pathway of differentiation.

    PubMed

    Braunstein, Marsela; Anderson, Michele K

    2011-03-01

    Early thymocytes possess multilineage potential, which is progressively restricted as cells transit through the double-negative stages of T-cell development. DN1 cells retain the ability to become natural killer cells, dendritic cells, B cells, and myeloid cells as well as T cells, but these options are lost by the DN3 stage. The Notch1 signaling pathway is indispensable for initiation of the T-cell lineage and inhibitory for the B-cell lineage, but the regulatory mechanisms by which the T-cell fate is locked in are largely undefined. Previously, we discovered that the E-protein transcription factor HEBAlt promoted T-cell specification. Here, we report that HEB(-/-) T-cell precursors have compromised Notch1 function and lose T-cell potential. Moreover, reconstituting HEB(-/-) precursors with Notch1 activity enforced fidelity to the T-cell fate. However, instead of becoming B cells, HEB(-/-) DN3 cells adopted a DN1-like phenotype and could be induced to differentiate into thymic NK cells. HEB(-/-) DN1-like cells retained GATA3 and Id2 expression but had lower levels of the Bcl11b gene, a Notch target gene. Therefore, our studies have revealed a new set of interactions between HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. PMID:21189289

  17. Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway.

    PubMed

    Cohen, Ethan David; Ihida-Stansbury, Kaori; Lu, Min Min; Panettieri, Reynold A; Jones, Peter Lloyd; Morrisey, Edward E

    2009-09-01

    Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and beta-catenin were required for expression of Pdgfralpha and Pdgfrbeta and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfralpha and Pdgfrbeta as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfralpha/beta expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung. PMID:19690384

  18. Rapid Capture Next-Generation Sequencing in Clinical Diagnostics of Kinase Pathway Aberrations in B-Cell Precursor ALL.

    PubMed

    Stadt, Udo Zur; Escherich, Gabriele; Indenbirken, Daniela; Alawi, Malik; Adao, Manuela; Horstmann, Martin A

    2016-07-01

    Comprehensive next-generation sequencing (NGS) applications have recently identified various recurrent kinase and cytokine receptor rearrangements in Ph-like B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) amenable to tyrosin kinase inhibitor treatment. For rapid diagnostics of kinase pathway aberrations in minimal residual disease (MRD) high-risk BCP-ALL, we developed a PCR-independent NGS custom enrichment capture panel targeting recurrent genomic alterations, which allows for the identification of unknown 5' fusion partner genes and precise mapping of variable genomic breakpoints. Using a standardized bioinformatics algorithm, we identified kinase and cytokine receptor rearrangements in the majority of ALL patients with high burden of postinduction MRD and enrichment of IKZF1 mutation or deletion (IKZF1(del) ). PMID:27007619

  19. Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3β signaling pathway.

    PubMed

    Deng, Juan; Habib, Ahsan; Obregon, Demian F; Barger, Steven W; Giunta, Brian; Wang, Yan-Jiang; Hou, Huayan; Sawmiller, Darrell; Tan, Jun

    2015-11-01

    We recently found that sAPPα decreases amyloid-beta generation by directly associating with β-site amyloid precursor protein (APP)-converting enzyme 1 (BACE1), thereby modulating APP processing. Because inhibition of BACE1 decreases glycogen synthase kinase 3 beta (GSK3β)-mediated Alzheimer's disease (AD)-like tau phosphorylation in AD patient-derived neurons, we determined whether sAPPα also reduces GSK3β-mediated tau phosphorylation. We initially found increased levels of inhibitory phosphorylation of GSK3β (Ser9) in primary neurons from sAPPα over-expressing mice. Further, recombinant human sAPPα evoked the same phenomenon in SH-SY5Y cells. Further, in SH-SY5Y cells over-expressing BACE1, and HeLa cells over-expressing human tau, sAPPα reduced GSK3β activity and tau phosphorylation. Importantly, the reductions in GSK3β activity and tau phosphorylation elicited by sAPPα were prevented by BACE1 but not γ-secretase inhibition. In accord, AD mice over-expressing human sAPPα had less GSK3β activity and tau phosphorylation compared with controls. These results implicate a direct relationship between APP β-processing and GSK3β-mediated tau phosphorylation and further define the central role of sAPPα in APP autoregulation and AD pathogenesis. PMID:26342176

  20. Pathway engineering to improve ethanol production by thermophilic bacteria

    SciTech Connect

    Lynd, L.R.

    1998-12-31

    Continuation of a research project jointly funded by the NSF and DOE is proposed. The primary project goal is to develop and characterize strains of C. thermocellum and C. thermosaccharolyticum having ethanol selectivity similar to more convenient ethanol-producing organisms. An additional goal is to document the maximum concentration of ethanol that can be produced by thermophiles. These goals build on results from the previous project, including development of most of the genetic tools required for pathway engineering in the target organisms. As well, we demonstrated that the tolerance of C. thermosaccharolyticum to added ethanol is sufficiently high to allow practical utilization should similar tolerance to produced ethanol be demonstrated, and that inhibition by neutralizing agents may explain the limited concentrations of ethanol produced in studies to date. Task 1 involves optimization of electrotransformation, using either modified conditions or alternative plasmids to improve upon the low but reproducible transformation, frequencies we have obtained thus far.

  1. Metformin Acts on Two Different Molecular Pathways to Enhance Adult Neural Precursor Proliferation/Self-Renewal and Differentiation

    PubMed Central

    Fatt, Michael; Hsu, Karolynn; He, Ling; Wondisford, Fredric; Miller, Freda D.; Kaplan, David R.; Wang, Jing

    2015-01-01

    Summary The recruitment of endogenous adult neural stem cells for brain repair is a promising regenerative therapeutic strategy. This strategy involves stimulation of multiple stages of adult neural stem cell development, including proliferation, self-renewal, and differentiation. Currently, there is a lack of a single therapeutic approach that can act on these multiple stages of adult neural stem cell development to enhance neural regeneration. Here we show that metformin, an FDA-approved diabetes drug, promotes proliferation, self-renewal, and differentiation of adult neural precursors (NPCs). Specifically, we show that metformin enhances adult NPC proliferation and self-renewal dependent upon the p53 family member and transcription factor TAp73, while it promotes neuronal differentiation of these cells by activating the AMPK-aPKC-CBP pathway. Thus, metformin represents an optimal candidate neuro-regenerative agent that is capable of not only expanding the adult NPC population but also subsequently driving them toward neuronal differentiation by activating two distinct molecular pathways. PMID:26677765

  2. Rhamnose biosynthesis pathway supplies precursors for primary and secondary metabolism in Saccharopolyspora spinosa.

    PubMed

    Madduri, K; Waldron, C; Merlo, D J

    2001-10-01

    Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products. PMID:11544225

  3. Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

    PubMed

    Bormann, Sebastian; Baer, Zachary C; Sreekumar, Sanil; Kuchenreuther, Jon M; Dean Toste, F; Blanch, Harvey W; Clark, Douglas S

    2014-09-01

    Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was

  4. Engineered Biosynthesis of Plant Polyketides: Structure-Based and Precursor-Directed Approach

    NASA Astrophysics Data System (ADS)

    Abe, Ikuro

    benzoyl-CoA as a starter substrate. These results suggested that the engineered biosynthesis of plant polyketides by combination of the structure-based and the precursor-directed approach would lead to further production of chemically and structurally divergent unnatural novel polyketides.

  5. New Biosynthetic Step in the Melanin Pathway of Wangiella (Exophiala) dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced as a pentaketide directly by an iter...

  6. Scaling up: Taking the Academic Pathways of People Learning Engineering Survey (APPLES) National. Research Brief

    ERIC Educational Resources Information Center

    Donaldson, Krista M.; Chen, Helen L.; Toye, George; Clark, Mia; Sheppard, Sheri D.

    2008-01-01

    The Academic Pathways of People Learning Engineering Survey (APPLES) was deployed for a second time in spring 2008 to undergraduate engineering students at 21 US universities. The goal of the second deployment of APPLES was to corroborate and extend findings from the Academic Pathways Study (APS; 2003-2007) and the first deployment of APPLES…

  7. Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways.

    PubMed

    Hansen, Immo A; Attardo, Geoffrey M; Rodriguez, Stacy D; Drake, Lisa L

    2014-01-01

    Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level. PMID:24688471

  8. Combining chemoinformatics with bioinformatics: in silico prediction of bacterial flavor-forming pathways by a chemical systems biology approach "reverse pathway engineering".

    PubMed

    Liu, Mengjin; Bienfait, Bruno; Sacher, Oliver; Gasteiger, Johann; Siezen, Roland J; Nauta, Arjen; Geurts, Jan M W

    2014-01-01

    The incompleteness of genome-scale metabolic models is a major bottleneck for systems biology approaches, which are based on large numbers of metabolites as identified and quantified by metabolomics. Many of the revealed secondary metabolites and/or their derivatives, such as flavor compounds, are non-essential in metabolism, and many of their synthesis pathways are unknown. In this study, we describe a novel approach, Reverse Pathway Engineering (RPE), which combines chemoinformatics and bioinformatics analyses, to predict the "missing links" between compounds of interest and their possible metabolic precursors by providing plausible chemical and/or enzymatic reactions. We demonstrate the added-value of the approach by using flavor-forming pathways in lactic acid bacteria (LAB) as an example. Established metabolic routes leading to the formation of flavor compounds from leucine were successfully replicated. Novel reactions involved in flavor formation, i.e. the conversion of alpha-hydroxy-isocaproate to 3-methylbutanoic acid and the synthesis of dimethyl sulfide, as well as the involved enzymes were successfully predicted. These new insights into the flavor-formation mechanisms in LAB can have a significant impact on improving the control of aroma formation in fermented food products. Since the input reaction databases and compounds are highly flexible, the RPE approach can be easily extended to a broad spectrum of applications, amongst others health/disease biomarker discovery as well as synthetic biology. PMID:24416282

  9. Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

    PubMed

    Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

    2011-01-01

    NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins. PMID:21589930

  10. Precursor engineering and controlled conversion for the synthesis of monodisperse thiolate-protected metal nanoclusters

    NASA Astrophysics Data System (ADS)

    Yu, Yong; Yao, Qiaofeng; Luo, Zhentao; Yuan, Xun; Lee, Jim Yang; Xie, Jianping

    2013-05-01

    In very recent years, thiolate-protected metal nanoclusters (or thiolated MNCs) with core sizes smaller than 2 nm have emerged as a new direction in nanoparticle research due to their discrete and size dependent electronic structures and molecular-like properties, such as HOMO-LUMO transitions in optical absorptions, quantized charging, and strong luminescence. Synthesis of monodisperse thiolated MNCs in sufficiently large quantities (up to several hundred micrograms) is necessary for establishing reliable size-property relationships and exploring potential applications. This Feature Article reviews recent progress in the development of synthetic strategies for the production of monodisperse thiolated MNCs. The preparation of monodisperse thiolated MNCs is viewed as an engineerable process where both the precursors (input) and their conversion chemistry (processing) may be rationally designed to achieve the desired outcome - monodisperse thiolated MNCs (output). Several strategies for tailoring the precursor and the conversion process are analyzed to arrive at a unifying understanding of the processes involved.

  11. Enhanced production of fatty alcohols by engineering the TAGs synthesis pathway in Saccharomyces cerevisiae.

    PubMed

    Tang, Xiaoling; Chen, Wei Ning

    2015-02-01

    The production of fatty acid-derived chemicals has received a great deal of attention in recent years. In yeast cells, the main storage forms of fatty acids are TAGs. However, the conversion of TAGs into fatty acid derivatives suffers from a practical standpoint. Herein, a more direct strategy was applied to accumulate cellular fatty acyl-CoAs in Saccharomyces cerevisiae, which are the activated forms of fatty acids and used as important precursors for various converting enzymes. The dga1 gene was deleted to block the fatty acyl-CoAs dependent pathway of TAGs synthesis and a significant decrease in lipid content was observed. The FAR gene was cloned and overexpressed in the wild type strain and gene disrupted strain, to convert the fatty acyl-CoAs to the corresponding fatty acid derivatives. The metabolic engineered pathway resulted in enhanced production of fatty alcohols. Compared with the wild type strain with overexpressed FAR gene, the yield of fatty alcohols in the Δdga1 strain with FAR was dramatically increased: the intracellular fatty alcohols increased from 26 mg/L to 45 mg/L, while the extracellular fatty alcohols increased from 2.2 mg/L to 4.3 mg/L. By optimizing the culture medium with increased carbon concentration and limited nitrogen concentration, the fatty alcohols yield in the Δdga1 strain with FAR was further increased to 84 mg/L in cells and 14 mg/L secreted in broth. The results in this study demonstrated the feasibility of using the designed strategy to solve the bottleneck in utilizing TAGs for fatty acid derivatives production. PMID:25116045

  12. A Fail-safe Mechanism for Negative Selection of Isotype-switched B Cell Precursors Is Regulated by the Fas/FasL Pathway

    PubMed Central

    Seagal, Jane; Edry, Efrat; Keren, Zohar; Leider, Nira; Benny, Ofra; Machluf, Marcelle; Melamed, Doron

    2003-01-01

    In B lymphocytes, immunoglobulin (Ig)M receptors drive development and construction of naive repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotype switching process requires appropriate B cell activation and T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a peripheral mechanism contributing to the establishment of self-tolerance. Using Igμ-deficient μMT mouse model, where B cell development is blocked at pro-B stage, here we show an alternative developmental pathway used by isotype-switched B cell precursors. We find that isotype switching occurs normally in B cell precursors and is T independent. Ongoing isotype switching was found in both normal and μMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells. These isotype-switched B cells are negatively selected by Fas pathway, as blocking the Fas/FasL interaction rescues the development of isotype-switched B cells in vivo and in vitro. Similar to memory B cells, isotype-switched B cells have a marginal zone phenotype. We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements. This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity. PMID:14623914

  13. Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alchohol

    SciTech Connect

    Cai, Yuanheng; Shanklin, John; Mohammad -Wadud Bhuiya; Liu, Chang -Jun

    2015-09-16

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.

  14. Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alchohol

    DOE PAGESBeta

    Cai, Yuanheng; Shanklin, John; Mohammad -Wadud Bhuiya; Liu, Chang -Jun

    2015-09-16

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create anmore » enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.« less

  15. Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.

    PubMed

    Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

    2015-10-30

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. PMID:26378240

  16. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  17. Dual-tagged amyloid-β precursor protein reveals distinct transport pathways of its N- and C-terminal fragments.

    PubMed

    Villegas, Christine; Muresan, Virgil; Ladescu Muresan, Zoia

    2014-03-15

    The amyloid-β precursor protein (APP), a type I transmembrane protein genetically associated with Alzheimer's disease, has a complex biology that includes proteolytic processing into potentially toxic fragments, extensive trafficking and multiple, yet poorly-defined functions. We recently proposed that a significant fraction of APP is proteolytically cleaved in the neuronal soma into N- and C-terminal fragments (NTFs and CTFs), which then target independently of each other to separate destinations in the cell. Here, we prove this concept with live imaging and immunolocalization of two dual, N- and C-termini-tagged APP constructs: CFP-APP-YFP [containing the fluorescent tags, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)] and FLAG-APP-Myc. When expressed at low levels in neuronal cells, these constructs are processed into differently tagged NTFs and CTFs that reveal distinct distributions and characteristics of transport. Like the endogenous N- and C-terminal epitopes of APP, the FLAG-tagged NTFs are present in trains of vesicles and tubules that localize to short filaments, which often immunostain for acetylated tubulin, whereas the Myc-tagged CTFs are detected on randomly distributed vesicle-like structures. The experimental treatments that selectively destabilize the acetylated microtubules abrogate the distribution of NTFs along filaments, without altering the random distribution of CTFs. These results indicate that the NTFs and CTFs are recruited to distinct transport pathways and reach separate destinations in neurons, where they likely accomplish functions independent of the parental, full-length APP. They also point to a compartment associated with acetylated microtubules in the neuronal soma--not the neurite terminals--as a major site of APP cleavage, and segregation of NTFs from CTFs. PMID:24203698

  18. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    ERIC Educational Resources Information Center

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  19. LWRS Fuels Pathway: Engineering Design and Fuels Pathway Initial Testing of the Hot Water Corrosion System

    SciTech Connect

    Dr. John Garnier; Dr. Kevin McHugh

    2012-09-01

    The Advanced LWR Nuclear Fuel Development R&D pathway performs strategic research focused on cladding designs leading to improved reactor core economics and safety margins. The research performed is to demonstrate the nuclear fuel technology advancements while satisfying safety and regulatory limits. These goals are met through rigorous testing and analysis. The nuclear fuel technology developed will assist in moving existing nuclear fuel technology to an improved level that would not be practical by industry acting independently. Strategic mission goals are to improve the scientific knowledge basis for understanding and predicting fundamental nuclear fuel and cladding performance in nuclear power plants, and to apply this information in the development of high-performance, high burn-up fuels. These will result in improved safety, cladding, integrity, and nuclear fuel cycle economics. To achieve these goals various methods for non-irradiated characterization testing of advanced cladding systems are needed. One such new test system is the Hot Water Corrosion System (HWCS) designed to develop new data for cladding performance assessment and material behavior under simulated off-normal reactor conditions. The HWCS is capable of exposing prototype rodlets to heated, high velocity water at elevated pressure for long periods of time (days, weeks, months). Water chemistry (dissolved oxygen, conductivity and pH) is continuously monitored. In addition, internal rodlet heaters inserted into cladding tubes are used to evaluate repeated thermal stressing and heat transfer characteristics of the prototype rodlets. In summary, the HWCS provides rapid ex-reactor evaluation of cladding designs in normal (flowing hot water) and off-normal (induced cladding stress), enabling engineering and manufacturing improvements to cladding designs before initiation of the more expensive and time consuming in-reactor irradiation testing.

  20. Targeting Undergraduate Students for Surveys: Lessons from the Academic Pathways of People Learning Engineering Survey (APPLES). Research Brief

    ERIC Educational Resources Information Center

    Donaldson, Krista M.; Chen, Helen L.; Toye, George; Sheppard, Sheri D.

    2007-01-01

    The Academic Pathways of People Learning Engineering Survey (APPLES or APPLE survey) is a component of the Academic Pathways Study (APS) of the Center for the Advancement of Engineering Education (CAEE). The APS aims to provide a comprehensive account of how people become engineers by exploring key questions around the engineering learning…

  1. Partitioning the effects of an ecosystem engineer: kangaroo rats control community structure via multiple pathways.

    PubMed

    Prugh, Laura R; Brashares, Justin S

    2012-05-01

    1. Ecosystem engineers impact communities by altering habitat conditions, but they can also have strong effects through consumptive, competitive and other non-engineering pathways. 2. Engineering effects can lead to fundamentally different community dynamics than non-engineering effects, but the relative strengths of these interactions are seldom quantified. 3. We combined structural equation modelling and exclosure experiments to partition the effects of a keystone engineer, the giant kangaroo rat (Dipodomys ingens), on plants, invertebrates and vertebrates in a semi-arid California grassland. 4. We separated the effects of burrow creation from kangaroo rat density and found that kangaroo rats increased the diversity and abundance of other species via both engineering and non-engineering pathways. 5. Engineering was the primary factor structuring plant and small mammal communities, whereas non-engineering effects structured invertebrate communities and increased lizard abundance. 6. These results highlight the importance of the non-engineering effects of ecosystem engineers and shed new light on the multiple pathways by which strong-interactors shape communities. PMID:22098534

  2. Career Pathways of Science, Engineering and Technology Research Postgraduates

    ERIC Educational Resources Information Center

    Giles, Marnie; Ski, Chantal; Vrdoljak, Davorin

    2009-01-01

    Suitably qualified scientists and engineers are essential for research and development, innovation and, in turn, the growth of the economy. Science, engineering and technology skills are therefore necessary for Australia to remain competitive in a global market. This article reports findings from a nationwide study investigating the career…

  3. Pathways to Engineering: The Validation Experiences of Transfer Students

    ERIC Educational Resources Information Center

    Zhang, Yi; Ozuna, Taryn

    2015-01-01

    Community college engineering transfer students are a critical student population of engineering degree recipients and technical workforce in the United States. Focusing on this group of students, we adopted Rendón's (1994) validation theory to explore the students' experiences in community colleges prior to transferring to a four-year…

  4. A Survey of Career Pathways of Engineering Deans in the United States: Strategies for Leadership Development

    ERIC Educational Resources Information Center

    Hargrove, S. Keith

    2015-01-01

    The career pathways of deans in higher education seem to follow the traditional model in academia from a senior faculty position and/or department chair. This however may be different from deans in engineering education. The goal of this survey research is to assess the career paths of current Deans of Colleges/Schools of Engineering in the United…

  5. An engineered pathway for the biosynthesis of renewable propane

    PubMed Central

    Kallio, Pauli; Pásztor, András; Thiel, Kati; Akhtar, M. Kalim; Jones, Patrik R.

    2014-01-01

    The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O2 supply and removal of native aldehyde reductases. PMID:25181600

  6. An engineered pathway for the biosynthesis of renewable propane.

    PubMed

    Kallio, Pauli; Pásztor, András; Thiel, Kati; Akhtar, M Kalim; Jones, Patrik R

    2014-01-01

    The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O2 supply and removal of native aldehyde reductases. PMID:25181600

  7. Combination of Entner-Doudoroff pathway with MEP increases isoprene production in engineered Escherichia coli.

    PubMed

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

    2013-01-01

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

  8. Metabolic engineering of a synergistic pathway for n-butanol production in Saccharomyces cerevisiae

    PubMed Central

    Shi, Shuobo; Si, Tong; Liu, Zihe; Zhang, Hongfang; Ang, Ee Lui; Zhao, Huimin

    2016-01-01

    n-Butanol has several favourable properties as an advanced fuel or a platform chemical. Bio-based production of n-butanol is becoming increasingly important for sustainable chemical industry. Synthesis of n-butanol can be achieved via more than one metabolic pathway. Here we report the metabolic engineering of Saccharomyces cerevisiae to produce n-butanol through a synergistic pathway: the endogenous threonine pathway and the introduced citramalate pathway. Firstly, we characterized and optimized the endogenous threonine pathway; then, a citramalate synthase (CimA) mediated pathway was introduced to construct the synergistic pathway; next, the synergistic pathway was optimized by additional overexpression of relevant genes identified previously; meanwhile, the n-butanol production was also improved by overexpression of keto-acid decarboxylases (KDC) and alcohol dehydrogenase (ADH). After combining these strategies with co-expression of LEU1 (two copies), LEU4, LEU2 (two copies), LEU5, CimA, NFS1, ADH7 and ARO10*, we achieved an n-butanol production of 835 mg/L in the final engineered strain, which is almost 7-fold increase compared to the initial strain. Furthermore, the production showed a 3-fold of the highest titer ever reported in yeast. Therefore, the engineered yeast strain represents a promising alternative platform for n-butanol production. PMID:27161023

  9. Metabolic engineering of a synergistic pathway for n-butanol production in Saccharomyces cerevisiae.

    PubMed

    Shi, Shuobo; Si, Tong; Liu, Zihe; Zhang, Hongfang; Ang, Ee Lui; Zhao, Huimin

    2016-01-01

    n-Butanol has several favourable properties as an advanced fuel or a platform chemical. Bio-based production of n-butanol is becoming increasingly important for sustainable chemical industry. Synthesis of n-butanol can be achieved via more than one metabolic pathway. Here we report the metabolic engineering of Saccharomyces cerevisiae to produce n-butanol through a synergistic pathway: the endogenous threonine pathway and the introduced citramalate pathway. Firstly, we characterized and optimized the endogenous threonine pathway; then, a citramalate synthase (CimA) mediated pathway was introduced to construct the synergistic pathway; next, the synergistic pathway was optimized by additional overexpression of relevant genes identified previously; meanwhile, the n-butanol production was also improved by overexpression of keto-acid decarboxylases (KDC) and alcohol dehydrogenase (ADH). After combining these strategies with co-expression of LEU1 (two copies), LEU4, LEU2 (two copies), LEU5, CimA, NFS1, ADH7 and ARO10(*), we achieved an n-butanol production of 835 mg/L in the final engineered strain, which is almost 7-fold increase compared to the initial strain. Furthermore, the production showed a 3-fold of the highest titer ever reported in yeast. Therefore, the engineered yeast strain represents a promising alternative platform for n-butanol production. PMID:27161023

  10. Metabolic and pathway engineering to influence native and altered erythromycin production through E. coli.

    PubMed

    Jiang, Ming; Pfeifer, Blaine A

    2013-09-01

    The heterologous production of the complex antibiotic erythromycin through Escherichia coli provides a unique challenge in metabolic engineering. In addition to introducing the 19 foreign genes needed for heterologous biosynthesis, E. coli metabolism must be engineered to provide the propionyl-CoA and (2S)-methylmalonyl-CoA substrates required to allow erythromycin formation. In this work, three different pathways to propionyl-CoA were compared in the context of supporting E. coli erythromycin biosynthesis. The comparison revealed that alternative citramalate and threonine metabolic pathways (both starting from exogenous glycerol) were capable of supporting final compound formation equal to a proven pathway reliant upon exogenous propionate. Furthermore, two pathways to (2S)-methylmalonyl-CoA were compared in the production of a novel benzyl-erythromycin analog. A pathway dependent upon exogenous methylmalonate improved selectivity and facilitated antibiotic assessment of this new analog. PMID:23747605

  11. Engineering precursor metabolite pools for increasing production of antitumor mithramycins in Streptomyces argillaceus.

    PubMed

    Zabala, Daniel; Braña, Alfredo F; Flórez, Ana B; Salas, José A; Méndez, Carmen

    2013-11-01

    Mithramycin (MTM) is a polyketide antitumor compound produced by Streptomyces argillaceus constituted by a tricyclic aglycone with two aliphatic side chains, a trisaccharide and a disaccharide chain. The biosynthesis of the polyketide aglycone is initiated by the condensation of ten malonyl-CoA units to render a carbon chain that is modified to a tetracyclic intermediate and sequentially glycosylated by five deoxysugars originated from glucose-1-phosphate. Further oxidation and reduction render the final compound. We aimed to increase the precursor supply of malonyl-CoA and/or glucose-1-phosphate in S. argillaceus to enhance MTM production. We have shown that by overexpressing either the S. coelicolor phosphoglucomutase gene pgm or the acetyl-CoA carboxylase ovmGIH genes from the oviedomycin biosynthesis gene cluster in S. argillaceus, we were able to increase the intracellular pool of glucose-1-phosphate and malonyl-CoA, respectively. Moreover, we have cloned the S. argillaceus ADP-glucose pyrophosphorylase gene glgCa and the acyl-CoA:diacylglycerol acyltransferase gene aftAa, and we showed that by inactivating them, an increase of the intracellular concentration of glucose-1-phosphate/glucose-6-phosphate and malonyl-CoA/acetyl-CoA was observed, respectively. Each individual modification resulted in an enhancement of MTM production but the highest production level was obtained by combining all strategies together. In addition, some of these strategies were successfully applied to increase production of four MTM derivatives with improved pharmacological properties: demycarosyl-mithramycin, demycarosyl-3D-β-D-digitoxosyl-mithramycin, mithramycin SK and mithramycin SDK. PMID:24148183

  12. Using decision pathway surveys to inform climate engineering policy choices.

    PubMed

    Gregory, Robin; Satterfield, Terre; Hasell, Ariel

    2016-01-19

    Over the coming decades citizens living in North America and Europe will be asked about a variety of new technological and behavioral initiatives intended to mitigate the worst impacts of climate change. A common approach to public input has been surveys whereby respondents' attitudes about climate change are explained by individuals' demographic background, values, and beliefs. In parallel, recent deliberative research seeks to more fully address the complex value tradeoffs linked to novel technologies and difficult ethical questions that characterize leading climate mitigation alternatives. New methods such as decision pathway surveys may offer important insights for policy makers by capturing much of the depth and reasoning of small-group deliberations while meeting standard survey goals including large-sample stakeholder engagement. Pathway surveys also can help participants to deepen their factual knowledge base and arrive at a more complete understanding of their own values as they apply to proposed policy alternatives. The pathway results indicate more fully the conditional and context-specific nature of support for several "upstream" climate interventions, including solar radiation management techniques and carbon dioxide removal technologies. PMID:26729883

  13. Using decision pathway surveys to inform climate engineering policy choices

    PubMed Central

    Gregory, Robin; Satterfield, Terre; Hasell, Ariel

    2016-01-01

    Over the coming decades citizens living in North America and Europe will be asked about a variety of new technological and behavioral initiatives intended to mitigate the worst impacts of climate change. A common approach to public input has been surveys whereby respondents’ attitudes about climate change are explained by individuals’ demographic background, values, and beliefs. In parallel, recent deliberative research seeks to more fully address the complex value tradeoffs linked to novel technologies and difficult ethical questions that characterize leading climate mitigation alternatives. New methods such as decision pathway surveys may offer important insights for policy makers by capturing much of the depth and reasoning of small-group deliberations while meeting standard survey goals including large-sample stakeholder engagement. Pathway surveys also can help participants to deepen their factual knowledge base and arrive at a more complete understanding of their own values as they apply to proposed policy alternatives. The pathway results indicate more fully the conditional and context-specific nature of support for several “upstream” climate interventions, including solar radiation management techniques and carbon dioxide removal technologies. PMID:26729883

  14. Engineering key components in a synthetic eukaryotic signal transduction pathway

    PubMed Central

    Antunes, Mauricio S; Morey, Kevin J; Tewari-Singh, Neera; Bowen, Tessa A; Smith, J Jeff; Webb, Colleen T; Hellinga, Homme W; Medford, June I

    2009-01-01

    Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB-VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB-VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved-signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways. PMID:19455134

  15. Learning from nature: new approaches to the metabolic engineering of plant defense pathways.

    PubMed

    Jirschitzka, Jan; Mattern, Derek Joseph; Gershenzon, Jonathan; D'Auria, John Charles

    2013-04-01

    Biotechnological manipulation of plant defense pathways can increase crop resistance to herbivores and pathogens while also increasing yields of medicinal, industrial, flavor and fragrance compounds. The most successful achievements in engineering defense pathways can be attributed to researchers striving to imitate natural plant regulatory mechanisms. For example, the introduction of transcription factors that control several genes in one pathway is often a valuable strategy to increase flux in that pathway. The use of multi-gene cassettes which mimic natural gene clusters can facilitate coordinated regulation of a pathway and speed transformation efforts. The targeting of defense pathway genes to organs and tissues in which the defensive products are typically made and stored can also increase yield as well as defensive potential. PMID:23141769

  16. Detection of Greenhouse Gas Precursors from Diesel Engines Using Electrochemical and Photoacoustic Sensors

    PubMed Central

    Mothé, Geórgia; Castro, Maria; Sthel, Marcelo; Lima, Guilherme; Brasil, Laisa; Campos, Layse; Rocha, Aline; Vargas, Helion

    2010-01-01

    Atmospheric pollution is one of the worst threats to modern society. The consequences derived from different forms of atmospheric pollution vary from the local to the global scale, with deep impacts on climate, environment and human health. Several gaseous pollutants, even when present in trace concentrations, play a fundamental role in important processes that occur in atmosphere. Phenomena such as global warming, photochemical smog formation, acid rain and the depletion of the stratospheric ozone layer are strongly related to the increased concentration of certain gaseous species in the atmosphere. The transport sector significantly produces atmospheric pollution, mainly when diesel oil is used as fuel. Therefore, new methodologies based on selective and sensitive gas detection schemes must be developed in order to detect and monitor pollutant gases from this source. In this work, CO2 Laser Photoacoustic Spectroscopy was used to evaluate ethylene emissions and electrochemical analyzers were used to evaluate the emissions of CO, NOx and SO2 from the exhaust of diesel powered vehicles (rural diesel with 5% of biodiesel, in this paper called only diesel) at different engine rotation speeds. Concentrations in the range 6 to 45 ppmV for ethylene, 109 to 1,231 ppmV for carbon monoxide, 75 to 868 ppmV for nitrogen oxides and 3 to 354 ppmV for sulfur dioxide were obtained. The results indicate that the detection techniques used were sufficiently selective and sensitive to detect the gaseous species mentioned above in the ppmV range. PMID:22163437

  17. Detection of greenhouse gas precursors from diesel engines using electrochemical and photoacoustic sensors.

    PubMed

    Mothé, Geórgia; Castro, Maria; Sthel, Marcelo; Lima, Guilherme; Brasil, Laisa; Campos, Layse; Rocha, Aline; Vargas, Helion

    2010-01-01

    Atmospheric pollution is one of the worst threats to modern society. The consequences derived from different forms of atmospheric pollution vary from the local to the global scale, with deep impacts on climate, environment and human health. Several gaseous pollutants, even when present in trace concentrations, play a fundamental role in important processes that occur in atmosphere. Phenomena such as global warming, photochemical smog formation, acid rain and the depletion of the stratospheric ozone layer are strongly related to the increased concentration of certain gaseous species in the atmosphere. The transport sector significantly produces atmospheric pollution, mainly when diesel oil is used as fuel. Therefore, new methodologies based on selective and sensitive gas detection schemes must be developed in order to detect and monitor pollutant gases from this source. In this work, CO(2) Laser Photoacoustic Spectroscopy was used to evaluate ethylene emissions and electrochemical analyzers were used to evaluate the emissions of CO, NO(x) and SO(2) from the exhaust of diesel powered vehicles (rural diesel with 5% of biodiesel, in this paper called only diesel) at different engine rotation speeds. Concentrations in the range 6 to 45 ppmV for ethylene, 109 to 1,231 ppmV for carbon monoxide, 75 to 868 ppmV for nitrogen oxides and 3 to 354 ppmV for sulfur dioxide were obtained. The results indicate that the detection techniques used were sufficiently selective and sensitive to detect the gaseous species mentioned above in the ppmV range. PMID:22163437

  18. An Engineered Rare Codon Device for Optimization of Metabolic Pathways

    PubMed Central

    Wang, You; Li, Chunying; Khan, Md. Rezaul Islam; Wang, Yushu; Ruan, Yunfeng; Zhao, Bin; Zhang, Bo; Ma, Xiaopan; Zhang, Kaisi; Zhao, Xiwen; Ye, Guanhao; Guo, Xizhi; Feng, Guoyin; He, Lin; Ma, Gang

    2016-01-01

    Rare codons generally arrest translation due to rarity of their cognate tRNAs. This property of rare codons can be utilized to regulate protein expression. In this study, a linear relationship was found between expression levels of genes and copy numbers of rare codons inserted within them. Based on this discovery, we constructed a molecular device in Escherichia coli using the rare codon AGG, its cognate tRNA (tRNAArg (CCU)), modified tRNAAsp (GUC → CCU), and truncated aspartyl-tRNA synthetase (TDRS) to switch the expression of reporter genes on or off as well as to precisely regulate their expression to various intermediate levels. To underscore the applicability of our work, we used the rare codon device to alter the expression levels of four genes of the fatty acid synthesis II (FASII) pathway (i.e. fabZ, fabG, fabI, and tesA’) in E. coli to optimize steady-state kinetics, which produced nearly two-fold increase in fatty acid yield. Thus, the proposed method has potential applications in regulating target protein expression at desired levels and optimizing metabolic pathways by precisely tuning in vivo molar ratio of relevant enzymes. PMID:26852704

  19. Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

    NASA Astrophysics Data System (ADS)

    Wittmann, Christoph

    The Gram-positive soil bacterium Corynebacterium glutamicum was discovered as a natural overproducer of glutamate about 50 years ago. Linked to the steadily increasing economical importance of this microorganism for production of glutamate and other amino acids, the quest for efficient production strains has been an intense area of research during the past few decades. Efficient production strains were created by applying classical mutagenesis and selection and especially metabolic engineering strategies with the advent of recombinant DNA technology. Hereby experimental and computational approaches have provided fascinating insights into the metabolism of this microorganism and directed strain engineering. Today, C. glutamicum is applied to the industrial production of more than 2 million tons of amino acids per year. The huge achievements in recent years, including the sequencing of the complete genome and efficient post genomic approaches, now provide the basis for a new, fascinating era of research - analysis of metabolic and regulatory properties of C. glutamicum on a global scale towards novel and superior bioprocesses.

  20. Photosynthetic biomaterials: a pathway towards autotrophic tissue engineering.

    PubMed

    Schenck, Thilo Ludwig; Hopfner, Ursula; Chávez, Myra Noemi; Machens, Hans-Günther; Somlai-Schweiger, Ian; Giunta, Riccardo Enzo; Bohne, Alexandra Viola; Nickelsen, Jörg; Allende, Miguel L; Egaña, José Tomás

    2015-03-01

    Engineered tissues are highly limited by poor vascularization in vivo, leading to hypoxia. In order to overcome this challenge, we propose the use of photosynthetic biomaterials to provide oxygen. Since photosynthesis is the original source of oxygen for living organisms, we suggest that this could be a novel approach to provide a constant source of oxygen supply independently of blood perfusion. In this study we demonstrate that bioartificial scaffolds can be loaded with a solution containing the photosynthetic microalgae Chlamydomonas reinhardtii, showing high biocompatibility and photosynthetic activity in vitro. Furthermore, when photosynthetic biomaterials were engrafted in a mouse full skin defect, we observed that the presence of the microalgae did not trigger a native immune response in the host. Moreover, the analyses showed that the algae survived for at least 5 days in vivo, generating chimeric tissues comprised of algae and murine cells. The results of this study represent a crucial step towards the establishment of autotrophic tissue engineering approaches and suggest the use of photosynthetic cells to treat a broad spectrum of hypoxic conditions. PMID:25536030

  1. Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.

    PubMed

    Hübner, Juwita; Hoseini, Shahabuddin S; Suerth, Julia D; Hoffmann, Dirk; Maluski, Marcel; Herbst, Jessica; Maul, Holger; Ghosh, Arnab; Eiz-Vesper, Britta; Yuan, Qinggong; Ott, Michael; Heuser, Michael; Schambach, Axel; Sauer, Martin G

    2016-08-01

    Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer. PMID:27138041

  2. Proteome Profiling Reveals Potential Toxicity and Detoxification Pathways Following Exposure of BEAS-2B Cells to Engineered Titanium Dioxide Nanoparticles

    EPA Science Inventory

    Oxidative stress is known to play important roles in engineered nanomaterial induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial mediated oxidative stress and toxicity are largely unknown. To identify these toxicity ...

  3. Engineering of Metabolic Pathways by Artificial Enzyme Channels

    PubMed Central

    Pröschel, Marlene; Detsch, Rainer; Boccaccini, Aldo R.; Sonnewald, Uwe

    2015-01-01

    Application of industrial enzymes for production of valuable chemical compounds has greatly benefited from recent developments in Systems and Synthetic Biology. Both, in vivo and in vitro systems have been established, allowing conversion of simple into complex compounds. Metabolic engineering in living cells needs to be balanced which is achieved by controlling gene expression levels, translation, scaffolding, compartmentation, and flux control. In vitro applications are often hampered by limited protein stability/half-life and insufficient rates of substrate conversion. To improve stability and catalytic activity, proteins are post-translationally modified and arranged in artificial metabolic channels. Within the review article, we will first discuss the supramolecular organization of enzymes in living systems and second summarize current and future approaches to design artificial metabolic channels by additive manufacturing for the efficient production of desired products. PMID:26557643

  4. Systems Genetics Analysis of a Recombinant Inbred Mouse Cell Culture Panel Reveals Wnt Pathway Member Lrp6 as a Regulator of Adult Hippocampal Precursor Cell Proliferation.

    PubMed

    Kannan, Suresh; Nicola, Zeina; Overall, Rupert W; Ichwan, Muhammad; Ramírez-Rodríguez, Gerardo; N Grzyb, Anna; Patone, Giannino; Saar, Kathrin; Hübner, Norbert; Kempermann, Gerd

    2016-03-01

    In much animal research, genetic variation is rather avoided than used as a powerful tool to identify key regulatory genes in complex phenotypes. Adult hippocampal neurogenesis is one such highly complex polygenic trait, for which the understanding of the molecular basis is fragmented and incomplete, and for which novel genetic approaches are needed. In this study, we aimed at marrying the power of the BXD panel, a mouse genetic reference population, with the flexibility of a cell culture model of adult neural precursor proliferation and differentiation. We established adult-derived hippocampal precursor cell cultures from 20 strains of the BXD panel, including the parental strains C57BL/6J and DBA/2J. The rates of cell proliferation and neuronal differentiation were measured, and transcriptional profiles were obtained from proliferating cultures. Together with the published genotypes of all lines, these data allowed a novel systems genetics analysis combining quantitative trait locus analysis with transcript expression correlation at a cellular level to identify genes linked with the differences in proliferation. In a proof-of-principle analysis, we identified Lrp6, the gene encoding the coreceptor to Frizzled in the Wnt pathway, as a potential negative regulator of precursor proliferation. Overexpression and siRNA silencing confirmed the regulatory role of Lrp6. As well as adding to our knowledge of the pathway surrounding Wnt in adult hippocampal neurogenesis, this finding allows the new appreciation of a negative regulator within this system. In addition, the resource and associated methodology will allow the integration of regulatory mechanisms at a systems level. Stem Cells 2016;34:674-684. PMID:26840599

  5. A search engine to identify pathway genes from expression data on multiple organisms

    PubMed Central

    Chen, Chunnuan; Weirauch, Matthew T; Powell, Corey C; Zambon, Alexander C; Stuart, Joshua M

    2007-01-01

    Background The completion of several genome projects showed that most genes have not yet been characterized, especially in multicellular organisms. Although most genes have unknown functions, a large collection of data is available describing their transcriptional activities under many different experimental conditions. In many cases, the coregulatation of a set of genes across a set of conditions can be used to infer roles for genes of unknown function. Results We developed a search engine, the Multiple-Species Gene Recommender (MSGR), which scans gene expression datasets from multiple organisms to identify genes that participate in a genetic pathway. The MSGR takes a query consisting of a list of genes that function together in a genetic pathway from one of six organisms: Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Arabidopsis thaliana, and Helicobacter pylori. Using a probabilistic method to merge searches, the MSGR identifies genes that are significantly coregulated with the query genes in one or more of those organisms. The MSGR achieves its highest accuracy for many human pathways when searches are combined across species. We describe specific examples in which new genes were identified to be involved in a neuromuscular signaling pathway and a cell-adhesion pathway. Conclusion The search engine can scan large collections of gene expression data for new genes that are significantly coregulated with a pathway of interest. By integrating searches across organisms, the MSGR can identify pathway members whose coregulation is either ancient or newly evolved. PMID:17477880

  6. Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia

    PubMed Central

    Maude, Shannon L.; Dolai, Sibasish; Delgado-Martin, Cristina; Vincent, Tiffaney; Robbins, Alissa; Selvanathan, Arthavan; Ryan, Theresa; Hall, Junior; Wood, Andrew C.; Tasian, Sarah K.; Hunger, Stephen P.; Loh, Mignon L.; Mullighan, Charles G.; Wood, Brent L.; Hermiston, Michelle L.; Grupp, Stephan A.; Lock, Richard B.

    2015-01-01

    Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed. PMID:25645356

  7. The PI3K-Akt pathway inhibits senescence and promotes self-renewal of human skin-derived precursors in vitro.

    PubMed

    Liu, Shuang; Liu, Shu; Wang, Xinyue; Zhou, Jiaxi; Cao, Yujing; Wang, Fei; Duan, Enkui

    2011-08-01

    Skin-derived precursors (SKPs) are embryonic neural crest- or somite-derived multipotent progenitor cells with properties of dermal stem cells. Although a large number of studies deal with their differentiation ability and potential applications in tissue damage repair, only a few studies have concentrated on the regulation of SKP self-renewal. Here, we found that after separation from their physiological microenvironment, human foreskin-derived SKPs (hSKPs) quickly senesced and lost their self-renewal ability. We observed a sharp decrease in Akt activity during this process, suggesting a possible role of the PI3K-Akt pathway in hSKP maintenance in vitro. Blocking this pathway with several inhibitors inhibited hSKP proliferation and sphere formation and increased hSKP senescence. In contrast, activating this pathway with PDGF-AA and a PTEN inhibitor, bpV(pic), promoted proliferation, improved sphere formation, and alleviated senescence of hSKPs, without altering their differentiation potential. Data also implied that this effect was associated with altered actions of FoxO3 and GSK-3β. Our results suggest an important role of the PI3K-Akt pathway in the senescence and self-renewal of hSKPs. These findings also provide a better understanding of the cellular mechanisms underlying hSKP self-renewal and stem cell senescence to allow more efficient expansion of hSKPs for regenerative medical applications. PMID:21418510

  8. Development and application of efficient pathway enumeration algorithms for metabolic engineering applications.

    PubMed

    Liu, F; Vilaça, P; Rocha, I; Rocha, M

    2015-02-01

    Metabolic Engineering (ME) aims to design microbial cell factories towards the production of valuable compounds. In this endeavor, one important task relates to the search for the most suitable heterologous pathway(s) to add to the selected host. Different algorithms have been developed in the past towards this goal, following distinct approaches spanning constraint-based modeling, graph-based methods and knowledge-based systems based on chemical rules. While some of these methods search for pathways optimizing specific objective functions, here the focus will be on methods that address the enumeration of pathways that are able to convert a set of source compounds into desired targets and their posterior evaluation according to different criteria. Two pathway enumeration algorithms based on (hyper)graph-based representations are selected as the most promising ones and are analyzed in more detail: the Solution Structure Generation and the Find Path algorithms. Their capabilities and limitations are evaluated when designing novel heterologous pathways, by applying these methods on three case studies of synthetic ME related to the production of non-native compounds in E. coli and S. cerevisiae: 1-butanol, curcumin and vanillin. Some targeted improvements are implemented, extending both methods to address limitations identified that impair their scalability, improving their ability to extract potential pathways over large-scale databases. In all case-studies, the algorithms were able to find already described pathways for the production of the target compounds, but also alternative pathways that can represent novel ME solutions after further evaluation. PMID:25580014

  9. Visualization of Intracellular Pathways of Engineered Baculovirus in Mammalian Cells

    PubMed Central

    Liu, Yarong; Joo, Kye-Il; Lei, Yuning; Wang, Pin

    2014-01-01

    Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy. PMID:24457070

  10. Reverse engineering of Alzheimer's disease based on biomarker pathways analysis.

    PubMed

    Richens, Joanna L; Morgan, Kevin; O'Shea, Paul

    2014-09-01

    Alzheimer's disease (AD) poses an increasingly profound problem to society, yet progress toward a genuine understanding of the disease remains worryingly slow. Perhaps, the most outstanding problem with the biology of AD is the question of its mechanistic origins, that is, it remains unclear wherein the molecular failures occur that underlie the disease. We demonstrate how molecular biomarkers could help define the nature of AD in terms of the early biochemical events that correlate with disease progression. We use a novel panel of biomolecules that appears in cerebrospinal fluid of AD patients. As changes in the relative abundance of these molecular markers are associated with progression to AD from mild cognitive impairment, we make the assumption that by tracking their origins we can identify the biochemical conditions that predispose their presence and consequently cause the onset of AD. We couple these protein markers with an analysis of a series of genetic factors and together this hypothesis essentially allows us to redefine AD in terms of the molecular pathways that underlie the disease. PMID:24684789

  11. Metabolic engineering of cottonseed oil biosynthesis pathway via RNA interference.

    PubMed

    Xu, Zhongping; Li, Jingwen; Guo, Xiaoping; Jin, Shuangxia; Zhang, Xianlong

    2016-01-01

    Cottonseed oil is recognized as an important oil in food industry for its unique characters: low flavor reversion and the high level of antioxidants (VitaminE) as well as unsaturated fatty acid. However, the cottonseed oil content of cultivated cotton (Gossypium hirsutum) is only around 20%. In this study, we modified the accumulation of oils by the down-regulation of phosphoenolpyruvate carboxylase 1 (GhPEPC1) via RNA interference in transgenic cotton plants. The qRT-PCR and enzyme activity assay revealed that the transcription and expression of GhPEPC1 was dramatically down-regulated in transgenic lines. Consequently, the cottonseed oil content in several transgenic lines showed a significant (P < 0.01) increase (up to 16.7%) without obvious phenotypic changes under filed condition when compared to the control plants. In order to elucidate the molecular mechanism of GhPEPC1 in the regulation of seed oil content, we quantified the expression of the carbon metabolism related genes of transgenic GhPEPC1 RNAi lines by transcriptome analysis. This analysis revealed the decrease of GhPEPC1 expression led to the increase expression of triacylglycerol biosynthesis-related genes, which eventually contributed to the lipid biosynthesis in cotton. This result provides a valuable information for cottonseed oil biosynthesis pathway and shows the potential of creating high cottonseed oil germplasm by RNAi strategy for cotton breeding. PMID:27620452

  12. Engineering Emergency: African Americans and Hispanics Lack Pathways to Engineering. Vital Signs: Reports on the Condition of STEM Learning in the U.S.

    ERIC Educational Resources Information Center

    Change the Equation, 2014

    2014-01-01

    A quality education that leads to good jobs offers a reliable pathway to economic security, yet the first step on that pathway remains inaccessible to far too many Americans, especially Americans of color. Nowhere is this inequity more apparent than in engineering. On average, people with engineering bachelor's degrees earn higher salaries than…

  13. Genetic engineering of the complete carotenoid pathway towards enhanced astaxanthin formation in Xanthophyllomyces dendrorhous starting from a high-yield mutant.

    PubMed

    Gassel, Sören; Breitenbach, Jürgen; Sandmann, Gerhard

    2014-01-01

    The yeast Xanthophyllomyces dendrorhous is one of the rare organisms which can synthesize the commercially interesting carotenoid astaxanthin. However, astaxanthin yield in wild-type and also in classical mutants is still too low for an attractive bioprocess. Therefore, we combined classical mutagenesis with genetic engineering of the complete pathway covering improved precursor supply for carotenogenesis, enhanced metabolite flow into the pathway, and efficient conversion of intermediates into the desired end product astaxanthin. We also constructed new transformation plasmids for the stepwise expression of the genes of 3-hydroxymethyl-3-glutaryl coenzyme A reductase, geranylgeranyl pyrophosphate synthase, phytoene synthase/lycopene cyclase, and astaxanthin synthase. Starting from two mutants with a 15-fold higher astaxanthin, we obtained transformants with an additional 6-fold increase in the final step of pathway engineering. Thus, a maximum astaxanthin content of almost 9 mg per g dry weight was reached in shaking cultures. Under optimized fermenter conditions, astaxanthin production with these engineered transformants should be comparable to Haematococcus pluvialis, the leading commercial producer of natural astaxanthin. PMID:24241897

  14. Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells.

    PubMed

    Seth, Rohit; Corniola, Rikki S; Gower-Winter, Shannon D; Morgan, Thomas J; Bishop, Brian; Levenson, Cathy W

    2015-04-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-μ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency. PMID:25467851

  15. Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

    PubMed

    Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

    2016-01-01

    Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways. PMID:27417934

  16. Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol.

    PubMed

    Pereira, Brian; Zhang, Haoran; De Mey, Marjan; Lim, Chin Giaw; Li, Zheng-Jun; Stephanopoulos, Gregory

    2016-02-01

    Ethylene glycol (EG) is an important commodity chemical with broad industrial applications. It is presently produced from petroleum or natural gas feedstocks in processes requiring consumption of significant quantities of non-renewable resources. Here, we report a novel pathway for biosynthesis of EG from the renewable sugar glucose in metabolically engineered Escherichia coli. Serine-to-EG conversion was first achieved through a pathway comprising serine decarboxylase, ethanolamine oxidase, and glycolaldehyde reductase. Serine provision in E. coli was then enhanced by overexpression of the serine-biosynthesis pathway. The integration of these two parts into the complete EG-biosynthesis pathway in E. coli allowed for production of 4.1 g/L EG at a cumulative yield of 0.14 g-EG/g-glucose, establishing a foundation for a promising biotechnology. PMID:26221864

  17. Engineering and comparison of non-natural pathways for microbial phenol production.

    PubMed

    Thompson, Brian; Machas, Michael; Nielsen, David R

    2016-08-01

    The non-renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non-natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediate p-hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL-dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745-1754. © 2016 Wiley Periodicals, Inc. PMID:26804162

  18. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

    PubMed Central

    Xu, Peng; Li, Lingyun; Zhang, Fuming; Stephanopoulos, Gregory; Koffas, Mattheos

    2014-01-01

    Global energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7- and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA–derived compounds. PMID:25049420

  19. Utilization of economical substrate-derived carbohydrates by solventogenic clostridia: pathway dissection, regulation and engineering.

    PubMed

    Gu, Yang; Jiang, Yu; Yang, Sheng; Jiang, Weihong

    2014-10-01

    Solventogenic clostridia can produce acetone, butanol and ethanol (ABE) by using different carbohydrates. For economical reasons, the utilization of cheap and renewable biomass in clostridia-based ABE fermentation has recently attracted increasing interests. With the study of molecular microbiology and development of genetic tools, the understanding of carbohydrate metabolism in clostridia has increased in recent years. Here, we review the pioneering work in this field, with a focus on dissecting the pathways and describing the regulation of the metabolism of economical substrate-derived carbohydrates by clostridia. Recent progress in the metabolic engineering of carbohydrate utilization pathways is also described. PMID:24769507

  20. Protein engineering strategies with potential applications for altering clinically relevant cellular pathways at the protein level.

    PubMed

    Regan, Lynne; Hinrichsen, Michael R; Oi, Curran

    2016-05-01

    All diseases can be fundamentally viewed as the result of malfunctioning cellular pathways. Protein engineering offers the potential to develop new tools that will allow these dysfunctional pathways to be better understood, in addition to potentially providing new routes to restore proper function. Here we discuss different approaches that can be used to change the intracellular activity of a protein by intervening at the protein level: targeted protein sequestration, protein recruitment, protein degradation, and selective inhibition of binding interfaces. The potential of each of these tools to be developed into effective therapeutic treatments will also be discussed, along with any major barriers that currently block their translation into the clinic. PMID:27031866

  1. Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

    PubMed Central

    Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

    2015-01-01

    The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

  2. An Amphipathic Alpha-Helix in the Prodomain of Cocaine and Amphetamine Regulated Transcript Peptide Precursor Serves as Its Sorting Signal to the Regulated Secretory Pathway

    PubMed Central

    Blanco, Elías H.; Lagos, Carlos F.; Andrés, María Estela; Gysling, Katia

    2013-01-01

    Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1–41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. In silico modeling predicted an alpha-helix structure located between residues 24–37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCARTL34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCARTL34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting. PMID:23527253

  3. More to Say: Analyzing Open-Ended Student Responses to the Academic Pathways of People Learning Engineering Survey. Research Brief

    ERIC Educational Resources Information Center

    Lande, Micah; Parikh, Sarah; Sheppard, Sheri; Toye, George; Chen, Helen; Donaldson, Krista

    2009-01-01

    The Academic Pathways of People Learning Engineering Survey (APPLES2) was administered in Spring 2008 to undergraduate engineering students at 21 American universities. Students took the 10-minute online survey that asked mainly multiple choice questions related to their undergraduate engineering experience. A final optional open-ended question…

  4. From PIE to APPLES: The Evolution of a Survey Instrument to Explore Engineering Student Pathways. Research Brief

    ERIC Educational Resources Information Center

    Chen, Helen; Donaldson, Krista; Eris, Ozgur; Chachra, Debbie; Lichtenstein, Gary; Sheppard, Sheri; Toye, George

    2008-01-01

    The Academic Pathways Study (APS) of the NSF-funded Center for the Advancement of Engineering Education (CAEE) is a cross-university study that systematically examines how engineering students navigate their education, and how engineering skills and identity develop during their undergraduate careers. The APS has utilized a variety of methods…

  5. [Engineering of the xylose metabolic pathway for microbial production of bio-based chemicals].

    PubMed

    Liu, Weixi; Fu, Jing; Zhang, Bo; Chen, Tao

    2013-08-01

    As the rapid development of economy necessitates a large number of oil, the contradiction between energy supply and demand is further exacerbated by the dwindling reserves of petroleum resource. Therefore, the research of the renewable cellulosic biomass resources is gaining unprecedented momentum. Because xylose is the second most abundant monosaccharide after glucose in lignocellulose hydrolyzes, high-efficiency bioconversion of xylose becomes one of the vital factors that affect the industrial prospects of lignocellulose application. According to the research progresses in recent years, this review summarized the advances in bioconversion of xylose, which included identification and redesign of the xylose metabolic pathway, engineering the xylose transport pathway and bio-based chemicals production. In order to solve the energy crisis and environmental pollution issues, the development of advanced bio-fuel technology, especially engineering the microbe able to metabolize xylose and produce ethanol by synthetic biology, is environmentally benign and sustainable. PMID:24364352

  6. Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

    PubMed

    Sirobhushanam, Sirisha; Galva, Charitha; Sen, Suranjana; Wilkinson, Brian J; Gatto, Craig

    2016-09-01

    Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis. PMID:27320015

  7. Neuronal production and precursor proliferation defects in the neocortex of mice with loss of function in the canonical Wnt signaling pathway.

    PubMed

    Zhou, C-J; Borello, U; Rubenstein, J L R; Pleasure, S J

    2006-11-01

    To better understand the function of the Wnt pathway in the developing telencephalon, we analyzed neocortical development in low density lipoprotein receptor-related protein (LRP) 6 mutants. LRP6 mutant mice are hypomorphic for the canonical Wnt signaling pathway and have hypoplasia of the developing neocortex. While early telencephalic morphogenesis is largely intact in these mice, probably due to compensation by LRP5, the mutant mice develop a dramatically thinner cortical plate. There is a prominent reduction of neurogenesis leading to a thin cortical plate. Reduced proliferation late in gestation probably also contributes to the hypoplasia. Although there are marked decreases in the numbers of layer 6 and layers 2-4 neurons all laminar identities are generated and there is no evidence of compensatory increases in layer 5 neurons. In addition, LRP6 mutants have partial penetrance of a complex of cortical dysmorphologies resembling those found in patients with developmental forms of epilepsy and mental retardation. These include ventricular and marginal zone heterotopias and cobblestone lissencephaly. This analysis demonstrates that canonical Wnt signaling is required for a diverse array of developmental processes in the neocortex in addition to the previously known roles in regulating precursor proliferation and patterning. PMID:16920270

  8. Reverse engineering an amyloid aggregation pathway with dimensional analysis and scaling

    NASA Astrophysics Data System (ADS)

    Bailey, J.; Potter, K. J.; Verchere, C. B.; Edelstein-Keshet, L.; Coombs, D.

    2011-12-01

    Human islet amyloid polypeptide (hIAPP) is a cytotoxic protein that aggregates into oligomers and fibrils that kill pancreatic β-cells. Here we analyze hIAPP aggregation in vitro, measured via thioflavin-T fluorescence. We use mass-action kinetics and scaling analysis to reconstruct the aggregation pathway, and find that the initiation step requires four hIAPP monomers. After this step, monomers join the nucleus in pairs, until the first stable nucleus (of size approximately 20 monomers) is formed. This nucleus then elongates by successive addition of single monomers. We find that the best-fit of our data is achieved when we include a secondary fibril-dependent nucleation pathway in the reaction scheme. We predict how interventions that change rates of fibril elongation or nucleation rates affect the accumulation of potentially cytotoxic oligomer species. Our results demonstrate the power of scaling analysis in reverse engineering biochemical aggregation pathways.

  9. A portable expression resource for engineering cross-species genetic circuits and pathways

    PubMed Central

    Kushwaha, Manish; Salis, Howard M.

    2015-01-01

    Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts. PMID:26184393

  10. A portable expression resource for engineering cross-species genetic circuits and pathways.

    PubMed

    Kushwaha, Manish; Salis, Howard M

    2015-01-01

    Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts. PMID:26184393

  11. Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid β-protein precursor (APP) transport into the late secretory pathway and facilitates APP cleavage.

    PubMed

    Takei, Norio; Sobu, Yuriko; Kimura, Ayano; Urano, Satomi; Piao, Yi; Araki, Yoichi; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Nakaya, Tadashi; Suzuki, Toshiharu

    2015-01-01

    The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo. PMID:25406318

  12. Cytoplasmic Fragment of Alcadein α Generated by Regulated Intramembrane Proteolysis Enhances Amyloid β-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage*

    PubMed Central

    Takei, Norio; Sobu, Yuriko; Kimura, Ayano; Urano, Satomi; Piao, Yi; Araki, Yoichi; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Nakaya, Tadashi; Suzuki, Toshiharu

    2015-01-01

    The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo. PMID:25406318

  13. Amyloid-β precursor protein induces glial differentiation of neural progenitor cells by activation of the IL-6/gp130 signaling pathway.

    PubMed

    Kwak, Young-Don; Dantuma, Elise; Merchant, Stephanie; Bushnev, Sergey; Sugaya, Kiminobu

    2010-11-01

    Although amyloid precursor protein (APP) due to the cytotoxicity of Aβ peptides, has been intensively studied, the physiological role of APP still remains wrapped up in veil. In this article, we propose that α-cleaved ectodomain of APP (sAPPα) stimulates the IL-6/gp130 signaling pathway for induction of gliogenesis within neural progenitor cells (NPCs). In our previous study, a high dose of APP differentiated NPCs into glial fibrillary acidic protein (GFAP) positive cells. In order to elucidate the mechanism of APP-induced glial differentiation, we examined the effects of sAPPα on the IL-6/gp130 signaling pathway. Application of sAPPα promoted mRNA expression of gp130, ciliary neurotrophic factor (CNTF), and Janus kinase 1 (JAK1). sAPPα stimulated the glial differentiation by upregulating the expression and phosphorylation of gp130. While mRNA expression of STAT3 was unchanged, phosphorylation of STAT3-Tyr705 gradually increased. Application of small interference RNA (siRNA) for STAT3 suppressed GFAP expression even in the presence of APP. Treatment with siRNA or inhibitor, AG490, of JAK1 efficiently suppressed STAT3 phosphorylation and GFAP expression. Upregulation of CNTF was observed in either short- or long-term treatment with sAPPα. RNA's interference of CNTF dose-dependently inhibited GFAP expression upregulated by treatment with sAPPα. This study suggests that the IL-6/gp130 signaling pathway is involved in sAPPα-induced glial differentiation of NPCs. Although further investigation is needed, this study may provide insight into the mechanism of glial differentiation of NPCs under pathological conditions in Alzheimer's disease or Down syndrome. PMID:20309664

  14. Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria.

    PubMed

    Nozzi, Nicole E; Atsumi, Shota

    2015-11-20

    Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter P(L)lacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, P(L)lacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene. PMID:25974153

  15. Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival.

    PubMed

    Astier, Anne Laurence; Xu, Ronghui; Svoboda, Marek; Hinds, Esther; Munoz, Olivier; de Beaumont, Rosalie; Crean, Colin Daniel; Gabig, Theodore; Freedman, Arnold Stephen

    2003-02-01

    The physical interactions between B cells and stromal cells from the lymphoid tissue microenvironment are critical to the survival of normal and malignant B cells. They are principally mediated by integrins expressed on B cells and counterreceptors on stromal cells. Specifically, alpha4beta1 integrin engagement rescues B cells from physiological or drug-induced apoptosis. Therefore, in order to understand the mechanisms by which integrins prevent apoptosis in leukemia B cells, we compared the temporal gene expression profiles induced by beta1-integrin ligation with fibronectin (Fn) or adhesion by poly-L-Lysine in serum-starved precursor B leukemia cells. Among the 38 selected differentially expressed genes, 6 genes involved in adhesion (VAV2, EPB41L1, CORO1A), proliferation (FRAP1, CCT4), and intercellular communication (GJB3) were validated by real-time quantitative polymerase chain reaction (RT-Q-PCR). Gene expression modulation could also be validated at the protein level for 5 other genes. We show that integrin stimulation up-regulated FBI-1 expression but inhibited CD79a, Requiem, c-Fos, and caspase 7 induction when the cells underwent apoptosis. We further demonstrate that Fn stimulation also inhibits caspase 3 activation but increases XIAP and survivin expression. Moreover, integrin stimulation also prevents caspase activation induced by doxorubicin. Therefore, we identified genes modulated by adhesion of human precursor B leukemia cells that regulate proliferation and apoptosis, highlighting new pathways that might provide insights into future therapy aiming at targeting apoptosis of leukemia cells. PMID:12393420

  16. Implication of the calcium sensing receptor and the Phosphoinositide 3-kinase/Akt pathway in the extracellular calcium-mediated migration of RAW 264.7 osteoclast precursor cells.

    PubMed

    Boudot, Cédric; Saidak, Zuzana; Boulanouar, Abdel Krim; Petit, Laurent; Gouilleux, Fabrice; Massy, Ziad; Brazier, Michel; Mentaverri, Romuald; Kamel, Saïd

    2010-05-01

    While the processes involved in the formation, maturation and apoptosis of osteoclasts have been investigated extensively in previous studies, little is known about the mechanisms responsible for the localization and homing of osteoclast precursor cells to the bone environment in order to initiate the bone remodeling process. Recent studies have suggested that the extracellular Ca(2+) (Ca(2+)(o)) concentration gradient present near the bone environment may be one of the participating factors, producing a chemoattractant effect on osteoclast precursors. Using the murine osteoclast precursor cells of the monocyte-macrophage lineage, the RAW 264.7 cell line, we have shown that Ca(2+)(o) increases the migration of these cells in a directional manner. The participation of the calcium sensing receptor (CaR) in this effect was tested by knocking down its expression through RNA interference, which resulted in an abolition of the migratory response. By the use of specific pathway inhibitors and western blot analysis, the phosphoinositide 3-kinase (PI3K)/Akt and phospholipase Cbeta pathways were shown to be implicated in the migratory effect. The implication of the Akt pathway in the Ca(2+)(o)-induced chemoattraction of RAW 264.7 cells was also confirmed by transducing the cells with the fusion protein TAT-dominant negative-Akt, which decreased the migratory effect. In contrast, the MAPK pathways (ERK1/2, p38 and JNK) were not involved in the production of the migratory effect. We conclude that through the activation of the CaR and subsequent signaling via the PI3K/Akt pathway, Ca(2+)(o) produces a chemoattractant effect on the osteoclast precursor RAW 264.7 cells. These results suggest that the Ca(2+)(o) gradient present near the bone may be one of the initiating factors for the homing of osteoclast precursors to bone, thus possibly playing a role in the initiation of bone remodeling. PMID:20149906

  17. Combinatorial pathway engineering for optimized production of the anti-malarial FR900098.

    PubMed

    Freestone, Todd S; Zhao, Huimin

    2016-02-01

    As resistance to current anti-malarial therapeutics spreads, new compounds to treat malaria are increasingly needed. One promising compound is FR900098, a naturally occurring phosphonate. Due to limitations in both chemical synthesis and biosynthetic methods for FR900098 production, this potential therapeutic has yet to see widespread implementation. Here we applied a combinatorial pathway engineering strategy to improve the production of FR900098 in Escherichia coli by modulating each of the pathway's nine genes with four promoters of different strengths. Due to the large size of the library and the low screening throughput, it was necessary to develop a novel screening strategy that significantly reduced the sample size needed to find an optimal strain. This was done by using biased libraries that localize searching around top hits and home in on high-producing strains. By incorporating this strategy, a significantly improved strain was found after screening less than 3% of the entire library. When coupled with culturing optimization, a strain was found to produce 96 mg/L, a 16-fold improvement over the original strain. We believe the enriched library method developed here can be used on other large pathways that may be difficult to engineer by combinatorial methods due to low screening throughput. PMID:26245694

  18. Moving from Pipeline Thinking to Understanding Pathways: Findings from the Academic Pathways Study of Engineering Undergraduates. Research Brief

    ERIC Educational Resources Information Center

    Atman, Cindy; Sheppard, Sheri; Fleming, Lorraine; Miller, Ron; Smith, Karl; Stevens, Reed; Streveler, Ruth; Loucks-Jaret, Tina; Lund, Dennis

    2008-01-01

    While engineering educators have engaged in many endeavors aimed at advancing engineering education and practice, much of this work has focused on broad curricular issues. Few studies focus on what it means to be an engineer or the process of what it takes to learn to engineer. In the last decade engineering educators have begun to focus on…

  19. Quercetin protects oligodendrocyte precursor cells from oxygen/glucose deprivation injury in vitro via the activation of the PI3K/Akt signaling pathway.

    PubMed

    Wang, X-Q; Yao, R-Q; Liu, X; Huang, J-J; Qi, D-S; Yang, L-H

    2011-10-10

    The aim of this study was to investigate the protection of quercetin (QUE) on oligodendrocyte precursor cells (OPCs) from oxygen/glucose deprivation (OGD)-induced injury in vitro and explore whether the PI3K/Akt signaling pathway contributed to the protection provided by quercetin. The OGD condition was induced by including 2mM sodium dithionite (Na(2)S(2)O(4)) in glucose-free DMEM medium. The concentration of QUE in this study ranged from 3μM to 81μM. OPCs were identified by immunocytochemical staining. Cell viability was analyzed using the water soluble tetrazolium salt-8 (WST-8) and lactate dehydrogenase assay (LDH). The morphological changes of the nucleus were measured using Hoechst 33258 nuclear staining, and the ratio of apoptotic cells was determined by FITC annexin V- and propidium iodide (PI) flow cytometry assay kit. In addition, the levels of pro-apoptotic proteins such as cleaved-caspase-3 and Bax and the anti-apoptotic proteins p-Akt and Bcl-2 were quantified using western blotting. The results showed that the OPC cell survival rate was significantly increased by incubation in conditioned medium supplemented with QUE as measured by the WST-8 assay, while the LDH release rate was significantly decreased as analyzed by the LDH assay. Furthermore, apoptosis assay showed that the apoptosis ratio of OPCs was also dramatically reduced by QUE. Western blotting showed that the expression levels of Bax and cleaved-caspase-3 proteins were down-regulated, while Bcl-2 and p-Akt were up-regulated. Further study showed that the increase in p-Akt by QUE was reduced by the PI3K inhibitor LY294002. These results indicated that QUE effectively protected OPCs from OGD-induced injury and that the mechanism might be related to the activation of the PI3K/Akt signaling pathway. PMID:21803128

  20. Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor.

    PubMed

    Biemel, K M; Reihl, O; Conrad, J; Lederer, M O

    2001-06-29

    Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-links N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOGDIC 12), N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOPDIC 13), and 6-((6S)-2-([(4S)-4-ammonio-5-oxido-5-oxopentyl] amino)-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9-13. The bicyclic structures 9-11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9-13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links. PMID:11279247

  1. Effect and mechanism of fuzhisan and donepezil on the sirtuin 1 pathway and amyloid precursor protein metabolism in PC12 cells.

    PubMed

    Guo, Peng; Wang, Desheng; Wang, Xiaomin; Feng, Honglin; Tang, Ying; Sun, Ruihong; Zheng, Yan; Dong, Lin; Zhao, Jiaying; Zhang, Xin; Wang, Shuyu; Sun, Hongxu

    2016-04-01

    The present study aimed to determine the effect and mechanism of fuzhisan (FZS) and donepezil on the SIRT1 signaling pathway and the metabolism of the amyloid precursor protein (APP) in PC12 cells. An experimental cell model of PC12 cells with Aβ25‑35‑induced neurotoxicity was established and cell proliferation was determined by the MTT assay following treatment with donepezil and FZS. In addition, cell apoptosis was determined using DAPI staining and light microscopy. Furthermore, western blot analysis and ELISA were utilized to evaluate the expression levels of associated APP, Aβ40, Aβ42, sAPPα, sAPPβ, ADAM10, sirtuin 1 (SIRT1) and forkhead box O (FoxO) protein. The results indicated that the cell model was successfully established and FZS protected the PC12 cells from the neurotoxic effects of Aβ25‑35, in a similar effect to donepezil, in a dose‑dependent manner. The expression of APP remained at the same level during the experimental period. The levels of Aβ40, Aβ42 and sAPPβ were downregulated, where as sAPPα, ADAM10, SIRT1 and FoxO expression levels were upregulated. In conclusion, FZS treatment attenuated the Aβ25‑35‑induced neurotoxicity in vitro. The neuroprotective mechanism of FZS was determined, including the induction of ADAM10 and SIRT1‑FoxO pathway, which participated in the process of neuroprotection. The present study identified the neuroprotective function of FZS, which may protect against Aβ‑induced toxicity. Therefore, FZS may be used clinically as a beneficial therapeutic drug for the development or progression of Alzheimer's disease. PMID:26936536

  2. Statins and the squalene synthase inhibitor zaragozic acid stimulate the non-amyloidogenic pathway of amyloid-beta protein precursor processing by suppression of cholesterol synthesis.

    PubMed

    Kojro, Elzbieta; Füger, Petra; Prinzen, Claudia; Kanarek, Anna Maria; Rat, Dorothea; Endres, Kristina; Fahrenholz, Falk; Postina, Rolf

    2010-01-01

    Cholesterol-lowering drugs such as statins influence the proteolytic processing of the amyloid-beta protein precursor (AbetaPP) and are reported to stimulate the activity of alpha-secretase, the major preventive secretase of Alzheimer's disease. Statins can increase the alpha-secretase activity by their cholesterol-lowering properties as well as by impairment of isoprenoids synthesis. In the present study, we elucidate the contribution of these pathways in alpha-secretase activation. We demonstrate that zaragozic acid, a potent inhibitor of squalene synthase which blocks cholesterol synthesis but allows synthesis of isoprenoids, also stimulates alpha-secretase activity. Treatment of human neuroblastoma cells with 50 microM zaragozic acid resulted in a approximately 3 fold increase of alpha-secretase activity and reduced cellular cholesterol by approximately 30%. These effects were comparable to results obtained from cells treated with a low lovastatin concentration (2 microM). Zaragozic acid-stimulated secretion of alpha-secretase-cleaved soluble AbetaPP was dose dependent and saturable. Lovastatin- or zaragozic acid-stimulated increase of alpha-secretase activity was completely abolished by a selective ADAM10 inhibitor. By targeting the alpha-secretase ADAM10 to lipid raft domains via a glycosylphosphatidylinositol anchor, we demonstrate that ADAM10 is unable to cleave AbetaPP in a cholesterol-rich environment. Our results indicate that inhibition of cholesterol biosynthesis by a low lovastatin concentration is sufficient for alpha-secretase activation. PMID:20413873

  3. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production.

    PubMed

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L(-1) L-arginine with yield up to 0.431 g L-arginine g(-1) glucose in fed-batch fermentation. PMID:27338253

  4. Metabolic Engineering of the Phenylpropanoid Pathway Enhances the Antioxidant Capacity of Saussurea involucrata

    PubMed Central

    Li, Chonghui; Han, Xiaoyan; Zhao, Qiao; Zhao, Dexiu; Hua, Xuejun; Pang, Yongzhen

    2013-01-01

    The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc) into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2′-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing anti-oxidant power) assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering. PMID:23976949

  5. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

    PubMed Central

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation. PMID:27338253

  6. Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae

    PubMed Central

    Qi, Xin; Zha, Jian; Liu, Gao-Gang; Zhang, Weiwen; Li, Bing-Zhi; Yuan, Ying-Jin

    2015-01-01

    Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003. PMID:26539187

  7. Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

    PubMed

    Qi, Xin; Zha, Jian; Liu, Gao-Gang; Zhang, Weiwen; Li, Bing-Zhi; Yuan, Ying-Jin

    2015-01-01

    Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003. PMID:26539187

  8. A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering

    PubMed Central

    Dhar, Niha; Razdan, Sumeer; Rana, Satiander; Bhat, Wajid W.; Vishwakarma, Ram; Lattoo, Surrinder K.

    2015-01-01

    Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well-characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomics, and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude toward development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool toward enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. somnifera for understanding various molecular network interactions in entirety. PMID:26640469

  9. Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering.

    PubMed

    Madzak, Catherine

    2015-06-01

    The oleaginous yeast Yarrowia lipolytica has become a recognized system for expression/secretion of heterologous proteins. This non-conventional yeast is currently being developed as a workhorse for biotechnology by several research groups throughout the world, especially for single-cell oil production, whole cell bioconversion and upgrading of industrial wastes. This mini-review presents established tools for protein expression in Y. lipolytica and highlights novel developments in the areas of promoter design, surface display, and host strain or metabolic pathway engineering. An overview of the industrial and commercial biotechnological applications of Y. lipolytica is also presented. PMID:25947247

  10. Rhamnolipids in perspective: gene regulatory pathways, metabolic engineering, production and technological forecasting.

    PubMed

    Dobler, Leticia; Vilela, Leonardo F; Almeida, Rodrigo V; Neves, Bianca C

    2016-01-25

    Rhamnolipids have emerged as a very promising class of biosurfactants in the last decades, exhibiting properties of great interest in several industrial applications, and have represented a suitable alternative to chemically-synthesized surfactants. This class of biosurfactants has been extensively studied in recent years, aiming at their large-scale production based on renewable resources, which still require high financial costs. Development of non-pathogenic, high-producing strains has been the focus of a number of studies involving heterologous microbial hosts as platforms. However, the intricate gene regulation network controlling rhamnolipid biosynthesis represents a challenge to metabolic engineering and remains to be further understood and explored. This article provides an overview of the biosynthetic pathways and the main gene regulatory factors involved in rhamnolipid production within Pseudomonas aeruginosa, the prototypal producing species. In addition, we provide a perspective view into the main strategies applied to metabolic engineering and biotechnological production. PMID:26409933

  11. Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine.

    PubMed

    Liu, Yanfeng; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-09-01

    Glucosamine (GlcN) and its acetylated derivative, N-acetylglucosamine (GlcNAc), are widely used in nutraceutical and pharmaceutical industries. Currently, GlcN and GlcNAc are mainly produced by hydrolysis from crab and shrimp shells, which can cause severe environmental pollution and carries the potential risk of allergic reactions. In this study, we attempted to achieve microbial production of GlcNAc by pathway engineering of Bacillus subtilis 168. Specifically, glmS (encoding GlcN-6-phosphate synthase) from B. subtilis 168 and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) from Saccharomyces cerevisiae S288C were firstly co-overexpressed in B. subtilis; the level of GlcNAc reached 240mg/L in shake flask culture. Next, nagP, encoding the GlcNAc-specific enzyme of phosphotransferase system, was deleted to block the importation of extracellular GlcNAC, thus improving GlcNAc production to 615mg/L in shake flask culture. Then, nagA (encoding GlcNAc-6-phosphate deacetylase), gamA (encoding GlcN-6-phosphate deaminase), and nagB (encoding GlcN-6-phosphate deaminase) were deleted to block the catabolism of intracellular GlcNAc, thereby further increasing the GlcNAc titer to 1.85g/L in shake flask culture. Finally, microbial production of GlcNAc by the engineered B. subtilis 168 was conducted in a 3-L fed-batch bioreactor, and the GlcNAc titer reached 5.19g/L, which was 2.8-fold of that in shake flask culture. This is the first report regarding the pathway engineering of B. subtilis for microbial production of GlcNAc, and provides a good starting point for further metabolic engineering to achieve the industrial production of GlcNAc by a generally regarded as safe strain. PMID:23876412

  12. Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.

    PubMed

    Wlodarczyk, Artur; Gnanasekaran, Thiyagarajan; Nielsen, Agnieszka Zygadlo; Zulu, Nodumo Nokolunga; Mellor, Silas Busck; Luckner, Manja; Thøfner, Jens Frederik Bang; Olsen, Carl Erik; Mottawie, Mohammed Saddik; Burow, Meike; Pribil, Mathias; Feussner, Ivo; Møller, Birger Lindberg; Jensen, Poul Erik

    2016-01-01

    Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation. PMID:26548317

  13. Pathway engineering of Propionibacterium jensenii for improved production of propionic acid.

    PubMed

    Liu, Long; Guan, Ningzi; Zhu, Gexin; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian

    2016-01-01

    Propionic acid (PA) is an important chemical building block widely used in the food, pharmaceutical, and chemical industries. In our previous study, a shuttle vector was developed as a useful tool for engineering Propionibacterium jensenii, and two key enzymes-glycerol dehydrogenase and malate dehydrogenase-were overexpressed to improve PA titer. Here, we aimed to improve PA production further via the pathway engineering of P. jensenii. First, the phosphoenolpyruvate carboxylase gene (ppc) from Klebsiella pneumoniae was overexpressed to access the one-step synthesis of oxaloacetate directly from phosphoenolpyruvate without pyruvate as intermediate. Next, genes encoding lactate dehydrogenase (ldh) and pyruvate oxidase (poxB) were deleted to block the synthesis of the by-products lactic acid and acetic acid, respectively. Overexpression of ppc and deleting ldh improved PA titer from 26.95 ± 1.21 g·L(-1) to 33.21 ± 1.92 g·L(-1) and 30.50 ± 1.63 g·L(-1), whereas poxB deletion decreased it. The influence of this pathway engineering on gene transcription, enzyme expression, NADH/NAD(+) ratio, and metabolite concentration was also investigated. Finally, PA production in P. jensenii with ppc overexpression as well as ldh deletion was investigated, which resulted in further increases in PA titer to 34.93 ± 2.99 g·L(-1) in a fed-batch culture. PMID:26814976

  14. Pathway engineering of Propionibacterium jensenii for improved production of propionic acid

    PubMed Central

    Liu, Long; Guan, Ningzi; Zhu, Gexin; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Chen, Jian

    2016-01-01

    Propionic acid (PA) is an important chemical building block widely used in the food, pharmaceutical, and chemical industries. In our previous study, a shuttle vector was developed as a useful tool for engineering Propionibacterium jensenii, and two key enzymes—glycerol dehydrogenase and malate dehydrogenase—were overexpressed to improve PA titer. Here, we aimed to improve PA production further via the pathway engineering of P. jensenii. First, the phosphoenolpyruvate carboxylase gene (ppc) from Klebsiella pneumoniae was overexpressed to access the one-step synthesis of oxaloacetate directly from phosphoenolpyruvate without pyruvate as intermediate. Next, genes encoding lactate dehydrogenase (ldh) and pyruvate oxidase (poxB) were deleted to block the synthesis of the by-products lactic acid and acetic acid, respectively. Overexpression of ppc and deleting ldh improved PA titer from 26.95 ± 1.21 g·L−1 to 33.21 ± 1.92 g·L−1 and 30.50 ± 1.63 g·L−1, whereas poxB deletion decreased it. The influence of this pathway engineering on gene transcription, enzyme expression, NADH/NAD+ ratio, and metabolite concentration was also investigated. Finally, PA production in P. jensenii with ppc overexpression as well as ldh deletion was investigated, which resulted in further increases in PA titer to 34.93 ± 2.99 g·L−1 in a fed-batch culture. PMID:26814976

  15. Disruption of kif3a results in defective osteoblastic differentiation in dental mesenchymal stem/precursor cells via the Wnt signaling pathway

    PubMed Central

    Jiang, Sicong; Chen, Guoqing; Feng, Lian; Jiang, Zongting; Yu, Mei; Bao, Jinku; Tian, Weidong

    2016-01-01

    The anterograde intraflagellar transport motor protein, kif3a, regulates the integrity of primary cilia and various cellular functions, however, the role of kif3a in dental mesenchymal stem/precursor cell differentiation remains to be fully elucidated. In the present study, the expression of kif3a was knocked down in human dental follicle cells (hDFCs) and human dental pulp cells (hDPCs) using short hairpin RNA. The results of subsequent immunofluorescence revealed that knocking down kif3a resulted in the loss of primary cilia, which led to impairment of substantial mineralization and expression of the differentiation-associated markers, including alkaline phosphatase, Runt-related transcription factor 2, dentin matrix protein 1 and dentin sialophosphoprotein in the hDFCs and hDPCs. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses showed that the expression levels of Wnt3a-mediated active β-catenin and lymphoid enhancer-binding factor 1 were attenuated, whereas the expression of phosphorylated glycogen synthase kinase 3β was enhanced, in the kif3a-knockdown cells. In addition, exogenous Wnt3a partially rescued osteoblastic differentiation in the hDFCs and hDPCs. These results demonstrated that inhibition of kif3a in the hDFCs and hDPCs disrupted primary cilia formation and/or function, and indicated that kif3a is important in the differentiation of hDFCs and hDPCs through the Wnt pathway. These findings not only enhance current understanding of tooth development and diseases of tooth mineralization, but also indicate possible strategies to regulate mineralization during tooth repair and regeneration. PMID:27432616

  16. Disruption of kif3a results in defective osteoblastic differentiation in dental mesenchymal stem/precursor cells via the Wnt signaling pathway.

    PubMed

    Jiang, Sicong; Chen, Guoqing; Feng, Lian; Jiang, Zongting; Yu, Mei; Bao, Jinku; Tian, Weidong

    2016-09-01

    The anterograde intraflagellar transport motor protein, kif3a, regulates the integrity of primary cilia and various cellular functions, however, the role of kif3a in dental mesenchymal stem/precursor cell differentiation remains to be fully elucidated. In the present study, the expression of kif3a was knocked down in human dental follicle cells (hDFCs) and human dental pulp cells (hDPCs) using short hairpin RNA. The results of subsequent immunofluorescence revealed that knocking down kif3a resulted in the loss of primary cilia, which led to impairment of substantial mineralization and expression of the differentiation‑associated markers, including alkaline phosphatase, Runt‑related transcription factor 2, dentin matrix protein 1 and dentin sialophosphoprotein in the hDFCs and hDPCs. The results of reverse transcription‑quantitative polymerase chain reaction and western blot analyses showed that the expression levels of Wnt3a‑mediated active β‑catenin and lymphoid enhancer‑binding factor 1 were attenuated, whereas the expression of phosphorylated glycogen synthase kinase 3β was enhanced, in the kif3a‑knockdown cells. In addition, exogenous Wnt3a partially rescued osteoblastic differentiation in the hDFCs and hDPCs. These results demonstrated that inhibition of kif3a in the hDFCs and hDPCs disrupted primary cilia formation and/or function, and indicated that kif3a is important in the differentiation of hDFCs and hDPCs through the Wnt pathway. These findings not only enhance current understanding of tooth development and diseases of tooth mineralization, but also indicate possible strategies to regulate mineralization during tooth repair and regeneration. PMID:27432616

  17. Enhanced production of coenzyme Q10 by self-regulating the engineered MEP pathway in Rhodobacter sphaeroides.

    PubMed

    Lu, Wenqiang; Ye, Lidan; Xu, Haoming; Xie, Wenping; Gu, Jiali; Yu, Hongwei

    2014-04-01

    Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10 ) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild-type (48.25 mg/L, 3.24 mg/g DCW). PMID:24122603

  18. Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering.

    PubMed

    Feng, Jun; Gu, Yanyan; Quan, Yufen; Cao, Mingfeng; Gao, Weixia; Zhang, Wei; Wang, Shufang; Yang, Chao; Song, Cunjiang

    2015-11-01

    A Bacillus amyloliquefaciens strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the epsA-O (responsible for extracellular polysaccharide synthesis), sac (responsible for levan synthesis), lps (responsible for lipopolysaccharide synthesis) and pta (encoding phosphotransacetylase) genes, showed increased γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15 g/L. The γ-PGA degrading genes pgdS (encoding poly-gamma-glutamate depolymerase) and cwlO (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased γ-PGA production from 4.15 to 9.18 g/L. The autoinducer AI-2 synthetase gene luxS was deleted in NK-E10 strain and the resulting NK-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54 g/L). In addition, we overexpressed the pgsBCA genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-PGA production. Finally, the rocG gene (encoding glutamate dehydrogenase) and the glnA gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The rocG-repressed NK-anti-rocG strain exhibited the highest γ-PGA titer (11.04 g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final γ-PGA titer of 20.3g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced γ-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are

  19. Pathways to space: A mission to foster the next generation of scientists and engineers

    NASA Astrophysics Data System (ADS)

    Dougherty, Kerrie; Oliver, Carol; Fergusson, Jennifer

    2014-06-01

    The first education project funded under the Australian Government's Australian Space Research Program (ASRP), Pathways to Space was a unique project combining education, science communication research and research in astrobiology and robotics. It drew upon the challenges of space exploration to inspire students to consider study and careers in science and engineering. A multi-faceted program, Pathways to Space provided hands-on opportunities for high school and university students to participate in realistic simulations of a robotic Mars exploration mission for astrobiology. Its development was a collaboration between the Australian Centre for Astrobiology (University of New South Wales), the Australian Centre for Field Robotics (University of Sydney), the Powerhouse Museum and industry partner, Cisco. Focused on students in Years 9-10 (15-16 years of age), this program provided them with the opportunity to engage directly with space engineers and astrobiologists, while carrying out a simulated Mars mission using the digital learning facilities available at the Powerhouse Museum. As a part of their program, the students operated robotic mini-rovers in the Powerhouse Museum's “Mars Yard”, a highly accurate simulation of the Martian surface, where university students also carry out the development and testing of experimental Mars roving vehicles. This aspect of the program has brought real science and engineering research into the public space of the museum. As they undertook the education program, the students participated in a research study aimed at understanding the effectiveness of the project in achieving its key objective - encouraging students to consider space related courses and careers. This paper outlines the development and operation of the Pathways to Space project over its 3-year funding period, during which it met and exceeded all the requirements of its ASRP grant. It will look at the goals of the project, the rationale behind the education and

  20. Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine.

    PubMed

    Herrero, Oscar; Ramón, Daniel; Orejas, Margarita

    2008-03-01

    Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising. PMID:18155949

  1. Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introuduced into activated sludge microcosms

    SciTech Connect

    Nublein, K.; Maris, D.; Timmis, K.; Dwyer, D.F. )

    1992-10-01

    Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA.

  2. Reverse engineering of metabolic pathways from observed data using genetic programming.

    PubMed

    Koza, J R; Mydlowec, W; Lanza, G; Yu, J; Keane, M A

    2001-01-01

    Recent work has demonstrated that genetic programming is capable of automatically creating complex networks (such as analog electrical circuits and controllers) whose behavior is modeled by linear and non-linear continuous-time differential equations and whose behavior matches prespecified output values. The concentrations of substances participating in networks of chemical reactions are also modeled by non-linear continuous-time differential equations. This paper demonstrates that it is possible to automatically create (reverse engineer) a network of chemical reactions from observed time-domain data. Genetic programming starts with observed time-domain concentrations of input substances and automatically creates both the topology of the network of chemical reactions and the rates of each reaction within the network such that the concentration of the final product of the automatically created network matches the observed time-domain data. Specifically, genetic programming automatically created metabolic pathways involved in the phospholipid cycle and the synthesis and degradation of ketone bodies. PMID:11262962

  3. Heading in the right direction: thermodynamics-based network analysis and pathway engineering.

    PubMed

    Ataman, Meric; Hatzimanikatis, Vassily

    2015-12-01

    Thermodynamics-based network analysis through the introduction of thermodynamic constraints in metabolic models allows a deeper analysis of metabolism and guides pathway engineering. The number and the areas of applications of thermodynamics-based network analysis methods have been increasing in the last ten years. We review recent applications of these methods and we identify the areas that such analysis can contribute significantly, and the needs for future developments. We find that organisms with multiple compartments and extremophiles present challenges for modeling and thermodynamics-based flux analysis. The evolution of current and new methods must also address the issues of the multiple alternatives in flux directionalities and the uncertainties and partial information from analytical methods. PMID:26360871

  4. In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide

    PubMed Central

    Neamati, Nouri; Shekhtman, Alexander; Camarero, Julio A.

    2013-01-01

    The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions. PMID:23848581

  5. A structured approach for the engineering of biochemical network models, illustrated for signalling pathways.

    PubMed

    Breitling, Rainer; Gilbert, David; Heiner, Monika; Orton, Richard

    2008-09-01

    Quantitative models of biochemical networks (signal transduction cascades, metabolic pathways, gene regulatory circuits) are a central component of modern systems biology. Building and managing these complex models is a major challenge that can benefit from the application of formal methods adopted from theoretical computing science. Here we provide a general introduction to the field of formal modelling, which emphasizes the intuitive biochemical basis of the modelling process, but is also accessible for an audience with a background in computing science and/or model engineering. We show how signal transduction cascades can be modelled in a modular fashion, using both a qualitative approach--qualitative Petri nets, and quantitative approaches--continuous Petri nets and ordinary differential equations (ODEs). We review the major elementary building blocks of a cellular signalling model, discuss which critical design decisions have to be made during model building, and present a number of novel computational tools that can help to explore alternative modular models in an easy and intuitive manner. These tools, which are based on Petri net theory, offer convenient ways of composing hierarchical ODE models, and permit a qualitative analysis of their behaviour. We illustrate the central concepts using signal transduction as our main example. The ultimate aim is to introduce a general approach that provides the foundations for a structured formal engineering of large-scale models of biochemical networks. PMID:18573813

  6. Expanding the docosahexaenoic acid food web for sustainable production: engineering lower plant pathways into higher plants

    PubMed Central

    Petrie, James R.; Singh, Surinder P.

    2011-01-01

    Background Algae are becoming an increasingly important component of land plant metabolic engineering projects. Land plants and algae have similar enough genetics to allow relatively straightforward gene transfer and they also share enough metabolic similarities that algal enzymes often function in a plant cell environment. Understanding metabolic systems in algae can provide insights into homologous systems in land plants. As examples, algal models are currently being used by several groups to better understand starch and lipid metabolism and catabolism, fields which have relevance in land plants. Importantly, land plants and algae also have enough metabolic divergence that algal genes can often provide new metabolic traits to plants. Furthermore, many algal genomes have now been sequenced, with many more in progress, and this easy access to genome-wide information has revealed that algal genomes are often relatively simple when compared with plants. Scope One example of the importance of algal, and in particular microalgal, resources to land plant research is the metabolic engineering of long-chain polyunsaturated fatty acids into oilseed crops which typically uses microalgal genes to extend existing natural plant biosynthetic pathways. This review describes both recent progress and remaining challenges in this field. PMID:22476481

  7. Improved Precursor Directed Biosynthesis in E. coli via Directed Evolution

    PubMed Central

    Lee, Ho Young; Harvey, Colin J.B.; Cane, David E.; Khosla, Chaitan

    2010-01-01

    Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure-function relationships in the bacterial ribosome. PMID:21081955

  8. Engineering of Corynebacterium glutamicum with an NADPH-generating glycolytic pathway for L-lysine production.

    PubMed

    Takeno, Seiki; Murata, Ryosuke; Kobayashi, Ryosuke; Mitsuhashi, Satoshi; Ikeda, Masato

    2010-11-01

    A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP(+) to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis. PMID:20851994

  9. Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering1[W][OA

    PubMed Central

    Pang, Yongzhen; Abeysinghe, I. Sarath B.; He, Ji; He, Xianzhi; Huhman, David; Mewan, K. Mudith; Sumner, Lloyd W.; Yun, Jianfei; Dixon, Richard A.

    2013-01-01

    Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (−)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (−)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols. PMID:23288883

  10. Science, Technology, Engineering, and Mathematics (STEM) Pathways: High School Science and Math Coursework and Postsecondary Degree Attainment

    ERIC Educational Resources Information Center

    Tyson, Will; Lee, Reginald; Borman, Kathryn M.; Hanson, Mary Ann

    2007-01-01

    This article examines how high school science and mathematics course-taking creates pathways toward future baccalaureate degree attainment in science, technology, engineering, and mathematics (STEM) majors in Florida 4-year universities using Burkam and Lee's (2003) course-taking categories developed using national student datasets. This study…

  11. Alternative Pathways to Engineering Success--Using Academic and Social Integration to Understand Two-Year Engineering Student Success

    ERIC Educational Resources Information Center

    Marra, Rose M.; Tsai, Chia-Lin; Bogue, Barbara; Pytel, Jean Landa

    2015-01-01

    The need for educating engineers in the United States continues as the projected demand is rising the number of high school seniors planning to enter engineering careers has remained relatively stable (Sargent, 2014). Additionally, figures show that attrition rates in undergraduate engineering continue to be an area of concern, (Sargent, 2014;…

  12. An Evaluation of Active Learning Causal Discovery Methods for Reverse-Engineering Local Causal Pathways of Gene Regulation

    PubMed Central

    Ma, Sisi; Kemmeren, Patrick; Aliferis, Constantin F.; Statnikov, Alexander

    2016-01-01

    Reverse-engineering of causal pathways that implicate diseases and vital cellular functions is a fundamental problem in biomedicine. Discovery of the local causal pathway of a target variable (that consists of its direct causes and direct effects) is essential for effective intervention and can facilitate accurate diagnosis and prognosis. Recent research has provided several active learning methods that can leverage passively observed high-throughput data to draft causal pathways and then refine the inferred relations with a limited number of experiments. The current study provides a comprehensive evaluation of the performance of active learning methods for local causal pathway discovery in real biological data. Specifically, 54 active learning methods/variants from 3 families of algorithms were applied for local causal pathways reconstruction of gene regulation for 5 transcription factors in S. cerevisiae. Four aspects of the methods’ performance were assessed, including adjacency discovery quality, edge orientation accuracy, complete pathway discovery quality, and experimental cost. The results of this study show that some methods provide significant performance benefits over others and therefore should be routinely used for local causal pathway discovery tasks. This study also demonstrates the feasibility of local causal pathway reconstruction in real biological systems with significant quality and low experimental cost. PMID:26939894

  13. Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid

    PubMed Central

    Ro, Dae-Kyun; Ouellet, Mario; Paradise, Eric M; Burd, Helcio; Eng, Diana; Paddon, Chris J; Newman, Jack D; Keasling, Jay D

    2008-01-01

    Background Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Results Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL-1 in shake-flask cultures and 1 g L-1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by

  14. Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli.

    PubMed

    Ning, Yike; Wu, Xuejiao; Zhang, Chenglin; Xu, Qingyang; Chen, Ning; Xie, Xixian

    2016-07-01

    Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called "bacterial milking" efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l-threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR, a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8g/g DCW and 0.84gL(-)(1)h(-)(1) respectively. The overall ectoine yield was 0.11g/g of glucose. PMID:26969253

  15. Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology.

    PubMed

    Pandey, Ramesh Prasad; Parajuli, Prakash; Koffas, Mattheos A G; Sohng, Jae Kyung

    2016-01-01

    In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. PMID:26946281

  16. Engineering of Glarea lozoyensis for Exclusive Production of the Pneumocandin B0 Precursor of the Antifungal Drug Caspofungin Acetate

    PubMed Central

    Chen, Li; Yue, Qun; Li, Yan; Niu, Xuemei; Xiang, Meichun; Wang, Wenzhao; Bills, Gerald F.

    2014-01-01

    Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0 production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain. PMID:25527531

  17. Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

    PubMed Central

    John, Maliyakal E.; Keller, Greg

    1996-01-01

    Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry. PMID:11038522

  18. 5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway.

    PubMed

    Ramzi, Ahmad Bazli; Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-12-01

    ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C5 pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering. PMID:26453466

  19. Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants.

    PubMed

    Ruiz-López, Noemi; Sayanova, Olga; Napier, Johnathan A; Haslam, Richard P

    2012-04-01

    Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described. PMID:22291131

  20. Enhanced Diterpene Tanshinone Accumulation and Bioactivity of Transgenic Salvia miltiorrhiza Hairy Roots by Pathway Engineering.

    PubMed

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Li, Leilei; Huang, Shengxiong; Zhang, Tong; Wang, Huizhong; Kai, Guoyin

    2016-03-30

    Tanshinones are health-promoting diterpenoids found in Salvia miltiorrhiza and have wide applications. Here, SmGGPPS (geranylgeranyl diphosphate synthase) and SmDXSII (1-deoxy-d-xylulose-5-phosphate synthase) were introduced into hairy roots of S. miltiorrhiza. Overexpression of SmGGPPS and SmDXSII in hairy roots produces higher levels of tanshinone than control and single-gene transformed lines; tanshinone production in the double-gene transformed line GDII10 reached 12.93 mg/g dry weight, which is the highest tanshinone content that has been achieved through genetic engineering. Furthermore, transgenic hairy root lines showed higher antioxidant and antitumor activities than control lines. In addition, contents of chlorophylls, carotenoids, indoleacetic acid, and gibberellins were significantly elevated in transgenic Arabidopsis thaliana plants. These results demonstrate a promising method to improve the production of diterpenoids including tanshinone as well as other natural plastid-derived isoprenoids in plants by genetic manipulation of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. PMID:26753746

  1. Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity

    PubMed Central

    Liang, Fu-Sen; Ho, Wen Qi; Crabtree, Gerald R.

    2011-01-01

    Chemically induced proximity (CIP) systems use small molecules and engineered proteins to control and study biological processes. However, small molecule–based systems for controlling protein abundance or activities have been limited by toxicity, instability, cost, and slow clearance of the small molecules in vivo. To address these problems, we modified proteins of the plant abscisic acid (ABA) stress response pathway to control the proximity of cellular proteins and showed that the system could be used to regulate transcription, signal transduction, and subcellular localization of proteins in response to exogenously applied ABA. We also showed that the ABA CIP system can be combined with other CIP systems to simultaneously control multiple processes. We found that, when given to mice, ABA was orally available and had a 4-hour half-life. These properties, along with its lack of toxicity and low cost, suggest that ABA may be well suited for therapeutic applications and as an experimental tool to control diverse cellular activities in vivo. PMID:21406691

  2. Pathways to Science and Engineering Bachelor’s Degrees for Men and Women

    PubMed Central

    Legewie, Joscha; DiPrete, Thomas A.

    2014-01-01

    Despite the striking reversal of the gender gap in educational attainment and the near–gender parity in math performance, women pursue science and engineering (S/E) degrees at much lower rates than their male peers do. Current efforts to increase the number of women in these fields focus on different life-course periods but lack a clear understanding of the importance of these periods and how orientations toward S/E fields develop over time. In this article, we examine the gendered pathways to a S/E bachelor’s degree from middle school to high school and college based on a representative sample from the 1973 to 1974 birth cohort. Using a counterfactual decomposition analysis, we determine the relative importance of these different life-course periods and thereby inform the direction of future research and policy. Our findings confirm previous research that highlights the importance of early encouragement for gender differences in S/E degrees, but our findings also attest to the high school years as a decisive period for the gender gap, while challenging the focus on college in research and policy. Indeed, if female high school seniors had the same orientation toward and preparation for S/E fields as their male peers, the gender gap in S/E degrees would be closed by as much as 82 percent. PMID:25089284

  3. Metabolic engineering of Saccharomyces cerevisiae for the production of 2-phenylethanol via Ehrlich pathway.

    PubMed

    Kim, Bosu; Cho, Bo-Ram; Hahn, Ji-Sook

    2014-01-01

    2-Phenylethanol (2-PE), a fragrance compound with a rose-like odor, is widely used in perfumery and cosmetics. Here, we report the first metabolic engineering approach for 2-PE production in Saccharomyces cerevisiae. 2-PE can be produced from the catabolism of L-phenylalanine via Ehrlich pathway, consisting of transamination to phenylpyruvate by Aro9, decarboxylation to phenylacetaldehyde by Aro10, and reduction to 2-PE by alcohol dehydrogenases. We demonstrated that Ald3 is mainly responsible for phenylacetaldehyde oxidation, competing with 2-PE production. ALD3 deletion strain overexpressing ARO9 and ARO10 both by episomal overexpression and by induction of the endogenous genes through overexpression of Aro80 transcription factor, produced 4.8 g/L 2-PE in a medium containing 10 g/L L-phenylalanine as a sole nitrogen source. Considering the cytotoxicity of 2-PE, this production titer is almost the upper limit that can be reached in batch cultures, suggesting the great potential of this yeast strain for 2-PE production. 2-PE production was further increased by applying two-phase fermentation method with polypropylene glycol 1200 as an extractant, reaching 6.1 g/L 2-PE in organic phase with the molar yield of 82.5%, which is about ninefold increase compared with wild type. PMID:23836015

  4. Cytokine Combination Therapy Prediction for Bone Remodeling in Tissue Engineering Based on the Intracellular Signaling Pathway

    PubMed Central

    Sun, Xiaoqiang; Su, Jing; Bao, Jiguang; Peng, Tao; Zhang, Le; Zhang, Yuanyuan; Yang, Yunzhi; Zhou, Xiaobo

    2012-01-01

    The long-term performance of tissue-engineered bone grafts is determined by a dynamic balance between bone regeneration and resorption. We proposed using embedded cytokine slow-releasing hydrogels to tune this balance toward a desirable final bone density. In this study we established a systems biology model, and quantitatively explored the combinatorial effects of delivered cytokines from hydrogels on final bone density. We hypothesized that: 1) bone regeneration was driven by transcription factors Runx2 and Osterix, which responded to released cytokines, such as Wnt, BMP2, and TGFβ, drove the development of osteoblast lineage, and contributed to bone mass generation; and 2) the osteoclast lineage, on the other hand, governed the bone resorption, and communications between these two lineages determined the dynamics of bone remodeling. In our model, Intracellular signaling pathways were represented by ordinary differential equations, while the intercellular communications and cellular population dynamics were modeled by stochastic differential equations. Effects of synergistic cytokine combinations were evaluated by Loewe index and Bliss index. Simulation results revealed that the Wnt/BMP2 combinations released from hydrogels showed best control of bone regeneration and synergistic effects, and suggested optimal dose ratios of given cytokine combinations released from hydrogels to most efficiently control the long-term bone remodeling. We revealed the characteristics of cytokine combinations of Wnt/BMP2 which could be used to guide the design of in vivo bone scaffolds and the clinical treatment of some diseases such as osteoporosis. PMID:22910219

  5. Importance of understanding the main metabolic regulation in response to the specific pathway mutation for metabolic engineering of Escherichia coli

    PubMed Central

    Matsuoka, Yu; Shimizu, Kazuyuki

    2013-01-01

    Recent metabolic engineering practice was briefly reviewed in particular for the useful metabolite production such as natural products and biofuel productions. With the emphasis on systems biology approach, the metabolic regulation of the main metabolic pathways in E. coli was discussed from the points of view of enzyme level (allosteric and phosphorylation/ dephosphorylation) regulation, and gene level (transcriptional) regulation. Then the effects of the specific pathway gene knockout such as pts, pgi, zwf, gnd, pyk, ppc, pckA, lpdA, pfl gene knockout on the metabolism in E. coli were overviewed from the systems biology point of view with possible application for strain improvement point. PMID:24688678

  6. STEM Career Cluster Engineering and Technology Education pathway in Georgia: Perceptions of Georgia engineering and technology education high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education survey

    NASA Astrophysics Data System (ADS)

    Crenshaw, Mark VanBuren

    This study examined the perceptions held by Georgia Science, Technology, Engineering, and Mathematics (STEM) Career Cluster Engineering and Technology Education (ETE) high school pathway teachers and Georgia's Career, Technical and Agriculture Education (CTAE) administrators regarding the ETE pathway and its effect on implementation within their district and schools. It provides strategies for ETE teaching methods, curriculum content, STEM integration, and how to improve the ETE pathway program of study. Current teaching and curricular trends were examined in ETE as well as the role ETE should play as related to STEM education. The study, using the Characteristics of Engineering and Technology Education Survey, was conducted to answer the following research questions: (a) Is there a significant difference in the perception of ETE teaching methodology between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? (b) Is there a significant difference in the perception of ETE curriculum content between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? (c) Is there a significant difference in the perception of STEM integration in the ETE high school pathway between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? and (d) Is there a significant difference in the perception of how to improve the ETE high school pathway between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? Suggestions for further research also were offered.

  7. Reducing natural organic matter and disinfection by-product precursors by alternating oxic and anoxic conditions during engineered short residence time riverbank filtration: A laboratory-scale column study.

    PubMed

    Liu, Peng; Farré, Maria José; Keller, Jurg; Gernjak, Wolfgang

    2016-09-15

    Riverbank filtration (RBF) with days to months of residence time has been successfully used as treatment or pre-treatment process to improve water quality for decades. However, its feasibility depends on the local hydrogeological conditions. Therefore, for sites unsuitable to traditional RBF, a smaller engineered RBF may be an option. This study evaluates the performance of engineered short residence time RBF on improving water quality, focusing on the removal of natural organic matter (NOM) and the reduction of precursors of carbon and nitrogen disinfection by-products (DBP). Lab-scale experiments were conducted with surface feed water from a drinking water plant. The results showed that within 6days hydraulic retention time (HRT), 60-70% dissolved organic carbon (DOC) and 70-80% ultraviolet absorbance at 254nm (UV254) could be removed. During the whole filtration process, biodegradation was responsible for the removal of organic matter, and it was found that alternating redox condition between oxic and anoxic was beneficial for the overall performance of the RBF. Dissolved oxygen (DO) had a substantial impact on the removal of DBP precursors. For carbon-containing DBP (C-DBP) precursors' removal, re-aeration after a sequence of oxic and anoxic conditions could further increase the removal efficiencies from 50%, 60%, and 60% to 80%, 90%, and 80% for trihalomethanes (THMs), chloral hydrate (CH), and haloketones (HKs). Prolonged anoxic conditions were however beneficial for the removal of nitrogen-containing DBP (N-DBP) precursors. PMID:27203522

  8. Activation of glyoxylate pathway without the activation of its related gene in succinate-producing engineered Escherichia coli.

    PubMed

    Zhu, Li-Wen; Li, Xiao-Hong; Zhang, Lei; Li, Hong-Mei; Liu, Jian-Hua; Yuan, Zhan-Peng; Chen, Tao; Tang, Ya-Jie

    2013-11-01

    For the first time, glyoxylate pathway in the biosynthesis of succinate was activated without the genetic manipulations of any gene related with glyoxylate pathway. Furthermore, the inactivation of succinate biosynthesis by-products genes encoding acetate kinase (ackA) and phosphotransacetylase (pta) was proven to be the key factor to activate glyoxylate pathway in the metabolically engineered Escherichia coli under anaerobic conditions. In order to enhance the succinate biosynthesis specifically, the genes (i.e., ldhA, ptsG, ackA-pta, focA-pflB, adhE) that disrupt by-products biosynthesis pathways were combinatorially deleted, while the E. coli malate dehydrogenase (MDH) was overexpression. The highest succinate production of 150.78 mM was obtained with YJ003 (ΔldhA, ptsG, ackA-pta), which were 5-folds higher than that obtained with wild type control strain DY329 (25.13 mM). For further understand the metabolic response as a result of several genetic manipulations, an anaerobic stoichiometric model that takes into account the glyoxylate pathway have successfully been implemented to estimate the intracellular fluxes in various recombinant E. coli. The fraction to the glyoxylate pathway from OAA in DY329 was 0 and 31% in YJ003, which indicated that even without the absence of the iclR mutation; the glyoxylate pathway was also activated by deleting the by-products biosynthetic genes, and to be responsible for the higher succinate yields. For further strengthen glyoxylate pathway, a two-stage fed-batch fermentation process was developed by using a 600 g l(-1) glucose feed to achieve a cell growth rate of 0.07 h(-1) in aerobic fermentation, and using a 750 g l(-1) glucose feed to maintain the residual glucose concentration around 40 g l(-1) when its residual level decreased to 10gl(-1) in anaerobic fermentation. The best mutant strain YJ003/pTrc99A-mdh produces final succinate concentration of 274 mM by fed-batch culture, which was 10-folds higher than that obtained

  9. Tailoring pathway modularity in the biosynthesis of erythromycin analogs heterologously engineered in E. coli

    PubMed Central

    Zhang, Guojian; Li, Yi; Fang, Lei; Pfeifer, Blaine A.

    2015-01-01

    Type I modular polyketide synthases are responsible for potent therapeutic compounds that include avermectin (antihelinthic), rapamycin (immunosuppressant), pikromycin (antibiotic), and erythromycin (antibiotic). However, compound access and biosynthetic manipulation are often complicated by properties of native production organisms, prompting an approach (termed heterologous biosynthesis) illustrated in this study through the reconstitution of the erythromycin pathway through Escherichia coli. Using this heterologous system, 16 tailoring pathways were introduced, systematically producing eight chiral pairs of deoxysugar substrates. Successful analog formation for each new pathway emphasizes the remarkable flexibility of downstream enzymes to accommodate molecular variation. Furthermore, analogs resulting from three of the pathways demonstrated bioactivity against an erythromycin-resistant Bacillus subtilis strain. The approach and results support a platform for continued molecular diversification of the tailoring components of this and other complex natural product pathways in a manner that mirrors the modular nature of the upstream megasynthases responsible for aglycone polyketide formation. PMID:26601183

  10. Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae.

    PubMed

    Kondo, Takashi; Tezuka, Hironori; Ishii, Jun; Matsuda, Fumio; Ogino, Chiaki; Kondo, Akihiko

    2012-05-31

    The production of higher alcohols by engineered bacteria has received significant attention. The budding yeast, Saccharomyces cerevisiae, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis. Initially, we overexpressed 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) in S. cerevisiae to enhance the endogenous activity of the Ehrlich pathway. We then overexpressed Ilv2, which catalyzes the first step in the valine synthetic pathway, and deleted the PDC1 gene encoding a major pyruvate decarboxylase with the intent of altering the abundant ethanol flux via pyruvate. Through these engineering steps, along with modification of culture conditions, the isobutanol titer of S. cerevisiae was elevated 13-fold, from 11 mg/l to 143 mg/l, and the yield was 6.6 mg/g glucose, which is higher than any previously reported value for S. cerevisiae. PMID:22342368

  11. Becoming an Engineer in Public Universities: Pathways for Women and Minorities. Palgrave Studies in Urban Education

    ERIC Educational Resources Information Center

    Borman, Kathryn M., Ed.; Tyson, Will, Ed.; Halperin, Rhoda H., Ed.

    2010-01-01

    Based on research conducted in a three-year, mixed-method, multi-site National Science Foundation, Science, Technology, Engineering and Mathematics Talent Expansion Program (STEP) Project, this book offers a comprehensive look into how engineering department culture and climate impacts the successful retention of female and under-represented…

  12. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

    PubMed Central

    2012-01-01

    Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. PMID:23092390

  13. Metabolism of chlorofluorocarbons and polybrominated compounds by pseudomonas putida G786(pHG-2) via an engineered metabolic pathway

    SciTech Connect

    Hur, H.G.; Sadowsky, M.J.; Wackett, L.P.

    1994-11-01

    Polyhalogenated EPA Priority Pollutants are among the most toxic and persistent of the xenobiotic compounds found in the environment. In those instances when biodegradation does occure, it is typically via reductive dechlorination reactions in anaerobic sediments. These reactions are very slow and difficult to study. In this study, cytochrome P-450{sub cam} from Pseudomonas putida G786 and toluene dioxygenase from P. putida F1 were used to catalyze consecutive cometabolic dehalogenation reactions. New halogenated substrates for both were identified. The results demonstrate the metabolism of polybrominated compounds and chlorofluoroalkanes via the engineered metabolic pathway in P. putida G786(pHG-2). 26 refs., 5 figs., 2 tabs.

  14. Engineering Plant Shikimate Pathway for Production of Tocotrienol and Improving Herbicide Resistance1

    PubMed Central

    Rippert, Pascal; Scimemi, Claire; Dubald, Manuel; Matringe, Michel

    2004-01-01

    Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants. PMID:14684842

  15. Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.

    PubMed

    Pasoreck, Elise K; Su, Jin; Silverman, Ian M; Gosai, Sager J; Gregory, Brian D; Yuan, Joshua S; Daniell, Henry

    2016-09-01

    The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering. PMID:27507797

  16. Expanding the fluorine chemistry of living systems using engineered polyketide synthase pathways

    PubMed Central

    Walker, Mark C.; Thuronyi, Benjamin W.; Charkoudian, Louise K.; Lowry, Brian; Khosla, Chaitan; Chang, Michelle C. Y.

    2014-01-01

    Organofluorines represent a rapidly expanding proportion of molecules used in pharmaceuticals, diagnostics, agrochemicals, and materials. Despite the prevalence of fluorine in synthetic compounds, the known biological scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems and show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be introduced site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the production of complex fluorinated natural products using synthetic biology. PMID:24009388

  17. Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering.

    PubMed

    Quintana, Naira; Van der Kooy, Frank; Van de Rhee, Miranda D; Voshol, Gerben P; Verpoorte, Robert

    2011-08-01

    The need to develop and improve sustainable energy resources is of eminent importance due to the finite nature of our fossil fuels. This review paper deals with a third generation renewable energy resource which does not compete with our food resources, cyanobacteria. We discuss the current state of the art in developing different types of bioenergy (ethanol, biodiesel, hydrogen, etc.) from cyanobacteria. The major important biochemical pathways in cyanobacteria are highlighted, and the possibility to influence these pathways to improve the production of specific types of energy forms the major part of this review. PMID:21691792

  18. Ion-Ice Astrochemistry: Barrierless Low-Energy Deposition Pathways to HCOOH, CH3OH, and CO2 on Icy Grain Mantles from Precursor Cations

    NASA Technical Reports Server (NTRS)

    Woon, David E.

    2011-01-01

    A new family of very favorable reaction pathways is explored involving the deposition of ions on icy grain mantles with very low energies. Quantum chemical cluster calculations at the MP2/6-31+G** level in 4H2O clusters and at the B3LYP/6-31+G** level in 17H2O clusters indicate that HCO+ and CH3 + are able to react spontaneously with one of the water molecules in the cluster to form protonated formic acid (HCOOH2 +) and protonated methanol (CH3OH2 +), respectively. It is furthermore found that these initial adducts spontaneously transfer their excess protons to the cluster to form neutral formic acid and methanol, plus solvated hydronium, H3O+. In the final case, if a CO molecule is bound to the surface of the cluster, OH+ may react with it to form protonated carbon dioxide (HCO2 +), which then loses its proton to yield CO2 and H3O+. In the present model, all of these processes were found to occur with no barriers. Discussion includes the analogous gas phase processes, which have been considered in previous studies, as well as the competitive abstraction pathway for HCO(+) + H2O.

  19. Increased secreted amyloid precursor protein-α (sAPPα) in severe autism: proposal of a specific, anabolic pathway and putative biomarker.

    PubMed

    Ray, Balmiki; Long, Justin M; Sokol, Deborah K; Lahiri, Debomoy K

    2011-01-01

    Autism is a neurodevelopmental disorder characterized by deficits in verbal communication, social interactions, and the presence of repetitive, stereotyped and compulsive behaviors. Excessive early brain growth is found commonly in some patients and may contribute to disease phenotype. Reports of increased levels of brain-derived neurotrophic factor (BDNF) and other neurotrophic-like factors in autistic neonates suggest that enhanced anabolic activity in CNS mediates this overgrowth effect. We have shown previously that in a subset of patients with severe autism and aggression, plasma levels of the secreted amyloid-β (Aβ) precursor protein-alpha form (sAPPα) were significantly elevated relative to controls and patients with mild-to-moderate autism. Here we further tested the hypothesis that levels of sAPPα and sAPPβ (proteolytic cleavage products of APP by α- and β-secretase, respectively) are deranged in autism and may contribute to an anabolic environment leading to brain overgrowth. We measured plasma levels of sAPPα, sAPPβ, Aβ peptides and BDNF by corresponding ELISA in a well characterized set of subjects. We included for analysis 18 control, 6 mild-to-moderate, and 15 severely autistic patient plasma samples. We have observed that sAPPα levels are increased and BDNF levels decreased in the plasma of patients with severe autism as compared to controls. Further, we show that Aβ1-40, Aβ1-42, and sAPPβ levels are significantly decreased in the plasma of patients with severe autism. These findings do not extend to patients with mild-to-moderate autism, providing a biochemical correlate of phenotypic severity. Taken together, this study provides evidence that sAPPα levels are generally elevated in severe autism and suggests that these patients may have aberrant non-amyloidogenic processing of APP. PMID:21731612

  20. Potentially bioaccessible phenolics, antioxidant activity and nutritional quality of young buckwheat sprouts affected by elicitation and elicitation supported by phenylpropanoid pathway precursor feeding.

    PubMed

    Świeca, Michał

    2016-02-01

    This paper presents the study on impact of elicitation and the phenylpropanoid pathway feeding on the nutritional quality, the potentially bioaccessible phenolics and the antioxidant capacity of young buckwheat sprouts. Phenolics content was increased by elicitation and feeding with tyrosine and shikimic acid--an elevation of 30% and 17%, respectively. Antioxidant capacity was improved by feeding with tyrosine--an increase of 16.7% and 17.1% in both untreated and treated sprouts, respectively. The highest protein digestibility was determined for the control sprouts and those obtained after tyrosine feeding. The lowest starch digestibility was found for elicited sprouts obtained from seeds fed with tyrosine (a decrease by 52%). An increase of expected glycemic index by 38% was determined for elicited sprouts obtained after phenylalanine feeding. Starch and protein digestibility were negatively correlated with total phenolics (r = -0.55 and -0.58, respectively), however starch digestibility was also affected by resistant starch content. PMID:26304392

  1. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    PubMed

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. PMID:26392384

  2. Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst.

    PubMed

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long

    2016-03-01

    In our previous study, we produced phenylpyruvic acid (PPA) in one step from L-phenylalanine by using an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase (L-AAD) from Proteus mirabilis KCTC2566. However, the PPA titer was low due to the degradation of PPA and low substrate specificity of L-AAD. In this study, metabolic engineering of the L-phenylalanine degradation pathway in E. coli and protein engineering of L-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer from 3.3 ± 0.2 to 3.9 ± 0.1 g/L and the substrate conversion ratio to 97.5 %. Next, L-AAD was engineered via error-prone polymerase chain reaction, followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336M produced the highest PPA titer of 10.0 ± 0.4 g/L, with a substrate conversion ratio of 100 %, which was 3.0 times that of wild-type L-AAD. Comparative kinetics analysis showed that compared with wild-type L-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. Finally, an optimal fed-batch biotransformation process was developed to achieve a maximal PPA titer of 21 ± 1.8 g/L within 8 h. This study developed a robust whole-cell E. coli biocatalyst for PPA production by integrating metabolic and protein engineering, strategies that may be useful for the construction of other biotransformation biocatalysts. PMID:26552798

  3. Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals.

    PubMed

    Cam, Yvan; Alkim, Ceren; Trichez, Debora; Trebosc, Vincent; Vax, Amélie; Bartolo, François; Besse, Philippe; François, Jean Marie; Walther, Thomas

    2016-07-15

    A synthetic pathway for (d)-xylose assimilation was stoichiometrically evaluated and implemented in Escherichia coli strains. The pathway proceeds via isomerization of (d)-xylose to (d)-xylulose, phosphorylation of (d)-xylulose to obtain (d)-xylulose-1-phosphate (X1P), and aldolytic cleavage of the latter to yield glycolaldehyde and DHAP. Stoichiometric analyses showed that this pathway provides access to ethylene glycol with a theoretical molar yield of 1. Alternatively, both glycolaldehyde and DHAP can be converted to glycolic acid with a theoretical yield that is 20% higher than for the exclusive production of this acid via the glyoxylate shunt. Simultaneous expression of xylulose-1 kinase and X1P aldolase activities, provided by human ketohexokinase-C and human aldolase-B, respectively, restored growth of a (d)-xylulose-5-kinase mutant on xylose. This strain produced ethylene glycol as the major metabolic endproduct. Metabolic engineering provided strains that assimilated the entire C2 fraction into the central metabolism or that produced 4.3 g/L glycolic acid at a molar yield of 0.9 in shake flasks. PMID:26186096

  4. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway.

    PubMed

    Xu, Jianzhong; Han, Mei; Zhang, Junlan; Guo, Yanfeng; Zhang, Weiguo

    2014-09-01

    The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and L-lysine production drastically improved. Moreover, increasing the flux through L-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and L-methionine biosynthesis, further improved L-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the L-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45% by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., L-threonine, L-methionine and L-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce L-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The L-lysine productivity was 2.73 g l(-1) h(-1) and the α was 47.06% after 48 h. However, the attenuation of MurE was not beneficial to increase the L-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through L-lysine biosynthetic pathway and DCW are beneficial to improve L-lysine production in C. glutamicum. PMID:24879631

  5. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

    PubMed

    Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

    2016-03-01

    Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1. PMID:26521242

  6. Engineering industrial oil biosynthesis: cloning and characterization of Kennedy pathway acyltransferases from novel oilseed species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For more than twenty years, various industrial, governmental, and academic laboratories have developed and refined genetic engineering strategies aimed at manipulating lipid metabolism in plants and microbes. The goal of these projects is to produce renewable specialized oils that can effectively c...

  7. Detection of Precursor Lesions of Pancreatic Adenocarcinoma in PET-CT in a Genetically Engineered Mouse Model of Pancreatic Cancer1

    PubMed Central

    Fendrich, Volker; Schneider, Ralph; Maitra, Anirban; Jacobsen, Ilse D; Opfermann, Thomas; Bartsch, Detlef K

    2011-01-01

    Background Pancreatic cancer is among the most dismal of human malignancies. The 5-year survival rate is lower than 5%. The identification of precursor lesions would be the main step to improve this fatal outcome. One precursor lesions are called pancreatic intraepithelial neoplasia (PanIN) and are graduated in grade 1 to 3, whereas grade 3 is classified as carcinoma in situ. Currently, no reliable, noninvasive imaging technique (e.g., ultrasound, computed tomography, magnet resonance imaging) exists to verify PanINs. Methods Recently, a transgenic mouse model of pancreatic cancer was established in which the tumor progression of human pancreatic carcinoma is reproduced. These so-called Pdx-1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+mice develop PanINs, which transform to invasive growing pancreatic carcinoma. The pancreata of mice of different ages were immunohistochemically stained using α-GLUT-2 antibodies. Furthermore, mice underwent positron emission tomography (PET)-computed tomography (CT) with 18F-fluorodeoxyglucose (FDG) to evaluate early detection of PanIN lesions. Results An expression of GLUT-2 in murine PanINs was found in PanINs of grade 1B and higher. This finding is associated with an elevated glucose metabolism, leading to the detection of precursor lesions of pancreatic cancer in the FDG PET-CT scan. In addition, immunohistochemical staining of GLUT-2 was detectable in 45 (75%) of 60 human PanINs, whereas PanINs of grade 1B and higher showed a very extensive expression. Conclusions In conclusion, we demonstrate for the first time that an elevated glucose metabolism occurs already in precursor lesions of murine and human pancreatic carcinoma. These findings are the basis for the detection of precursor lesions by PET-CT, thereby helping improving the prognosis of this devastating disease. PMID:21403843

  8. Exploring the folding pathway of green fluorescent protein through disulfide engineering

    PubMed Central

    Pitman, Derek J; Banerjee, Shounak; Macari, Stephen J; Castaldi, Christopher A; Crone, Donna E; Bystroff, Christopher

    2015-01-01

    We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C-termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m-value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink-induced perturbations in kinetics are useful for mapping the protein folding pathway. PMID:25516354

  9. Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway.

    PubMed

    Naim, Fatima; Shrestha, Pushkar; Singh, Surinder P; Waterhouse, Peter M; Wood, Craig C

    2016-06-01

    Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application. PMID:26628000

  10. Structure and Properties of Precursor/Successor Complex and Transition State of the FeCl(2+)/Fe(2+) Electron Self-Exchange Reaction via the Inner-Sphere Pathway.

    PubMed

    Rotzinger, François P

    2015-11-01

    The electron self-exchange reaction FeCl(OH2)5(2+) + Fe(OH2)6(2+) → Fe(OH2)6(2+) + FeCl(OH2)5(2+), proceeding via the inner-sphere pathway, was investigated with quantum chemical methods. Geometry and vibrational frequencies of the precursor/successor complex, (H2O)5Fe(III)ClFe(II)(OH2)5(4+)/(H2O)5Fe(II)ClFe(III)(OH2)5(4+) (P/S), and the transition state, (H2O)5FeClFe(OH2)5(4+⧧) (TS), were computed with the LC-BOP functional and CPCM hydration. Bent and linear structures were computed for the TS and P/S. The electronic coupling matrix element (Hab) and the electronic energies were calculated with multistate extended general multiconfiguration quasi-degenerate second-order perturbation theory (XGMC-QDPT2) and spin-orbit configuration interaction (SO-CI). Since the Fe···Fe distance changes considerably along the electron transfer step, the transformation P → TS → S, equations based on the hypothesis of a fixed donor-acceptor distance cannot be applied. Hence, the rate constant for the electron transfer step (ket) was calculated as described previously (Rotzinger, F. P. Inorg. Chem. 2014, 53, 9923). ket is very fast, ∼9.4 × 10(8)-6.6 × 10(9) s(-1) at 0 °C. The experimental rate constant of the title reaction (k) is much slower and controlled by the formation of the precursor complex. The substitution of a water ligand by FeCl(OH2)5(2+) at Fe(OH2)6(2+) is rate-determining. PMID:26479082

  11. Engineering PQS Biosynthesis Pathway for Enhancement of Bioelectricity Production in Pseudomonas aeruginosa Microbial Fuel Cells

    PubMed Central

    Cao, Bin; Seviour, Thomas; Nesatyy, Victor J.; Marsili, Enrico; Kjelleberg, Staffan; Givskov, Michael; Tolker-Nielsen, Tim; Song, Hao; Loo, Joachim Say Chye; Yang, Liang

    2013-01-01

    The biosynthesis of the redox shuttle, phenazines, in Pseudomonas aeruginosa, an ubiquitous microorganism in wastewater microflora, is regulated by the 2-heptyl-3,4-dihydroxyquinoline (PQS) quorum-sensing system. However, PQS inhibits anaerobic growth of P. aeruginosa. We constructed a P. aeruginosa strain that produces higher concentrations of phenazines under anaerobic conditions by over-expressing the PqsE effector in a PQS negative ΔpqsC mutant. The engineered strain exhibited an improved electrical performance in microbial fuel cells (MFCs) and potentiostat-controlled electrochemical cells with an approximate five-fold increase of maximum current density relative to the parent strain. Electrochemical analysis showed that the current increase correlates with an over-synthesis of phenazines. These results therefore demonstrate that targeting microbial cell-to-cell communication by genetic engineering is a suitable technique to improve power output of bioelectrochemical systems. PMID:23700414

  12. Pathways to excellence: A Federal strategy for science, mathematics, engineering, and technology education

    NASA Technical Reports Server (NTRS)

    1993-01-01

    This Strategic Plan was developed by the Federal Coordinating Council for Science, Engineering, and Technology (FCCSET) through its Committee on Education and Human Resources (CEHR), with representatives from 16 Federal agencies. Based on two years of coordinated interagency effort, the Plan confirms the Federal Government's commitment to ensuring the health and well-being of science, mathematics, engineering, and technology education at all levels and in all sectors (i.e., elementary and secondary, undergraduate, graduate, public understanding of science, and technology education). The Plan represents the Federal Government's efforts to develop a five-year planning framework and associated milestones that focus Federal planning and the resources of the participating agencies toward achieving the requisite or expected level of mathematics and science competence by all students. The priority framework outlines the strategic objectives, implementation priorities, and components for the Strategic Plan and serves as a road map for the Plan. The Plan endorses a broad range of ongoing activities, including continued Federal support for graduate education as the backbone of our country's research and development enterprise. The Plan also identifies three tiers of program activities with goals that address issues in science, mathematics, engineering, and technology education meriting special attention. Within each tier, individual agency programs play important and often unique roles that strengthen the aggregate portfolio. The three tiers are presented in descending order of priority: (1) reforming the formal education system; (2) expanding participation and access; and (3) enabling activities.

  13. Pathways to excellence: A Federal strategy for science, mathematics, engineering, and technology education

    NASA Astrophysics Data System (ADS)

    This Strategic Plan was developed by the Federal Coordinating Council for Science, Engineering, and Technology (FCCSET) through its Committee on Education and Human Resources (CEHR), with representatives from 16 Federal agencies. Based on two years of coordinated interagency effort, the Plan confirms the Federal Government's commitment to ensuring the health and well-being of science, mathematics, engineering, and technology education at all levels and in all sectors (i.e., elementary and secondary, undergraduate, graduate, public understanding of science, and technology education). The Plan represents the Federal Government's efforts to develop a five-year planning framework and associated milestones that focus Federal planning and the resources of the participating agencies toward achieving the requisite or expected level of mathematics and science competence by all students. The priority framework outlines the strategic objectives, implementation priorities, and components for the Strategic Plan and serves as a road map for the Plan. The Plan endorses a broad range of ongoing activities, including continued Federal support for graduate education as the backbone of our country's research and development enterprise. The Plan also identifies three tiers of program activities with goals that address issues in science, mathematics, engineering, and technology education meriting special attention. Within each tier, individual agency programs play important and often unique roles that strengthen the aggregate portfolio. The three tiers are presented in descending order of priority: (1) reforming the formal education system; (2) expanding participation and access; and (3) enabling activities.

  14. Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways.

    PubMed

    Liu, Z Lewis; Ma, Menggen; Song, Mingzhou

    2009-09-01

    Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)(+) for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under

  15. METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

    SciTech Connect

    John J. Kilbane III

    2003-12-01

    The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project will focus on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate amidase. The objective of the final phase of the project will be to develop derivative CN bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. The project is on schedule and no major difficulties have been encountered. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments have resulted in the isolation of promising cultures that may be capable of cleaving C-N bonds in aromatic amides, several amidase genes have been cloned and are currently undergoing directed evolution to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. Future research will address expression of these genes in Rhodococcus erythropolis. Enrichment culture experiments and directed evolution experiments continue to be a main focus of research activity and further work is required to obtain an appropriate amidase that will selectively cleave C-N bonds in aromatic substrates. Once an appropriate amidase gene is obtained it must be combined with genes encoding an enzyme capable of converting carbazole to 2'aminobiphenyl-2,3-diol: specifically carA genes. The carA genes from two sources have been cloned and are ready for construction of C-N bond cleavage pathway

  16. Improving 2-phenylethanol production via Ehrlich pathway using genetic engineered Saccharomyces cerevisiae strains.

    PubMed

    Yin, Sheng; Zhou, Hui; Xiao, Xiao; Lang, Tiandan; Liang, Jingru; Wang, Chengtao

    2015-05-01

    2-phenylethanol (2-PE) is an important aromatic compound with a rose-like fragrance widely used in food industry and cosmetic manufacture. In order to obtain "natural" 2-PE, the genetically modified budding yeasts were developed and applied for the 2-PE production. The gene ARO8 encoding transaminase and the gene ARO10 encoding decarboxylase in the Ehrlich pathway were expressed in Saccharomyces cerevisiae S288c. The activities of transaminase and decarboxylase were both enhanced in the corresponding recombinant strains. Consequently, the 2-PE yield in the recombinant strains with ARO8 and ARO10 were increased by 9.3 and 16.3 %, respectively, than that in the wild strain. A co-expression vector harboring ARO8 and ARO10 was then introduced into S. cerevisiae S288c, generating the recombinant strain SPO810. The fed-batch fermentation results indicated that the 2-PE yield in SPO810 reached 2.61 g L(-1) after 60 h of cultivation, which was 36.8 % higher than that in the wild strain. These results demonstrated that the 2-PE production was significantly improved by enhanced expression of the two key enzymes encoded by ARO8 and ARO10 in the Ehrlich pathway, providing new perspectives for enhancing "natural" 2-PE production in S. cerevisiae. PMID:25681107

  17. High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers*

    PubMed Central

    Faries, Kaitlyn M.; Kressel, Lucas L.; Wander, Marc J.; Holten, Dewey; Laible, Philip D.; Kirmaier, Christine; Hanson, Deborah K.

    2012-01-01

    Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization. PMID:22247556

  18. Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

    SciTech Connect

    John J. Kilbane II

    2005-10-01

    The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been the focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen.

  19. METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

    SciTech Connect

    John J. Kilbane II

    2004-10-01

    The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project was focused on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate deaminase. The objective of the final phase of the project will be to develop derivative C-N bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments resulted in the isolation of microbial cultures that utilize aromatic amides as sole nitrogen sources, several amidase genes were cloned and were included in directed evolution experiments to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. During the second year of the project (October, 2003-September, 2004) enrichment culture experiments succeeded in isolating a mixed bacterial culture that can utilize 2-aminobiphenyl as a sole nitrogen source, directed evolution experiments were focused on the aniline dioxygenase enzyme that is capable of deaminating aniline, and expression vectors were constructed to enable the expression of genes encoding C-N bond cleaving enzymes in Rhodococcus hosts. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the

  20. Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

    PubMed

    de Jong, Bouke Wim; Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2015-03-01

    Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae. PMID:25422103

  1. Niche inheritance: a cooperative pathway to enhance cancer cell fitness through ecosystem engineering.

    PubMed

    Yang, Kimberline R; Mooney, Steven M; Zarif, Jelani C; Coffey, Donald S; Taichman, Russell S; Pienta, Kenneth J

    2014-09-01

    Cancer cells can be described as an invasive species that is able to establish itself in a new environment. The concept of niche construction can be utilized to describe the process by which cancer cells terraform their environment, thereby engineering an ecosystem that promotes the genetic fitness of the species. Ecological dispersion theory can then be utilized to describe and model the steps and barriers involved in a successful diaspora as the cancer cells leave the original host organ and migrate to new host organs to successfully establish a new metastatic community. These ecological concepts can be further utilized to define new diagnostic and therapeutic areas for lethal cancers. PMID:24700698

  2. The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application

    PubMed Central

    Xu, Leyuan; Sheybani, Natasha; Yeudall, W. Andrew; Yang, Hu

    2015-01-01

    Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators. PMID:25709809

  3. Nitric oxide and nitrous oxide turnover in natural and engineered microbial communities: biological pathways, chemical reactions, and novel technologies

    PubMed Central

    Schreiber, Frank; Wunderlin, Pascal; Udert, Kai M.; Wells, George F.

    2012-01-01

    Nitrous oxide (N2O) is an environmentally important atmospheric trace gas because it is an effective greenhouse gas and it leads to ozone depletion through photo-chemical nitric oxide (NO) production in the stratosphere. Mitigating its steady increase in atmospheric concentration requires an understanding of the mechanisms that lead to its formation in natural and engineered microbial communities. N2O is formed biologically from the oxidation of hydroxylamine (NH2OH) or the reduction of nitrite (NO−2) to NO and further to N2O. Our review of the biological pathways for N2O production shows that apparently all organisms and pathways known to be involved in the catabolic branch of microbial N-cycle have the potential to catalyze the reduction of NO−2 to NO and the further reduction of NO to N2O, while N2O formation from NH2OH is only performed by ammonia oxidizing bacteria (AOB). In addition to biological pathways, we review important chemical reactions that can lead to NO and N2O formation due to the reactivity of NO−2, NH2OH, and nitroxyl (HNO). Moreover, biological N2O formation is highly dynamic in response to N-imbalance imposed on a system. Thus, understanding NO formation and capturing the dynamics of NO and N2O build-up are key to understand mechanisms of N2O release. Here, we discuss novel technologies that allow experiments on NO and N2O formation at high temporal resolution, namely NO and N2O microelectrodes and the dynamic analysis of the isotopic signature of N2O with quantum cascade laser absorption spectroscopy (QCLAS). In addition, we introduce other techniques that use the isotopic composition of N2O to distinguish production pathways and findings that were made with emerging molecular techniques in complex environments. Finally, we discuss how a combination of the presented tools might help to address important open questions on pathways and controls of nitrogen flow through complex microbial communities that eventually lead to N2O build

  4. Engineering Salidroside Biosynthetic Pathway in Hairy Root Cultures of Rhodiola crenulata Based on Metabolic Characterization of Tyrosine Decarboxylase

    PubMed Central

    Zeng, Lingjiang; Liu, Xiaoqiang; Qiu, Fei; Zheng, Weilie; Quan, Hong; Liao, Zhihua; Chen, Min; Huang, Wenlin; Liu, Wanhong; Wang, Qiang

    2013-01-01

    Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside. PMID:24124492

  5. Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance.

    PubMed

    Bai, Chao; Capell, Teresa; Berman, Judit; Medina, Vicente; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2016-01-01

    The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor-product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce β-carotene in the presence of endogenous lycopene β-cyclase. We combined this mini-pathway with the Arabidopsis thaliana genes AtDXS (encoding 1-deoxy-D-xylulose 5-phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up-regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles. PMID:25857664

  6. Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase C(alpha), src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.

    PubMed

    Canet-Aviles, Rosa-Maria; Anderton, Mark; Hooper, Nigel M; Turner, Anthony J; Vaughan, Peter F T

    2002-06-15

    The signalling pathways by which muscarine and epidermal growth factor (EGF) regulate the secretion of the alpha-secretase cleavage product (sAPPalpha) of the amyloid precursor protein (APP) were examined in the human neuroblastoma SH-SY5Y. Using specific inhibitors it was found that over 80% of sAPPalpha secretion, enhanced by muscarine, occurred via the extracellular signal-regulated kinase (ERK1/2) member of the mitogen-activated protein kinase (MAPK) family and was dependent on protein kinase Calpha (PKCalpha) and a member of the Src family of non-receptor tyrosine kinases (Src-TK). In contrast the stimulation of sAPPalpha secretion by EGF was not affected by inhibitors of PKC nor Src-TK but was dependent on ERK1/2. In addition muscarine-enhanced sAPPalpha secretion and ERK1/2 activation were inhibited 60 and 80%, respectively, by micromolar concentrations of the phosphatidylinositol 3 kinase (PI-3K) inhibitor wortmannin. In comparison wortmannin decreased EGF stimulation of sAPPalpha secretion and ERK 1/2 activation by approximately 40%. Unexpectedly, U73122, an inhibitor of phosphoinositide-specific phospholipase C, did not inhibit muscarine enhancement of sAPPalpha secretion. These data are discussed in relation to a pathway for the enhancement of sAPPalpha secretion by muscarine which involves the activation of a Src-TK by G-protein beta/gamma-subunits leading to activation of PKCalpha, and ERK1/2 by a mechanism not involving phospholipase C. PMID:12191495

  7. Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum.

    PubMed

    Rittmann, Doris; Lindner, Steffen N; Wendisch, Volker F

    2008-10-01

    The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures. PMID:18757581

  8. Violet/blue chrysanthemums--metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors.

    PubMed

    Brugliera, Filippa; Tao, Guo-Qing; Tems, Ursula; Kalc, Gianna; Mouradova, Ekaterina; Price, Kym; Stevenson, Kim; Nakamura, Noriko; Stacey, Iolanda; Katsumoto, Yukihisa; Tanaka, Yoshikazu; Mason, John G

    2013-10-01

    Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color. PMID:23926066

  9. Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway

    PubMed Central

    Jawed, Kamran; Mattam, Anu Jose; Fatma, Zia; Wajid, Saima; Abdin, Malik Z.; Yazdani, Syed Shams

    2016-01-01

    Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product. PMID:27466817

  10. Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

    PubMed

    Jawed, Kamran; Mattam, Anu Jose; Fatma, Zia; Wajid, Saima; Abdin, Malik Z; Yazdani, Syed Shams

    2016-01-01

    Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product. PMID:27466817

  11. Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells

    SciTech Connect

    Shafarman, William N.

    2015-10-12

    This project “Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells”, completed by the Institute of Energy Conversion (IEC) at the University of Delaware in collaboration with the Department of Chemical Engineering at the University of Florida, developed the fundamental understanding and technology to increase module efficiency and improve the manufacturability of Cu(InGa)(SeS)2 films using the precursor reaction approach currently being developed by a number of companies. Key results included: (1) development of a three-step H2Se/Ar/H2S reaction process to control Ga distribution through the film and minimizes back contact MoSe2 formation; (2) Ag-alloying to improve precursor homogeneity by avoiding In phase agglomeration, faster reaction and improved adhesion to allow wider reaction process window; (3) addition of Sb, Bi, and Te interlayers at the Mo/precursor junction to produce more uniform precursor morphology and improve adhesion with reduced void formation in reacted films; (4) a precursor structure containing Se and a reaction process to reduce processing time to 5 minutes and eliminate H2Se usage, thereby increasing throughput and reducing costs. All these results were supported by detailed characterization of the film growth, reaction pathways, thermodynamic assessment and device behavior.

  12. Engineering of an L-arabinose metabolic pathway in Rhodococcus jostii RHA1 for biofuel production.

    PubMed

    Xiong, Xiaochao; Wang, Xi; Chen, Shulin

    2016-07-01

    The oleaginous bacterium, Rhodococcus jostii RHA1 has attracted considerable attention due to its capability to accumulate significant levels of triacylglycerol as renewable hydrocarbon. To enable the strain to utilize arabinose derived from lignocellulosic biomass, the metabolic pathway of L-arabinose utilization was introduced into R. jostii RHA1 by heterogenous expression of the operon, araBAD from Escherichia coli. The results showed that recombinant bearing araBAD could grow on L-arabinose as the sole carbon source, and additional expression of araFGH encoding the arabinose transporter from E. coli could improve the cell biomass yield from high contents of arabinose. We further increased the content of lipid produced from arabinose in the recombinants from 47.9 to 56.8 % of the cell dry weight (CDW) by overexpression of a gene, atf1 encoding a diglyceride acyltransferase from R. opacus PD630. This work demonstrated the feasibility of producing lipid from arabinose by genetic modification of the rhodococci strain. PMID:27143134

  13. Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

    SciTech Connect

    Dien, S.J. van; Keasling, J.D.

    1998-09-20

    Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains over-expressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction.

  14. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

    PubMed Central

    Wu, T C; Guarnieri, F G; Staveley-O'Carroll, K F; Viscidi, R P; Levitsky, H I; Hedrick, L; Cho, K R; August, J T; Pardoll, D M

    1995-01-01

    The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency. Images Fig. 2 Fig. 3 PMID:8524826

  15. Hydrology without hillslopes: runoff controls and pathways on a near-level, engineered landscape

    NASA Astrophysics Data System (ADS)

    Petzold, Halya; Ali, Genevieve

    2015-04-01

    Low relief, artificially drained landscapes like those of the Prairies in south-central Canada have received little consideration in hydrologic study. While topography is generally asserted as the main control of runoff generation, it is unknown whether this is also the case where relief is low, or if in the absence of high relief other landscape characteristics become relatively more influential. To address this knowledge gap, runoff behaviour was analysed via event rainfall-runoff hydrographs and perched water table level at 6 study sites to infer dominant runoff processes and their control factors. Instrumentation was deployed in the Catfish Creek watershed, a 642 km2 near-level, mixed land use and engineered Prairie watershed located 90 km northeast of Winnipeg, Manitoba, Canada. Specifically, surface water levels were measured at the outlet of six sub-watersheds while perched water table levels were monitored in riparian areas. At each site, rainfall events were delineated and rainfall-runoff parameters, perched water table maximum rise and antecedent rainfall variables (as surrogates for antecedent moisture conditions or AMCs) were calculated on an event basis. Landscape characteristics, including elevation, slope, and land use and land cover statistics, were also determined for each sub-watershed. Correlation analysis and principal component analysis were then carried out including all variables. Rainfall-runoff responses were highly variable across sites. Event hydrographs were generally characterized by short lag times and initial abstractions which correlated moderately to total event rainfall. However, sub-watersheds characteristics did not influence the different hydrograph characteristics recorded across sites. Threshold behaviour was observed only in the two study sub-watersheds of greatest relief. Runoff generation was hypothesized to occur predominantly as Hortonian overland flow, although the dominant runoff process shifted under certain moisture

  16. Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

    PubMed Central

    Zuroff, Trevor R.; Ramya, Mohandass; Boutard, Magali; Cerisy, Tristan; Curtis, Wayne R.

    2015-01-01

    Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance. PMID:26048945

  17. Understanding the conditions that encourage the persistence of women in science, mathematics, and engineering career pathways

    NASA Astrophysics Data System (ADS)

    Kondrick, Linda C.

    The purpose of this study was to determine which factors encourage the persistence of women in the pursuit of Science, Math, and Engineering (SME) careers. Surveys with 36 parallel pairs of theory and history questions regarding the importance and the aptness of variables identified in the literature were completed by 205 SME career women. The variables covered three educational levels: High School, Undergraduate and Graduate. Results reveal which variables fit the experiences of these women and were also believed by them to be important to women in the pursuit of an SME career goal. False Negatives, women who according to the SME literature should not have persisted but did, were identified. Their existence, together with the false positives identified in the SME literature, is evidence, according to Confirmation/Disconfirmation Theory, that important variables in SME persistence are yet to be discovered. Follow-up telephone interviews with nineteen respondents identified important affective variables. Love of math or science was in itself a powerful motivator. Respondents made suggestions for intervention programs that may help to develop that abiding interest. Mentors, role models, and social support networks were identified as important in building the confidence and sustaining the focus needed to cope with the rigorous curriculum and negative sex-bias encountered in SME programs. The qualitative and quantitative results were synthesized in a Causal Event Flow Network, a cognitive map of the longitudinal effects of both positive and negative push/pull vectors operating on women in pursuit of an SME career goal.

  18. Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation

    PubMed Central

    Vetcher, Leandro; Tian, Zong-Qiang; McDaniel, Robert; Rascher, Andreas; Revill, W. Peter; Hutchinson, C. Richard; Hu, Zhihao

    2005-01-01

    Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog. PMID:15812008

  19. Pathway-Specific Engineered Mouse Allograft Models Functionally Recapitulate Human Serous Epithelial Ovarian Cancer

    PubMed Central

    Szabova, Ludmila; Bupp, Sujata; Kamal, Muhaymin; Householder, Deborah B.; Hernandez, Lidia; Schlomer, Jerome J.; Baran, Maureen L.; Yi, Ming; Stephens, Robert M.; Annunziata, Christina M.; Martin, Philip L.; Van Dyke, Terry A.

    2014-01-01

    The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1–deficient tumors and development of relevant biomarkers. PMID:24748377

  20. Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans.

    PubMed

    Tolonen, Andrew C; Zuroff, Trevor R; Ramya, Mohandass; Boutard, Magali; Cerisy, Tristan; Curtis, Wayne R

    2015-08-15

    Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance. PMID:26048945

  1. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

    PubMed

    Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

    2016-06-01

    The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. PMID:26990396

  2. Evolution of hydrological pathways in engineered hillslopes due to soil and vegetation development

    NASA Astrophysics Data System (ADS)

    Appels, Willemijn M.; Ireson, Andrew M.; McDonnell, Jeffrey J.; Barbour, S. Lee

    2015-04-01

    The structure and hydraulic properties of soils and bedrock within a hillslope combined with the timing and rates of water availability control the partitioning of precipitation into vertical and lateral flowpaths. In natural hillslope sites, heterogeneity in both soil texture and structure are the result of long-term landscape evolution processes and consequently can be assumed to be static relative to the timescale of rainfall-runoff processes. However; engineered hillslopes, constructed commonly as reclamation covers overlying mine waste, have been observed to undergo rapid changes in hydraulic properties over relatively short timescales (i.e. 3-5 years) as a result of weathering (e.g. freeze-thaw and wet-dry cycles) and vegetation growth (e.g. increasing rooting depth and density). Rainfall-runoff responses on such hillslopes would therefore not only be expected to reflect seasonal dynamics, but also the evolution of the system from a relatively homogeneous initial condition to a system with increasing heterogeneity of soil texture and structure. We present results of a combined field and modeling study of three prototype soil covers on a saline-sodic shale overburden dump at the Syncrude Canada Ltd. Mildred Lake mine, north of Fort McMurray, Canada. Since their construction in 1999, soil properties, hydrological response to atmospheric and vegetative demands, and vegetation properties have been extensively monitored. The three covers have undergone substantial evolution due to freeze-thaw processes and aggrading vegetation. In this work, we quantify hydrological processes in the reclamation covers, focusing on inter- and intra-annual patterns. To this purpose we analyzed the long-term hydrometric data with field sampling of the distribution of salts and the stable isotopes of water within soil water and subsurface flow in the base of the cover. We use a 2D Hydrus model to explore the co-evolution of soil and vegetation and quantify its effect on flow

  3. Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane.

    PubMed

    Mena-Benitez, Gilda L; Gandia-Herrero, Fernando; Graham, Stuart; Larson, Tony R; McQueen-Mason, Simon J; French, Christopher E; Rylott, Elizabeth L; Bruce, Neil C

    2008-07-01

    Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater. PMID:18467461

  4. Metabolic Engineering and Modeling of Metabolic Pathways to Improve Hydrogen Production by Photosynthetic Bacteria

    SciTech Connect

    Jiao, Y.; Navid, A.

    2014-12-19

    traits act as the biocatalysts of the process designed to both enhance the system efficiency of CO2 fixation and the net hydrogen production rate. Additionally we applied metabolic engineering approaches guided by computational modeling for the chosen model microorganisms to enable efficient hydrogen production.

  5. Amyloid precursor protein cooperates with c-KIT mutation/overexpression to regulate cell apoptosis in AML1-ETO-positive leukemia via the PI3K/AKT signaling pathway.

    PubMed

    Yu, Guopan; Yin, Changxin; Jiang, Ling; Zheng, Zhongxin; Wang, Zhixiang; Wang, Chunli; Zhou, Hongsheng; Jiang, Xuejie; Liu, Qifa; Meng, Fanyi

    2016-09-01

    It has been reported that amyloid precursor protein (APP) promotes cell proliferation and metastasis in various types of solid cancers. In our previous study, we showed that APP is highly expressed and regulates leukemia cell migration in AML1‑ETO-positive (AE) leukemia. Whether APP is involved in the regulation of AE leukemia cell proliferation or apoptosis is unclear. In the present study we focused on the correlation of APP with c-KIT mutation/overexpression and cell proliferation and apoptosis in AE leukemia. APP and c-KIT expression detected by quantitative real-time (qPCR) method, and c-KIT mutations screened using PCR in bone marrow cells from 65 patients with AE leukemia before their first chemotherapy, were simultaneously assessed. Furthermore, the Kasumi-1 cell line was chosen as the cell model, and the APP gene was knocked down using siRNA technology. The correlation of cell cycle distribution and apoptosis and c-Kit expression with APP expression levels, as well as the regulation of the PI3K/AKT signaling pathway by APP were analyzed in the Kasumi-1 cell line. The results showed that peripheral white blood cell counts (P=0.008) and bone marrow cellularity (P=0.031), but not bone marrow blasts, were correlated with APP expression. Moreover, the patients with APP high expression had a significantly higher incidence of c-KIT mutations (P<0.001) and increased levels of c-KIT expression (P=0.001) and poorer disease outcome. In the Kasumi-1 cell line, as compared with the wild-type and negative control cells, cell apoptosis, both early (P<0.001) and late (P<0.001), was significantly increased when the APP gene was knocked down, concomitant with reduced levels of anti-apoptotic protein Bcl-2 and increased levels of caspase-3 and -9, however, no apparent change was observed in the cell cycle distribution (P>0.05). Moreover, the knockdown of APP markedly decreased c-KIT expression at both the transcription (as evidenced by qPCR analysis) and translation

  6. Efficient biosynthesis of (2S)-pinocembrin from d-glucose by integrating engineering central metabolic pathways with a pH-shift control strategy.

    PubMed

    Wu, Junjun; Zhang, Xia; Zhou, Jingwen; Dong, Mingsheng

    2016-10-01

    Microbial fermentations promise to revolutionize the conventional extraction of (2S)-pinocembrin from natural plant sources. Previously an Escherichia coli fermentation system was developed for one-step (2S)-pinocembrin production. However, this fermentation platform need supplementation of expensive malonyl-CoA precursor malonate and requires morpholinopropane sulfonate to provide buffering capacity. Here, a clustered regularly interspaced short palindromic repeats interference was constructed to efficiently channel carbon flux toward malonyl-CoA. By exploring the effects of different culture pH on microbial fermentation, it was found that high pH values favored upstream pathway catalysis, while low pH values favored downstream pathway catalysis. Based on this theory, a two-stage pH control strategy was proposed. The pH was controlled at 7.0 during 0-10h, and was shifted to 6.5 after 10h. Finally, the (2S)-pinocembrin titers increased to 525.8mg/L. These results were attained in minimal medium without additional precursor supplementation, thus offering opportunities for industrial scale low-cost production of flavonoids. PMID:27450982

  7. Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.

    PubMed

    Jensen, Jaide V K; Eberhardt, Dorit; Wendisch, Volker F

    2015-11-20

    The glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. Corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. Here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and arginine, and the diamine putrescine was demonstrated. Feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of glutamate dehydrogenase gene gdh, lowering expression of phosphoglucoisomerase gene pgi, along with the introduction of a second copy of the arginine biosynthesis operon argCJB(A49V,M54V)D into the chromosome resulted in a C. glutamicum strain producing ornithine with a yield of 0.52 g ornithine per g glucose, an increase of 71% as compared to the parental ΔargFRG strain. Strains capable of producing 0.41 g citrulline per g glucose, 0.29 g proline per g glucose, 0.30 g arginine per g glucose, and 0.17 g putrescine per g glucose were derived from the ornithine-producing platform strain by plasmid-based overexpression of appropriate pathway modules with one to three genes. PMID:26393954

  8. New insights into precursors of renal endothelium.

    PubMed

    Sequeira-Lopez, Maria Luisa S; Torban, Elena

    2016-08-01

    The kidney vasculature is extremely complex, yet, despite recent progress, our understanding of how the renal vascular system develops is limited. By using advanced tissue engineering techniques and in vivo and in vitro depletion of specific populations of endothelial cell precursors, Halt et al. have identified a CD146-expressing precursor as an important player in the development of the renal vasculature. PMID:27418087

  9. Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition.

    PubMed

    Chwa, Jun-Won; Kim, Wook Jin; Sim, Sang Jun; Um, Youngsoon; Woo, Han Min

    2016-08-01

    Capture and conversion of CO2 to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry-relevant chemicals from CO2 . However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO2 to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP-driven malonyl-CoA synthesis pathway and heterologous phosphoketolase (PHK)-phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl-CoA directly from CO2 in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl-CoA-derived biochemicals. PMID:26879003

  10. Proteome Profiling Reveals Potential Toxicity and Detoxification Pathways Following Exposure of BEAS-2B Cells to Engineered Nanoparticle Titanium Dioxide

    EPA Science Inventory

    Identification of toxicity pathways linked to chemical -exposure is critical for a better understanding of biological effects of the exposure, toxic mechanisms, and for enhancement of the prediction of chemical toxicity and adverse health outcomes. To identify toxicity pathways a...

  11. Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc engineered monoclonal antibody targeting the BAFF-R

    PubMed Central

    Parameswaran, Reshmi; Lim, Min; Fei, Fei; Abdel-Azim, Hisham; Arutyunyan, Anna; Schiffer, Isabelle; McLaughlin, Margaret E.; Gram, Hermann; Huet, Heather; Groffen, John; Heisterkamp, Nora

    2014-01-01

    B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia ALL (pre-B ALL) cells but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild type cells but were more sensitive to drug treatment. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated NK cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted. PMID:24825858

  12. Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift

    PubMed Central

    Piatkevich, Kiryl D.; Malashkevich, Vladimir N.; Almo, Steven C.; Verkhusha, Vladislav V.

    2010-01-01

    LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs) which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 Å and 1.5 Å, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis analyses, as well as isotope and temperature dependences suggest that an excited state proton transfer (ESPT) is responsible for the LSSs observed in LSSmKates. Hydrogen bonding between the chromophore hydroxyl and Glu160 in LSSmKate1 and a proton relay involving the chromophore tyrosine hydroxyl, Ser158 and the Asp160 carboxylate in LSSmKate2 represent the putative ESPT pathways. Comparisons with mKeima LSS RFP suggest that similar proton relays could be engineered in other FPs. Accordingly, we mutated positions 158 and 160 in several conventional red-shifted FPs, including mNeptune, mCherry, mStrawberry, mOrange and mKO, and the resulting FP variants exhibited LSS fluorescence emission in a wide range of wavelengths from 560 to 640 nm. These data suggest that different chromophores formed by distinct tripeptides in different environments can be rationally modified to yield RFPs with novel photochemical properties. PMID:20681709

  13. Advances in the engineering science of immiscible polymer blends: A powder route for delicate polymer precursors and a highly renewable polyamide/terephthalate blend system

    NASA Astrophysics Data System (ADS)

    Giancola, Giorgiana

    Powder processing of thermoplastic polymer composites is an effective way to achieve a high level of component homogenization in raw blends prior to melt processing, thus reducing the thermal and shear stress on the components. Polymer blends can be prepared that would otherwise not be possible due to thermodynamic incompatibility. Evaluation of this concept was conducted by processing PMMA and HDPE micron sized powders which were characterized using DSC and rheology. Optical microscopy and SEM, showed that high-quality, fine domain sized blends can be made by the compression molding process. Silica marker spheres were used to qualitatively assess the level dispersive mixing. EDS chemical analysis was effective in providing image contrast between PMMA and HDPE based on the carbonyl and ester oxygen. EDS image maps, combined with secondary electron images show that compression molding of blended powder precursors produces composites of comparable homogeneity and domain size as extrusion processing. FTIR proved valuable when assessing the intimacy of the constituents at the interface of the immiscible domains. The formation of an in-situ, PMMA nano-network structure resulting from solvent extraction and redeposition using DMF was uniquely found on the surface of these immiscible polymer blends. This work has shown that powder processing of polymers is an effective means to melt processed fragile polymers to high quality blends. Recently, efforts towards the development of sustainable materials have evolved due in part to the increase in price and limited supply of crude oil. Immiscible polymer blending is a paradigm that enables synergistic material performance in certain instances where the composite properties are superior to the sum of the constituents. The addition of PA6,10 to PTT offers an opportunity to increase the bio-based content of PTT while simultaneously maintaining or improving mechanical properties. PA6,10 and PTT are immiscible polymers that can be

  14. Exploiting mixtures of H2, CO2, and O2 for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered Cupriavidus necator strains.

    PubMed

    Przybylski, Denise; Rohwerder, Thore; Dilßner, Cornelia; Maskow, Thomas; Harms, Hauke; Müller, Roland H

    2015-03-01

    Current manufacturing of most bulk chemicals through petrochemical routes considerably contributes to common concerns over the depletion of fossil carbon sources and greenhouse gas emissions. Sustainable future production of commodities thus requires the shift to renewable feedstocks in combination with established or newly developed synthesis routes. In this study, the potential of Cupriavidus necator H16 for autotrophic synthesis of the building block chemical 2-hydroxyisobutyric acid (2-HIBA) is evaluated. A novel biosynthetic pathway was implemented by heterologous expression of the 2-hydroxyisobutyryl-coenzyme A (2-HIB-CoA) mutase from Aquincola tertiaricarbonis L108, relying on a main intermediate of strain H16's C4 overflow metabolism, 3-hydroxybutyryl-CoA. The intention was to direct the latter to 2-HIBA instead or in addition to poly-3-hydroxybutyrate (PHB). Autotrophic growth and 2-HIBA (respectively, PHB) synthesis of wild-type and PHB-negative mutant strains were investigated producing maximum 2-HIBA titers of 3.2 g L(-1) and maximum specific 2-HIBA synthesis rates (q 2-HIBA) of about 16 and 175 μmol g(-1) h(-1), respectively. The obtained specific productivity was the highest reported to date for mutase-dependent 2-HIBA synthesis from heterotrophic and autotrophic substrates. Furthermore, expression of a G protein chaperone (MeaH) in addition to the 2-HIB-CoA mutase subunits yielded improved productivity. Analyzing the inhibition of growth and product synthesis due to substrate availability and product accumulation revealed a strong influence of 2-HIBA, when cells were cultivated at high titers. Nevertheless, the presented results imply that at the time the autotrophic synthesis route is superior to thus far established heterotrophic routes for production of 2-HIBA with C. necator. PMID:25503317

  15. Diverse Pathways to the Phd: a Study of Women Faculty in the Sciences and Engineering at a Hispanic-Serving Institution

    NASA Astrophysics Data System (ADS)

    Ryabov, Igor; Witherspoon, Patricia D.

    The National Science Foundation's ADVANCE grants for Institutional Transformation have been awarded to institutions to study, and to implement programs to improve, the number of women who are recruited, retained, and promoted as faculty in the sciences and engineering at American universities. At one ADVANCE institution, 57 women faculty in the social sciences, natural sciences, and engineering were interviewed to determine their pathways to the doctoral degree. Through the use of qualitative analyses, this study identified major themes that emerged from the interviews of Anglo, international, and Latina faculty. The findings of the study should contribute to the discourse on underrepresentation of women faculty in the sciences and engineering in general, and Latina faculty in particular.

  16. Bioinformatic analysis of peptide precursor proteins.

    PubMed

    Baggerman, G; Liu, F; Wets, G; Schoofs, L

    2005-04-01

    Neuropeptides are among the most important signal molecules in animals. Traditional identification of peptide hormones through peptide purification is a tedious and time-consuming process. With the advent of the genome sequencing projects, putative peptide precursor can be mined from the genome. However, because bioactive peptides are usually quite short in length and because the active core of a peptide is often limited to only a few amino acids, using the BLAST search engine to identify neuropeptide precursors in the genome is difficult and sometimes impossible. To overcome these shortcomings, we subject the entire set of all known Drosophila melanogaster peptide precursor sequences to motif-finding algorithms in search of a motif that is common for all prepropeptides and that could be used in the search for new peptide precursors. PMID:15891006

  17. EXPANSION OF BISINDOLE BIOSYNTHETIC PATHWAYS BY COMBINATORIAL CONSTRUCTION

    PubMed Central

    Du, Yi-Ling; Ryan, Katherine S.

    2015-01-01

    Cladoniamides are indolotryptoline natural products that derive from indolocarbazole precursors. Here, we present a microbial platform to artificially redirect the cladoniamide pathway to generate unnatural bisindoles for drug discovery. Specifically, we target glycosyltransferase, halogenase, and oxidoreductase genes from the phylogenetically-related indolocarbazole rebeccamycin and staurosporine pathways. We generate a series of novel compounds, reveal details about the substrate specificities of a number of enzymes, and set the stage for future efforts to develop new catalysts and compounds by engineering of bisindole genes. The strategy for structural diversification we use here could furthermore be applied to other natural product families with known biosynthetic genes. PMID:25548949

  18. Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

    PubMed Central

    Ruiz-Sola, M. Águila; Rodríguez-Concepción, Manuel

    2012-01-01

    Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology. PMID:22582030

  19. Polyimide Precursor Solid Residuum

    NASA Technical Reports Server (NTRS)

    Weiser, Erik S. (Inventor); St.Clair, Terry L. (Inventor); Echigo, Yoshiaki (Inventor); Kaneshiro, Hisayasu (Inventor)

    2001-01-01

    A polyimide precursor solid residuum is an admixture of an aromatic dianhydride or derivative thereof and an aromatic diamine or derivative thereof plus a complexing agent, which is complexed with the admixture by hydrogen bonding. The polyimide precursor solid residuum is effectively employed in the preparation of polyimide foam and the fabrication of polyimide foam structures.

  20. Yeast secretory expression of insulin precursors.

    PubMed

    Kjeldsen, T

    2000-09-01

    Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield. PMID:11030562

  1. Earthquakes: hydrogeochemical precursors

    USGS Publications Warehouse

    Ingebritsen, Steven E.; Manga, Michael

    2014-01-01

    Earthquake prediction is a long-sought goal. Changes in groundwater chemistry before earthquakes in Iceland highlight a potential hydrogeochemical precursor, but such signals must be evaluated in the context of long-term, multiparametric data sets.

  2. Data for rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase–xylitol dehydrogenase pathway

    PubMed Central

    Zhang, Biao; Zhang, Jia; Wang, Dongmei; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

    2015-01-01

    A thermo-tolerant NADP(H)-preferring xylose pathway was constructed in Kluyveromyces marxianus for ethanol production with xylose at elevated temperatures (Zhang et al., 2015 [25]). Ethanol production yield and efficiency was enhanced by pathway engineering in the engineered strains. The constructed strain, YZJ088, has the ability to co-ferment glucose and xylose for ethanol and xylitol production, which is a critical step toward enabling economic biofuel production from lignocellulosic biomass. This study contains the fermentation results of strains using the metabolic pathway engineering procedure. The ethanol-producing abilities of various yeast strains under various conditions were compared, and strain YZJ088 showed the highest production and fastest productivity at elevated temperatures. The YZJ088 xylose fermentation results indicate that it fermented well with xylose at either low or high inoculum size. When fermented with an initial cell concentration of OD600=15 at 37 °C, YZJ088 consumed 200 g/L xylose and produced 60.07 g/L ethanol; when the initial cell concentration was OD600=1 at 37 °C, YZJ088 consumed 98.96 g/L xylose and produced 33.55 g/L ethanol with a productivity of 0.47 g/L/h. When fermented with 100 g/L xylose at 42 °C, YZJ088 produced 30.99 g/L ethanol with a productivity of 0.65 g/L/h, which was higher than that produced at 37 °C. PMID:26543879

  3. Data for rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

    PubMed

    Zhang, Biao; Zhang, Jia; Wang, Dongmei; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

    2015-12-01

    A thermo-tolerant NADP(H)-preferring xylose pathway was constructed in Kluyveromyces marxianus for ethanol production with xylose at elevated temperatures (Zhang et al., 2015 [25]). Ethanol production yield and efficiency was enhanced by pathway engineering in the engineered strains. The constructed strain, YZJ088, has the ability to co-ferment glucose and xylose for ethanol and xylitol production, which is a critical step toward enabling economic biofuel production from lignocellulosic biomass. This study contains the fermentation results of strains using the metabolic pathway engineering procedure. The ethanol-producing abilities of various yeast strains under various conditions were compared, and strain YZJ088 showed the highest production and fastest productivity at elevated temperatures. The YZJ088 xylose fermentation results indicate that it fermented well with xylose at either low or high inoculum size. When fermented with an initial cell concentration of OD600=15 at 37 °C, YZJ088 consumed 200 g/L xylose and produced 60.07 g/L ethanol; when the initial cell concentration was OD600=1 at 37 °C, YZJ088 consumed 98.96 g/L xylose and produced 33.55 g/L ethanol with a productivity of 0.47 g/L/h. When fermented with 100 g/L xylose at 42 °C, YZJ088 produced 30.99 g/L ethanol with a productivity of 0.65 g/L/h, which was higher than that produced at 37 °C. PMID:26543879

  4. Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616

  5. Glutathione-conjugated sulfanylalkanols are substrates for ABCC11 and γ-glutamyl transferase 1: a potential new pathway for the formation of odorant precursors in the apocrine sweat gland

    PubMed Central

    Baumann, Tim; Bergmann, Sophia; Schmidt-Rose, Thomas; Max, Heiner; Martin, Annette; Enthaler, Bernd; Terstegen, Lara; Schweiger, Dorothea; Kalbacher, Hubert; Wenck, Horst; Jedlitschky, Gabriele; Jovanovic, Zorica

    2014-01-01

    We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH. PMID:24533866

  6. Amino acid precursors in lunar samples.

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Harada, K.; Hare, P. E.

    1972-01-01

    The use of hot water to extract lunar samples, followed by the hydrolysis of the aqueous extract, appears to be the method of choice for identification and quantitation of amino acid precursors in extraterrestrial sources. The net inferences from the analyses to date are (1) that amino acid precursors are verifiably present in lunar dust, and (2) that they are quite certainly not the consequence of contamination by terrestrial organisms, including man. It is suggested that prebiotic evolutionary pathways such as have been traversed on the earth were terminated on the moon for lack of sufficient water. Although some or all of the amino acid precursors may be indigenous, the low level observed suggests that they may also result from onfall of organic compounds from interstellar matter, comets, tails, solar wind, or meteorites.

  7. Engine

    SciTech Connect

    Shin, H.B.

    1984-02-28

    An internal combustion engine has a piston rack depending from each piston. This rack is connected to a power output shaft through a mechanical rectifier so that the power output shaft rotates in only one direction. A connecting rod is pivotally connected at one end to the rack and at the other end to the crank of a reduced function crankshaft so that the crankshaft rotates at the same angular velocity as the power output shaft and at the same frequency as the pistons. The crankshaft has a size, weight and shape sufficient to return the pistons back into the cylinders in position for the next power stroke.

  8. Dietary phytochemicals as potent chemotherapeutic agents against breast cancer: Inhibition of NF-κB pathway via molecular interactions in rel homology domain of its precursor protein p105

    PubMed Central

    Khan, Mohammad K. A.; Ansari, Irfan A.; Khan, M. Salman; Arif, Jamal M.

    2013-01-01

    Background: Dietary phytochemicals consist of a wide variety of biologically active compounds that are ubiquitous in plants, many of which have been reported to have anti-tumor as well as anti-inflammatory properties. Objective: In the present study, we aimed to validate these findings by using docking protocols and explicate the possible mechanism of action for a dataset of nine phytochemicals namely boswellic acid, 1-caffeoylquinic acid, ellagic acid, emodin, genistein, guggulsterone, quercetin, resveratrol, and sylibinin from different plants against the nuclear factor- kappaB (NF-κB) precursor protein p105, an important transcription factor reported to be overexpressed in breast cancer. Materials and Methods: 2-D structures of all phytochemicals were retrieved from PubChem Compound database and their subsequent conversion into 3-D structures was performed by using online software system CORINA. The X-ray crystallographic structure of the NF-κB precursor p105 was extracted from Brookhaven Protein Data Bank. Molecular docking simulation study was carried out by using AutoDock Tools 4.0. Results: Our results showed significant binding affinity of different phytochemicals with the Rel homology domain of the NF-κB precursor protein p105. Quercetin and 1-caffeoylquinic acid were found to be very effective inhibitors against target molecule as they showed binding energy of −12.11 and −11.50 Kcal/mol, respectively. The order of affinity of other ligands with p105 was found as follows: guggulsterone > sylibinin > emodin > resveratrol > genistein > boswellic acid > ellagic acid. Conclusion: Our in silico study has explored the possible chemopreventive mechanism of these phytochemicals against the NF-κB precursor protein p105 and deciphered that quercetin, 1-caffeoylquinic acid and guggulsterone were the potent inhibitors against target molecule. In addition, large scale preclinical and clinical trials are needed to explore the role of these chemotherapeutic

  9. Gender and Ethnic Differences in Precollege Mathematics Coursework Related to Science, Technology, Engineering, and Mathematics (STEM) Pathways

    ERIC Educational Resources Information Center

    You, Sukkyung

    2013-01-01

    In 2004, the pattern in academic pathways for high school students in the USA showed that students were completing more demanding mathematics courses. Despite the upward pattern in advanced-level mathematics course-taking, disparities among racial/ethnic groups persisted between 1982 and 2004. Using data from the Education Longitudinal Study of…

  10. The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

    PubMed Central

    Gao, Shouhong; Saechao, Saengking; Di, Peng; Chen, Junfeng; Chen, Wansheng

    2011-01-01

    Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors. PMID:22242141

  11. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids.

    PubMed

    Clomburg, James M; Blankschien, Matthew D; Vick, Jacob E; Chou, Alexander; Kim, Seohyoung; Gonzalez, Ramon

    2015-03-01

    An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6-10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C6-C10 carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C6-C10 dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C6-C10 ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media. PMID:25638687

  12. Thermal Studies of New Precursors to Indium-tin Oxides for Use as Sensor Materials in the Detection of NO(x)

    NASA Technical Reports Server (NTRS)

    Goldsby, J. C.; Kacik, T.; Hockensmith, C. M.

    1999-01-01

    Control of combustion product emissions in both sub and super-sonic jet engines can be facilitated by measurement of NO(x) levels with metal oxide sensors, In2O3, metal-doped SnO2, and SnO, (as well as other materials) show resistivity changes in the presence of NO(x), but often their sensitivity, stability, and selectivity are low. This study was designed to develop new synthetic pathways to precursors that produce high purity, two phase In2O3-SnO2. The precursors were formed by complexation of tin with any oxide ligands to give the ammonium salt (NH4). Thermal studies of these precursors were carried out by thermal gravimetry (TG) and differential scanning calorimetry (DSC). Further studies by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) were also conducted.

  13. Biochemical Removal of HAP Precursors from Coal

    SciTech Connect

    Olson, Gregory J

    1997-05-12

    Column biooxidation tests with Kentucky coal confirmed results of earlier shake flask tests showing significant removal from the coal of arsenic, selenium, cobalt, manganese, nickel and cadmium. Rates of pyrite biooxidation in Kentucky coal were only slightly more than half the rates found previously for Indiana and Pittsburgh coals. Removal of pyrite from Pittsburgh coal by ferric ion oxidation slows markedly as ferrous ions accumulate in solution, requiring maintenance of high redox potentials in processes designed for removal of pyrite and hazardous air pollutant (HAP) precursors by circulation of ferric solutions through coal. The pyrite oxidation rates obtained in these tests were used by Unifield Engineering to support the conceptual designs for alternative pyrite and HAP precursor bioleaching processes for the phase 2 pilot plant. Thermophilic microorganisms were tested to determine if mercury could be mobilized from coal under elevated growth temperatures. There was no evidence for mercury removal from coal under these conditions. However, the activity of the organisms may have liberated mercury physically. It is also possible that the organisms dissolved mercury and it readsorbed to the clay preferentially. Both of these possibilities are undergoing further testing. The Idaho National Engineering and Environmental Laboratory's (INEEL) slurry column reactor was operated and several batches of feed coal, product coal, waste solids and leach solutions were submitted to LBL for HAP precursor analysis. Results to date indicate significant removal of mercury, arsenic and other HAP precursors in the combined physical-biological process.

  14. OXIDANT-PRECURSOR RELATIONSHIPS

    EPA Science Inventory

    New methods of ambient air analysis were used to define more clearly the relationships between oxidants and their precursors. Non-methane hydrocarbons, NOx, O2, and oxidants were measured at the same time and location (Riverside, California). The ambient air data presented in thi...

  15. Untangling ENSO Precursors

    NASA Astrophysics Data System (ADS)

    Pegion, K.; Alexander, M. A.

    2014-12-01

    There are several proposed precursors to the El Nino-Southern Oscillation (ENSO) that may provide the ability to predict ENSO as much as one year in advance. Some of these precursors are associated with stochastic forcing from extratropical atmospheric variability. Two examples are the seasonal footprinting mechanism (SFM) and the Pacific meridional mode (PMM). Both of these ENSO precursors are thought to be forced by the North Pacific Oscillation (NPO), a north-south sea level pressure dipole in the north Pacific. Additionally, both the PMM and SFM are thought to impact the tropics through wind evaporation SST feedbacks and have a correlation with ENSO up to one year in advance. These two precursors are discussed interchangeably throughout the literature and various indices used to define them co-mingle them. As a result, whether they are independent of each other or are part of the same process has not been investigated. The research presented is focused on untangling the relationship between the PMM, SFM, NPO, and ENSO using observational datasets and model simulations. Observational results demonstrate that these two mechanisms are different, are forced by different atmospheric circulations, and result in different manifestations of ENSO. Modeling results highlight the extent to which climate models can simulate these relationships and their impact on the simulation of ENSO.

  16. Enhancing a Pathway-Genome Database (PGDB) to Capture Subcellular Localization of Metabolites and Enzymes: The Nucleotide-Sugar Biosynthetic Pathways of Populus trichocarpa

    SciTech Connect

    Nag, A.; Karpinets, T. V.; Chang, C. H.; Bar-Peled, M.

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s).

  17. Distributing a metabolic pathway among a microbial consortium enhances production of natural products

    PubMed Central

    Zhou, Kang; Qiao, Kangjian; Edgar, Steven; Stephanopoulos, Gregory

    2016-01-01

    Metabolic engineering of microorganisms such as Escherichia coli and Saccharomyces cerevisiae to produce high-value natural metabolites is often done through functional reconstitution of long metabolic pathways. Problems arise when parts of pathways require specialized environments or compartments for optimal function. Here we solve this problem through co-culture of engineered organisms, each of which contains the part of the pathway that it is best suited to hosting. In one example, we divided the synthetic pathway for the acetylated diol paclitaxel precursor into two modules, expressed in either S. cerevisiae or E. coli, neither of which can produce the paclitaxel precursor on their own. Stable co-culture in the same bioreactor was achieved by designing a mutualistic relationship between the two species in which a metabolic intermediate produced by E. coli was used and functionalized by yeast. This synthetic consortium produced 33 mg/L oxygenated taxanes, including a monoacetylated dioxygenated taxane. The same method was also used to produce tanshinone precursors and functionalized sesquiterpenes. PMID:25558867

  18. Improved Wood Properties Through Genetic Manipulation: Engineering of Syringyl Lignin in Softwood Species Through Xylem-Specific Expression of Hardwood Syringyl Monolignol Pathway Genes

    SciTech Connect

    Chandrashekhar P. Joshi; Vincent L. Chiang

    2009-01-29

    Project Objective: Our long-term goal is to genetically engineer higher value raw materials with desirable wood properties to promote energy efficiency, international competitiveness, and environmental responsiveness of the U.S. forest products industry. The immediate goal of this project was to produce the first higher value softwood raw materials engineered with a wide range of syringyl lignin quantities. Summary: The most important wood property affecting directly the levels of energy, chemical and bleaching requirements for kraft pulp production is lignin. Softwoods contain almost exclusively chemically resistant guaiacyl (G) lignin, whereas hardwoods have more reactive or easily degradable lignins of the guaiacyl (G)-syringyl (S) type. It is also well established that the reactive S lignin component is the key factor that permits much lower effective alkali and temperature, shorter pulping time and less bleaching stages for processing hardwoods than for softwoods. Furthermore, our pulping kinetic study explicitly demonstrated that every increase in one unit of the lignin S/G ratio would roughly double the rate of lignin removal. These are clear evidence that softwoods genetically engineered with S lignin are keys to revolutionizing the energy efficiency and enhancing the environmental performance of this industry. Softwoods and hardwoods share the same genetic mechanisms for the biosynthesis of G lignin. However, in hardwoods, three additional genes branch out from the G-lignin pathway and become specifically engaged in regulating S lignin biosynthesis. In this research, we simultaneously transferred aspen S-specific genes into a model softwood, black spruce, to engineer S lignin.

  19. Subsurface Pathway Flow and Transport Modeling for the Idaho National Engineering and Environmental Laboratory's Subsurface Disposal Area

    SciTech Connect

    Magnuson, Swen O

    2002-08-01

    Migration of contaminants through the complex subsurface at the Idaho National Engineering and Environmental Laboratory's Subsurface Disposal Area was simulated for an ongoing Comprehensive Environmental Response, Compensation, and Liability (CERCLA) assessment. A previously existing model for simulating flow and transport through the vadose zone for this site was updated to incorporate information obtained from recent characterization activities. Given the complexity of the subsurface at this site, the simulation results were acknowledged to be uncertain. Rather than attempt parametric approaches to quantify uncertainty, it was recognized that conceptual uncertainty involving the controlling processes was likely dominant. So, the effort focused on modeling different scenarios to evaluate the impact of the conceptual uncertainty.

  20. Subsurface Pathway Flow and Transport Modeling for the Idaho National Engineering and Environmental Laboratory's Subsurface Disposal Area

    SciTech Connect

    Magnuson, S.O.

    2002-05-10

    Migration of contaminants through the complex subsurface at the Idaho National Engineering and Environmental Laboratory's Subsurface Disposal Area was simulated for an ongoing Comprehensive Environmental Response, Compensation, and Liability (CERCLA) assessment. A previously existing model for simulating flow and transport through the vadose zone for this site was updated to incorporate information obtained from recent characterization activities. Given the complexity of the subsurface at this site, the simulation results were acknowledged to be uncertain. Rather than attempt parametric approaches to quantify uncertainty, it was recognized that conceptual uncertainty involving the controlling processes was likely dominant. So, the effort focused on modeling different scenarios to evaluate the impact of the conceptual uncertainty.

  1. Metabolic Engineering of a Glycerol-Oxidative Pathway in Lactobacillus panis PM1 for Utilization of Bioethanol Thin Stillage: Potential To Produce Platform Chemicals from Glycerol

    PubMed Central

    Kang, Tae Sun; Korber, Darren R.

    2014-01-01

    Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production. PMID:25281374

  2. Metabolic engineering of a glycerol-oxidative pathway in Lactobacillus panis PM1 for utilization of bioethanol thin stillage: potential to produce platform chemicals from glycerol.

    PubMed

    Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji

    2014-12-01

    Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production. PMID:25281374

  3. Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli.

    PubMed

    Cheng, Zhuan; Jiang, Jiaqi; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-01-01

    In this study, production of 3-HP via malonyl-CoA was investigated by using metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Three engineered E. coli strains with different host-vector systems were constructed and investigated. The results indicated that the combination of E. coli BL21(DE3) and pET28a was the most efficient host-vector system for 3-HP production, and the highest concentration of 3-HP attained in shake flask cultivation reached 1.80g/L by the strain BE-MDA with induction at 0.25mM IPTG and 25°C, and supplementation of NaHCO3 and biotin. In fed-batch fermentation performed in a 5-L reactor, the concentration of 3-HP achieved 10.08g/L in 36h. PMID:26606325

  4. Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae.

    PubMed

    Si, Tong; Luo, Yunzi; Xiao, Han; Zhao, Huimin

    2014-03-01

    Microbial production of higher alcohols from renewable feedstock has attracted intensive attention thanks to its potential as a source for next-generation gasoline substitutes. Here we report the discovery, characterization and engineering of an endogenous 1-butanol pathway in Saccharomyces cerevisiae. Upon introduction of a single gene deletion adh1Δ, S. cerevisiae was able to accumulate more than 120 mg/L 1-butanol from glucose in rich medium. Precursor feeding, ¹³C-isotope labeling and gene deletion experiments demonstrated that the endogenous 1-butanol production was dependent on catabolism of threonine in a manner similar to fusel alcohol production by the Ehrlich pathway. Specifically, the leucine biosynthesis pathway was engaged in the conversion of key 2-keto acid intermediates. Overexpression of the pathway enzymes and elimination of competing pathways achieved the highest reported 1-butanol titer in S. cerevisiae (242.8 mg/L). PMID:24412568

  5. The EM Earthquake Precursor

    NASA Astrophysics Data System (ADS)

    Jones, K. B., II; Saxton, P. T.

    2013-12-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After the 1989 Loma Prieta Earthquake, American earthquake investigators predetermined magnetometer use and a minimum earthquake magnitude necessary for EM detection. This action was set in motion, due to the extensive damage incurred and public outrage concerning earthquake forecasting; however, the magnetometers employed, grounded or buried, are completely subject to static and electric fields and have yet to correlate to an identifiable precursor. Secondly, there is neither a networked array for finding any epicentral locations, nor have there been any attempts to find even one. This methodology needs dismissal, because it is overly complicated, subject to continuous change, and provides no response time. As for the minimum magnitude threshold, which was set at M5, this is simply higher than what modern technological advances have gained. Detection can now be achieved at approximately M1, which greatly improves forecasting chances. A propagating precursor has now been detected in both the field and laboratory. Field antenna testing conducted outside the NE Texas town of Timpson in February, 2013, detected three strong EM sources along with numerous weaker signals. The antenna had mobility, and observations were noted for recurrence, duration, and frequency response. Next, two

  6. Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Navarro, Alicia; Arrillaga, Isabel; Segura, Juan

    2014-05-01

    Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used. PMID:24685653

  7. Assimilation of NAD(+) precursors in Candida glabrata.

    PubMed

    Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

    2007-10-01

    The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection. PMID:17725566

  8. Robotic precursor missions to the moon and Mars

    NASA Technical Reports Server (NTRS)

    Smith, William L.

    1993-01-01

    NASA's Office of Exploration has determined that both the global and the more focused data sets required for the engineering development of manned lunar and Martian exploration systems call for the running of robotic precursor missions. Accounts are presented of the nature of such robotic precursor efforts, which must conduct (1) resource characterization and location, (2) site selection, and (3) fundamental scientific data acquisition tasks. Attention is given to the configurations and instrument suites of prospective robotic lander designs.

  9. Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum

    PubMed Central

    Heider, Sabine A. E.; Wolf, Natalie; Hofemeier, Arne; Peters-Wendisch, Petra; Wendisch, Volker F.

    2014-01-01

    The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca. PMID:25191655

  10. Creating pathways towards aromatic building blocks and fine chemicals.

    PubMed

    Thompson, Brian; Machas, Michael; Nielsen, David R

    2015-12-01

    Aromatic compounds represent a broad class of chemicals with a range of industrial applications, all of which are conventionally derived from petroleum feedstocks. However, owing to a diversity of available pathway precursors along with natural and engineered enzyme 'parts', microbial cell factories can be engineered to create alternative, renewable routes to many of the same aromatic products. Drawing from the latest tools and strategies in metabolic engineering and synthetic biology, such efforts are becoming an increasingly systematic practice, while continued efforts promise to open new doors to an ever-expanding range and diversity of renewable chemical and material products. This short review will highlight recent and notable achievements related for the microbial production of aromatic chemicals. PMID:26264997

  11. Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

    PubMed Central

    Bajaj, Madhu S.; Ogueli, Godwin I.; Kumar, Yogesh; Vadivel, Kanagasabai; Lawson, Gregory; Shanker, Sreejesh; Schmidt, Amy E.; Bajaj, S. Paul

    2011-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R. PMID:21115497

  12. Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate.

    PubMed

    Vuoristo, Kiira S; Mars, Astrid E; Sangra, Jose Vidal; Springer, Jan; Eggink, Gerrit; Sanders, Johan P M; Weusthuis, Ruud A

    2015-12-01

    Itaconic acid, an unsaturated C5-dicarboxylic acid, is a biobased building block for the polymer industry. The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway of E. coli. E. coli BW25113 (DE3) and the phosphate acetyltransferase (pta) and lactate dehydrogenase (ldhA) deficient strain E. coli BW25113 (DE3) Δpta-ΔldhA were used to study anaerobic itaconate production in E. coli. Heterologous expression of the gene encoding cis-aconitate decarboxylase (cadA) from A. terreus in E. coli BW25113 (DE3) did not result in itaconate production under anaerobic conditions, but 0.08 mM of itaconate was formed when the genes encoding citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum were also expressed. The same amount was produced when cadA was expressed in E. coli BW25113 (DE3) Δpta-ΔldhA. The titre increased 8 times to 0.66 mM (1.2 % Cmol) when E. coli BW25113 (DE3) Δpta-ΔldhA also expressed gltA and acnA. In addition, this strain produced 8.5 mM (13 % Cmol) of glutamate. The use of a nitrogen-limited growth medium reduced the accumulation of glutamate by nearly 50 % compared to the normal medium, and also resulted in a more than 3-fold increase of the itaconate titre to 2.9 mM. These results demonstrated that E. coli has potential to produce itaconate and glutamate under anaerobic conditions, closing the redox balance by co-production of succinate or ethanol with H2 and CO2. PMID:26384341

  13. Vascular Precursor Cells

    PubMed Central

    Chaudhury, Hera; Goldie, Lauren C.

    2011-01-01

    Understanding the mechanisms that regulate the proliferation and differentiation of human stem and progenitor cells is critically important for the development and optimization of regenerative medicine strategies. For vascular regeneration studies, specifically, a true “vascular stem cell” population has not yet been identified. However, a number of cell types that exist endogenously, or can be generated or propagated ex vivo, function as vascular precursor cells and can participate in and/or promote vascular regeneration. Herein, we provide an overview of what is known about the regulation of their differentiation specifically toward a vascular endothelial cell phenotype. PMID:22866199

  14. Cardiac differentiation of mouse embryonic stem cells is influenced by a PPAR γ/PGC-1α-FNDC5 pathway during the stage of cardiac precursor cell formation.

    PubMed

    Zadegan, Faezeh Ghazvini; Ghaedi, Kamran; Kalantar, Seyed Mehdi; Peymani, Maryam; Hashemi, Motahare-Sadat; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-06-01

    Peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α) up-regulation induces FNDC5 expression in muscle and consequently causes browning of white adipose tissue (WAT). In addition to skeletal muscle, FNDC5 is mainly expressed in heart and brain tissues. Here, we demonstrate that FNDC5 expression increased during the process of cardiac differentiation of mouse embryonic stem cells (mESCs) similar to PGC-1α and PPARα. To testify the correlation between PGC-1α and FNDC5 in cardiac cell differentiation of mESCs, we utilized specific PPARγ agonist and antagonist in two stages of cardiac differentiation, during and post-cardiac precursor cells (CPCs) formation. Our results indicated that a reduction in PGC-1α expression, via treatment with GW9662 during CPCs formation stage, down-regulated FNDC5 transcript levels as well as mitochondrial markers which negatively influenced on the whole process of cardiac differentiation efficiency. On the other hand, increase PGC-1α expression during CPCs formation stage via rosiglitazone treatment increase FNDC5 and mitochondrial markers transcript levels which enhanced cardiac differentiation efficiency. Importantly, such alteration in PGC-1α expression at post-CPCs formation stage did not affect overall cardiac differentiation rate as expression of FNDC5 and mitochondrial markers were not significantly changed. We concluded that PPARγ agonist and antagonist induced up and down-regulation of PGC-1α and subsequently modulated the process of CPCs formation through an alteration in FNDC5 and mitochondrial markers expression. PMID:25936576

  15. Alternative isoleucine synthesis pathway in cyanobacterial species.

    PubMed

    Wu, Bing; Zhang, Baichen; Feng, Xueyang; Rubens, Jacob R; Huang, Rick; Hicks, Leslie M; Pakrasi, Himadri B; Tang, Yinjie J

    2010-02-01

    Cyanothece sp. ATCC 51142 is an aerobic N(2)-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids revealed an identical labelling profile for leucine and isoleucine when Cyanothece 51142 was grown mixotrophically using 2-(13)C-labelled glycerol as the main carbon source. This indicated that Cyanothece 51142 employs the atypical alternative citramalate pathway for isoleucine synthesis, with pyruvate and acetyl-CoA as precursors. Utilization of the citramalate pathway was confirmed by an enzyme assay and LC-MS/MS analysis. Furthermore, the genome sequence of Cyanothece 51142 shows that the gene encoding the key enzyme (threonine ammonia-lyase) in the normal isoleucine pathway is missing. Instead, the cce_0248 gene in Cyanothece 51142 exhibits 53 % identity to the gene encoding citramalate synthase (CimA, GSU1798) from Geobacter sulfurreducens. Reverse-transcription PCR indicated that the cce_0248 gene is expressed and its transcriptional level is lower in medium with isoleucine than in isoleucine-free medium. Additionally, a blast search for citramalate synthase and threonine ammonia-lyase implies that this alternative isoleucine synthesis pathway may be present in other cyanobacteria, such as Cyanothece and Synechococcus. This suggests that the pathway is more widespread than originally thought, as previous identifications of the citramalate pathway are limited to mostly anaerobic bacteria or archaea. Furthermore, this discovery opens the possibility that such autrotrophic micro-organisms may be engineered for robust butanol and propanol production from 2-ketobutyrate, which is an intermediate in the isoleucine biosynthesis pathway. PMID:19875435

  16. An engineered pathway for glyoxylate metabolism in tobacco plants aimed to avoid the release of ammonia in photorespiration

    PubMed Central

    2011-01-01

    Background The photorespiratory nitrogen cycle in C3 plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO2. Because of the loss of CO2 and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C3 plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle. Results When grown in ambient air, but not in elevated CO2, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation. Conclusions Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a

  17. Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii

    PubMed Central

    Nair, Sethu C.; Brooks, Carrie F.; Goodman, Christopher D.; Strurm, Angelika; McFadden, Geoffrey I.; Sundriyal, Sandeep; Anglin, Justin L.; Song, Yongcheng; Moreno, Silvia N.J.

    2011-01-01

    Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance. PMID:21690250

  18. Metabolic engineering of E.coli for the production of a precursor to artemisinin, an anti-malarial drug [Chapter 25 in Manual of Industrial Microbiology and Biotechnology, 3rd edition

    SciTech Connect

    Petzold, Christopher; Keasling, Jay

    2011-07-18

    This document is Chapter 25 in the Manual of Industrial Microbiology and Biotechnology, 3rd edition. Topics covered include: Incorporation of Amorpha-4,11-Diene Biosynthetic Pathway into E. coli; Amorpha-4,11-Diene Pathway Optimization; "-Omics" Analyses for Increased Amorpha-4,11-Diene Production; Biosynthetic Oxidation of Amorpha-4,11-Diene.

  19. Improved 1,3-propanediol production by engineering the 2,3-butanediol and formic acid pathways in integrative recombinant Klebsiella pneumoniae.

    PubMed

    Wu, Zhe; Wang, Zhe; Wang, Guoqing; Tan, Tianwei

    2013-10-20

    In the biotechnological process, insufficient cofactor NADH and multiple by-products restrain the final titer of 1,3-propanediol (1,3-PD). In this study, 1,3-PD production was improved by engineering the 2,3-butanediol (2,3-BD) and formic acid pathways in integrative recombinant Klebsiella pneumoniae. The formation of 2,3-BD is catalysed by acetoin reductase (AR). An inactivation mutation of the AR in K. pneumoniae CF was generated by insertion of a formate dehydrogenase gene. Inactivation of AR and expression of formate dehydrogenase reduced 2,3-BD formation and improved 1,3-PD production. Fermentation results revealed that intracellular metabolic flux was redistributed pronouncedly. The yield of 1,3-PD reached 0.74 mol/mol glycerol in flask fermentation, which is higher than the theoretical yield. In 5 L fed-batch fermentation, the final titer and 1,3-PD yield of the K. pneumoniae CF strain reached 72.2 g/L and 0.569 mol/mol, respectively, which were 15.9% and 21.7% higher than those of the wild-type strain. The titers of 2,3-BD and formic acid decreased by 52.2% and 73.4%, respectively. By decreasing the concentration of all nonvolatile by-products and by increasing the availability of NADH, this study demonstrates an important strategy in the metabolic engineering of 1,3-PD production by integrative recombinant hosts. PMID:23665191

  20. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    SciTech Connect

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    This project used the cyanobacterial species Synechocystis PCC 6803 to pursue two lines of inquiry, with each line addressing one of the two main factors affecting hydrogen (H2) production in Synechocystis PCC 6803: NADPH availability and O2 sensitivity. H2 production in Synechocystis PCC 6803 requires a very high NADPH:NADP+ ratio, that is, the NADP pool must be highly reduced, which can be problematic because several metabolic pathways potentially can act to raise or lower NADPH levels. Also, though the [NiFe]-hydrogenase in PCC 6803 is constitutively expressed, it is reversibly inactivated at very low O2 concentrations. Largely because of this O2 sensitivity and the requirement for high NADPH levels, a major portion of overall H2 production occurs under anoxic conditions in the dark, supported by breakdown of glycogen or other organic substrates accumulated during photosynthesis. Also, other factors, such as N or S limitation, pH changes, presence of other substances, or deletion of particular respiratory components, can affect light or dark H2 production. Therefore, in the first line of inquiry, under a number of culture conditions with wild type (WT) Synechocystis PCC 6803 cells and a mutant with impaired type I NADPH-dehydrogenase (NDH-1) function, we used H2 production profiling and metabolic flux analysis, with and without specific inhibitors, to examine systematically the pathways involved in light and dark H2 production. Results from this work provided rational bases for metabolic engineering to maximize photobiological H2 production on a 24-hour basis. In the second line of inquiry, we used site-directed mutagenesis to create mutants with hydrogenase enzymes exhibiting greater O2 tolerance. The research addressed the following four tasks: 1. Evaluate the effects of various culture conditions (N, S, or P limitation; light/dark; pH; exogenous organic carbon) on H2 production profiles of WT cells and an NDH-1 mutant; 2. Conduct metabolic flux analyses for

  1. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

    PubMed

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

    2015-11-01

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. PMID:26385918

  2. Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly of Two Key Enzymes in Pichia pastoris.

    PubMed

    Zhao, Chengcheng; Gao, Xin; Liu, Xinbin; Wang, Yong; Yang, Shengli; Wang, Fengqing; Ren, Yuhong

    2016-05-01

    Ginsenosides from the edible and medicinal plant ginseng have demonstrated various pharmacological activities. However, producing ginsenoside efficiently remains a challenge. Engineering metabolic pathways through protein assembly in yeast is a promising way for ginsenoside production. In the biosynthetic pathway of ginsenosides, dammarenediol-II synthase and squalene epoxidase are two key enzymes that determine the production rate of the dammarane-type ginsenoside precursor dammarenediol-II. In this work, a strategy to enhance the biosynthesis of dammarenediol-II in Pichia pastoris was developed by the self-assembly of the two key enzymes via protein-protein interaction. After being modified by interacting proteins, the two enzymes were successfully co-localized, resulting in a 2.1-fold enhancement in dammarenediol-II yields. PMID:27074597

  3. An interstellar precursor mission

    NASA Technical Reports Server (NTRS)

    Jaffe, L. D.; Ivie, C.; Lewis, J. C.; Lipes, R. G.; Norton, H. N.; Stearns, J. W.; Stimpson, L.; Weissman, P.

    1977-01-01

    A mission out of the planetary system, with launch about the year 2000, could provide valuable scientific data as well as test some of the technology for a later mission to another star. Primary scientific objectives for the precursor mission concern characteristics of the heliopause, the interstellar medium, stellar distances (by parallax measurements), low energy cosmic rays, interplanetary gas distribution, and mass of the solar system. Secondary objectives include investigation of Pluto. Candidate science instruments are suggested. Individual spacecraft systems for the mission were considered, technology requirements and problem areas noted, and a number of recommendations made for technology study and advanced development. The most critical technology needs include attainment of 50-yr spacecraft lifetime and development of a long-life NEP system.

  4. Identified EM Earthquake Precursors

    NASA Astrophysics Data System (ADS)

    Jones, Kenneth, II; Saxton, Patrick

    2014-05-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After a number of custom rock experiments, two hypotheses were formed which could answer the EM wave model. The first hypothesis concerned a sufficient and continuous electron movement either by surface or penetrative flow, and the second regarded a novel approach to radio transmission. Electron flow along fracture surfaces was determined to be inadequate in creating strong EM fields, because rock has a very high electrical resistance making it a high quality insulator. Penetrative flow could not be corroborated as well, because it was discovered that rock was absorbing and confining electrons to a very thin skin depth. Radio wave transmission and detection worked with every single test administered. This hypothesis was reviewed for propagating, long-wave generation with sufficient amplitude, and the capability of penetrating solid rock. Additionally, fracture spaces, either air or ion-filled, can facilitate this concept from great depths and allow for surficial detection. A few propagating precursor signals have been detected in the field occurring with associated phases using custom-built loop antennae. Field testing was conducted in Southern California from 2006-2011, and outside the NE Texas town of Timpson in February, 2013. The antennae have mobility and observations were noted for

  5. Metabolic engineering of Pediococcus acidilactici BD16 for production of vanillin through ferulic acid catabolic pathway and process optimization using response surface methodology.

    PubMed

    Kaur, Baljinder; Chakraborty, Debkumar; Kumar, Balvir

    2014-10-01

    Occurrence of feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase (ech) genes responsible for the bioconversion of ferulic acid to vanillin have been reported and characterized from Amycolatopsis sp., Streptomyces sp., and Pseudomonas sp. Attempts have been made to express these genes in Escherichia coli DH5α, E. coli JM109, and Pseudomonas fluorescens. However, none of the lactic acid bacteria strain having GRAS status was previously proposed for heterologous expression of fcs and ech genes for production of vanillin through biotechnological process. Present study reports heterologous expression of vanillin synthetic gene cassette bearing fcs and ech genes in a dairy isolate Pediococcus acidilactici BD16. After metabolic engineering, statistical optimization of process parameters that influence ferulic acid to vanillin biotransformation in the recombinant strain was carried out using central composite design of response surface methodology. After scale-up of the process, 3.14 mM vanillin was recovered from 1.08 mM ferulic acid per milligram of recombinant cell biomass within 20 min of biotransformation. From LCMS-ESI spectral analysis, a metabolic pathway of phenolic biotransformations was predicted in the recombinant P. acidilactici BD16 (fcs (+)/ech (+)). PMID:25077778

  6. Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

    PubMed

    Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

    2015-09-01

    Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP(+)-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway. PMID:26253204

  7. Increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway--metabolic engineering of the availability of succinyl-CoA in Corynebacterium glutamicum.

    PubMed

    Kind, Stefanie; Becker, Judith; Wittmann, Christoph

    2013-01-01

    In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum ΔsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with (13)C isotope studies confirmed that the increase in lysine production was mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upon deletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the α-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizes lysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bio-production in the future. PMID:22871505

  8. Generation of Nonlinear Vortex Precursors.

    PubMed

    Chen, Yue-Yue; Feng, Xun-Li; Liu, Chengpu

    2016-07-01

    We numerically study the propagation of a few-cycle pulse carrying orbital angular momentum (OAM) through a dense atomic system. Nonlinear precursors consisting of high-order vortex harmonics are generated in the transmitted field due to carrier effects associated with ultrafast Bloch oscillation. The nonlinear precursors survive to propagation effects and are well separated with the main pulse, which provides a straightforward way to measure precursors. By virtue of carrying high-order OAM, the obtained vortex precursors as information carriers have potential applications in optical information and communication fields where controllable loss, large information-carrying capacity, and high speed communication are required. PMID:27447507

  9. Generation of Nonlinear Vortex Precursors

    NASA Astrophysics Data System (ADS)

    Chen, Yue-Yue; Feng, Xun-Li; Liu, Chengpu

    2016-07-01

    We numerically study the propagation of a few-cycle pulse carrying orbital angular momentum (OAM) through a dense atomic system. Nonlinear precursors consisting of high-order vortex harmonics are generated in the transmitted field due to carrier effects associated with ultrafast Bloch oscillation. The nonlinear precursors survive to propagation effects and are well separated with the main pulse, which provides a straightforward way to measure precursors. By virtue of carrying high-order OAM, the obtained vortex precursors as information carriers have potential applications in optical information and communication fields where controllable loss, large information-carrying capacity, and high speed communication are required.

  10. The potential of the mevalonate pathway for enhanced isoprenoid production.

    PubMed

    Liao, Pan; Hemmerlin, Andréa; Bach, Thomas J; Chye, Mee-Len

    2016-01-01

    The cytosol-localised mevalonic acid (MVA) pathway delivers the basic isoprene unit isopentenyl diphosphate (IPP). In higher plants, this central metabolic intermediate is also synthesised by the plastid-localised methylerythritol phosphate (MEP) pathway. Both MVA and MEP pathways conspire through exchange of intermediates and regulatory interactions. Products downstream of IPP such as phytosterols, carotenoids, vitamin E, artemisinin, tanshinone and paclitaxel demonstrate antioxidant, cholesterol-reducing, anti-ageing, anticancer, antimalarial, anti-inflammatory and antibacterial activities. Other isoprenoid precursors including isoprene, isoprenol, geraniol, farnesene and farnesol are economically valuable. An update on the MVA pathway and its interaction with the MEP pathway is presented, including the improvement in the production of phytosterols and other isoprenoid derivatives. Such attempts are for instance based on the bioengineering of microbes such as Escherichia coli and Saccharomyces cerevisiae, as well as plants. The function of relevant genes in the MVA pathway that can be utilised in metabolic engineering is reviewed and future perspectives are presented. PMID:26995109

  11. Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

    PubMed

    Ramos, Kristine Rose M; Valdehuesa, Kris Niño G; Liu, Huaiwei; Nisola, Grace M; Lee, Won-Keun; Chung, Wook-Jin

    2014-12-01

    An engineered Escherichia coli strain was developed for enhanced isoprene production using D-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley-Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from D-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct D-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze D-galactose conversion to D-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD-MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD-MEP pathway. PMID:24928200

  12. Precursor-directed biosynthesis of novel triketide lactones.

    PubMed

    Regentin, Rika; Kennedy, Jonathan; Wu, Nicholas; Carney, John R; Licari, Peter; Galazzo, Jorge; Desai, Ruchir

    2004-01-01

    Precursor-directed biosynthesis was used to produce different triketide lactones (R-TKLs) in a fermentation process. Plasmids expressing engineered versions of the first subunit of 6-deoxyerythronolide B synthase (DEBS1) fused to the terminal DEBS thioesterase (TE) were introduced into three different Streptomyces strains. The DEBS1 protein fused to TE had either an inactivated ketosynthase domain (KS1 degrees ) or a partial DEBS1 lacking module 1 but containing module 2 (M2+TE). Different synthetic precursors were examined for their effect on R-TKL production. An overproducing strain of S. coelicolor expressing the M2+TE protein was found to be best for production of R-TKLs. Racemic precursors were as effective as enantiomerically pure precursors in the fermentation process. The R group on the precursor significantly affected titer (propyl > chloromethyl > vinyl). The R-TKLs were unstable in fermentation broth at pH 6-8. A two-phase fermentation with a pH shift was implemented to stabilize the products. The fermentation pH initially was controlled at optimal values for cell growth (pH 6.5) and then shifted to 5.5 during production. This doubled peak titers and stabilized the product. Finally, the concentration of synthetic precursor in the fermentation was optimized to improve production. A maximum titer of 500 mg/L 5-chloromethyl-TKL was obtained using 3.5 g/L precursor. PMID:14763833

  13. An interstellar precursor mission

    NASA Technical Reports Server (NTRS)

    Jaffe, L. D.; Ivie, C.; Lewis, J. C.; Lipes, R.; Norton, H. N.; Stearns, J. W.; Stimpson, L. D.; Weissman, P.

    1980-01-01

    A mission out of the planetary system, launched about the year 2000, could provide valuable scientific data as well as test some of the technology for a later mission to another star. Primary scientific objectives for the precursor mission concern characteristics of the heliopause, the interstellar medium, stellar distances (by parallax measurements), low-energy cosmic rays, interplanetary gas distribution, and the mass of the solar system. Secondary objectives include investigation of Pluto. The mission should extend to 400-1000 AU from the sun. A heliocentric hyperbolic escape velocity of 50-100 km/sec or more is needed to attain this distance within a reasonable mission duration (20-50 years). The trajectory should be toward the incoming interstellar gas. For a year 2000 launch, a Pluto encounter and orbiter can be included. A second mission targeted parallel to the solar axis would also be worthwhile. The mission duration is 20 years, with an extended mission to a total of 50 years. A system using one or two stages of nuclear electric propulsion (NEP) was selected as a possible baseline. The most promising alternatives are ultralight solar sails or laser sailing, with the lasers in earth orbit, for example. The NEP baseline design allows the option of carrying a Pluto orbiter as a daughter spacecraft.

  14. Identifying Branched Metabolic Pathways by Merging Linear Metabolic Pathways

    NASA Astrophysics Data System (ADS)

    Heath, Allison P.; Bennett, George N.; Kavraki, Lydia E.

    This paper presents a graph-based algorithm for identifying complex metabolic pathways in multi-genome scale metabolic data. These complex pathways are called branched pathways because they can arrive at a target compound through combinations of pathways that split compounds into smaller ones, work in parallel with many compounds, and join compounds into larger ones. While most previous work has focused on identifying linear metabolic pathways, branched metabolic pathways predominate in metabolic networks. Automatic identification of branched pathways has a number of important applications in areas that require deeper understanding of metabolism, such as metabolic engineering and drug target identification. Our algorithm utilizes explicit atom tracking to identify linear metabolic pathways and then merges them together into branched metabolic pathways. We provide results on two well-characterized metabolic pathways that demonstrate that this new merging approach can efficiently find biologically relevant branched metabolic pathways with complex structures.

  15. Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria.

    PubMed

    Park, Si Jae; Lee, Sang Yup; Kim, Tae Wan; Jung, Yu Kyung; Yang, Taek Ho

    2012-02-01

    Due to increasing concerns about environmental problems, climate change and limited fossil resources, bio-based production of chemicals and polymers is gaining attention as one of the solutions to these problems. Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microbial fermentation. PHAs are synthesized using monomer precursors provided from diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so-called bio-based polymers including polybutylene succinate, polytrimethylene terephthalate, and polylactic acid (PLA) are synthesized by a chemical process using monomers produced by fermentation. PLA, an attractive biomass-derived plastic, is currently synthesized by heavy metal-catalyzed ring opening polymerization of L-lactide that is made from fermentation-derived L-lactic acid. Recently, a complete biological process for the production of PLA and PLA copolymers from renewable resources has been developed by direct fermentation of recombinant bacteria employing PHA biosynthetic pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl-CoA and engineering PHA synthase to accept lactyl-CoA as a substrate combined with systems metabolic engineering. In this article, we review recent advances in the production of lactate-containing homo- and co-polyesters. Challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are discussed. PMID:22057878

  16. Designing and creating a modularized synthetic pathway in cyanobacterium Synechocystis enables production of acetone from carbon dioxide.

    PubMed

    Zhou, Jie; Zhang, Haifeng; Zhang, Yanping; Li, Yin; Ma, Yanhe

    2012-07-01

    Ketones are a class of important organic compounds. As the simplest ketone, acetone is widely used as solvents or precursors for industrial chemicals. Presently, million tonnes of acetone is produced worldwide annually, from petrochemical processes. Here we report a biotechnological process that can produce acetone from CO(2), by designing and creating a modularized synthetic pathway in engineered cyanobacterium Synechocystis sp. PCC 6803. The engineered Synechocystis cells are able to produce acetone (36.0 mgl(-1) culture medium) using CO(2) as the sole carbon source, thus opens the gateway for biosynthesis of ketones from CO(2). PMID:22475865

  17. Generation of Vibrationally Excited HCP from a Stable Synthetic Precursor

    NASA Astrophysics Data System (ADS)

    Hull, Alexander W.; Jiang, Jun; Erickson, Trevor J.; Womack, Carrie; Nava, Matthew; Cummins, Christopher; Field, Robert W.

    2015-06-01

    HCP belongs to a class of reactive small molecules with much interest to spectroscopists. It bears certain similarities to HCN, including a strong {A}(bent) - {X}(linear) ultraviolet transition, associated with the HCP-HPC isomerization pathway. HCP has traditionally been generated by the in situ reaction of PH_3 and acetylene. In this talk, we will discuss a recently developed synthetic precursor molecule, 1,1-((triphenylphosphoranylidene)methyl)-9,10-phosphanoanthracene. At temperatures above 200 degrees Celsius, this precursor is thought to release HCP in a vibrationally excited state. We will present preliminary spectra on this system obtained by LIF and chirped pulse millimeter wave spectroscopy.

  18. Preparation of superconductor precursor powders

    DOEpatents

    Bhattacharya, Raghunath

    1998-01-01

    A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.

  19. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine.

    PubMed

    Qin, Jiufu; Zhou, Yongjin J; Krivoruchko, Anastasia; Huang, Mingtao; Liu, Lifang; Khoomrung, Sakda; Siewers, Verena; Jiang, Bo; Nielsen, Jens

    2015-01-01

    Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l(-1) with a yield of 67 mg (g glucose)(-1) in shake-flask cultures and a titre of 5.1 g l(-1) in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals. PMID:26345617

  20. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

    PubMed Central

    Qin, Jiufu; Zhou, Yongjin J.; Krivoruchko, Anastasia; Huang, Mingtao; Liu, Lifang; Khoomrung, Sakda; Siewers, Verena; Jiang, Bo; Nielsen, Jens

    2015-01-01

    Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l−1 with a yield of 67 mg (g glucose)−1 in shake-flask cultures and a titre of 5.1 g l−1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals. PMID:26345617

  1. Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites.

    PubMed

    Lian, Jiazhang; Zhao, Huimin

    2015-03-01

    Fatty acids or their activated forms, fatty acyl-CoAs and fatty acyl-ACPs, are important precursors to synthesize a wide variety of fuels and chemicals, including but not limited to free fatty acids (FFAs), fatty alcohols (FALs), fatty acid ethyl esters (FAEEs), and alkanes. However, Saccharomyces cerevisiae, an important cell factory, does not naturally accumulate fatty acids in large quantities. Therefore, metabolic engineering strategies were carried out to increase the glycolytic fluxes to fatty acid biosynthesis in yeast, specifically to enhance the supply of precursors, eliminate competing pathways, and bypass the host regulatory network. This review will focus on the genetic manipulation of both structural and regulatory genes in each step for fatty acids overproduction in S. cerevisiae, including from sugar to acetyl-CoA, from acetyl-CoA to malonyl-CoA, and from malonyl-CoA to fatty acyl-CoAs. The downstream pathways for the conversion of fatty acyl-CoAs to the desired products will also be discussed. PMID:25306882

  2. A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop.

    PubMed

    Fuentes, Paulina; Zhou, Fei; Erban, Alexander; Karcher, Daniel; Kopka, Joachim; Bock, Ralph

    2016-01-01

    Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. PMID:27296645

  3. A phenomenological model for precursor volcanic eruptions.

    PubMed

    Menand, T; Tait, S R

    2001-06-01

    Intense explosions of relatively short duration frequently precede large explosive and effusive volcanic eruptions-by as much as weeks to months in the case of very viscous magmas. In some cases, such pre-eruption activity has served as a sufficient warning to those living in the vicinity to evacuate and avoid calamity. Precursor events seem to be related to the formation of a magma pathway to the surface, but their precise interpretation is a long-standing puzzle. It has been inferred from theoretical studies that exsolution of volatiles might create an almost separate gas pocket at the tip of a propagating dyke. Here we explain the role that such a process may have, using a laboratory study of the transient propagation of a liquid-filled crack with a gas pocket at its tip that grows with time. We show that once the gas pocket acquires sufficient buoyancy to overcome the fracture resistance of the host solid the dynamics of the gas pocket, rather than those of the liquid, determine the velocity of the crack tip. Furthermore, we find that the gas can ultimately separate from the liquid. We propose that fast-moving, gas-rich pockets reaching the surface ahead of the main liquid-filled fissure could be the origin of many precursor eruptions. PMID:11395766

  4. The challenges of cellular compartmentalization in plant metabolic engineering.

    PubMed

    Heinig, Uwe; Gutensohn, Michael; Dudareva, Natalia; Aharoni, Asaph

    2013-04-01

    The complex metabolic networks in plants are highly compartmentalized and biochemical steps of a single pathway can take place in multiple subcellular locations. Our knowledge regarding reactions and precursor compounds in the various cellular compartments has increased in recent years due to innovations in tracking the spatial distribution of proteins and metabolites. Nevertheless, to date only few studies have integrated subcellular localization criteria in metabolic engineering attempts. Here, we highlight the crucial factors for subcellular-localization-based strategies in plant metabolic engineering including substrate availability, enzyme targeting, the role of transporters, and multigene transfer approaches. The availability of compartmentalized metabolic network models for plants in the near future will greatly advance the integration of localization constraints in metabolic engineering experiments and aid in predicting their outcomes. PMID:23246154

  5. PECAM-1, apoptosis and CD34+ precursors.

    PubMed

    Zocchi, Maria R; Poggi, A

    2004-11-01

    Apoptosis is a physiological process that controls tissue homeostasis, in combination with survival signals delivered by distinct receptors that bind hormones, growth factors or extracellular matrix components. The extrinsic pathway of apoptosis is due to the triggering of death receptors and the activation of the caspase cascade; the intrinsic pathway is due to withdrawal of growth factors and mainly related to mitochondrial metabolism. The choice between survival or apoptosis, which is the result of such different integrated environmental signals, is crucial for the maintainance of bone marrow reservoir of hematopoietic precursors (HPC). CD34+ HPC can receive multiple survival signals during homing and maturation, due to different interactions with adhesion molecules expressed on endothelial and bone marrow stromal cells, proteins of the extracellular matrix and chemokines or growth factors. Among them, the signal delivered via platelet endothelial cell adhesion molecule-1 (PECAM-1) seems to contribute to the resistance of this cell population to starvation, and it is related to the maintainance of mitochondrial metabolism. Indeed, this molecule, originally described as an adhesion receptor belonging to the immunoglobulin superfamily, capable of homophilic and heterophilic interactions, turned out to be a signalling molecule, containing an immunoreceptor tyrosine-based inhibitory motifs (ITIM) within its cytoplasmic domain. In particular, it has been shown that PECAM-1 binds to different kinases and phosphatases, including the phosphatidylinositide-3-kinase that phosphorylates Akt, which, in turn can upregulate transcription and function of antiapoptotic proteins, such as Bcl-2 and Bcl-x or A1, responsible for the rescue from mitochondrial apoptosis. The possible role of PECAM-1 engagement in the prevention of starvation-induced apoptosis of HPC precursors and in the maintainance of their survival is discussed. PMID:15512808

  6. Precursors of Hypertension: A Review

    PubMed Central

    Thomas, John; Neser, William B.; Thomas, Johniene; Semenya, Kofi; Green, Donald R.

    1983-01-01

    Recent advances in hypertension therapy have been remarkable; however, much less is known about those precursors that facilitate preventive and early intervention measures. This review of the literature indicates that relevant precursors are early elevated casual systolic blood pressures, positive family history, and obesity in females. Additional predisposing or enhancing factors point to high sodium ingestion, heavy smoking, and high socioecologic stress. Evidence for a high-risk hypertensive personality is not conclusive. There is a paucity of longitudinal data on hypertension in the black population. PMID:6864814

  7. Fuel from Tobacco and Arundo Donax: Synthetic Crop for Direct Drop-in Biofuel Production through Re-routing the Photorespiration Intermediates and Engineering Terpenoid Pathways

    SciTech Connect

    2012-02-15

    PETRO Project: Biofuels offer renewable alternatives to petroleum-based fuels that reduce net greenhouse gas emissions to nearly zero. However, traditional biofuels production is limited not only by the small amount of solar energy that plants convert through photosynthesis into biological materials, but also by inefficient processes for converting these biological materials into fuels. Farm-ready, non-food crops are needed that produce fuels or fuel-like precursors at significantly lower costs with significantly higher productivity. To make biofuels cost-competitive with petroleum-based fuels, biofuels production costs must be cut in half.

  8. AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.

    PubMed

    Du, Qiang; Hou, Xiaojuan; Wang, Lei; Zhang, Yingqi; Xi, Xinping; Wang, Hui; Zhou, Mei; Duan, Jinao; Wei, Minjie; Chen, Tianbao; Shaw, Chris

    2015-02-01

    The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation. PMID:25626077

  9. AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities

    PubMed Central

    Du, Qiang; Hou, Xiaojuan; Wang, Lei; Zhang, Yingqi; Xi, Xinping; Wang, Hui; Zhou, Mei; Duan, Jinao; Wei, Minjie; Chen, Tianbao; Shaw, Chris

    2015-01-01

    The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation. PMID:25626077

  10. Preparation of superconductor precursor powders

    DOEpatents

    Bhattacharya, R.

    1998-08-04

    A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products. 7 figs.

  11. Preparation of superconductor precursor powders

    DOEpatents

    Bhattacharya, Raghunath; Blaugher, Richard D.

    1995-01-01

    A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals, such as nitrate salts of thallium, barium, calcium, and copper, which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of thallium in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.

  12. Perfluoroalkyl Acids (PFAAs) and Selected Precursors in the Baltic Sea Environment: Do Precursors Play a Role in Food Web Accumulation of PFAAs?

    PubMed

    Gebbink, Wouter A; Bignert, Anders; Berger, Urs

    2016-06-21

    The present study examined the presence of perfluoroalkyl acids (PFAAs) and selected precursors in the Baltic Sea abiotic environment and guillemot food web, and investigated the relative importance of precursors in food web accumulation of PFAAs. Sediment, water, zooplankton, herring, sprat, and guillemot eggs were analyzed for perfluoroalkane sulfonic acids (PFSAs; C4,6,8,10) and perfluoroalkyl carboxylic acids (PFCAs; C6-15) along with six perfluoro-octane sulfonic acid (PFOS) precursors and 11 polyfluoroalkyl phosphoric acid diesters (diPAPs). FOSA, FOSAA and its methyl and ethyl derivatives (Me- and EtFOSAA), and 6:2/6:2 diPAP were detected in sediment and water. While FOSA and the three FOSAAs were detected in all biota, a total of nine diPAPs were only detected in zooplankton. Concentrations of PFOS precursors and diPAPs exceeded PFOS and PFCA concentrations, respectively, in zooplankton, but not in fish and guillemot eggs. Although PFOS precursors were present at all trophic levels, they appear to play a minor role in food web accumulation of PFOS based on PFOS precursor/PFOS ratios and PFOS and FOSA isomer patterns. The PFCA pattern in fish could not be explained by the intake pattern based on PFCAs and analyzed precursors, that is, diPAPs. Exposure to additional precursors might therefore be a dominant exposure pathway compared to direct PFCA exposure for fish. PMID:27192404

  13. Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

    PubMed

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

    2015-01-01

    Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity. PMID:27250745

  14. Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli.

    PubMed

    Bokinsky, Gregory; Peralta-Yahya, Pamela P; George, Anthe; Holmes, Bradley M; Steen, Eric J; Dietrich, Jeffrey; Lee, Taek Soon; Tullman-Ercek, Danielle; Voigt, Christopher A; Simmons, Blake A; Keasling, Jay D

    2011-12-13

    One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels. PMID:22123987

  15. Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

    PubMed Central

    Bokinsky, Gregory; Peralta-Yahya, Pamela P.; George, Anthe; Holmes, Bradley M.; Steen, Eric J.; Dietrich, Jeffrey; Soon Lee, Taek; Tullman-Ercek, Danielle; Voigt, Christopher A.; Simmons, Blake A.; Keasling, Jay D.

    2011-01-01

    One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels. PMID:22123987

  16. Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy.

    PubMed

    Wu, Junjun; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2013-03-01

    Flavonoids are valuable natural products widely used in human health and nutrition. Recent advances in synthetic biology and metabolic engineering have yielded improved strain titers and yields. However, current fermentation strategies often require supplementation of expensive phenylpropanoic precursors in the media and separate evaluation of each strategy in turn as part of the flavonoid pathway, implicitly assuming the modifications are additive. In this study, an Escherichia coli fermentation system was developed to bypass both of these problems. An eight-step pathway, consisting of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydratase (CM/PDT), phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was assembled on four vectors in order to produce the flavonoid precursor (2S)-pinocembrin directly from glucose. Furthermore, a modular metabolic strategy was employed to identify conditions that optimally balance the four pathway modules. Once this metabolic balance was achieved, such strains were capable of producing 40.02mg/L (2S)-pinocembrin directly from glucose. These results were attained by culturing engineered cells in minimal medium without additional precursor supplementation. The fermentation platform described here paves the way for the development of an economical process for microbial production of flavonoids directly from glucose. PMID:23246524

  17. Pathways Intern Work Report

    NASA Technical Reports Server (NTRS)

    Midulla, Laura P.

    2014-01-01

    For the spring of 2014, I continued my position at NASA as a Pathways Engineering Student Trainee in the Engineering and Technology Directorate (NE), in the Test, Operations & Support Software Engineering Branch of the Control and Data Systems Division. The Control and Data Systems Division provides control, monitor, checkout, processing, display, and simulation capabilities to support vehicle element testing, checkout and launch. In addition the division provides significant leadership associated with engineering processes and Information Technology Security management. My assignment during this semester was to continue to support the Engineering Information Technology (IT) Security team, providing IT Security support to the Ground Systems Development and Operations Spaceport Command & Control System and Ground Systems, NE Labs and other systems.

  18. Engineering catechol 1, 2-dioxygenase by design for improving the performance of the cis, cis-muconic acid synthetic pathway in Escherichia coli.

    PubMed

    Han, Li; Liu, Pi; Sun, Jixue; Wu, Yuanqing; Zhang, Yuanyuan; Chen, Wujiu; Lin, Jianping; Wang, Qinhong; Ma, Yanhe

    2015-01-01

    Regulating and ameliorating enzyme expression and activity greatly affects the performance of a given synthetic pathway. In this study, a new synthetic pathway for cis, cis-muconic acid (ccMA) production was reconstructed without exogenous induction by regulating the constitutive expression of the important enzyme catechol 1,2-dioxygenase (CatA). Next, new CatAs with significantly improved activities were developed to enhance ccMA production using structure-assisted protein design. Nine mutations were designed, simulated and constructed based on the analysis of the CatA crystal structure. These results showed that mutations at Gly72, Leu73 and/or Pro76 in CatA could improve enzyme activity, and the activity of the most effective mutant was 10-fold greater than that of the wild-type CatA from Acinetobacter sp. ADP1. The most productive synthetic pathway with a mutated CatA increased the titer of ccMA by more than 25%. Molecular dynamic simulation results showed that enlarging the entrance of the substrate-binding pocket in the mutants contributed to their increased enzyme activities and thus improved the performance of the synthetic pathway. PMID:26306712

  19. Engineering catechol 1, 2-dioxygenase by design for improving the performance of the cis, cis-muconic acid synthetic pathway in Escherichia coli

    PubMed Central

    Han, Li; Liu, Pi; Sun, Jixue; Wu, Yuanqing; Zhang, Yuanyuan; Chen, Wujiu; Lin, Jianping; Wang, Qinhong; Ma, Yanhe

    2015-01-01

    Regulating and ameliorating enzyme expression and activity greatly affects the performance of a given synthetic pathway. In this study, a new synthetic pathway for cis, cis-muconic acid (ccMA) production was reconstructed without exogenous induction by regulating the constitutive expression of the important enzyme catechol 1,2-dioxygenase (CatA). Next, new CatAs with significantly improved activities were developed to enhance ccMA production using structure-assisted protein design. Nine mutations were designed, simulated and constructed based on the analysis of the CatA crystal structure. These results showed that mutations at Gly72, Leu73 and/or Pro76 in CatA could improve enzyme activity, and the activity of the most effective mutant was 10-fold greater than that of the wild-type CatA from Acinetobacter sp. ADP1. The most productive synthetic pathway with a mutated CatA increased the titer of ccMA by more than 25%. Molecular dynamic simulation results showed that enlarging the entrance of the substrate-binding pocket in the mutants contributed to their increased enzyme activities and thus improved the performance of the synthetic pathway. PMID:26306712

  20. Lysophosphatidic Acid Receptor Is a Functional Marker of Adult Hippocampal Precursor Cells

    PubMed Central

    Walker, Tara L.; Overall, Rupert W.; Vogler, Steffen; Sykes, Alex M.; Ruhwald, Susann; Lasse, Daniela; Ichwan, Muhammad; Fabel, Klaus; Kempermann, Gerd

    2016-01-01

    Summary Here, we show that the lysophosphatidic acid receptor 1 (LPA1) is expressed by a defined population of type 1 stem cells and type 2a precursor cells in the adult mouse dentate gyrus. LPA1, in contrast to Nestin, also marks the quiescent stem cell population. Combining LPA1-GFP with EGFR and prominin-1 expression, we have enabled the prospective separation of both proliferative and non-proliferative precursor cell populations. Transcriptional profiling of the isolated proliferative precursor cells suggested immune mechanisms and cytokine signaling as molecular regulators of adult hippocampal precursor cell proliferation. In addition to LPA1 being a marker of this important stem cell population, we also show that the corresponding ligand LPA is directly involved in the regulation of adult hippocampal precursor cell proliferation and neurogenesis, an effect that can be attributed to LPA signaling via the AKT and MAPK pathways. PMID:27050949

  1. The Innate Lymphoid Cell Precursor.

    PubMed

    Ishizuka, Isabel E; Constantinides, Michael G; Gudjonson, Herman; Bendelac, Albert

    2016-05-20

    The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages. PMID:27168240

  2. Precursor polymer compositions comprising polybenzimidazole

    SciTech Connect

    Klaehn, John R.; Peterson, Eric S.; Orme, Christopher J.

    2015-07-14

    Stable, high performance polymer compositions including polybenzimidazole (PBI) and a melamine-formaldehyde polymer, such as methylated, poly(melamine-co-formaldehyde), for forming structures such as films, fibers and bulky structures. The polymer compositions may be formed by combining polybenzimidazole with the melamine-formaldehyde polymer to form a precursor. The polybenzimidazole may be reacted and/or intertwined with the melamine-formaldehyde polymer to form the polymer composition. For example, a stable, free-standing film having a thickness of, for example, between about 5 .mu.m and about 30 .mu.m may be formed from the polymer composition. Such films may be used as gas separation membranes and may be submerged into water for extended periods without crazing and cracking. The polymer composition may also be used as a coating on substrates, such as metal and ceramics, or may be used for spinning fibers. Precursors for forming such polymer compositions are also disclosed.

  3. Serum levels of testosterone precursors, testosterone and estradiol in 10 animal species.

    PubMed

    Wichmann, U; Wichmann, G; Krause, W

    1984-05-01

    Blood levels of testosterone precursors, i.e. pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstendione, DHEA, and delta 5-androstendiol as well as testosterone and estradiol are measured in 10 animals each of 10 different species. The determination is done by radioimmunoassay with steroidspecific antibodies. Precursors of the delta 5-pathway (DHEA, androstendiol) are low in the red deer, dog, cat, rat and guinea pig. Precursors of the delta 4-pathway (progesterone, 17-hydroxprogesterone, androstendione) are lower in the bull, boar, ram, stallion and rabbit thus indicating a predominance of different pathways in the animal species studied herein. Pregnenolone concentrations are of equal height in all animals, testosterone is lowest in the cat and stallion. In the latter species the estradiol/testosterone ratio is spectacular high. PMID:6540697

  4. Nucleation precursors in protein crystallization

    PubMed Central

    Vekilov, Peter G.; Vorontsova, Maria A.

    2014-01-01

    Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Here, methods of detection and characterization of the precursors are reviewed: dynamic light scattering, atomic force microscopy and Brownian microscopy. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein-dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometres in size, the cluster population occupies from 10−7 to 10−3 of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e. undersaturated, solutions. The clusters exist owing to the conformation flexibility of the protein molecules, leading to exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. Investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics. PMID:24598910

  5. Tomato Fruits-A Platform for Metabolic Engineering of Terpenes.

    PubMed

    Gutensohn, M; Dudareva, N

    2016-01-01

    Terpenoids are a large and diverse class of plant metabolites including mono-, sesqui-, and diterpenes. They have numerous functions in basic physiological processes as well as the interaction of plants with their biotic and abiotic environment. Due to the tight regulation of biosynthetic pathways and the resulting limited natural availability of terpenes, there is a strong interest in increasing their production in plants by metabolic engineering for agricultural, pharmaceutical, and industrial applications. The tomato fruit system was developed as a platform for metabolic engineering of terpenes to overcome detrimental effects on overall plant growth and photosynthesis traits, which are affected when terpenoid engineering is performed in vegetative tissues. Here we describe how the use of fruit-specific promoters for transgene expression can avoid these unwanted effects. In addition, targeting the expression of the introduced terpene biosynthetic gene to fruit tissue can take advantage of the large precursor pool provided by the methylerythritol-phosphate (MEP) pathway, which is highly active during tomato fruit ripening to facilitate the accumulation of carotenoids. We also discuss how the production of high levels of target terpene compounds can be achieved in fruits by the expression of individual or a combination of (i) the MEP or mevalonic acid pathway enzymes, (ii) prenyltransferases, and/or (iii) terpene synthases. Finally, we provide a brief outline of how the emitted as well as internal pools of terpenes can be analyzed in transgenic tomato fruits. PMID:27480692

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Comprehensive Assessment of Transcriptional Regulation Facilitates Metabolic Engineering of Isoprenoid Accumulation in Arabidopsis1[OPEN

    PubMed Central

    Lange, Iris; Poirier, Brenton C.; Herron, Blake K.; Lange, Bernd Markus

    2015-01-01

    In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants. PMID:26282236

  8. Genetically engineered fusion of MAP-1 and factor H domains 1-5 generates a potent dual upstream inhibitor of both the lectin and alternative complement pathways.

    PubMed

    Nordmaj, Mie Anemone; Munthe-Fog, Lea; Hein, Estrid; Skjoedt, Mikkel-Ole; Garred, Peter

    2015-12-01

    Inhibition of the complement cascade has emerged as an option for treatment of a range of diseases. Mannose-binding lectin/ficolin/collectin-associated protein (MAP-1) is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway. The central regulator of the alternative pathway (AP) is complement factor H (FH). Our aim was to design a dual upstream inhibitor of both human lectin and APs by fusing MAP-1 with a part of FH. There were 2 different recombinant chimeric proteins comprising full-length human MAP-1 and the first 5 N-terminal domains of human FH designed. The FH domains were orientated either in the N- or C-terminal part of MAP-1. The complement inhibition potential in human serum was assessed. Both chimeric constructs displayed the characteristics of the native molecules and bound to the PRMs with an EC50 of ∼ 2 nM. However, when added to serum diluted 1:4 in a solid-phase functional assay, only the first 5 N-terminal domains of complement FH fused to the C-terminal part of full-length MAP-1 chimeric construct were able to combine inhibition of lectin and AP activation with an half maximal inhibitory concentration of ∼ 100 and 20 nM, respectively. No effect was seen on the classical pathway. Fusion of MAP-1 with FH domains represents a novel therapeutic approach for selective targeting upstream and central complement activation at sites of inflammation. PMID:26260032

  9. Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors

    PubMed Central

    Zakrzewski, Johannes L; Suh, David; Markley, John C; Smith, Odette M; King, Christopher; Goldberg, Gabrielle L; Jenq, Robert; Holland, Amanda M; Grubin, Jeremy; Cabrera-Perez, Javier; Brentjens, Renier J; Lu, Sydney X; Rizzuto, Gabrielle; Sant’Angelo, Derek B; Riviere, Isabelle; Sadelain, Michel; Heller, Glenn; Zúñiga-Pflücker, Juan Carlos; Lu, Chen; van den Brink, Marcel R M

    2009-01-01

    We present a strategy for adoptive immunotherapy using T-lineage committed lymphoid precursor cells generated by Notch1-based culture. We found that allogeneic T-cell precursors can be transferred to irradiated individuals irrespective of major histocompatibility complex (MHC) disparities and give rise to host-MHC restricted and host-tolerant functional allogeneic T cells, improving survival in irradiated recipients as well as enhancing anti-tumor responses. T-cell precursors transduced to express a chimeric receptor targeting hCD19 resulted in significant additional anti-tumor activity, demonstrating the feasibility of genetic engineering of these cells. We conclude that ex vivo generated MHC-disparate T-cell precursors from any donor can be used universally for ‘off-the-shelf’ immunotherapy, and can be further enhanced by genetic engineering for targeted immunotherapy. PMID:18376399

  10. Precursors in gamma-ray bursts detected by the Fermi-LAT and GBM

    NASA Astrophysics Data System (ADS)

    Zhu, Sylvia; Fermi Large Area Telescope Collaboration

    2015-04-01

    Many aspects of gamma-ray bursts (GRBs) remain mysterious more than 40 years after their initial discovery. However, observations of GRBs by the Fermi Large Area Telescope (LAT) and Gamma-ray Burst Monitor (GBM) have uncovered new information about the observed properties and the underlying physics. In a small minority (roughly 5-20%), a dim, temporally distinct precursor peak occurs before the brightest part of the prompt emission in the keV-MeV range. The origin of precursors is still unknown, and studies of precursors can probe the formation of the GRB central engine and/or the nature of the jets that produce the emission. We present a systematic search for precursor emission in LAT and GBM data, and the temporal and spectral properties and energetics of the population of GRBs with precursors.

  11. Metabolic engineering for isoprenoid-based biofuel production.

    PubMed

    Gupta, P; Phulara, S C

    2015-09-01

    Sustainable economic and industrial growth is the need of the hour and it requires renewable energy resources having better performance and compatibility with existing fuel infrastructure from biological routes. Isoprenoids (C ≥ 5) can be a potential alternative due to their diverse nature and physiochemical properties similar to that of petroleum based fuels. In the past decade, extensive research has been done to utilize metabolic engineering strategies in micro-organisms primarily, (i) to overcome the limitations associated with their natural and non-natural production and (ii) to develop commercially competent microbial strain for isoprenoid-based biofuel production. This review briefly describes the engineered isoprenoid biosynthetic pathways in well-characterized microbial systems for the production of several isoprenoid-based biofuels and fuel precursors. PMID:26095690

  12. Precursors of Short Gamma-Ray Bursts

    NASA Technical Reports Server (NTRS)

    Troja, E.; Rosswog, S.; Gehrels, N.

    2010-01-01

    We carried out a systematic search of precursors on the sample of short GRBs observed by Swift. We found that approx. 8-10% of short GRBs display such early episode of emission. One burst (GRB 090510) shows two precursor events, the former approx.13 s and the latter approx. 0.5 s before the GRB. We did not find any substantial difference between the precursor and the main GRB emission, and between short GRBs with and without precursors. We discuss possible mechanisms to reproduce the observed precursor emission within the scenario of compact object mergers. The implications of our results on quantum gravity constraints are also discussed.

  13. Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics*

    PubMed Central

    Lukacs, Peter; Gawali, Vaibhavkumar S.; Cervenka, Rene; Ke, Song; Koenig, Xaver; Rubi, Lena; Zarrabi, Touran; Hilber, Karlheinz; Stary-Weinzinger, Anna; Todt, Hannes

    2014-01-01

    Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels. PMID:24947510

  14. Metabolic engineering Corynebacterium glutamicum to produce triacylglycerols.

    PubMed

    Plassmeier, Jens; Li, Youyuan; Rueckert, Christian; Sinskey, Anthony J

    2016-01-01

    In this study, we metabolically engineered Corynebacterium glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (iii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis), and (iv) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38 ± 0.05 g/L intracellular fatty acids and 0.64 ± 0.09 g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular fatty acids content of 17.8 ± 0.5% of the dry cell. PMID:26645801

  15. Polymer precursors for ceramic composites

    NASA Technical Reports Server (NTRS)

    Hurwitz, Frances I.

    1986-01-01

    The fiber composite approach to reinforced ceramics provides the possibility of achieving ceramics with high fracture toughness relative to monolithics. Fabrication of ceramic composites, however, demands low processing temperatures to avoid fiber degradation. Formation of complex shapes further requires small diameter fibers as well as techniques for infiltrating the matrix between fibers. Polymers offer low temperature processability, control of rheology not available with ceramic powders, and should serve as precursors to matrix fibers. In recent years, a number of polysilanes and polysilezanes were investigated as potential presursors. A review of candidate polymers is presented, including recent studies of silsesquioxanes.

  16. Phospholipid biosynthesis in Candida albicans: Regulation by the precursors inositol and choline

    SciTech Connect

    Klig, L.S.; Friedli, L.; Schmid, E. )

    1990-08-01

    Phospholipid metabolism in the pathogenic fungus Candida albicans was examined. The phospholipid biosynthetic pathways of C. albicans were elucidated and were shown to be similar to those of Saccharomyces cerevisiae. However, marked differences were seen between these two fungi in the regulation of the pathways in response to exogenously provided precursors inositol and choline. In S. cerevisiae, the biosynthesis of phosphatidylcholine via methylation of phosphatidylethanolamine appears to be regulated in response to inositol and choline; provision of choline alone does not repress the activity of this pathway. The same pathway in C. albicans responds to the exogenous provision of choline. Possible explanations for the observed differences in regulation are discussed.

  17. Refining the pathway of carbide insertion into the nitrogenase M-cluster

    PubMed Central

    Wiig, Jared A.; Hu, Yilin; Ribbe, Markus W.

    2015-01-01

    Carbide insertion plays a pivotal role in the biosynthesis of M-cluster, the cofactor of nitrogenase. Previously, we proposed a carbide insertion pathway involving methyltransfer from SAM to a FeS precursor and hydrogen abstraction from this methyl group that initiates the radical-based precursor maturation. Here we demonstrate that the methyl group is transferred to a precursor-associated sulfur before hydrogen abstraction, thereby refining the initial steps of the carbide insertion pathway. PMID:26259825

  18. CathaCyc, a metabolic pathway database built from Catharanthus roseus RNA-Seq data.

    PubMed

    Van Moerkercke, Alex; Fabris, Michele; Pollier, Jacob; Baart, Gino J E; Rombauts, Stephane; Hasnain, Ghulam; Rischer, Heiko; Memelink, Johan; Oksman-Caldentey, Kirsi-Marja; Goossens, Alain

    2013-05-01

    The medicinal plant Madagascar periwinkle (Catharanthus roseus) synthesizes numerous terpenoid indole alkaloids (TIAs), such as the anticancer drugs vinblastine and vincristine. The TIA pathway operates in a complex metabolic network that steers plant growth and survival. Pathway databases and metabolic networks reconstructed from 'omics' sequence data can help to discover missing enzymes, study metabolic pathway evolution and, ultimately, engineer metabolic pathways. To date, such databases have mainly been built for model plant species with sequenced genomes. Although genome sequence data are not available for most medicinal plant species, next-generation sequencing is now extensively employed to create comprehensive medicinal plant transcriptome sequence resources. Here we report on the construction of CathaCyc, a detailed metabolic pathway database, from C. roseus RNA-Seq data sets. CathaCyc (version 1.0) contains 390 pathways with 1,347 assigned enzymes and spans primary and secondary metabolism. Curation of the pathways linked with the synthesis of TIAs and triterpenoids, their primary metabolic precursors, and their elicitors, the jasmonate hormones, demonstrated that RNA-Seq resources are suitable for the construction of pathway databases. CathaCyc is accessible online (http://www.cathacyc.org) and offers a range of tools for the visualization and analysis of metabolic networks and 'omics' data. Overlay with expression data from publicly available RNA-Seq resources demonstrated that two well-characterized C. roseus terpenoid pathways, those of TIAs and triterpenoids, are subject to distinct regulation by both developmental and environmental cues. We anticipate that databases such as CathaCyc will become key to the study and exploitation of the metabolism of medicinal plants. PMID:23493402

  19. Annealing of aromatic polyimide precursors

    NASA Technical Reports Server (NTRS)

    Wakelyn, N. T.

    1975-01-01

    A study has been made of the thermal behavior of polyimide precursors: an isomeric pair of crystals of the complex formed by p-phenylenediamine with the separated isomers of the di-isopropyl ester of pyromellitic acid. Specimens of this material were isothermally annealed in the temperature range 120 C to 170 C for periods of time up to 1 week. Although this temperature range is well below that customarily used for imidizations, the working hypothesis was that it would be more likely that a polymer embodying at least part of the precursor structure could be formed if the molecular motion was minimized to that actually required for the formation of the imide linkage. The progress of the annealing was followed by: infrared spectroscopy, differential thermal analysis, powder X-ray diffraction, and thermal gravimetric analysis. Single crystal X-ray analysis of the meta monomer yields a structure of chains of alternating acid and base and suggests that this monomer is amenable to polymerization with a minimum of geometrical disruption.

  20. A dynamic pathway analysis approach reveals a limiting futile cycle in N-acetylglucosamine overproducing Bacillus subtilis.

    PubMed

    Liu, Yanfeng; Link, Hannes; Liu, Long; Du, Guocheng; Chen, Jian; Sauer, Uwe

    2016-01-01

    Recent advances in genome engineering have further widened the gap between our ability to implement essentially any genetic change and understanding the impact of these changes on cellular function. We lack efficient methods to diagnose limiting steps in engineered pathways. Here, we develop a generally applicable approach to reveal limiting steps within a synthetic pathway. It is based on monitoring metabolite dynamics and simplified kinetic modelling to differentiate between putative causes of limiting product synthesis during the start-up phase of the pathway with near-maximal rates. We examine the synthetic N-acetylglucosamine (GlcNAc) pathway in Bacillus subtilis and find none of the acetyl-, amine- or glucose-moiety precursors to limit synthesis. Our dynamic metabolomics approach predicts an energy-dissipating futile cycle between GlcNAc6P and GlcNAc as the primary problem in the pathway. Deletion of the responsible glucokinase more than doubles GlcNAc productivity by restoring healthy growth of the overproducing strain. PMID:27324299

  1. Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds Along Two Different Reaction Pathways: Implica-tions for Engineering Bioscavengers

    SciTech Connect

    Wymore, Troy W; Langan, Paul; Smith, Jeremy C; Field, Martin J.; Parks, Jerry M

    2014-01-01

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ( bioscavengers ) is an emerging prophylactic approach to diminishing their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical (QM/MM) umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily due to the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed -propeller folds for more efficient degradation of OP compounds.

  2. Hydrolysis of DFP and the nerve agent (S)-sarin by DFPase proceeds along two different reaction pathways: implications for engineering bioscavengers.

    PubMed

    Wymore, Troy; Field, Martin J; Langan, Paul; Smith, Jeremy C; Parks, Jerry M

    2014-05-01

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ("bioscavengers") is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P-F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca(2+), donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds. PMID:24720808

  3. Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers

    PubMed Central

    2015-01-01

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents (“bioscavengers”) is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P–F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds. PMID:24720808

  4. Percolation-based precursors of transitions in extended systems

    PubMed Central

    Rodríguez-Méndez, Víctor; Eguíluz M, Víctor M.; Hernández-García, Emilio; Ramasco, José J.

    2016-01-01

    Abrupt transitions are ubiquitous in the dynamics of complex systems. Finding precursors, i.e. early indicators of their arrival, is fundamental in many areas of science ranging from electrical engineering to climate. However, obtaining warnings of an approaching transition well in advance remains an elusive task. Here we show that a functional network, constructed from spatial correlations of the system’s time series, experiences a percolation transition way before the actual system reaches a bifurcation point due to the collective phenomena leading to the global change. Concepts from percolation theory are then used to introduce early warning precursors that anticipate the system’s tipping point. We illustrate the generality and versatility of our percolation-based framework with model systems experiencing different types of bifurcations and with Sea Surface Temperature time series associated to El Niño phenomenon. PMID:27412567

  5. Percolation-based precursors of transitions in extended systems.

    PubMed

    Rodríguez-Méndez, Víctor; Eguíluz M, Víctor M; Hernández-García, Emilio; Ramasco, José J

    2016-01-01

    Abrupt transitions are ubiquitous in the dynamics of complex systems. Finding precursors, i.e. early indicators of their arrival, is fundamental in many areas of science ranging from electrical engineering to climate. However, obtaining warnings of an approaching transition well in advance remains an elusive task. Here we show that a functional network, constructed from spatial correlations of the system's time series, experiences a percolation transition way before the actual system reaches a bifurcation point due to the collective phenomena leading to the global change. Concepts from percolation theory are then used to introduce early warning precursors that anticipate the system's tipping point. We illustrate the generality and versatility of our percolation-based framework with model systems experiencing different types of bifurcations and with Sea Surface Temperature time series associated to El Niño phenomenon. PMID:27412567

  6. Percolation-based precursors of transitions in extended systems

    NASA Astrophysics Data System (ADS)

    Rodríguez-Méndez, Víctor; Eguíluz M, Víctor M.; Hernández-García, Emilio; Ramasco, José J.

    2016-07-01

    Abrupt transitions are ubiquitous in the dynamics of complex systems. Finding precursors, i.e. early indicators of their arrival, is fundamental in many areas of science ranging from electrical engineering to climate. However, obtaining warnings of an approaching transition well in advance remains an elusive task. Here we show that a functional network, constructed from spatial correlations of the system’s time series, experiences a percolation transition way before the actual system reaches a bifurcation point due to the collective phenomena leading to the global change. Concepts from percolation theory are then used to introduce early warning precursors that anticipate the system’s tipping point. We illustrate the generality and versatility of our percolation-based framework with model systems experiencing different types of bifurcations and with Sea Surface Temperature time series associated to El Niño phenomenon.

  7. A Fission-Powered Interstellar Precursor Mission

    SciTech Connect

    Lenard, R.X.; Lipinski, R.J.; West, J.L.; Wright, S.A.

    1998-10-28

    An 'interstellar precursor mission' lays the groundwork for eventual interstellar exploration by studying the interstellar medium and by stretching technologies that have potential application for eventual interstellar exploration. The numerous scientific goals for such a mission include generating a 3-D stellar map of our galaxy, studying Kuiper-belt and Oort cloud objects, and observing distant objects using the sun's gravitational lens as the primary of an enormous telescope. System equations are developed for a space tug which propels a 2500-kg scientific payload to 550 astronomical units in about 20 years. The tug to transport this payload uses electric propulsion with an Isp of 15,000 seconds and a fission reactor with a closed Brayton cycle to genemte the electricity. The optimal configuration may be to thrust for only about 6 years and then coast for the remaining 14 pars. This spacecraft does not require any physics breakthroughs or major advances in technology. The fission power syslem can be engineered and built by drawing upon known technologies developed for relatgd systems over the past 40 years. The tug system would eventually reach 1000 a.u in 33 years, and would have adequate power to relay large amounts of data throughout its journey.

  8. A fission-powered interstellar precursor mission

    SciTech Connect

    Lipinski, Ronald J.; Lenard, Roger X.; Wright, Steven A. West, John L.

    1999-01-01

    An {open_quotes}interstellar precursor mission{close_quotes} lays the groundwork for eventual interstellar exploration by studying the interstellar medium and by stretching technologies that have potential application for eventual interstellar exploration. The numerous scientific goals for such a mission include generating a 3-D stellar map of our galaxy, studying Kuiper-belt and Oort cloud objects, and observing distant objects using the sun{close_quote}s gravitational lens as the primary of an enormous telescope. System equations are developed for a space tug which propels a 2500-kg scientific payload to 550 astronomical units in about 20 years. The tug to transport this payload uses electric propulsion with an lsp of 15,000 seconds and a fission reactor with a closed Brayton cycle to generate the electricity. The optimal configuration may be to thrust for only about 6 years and then coast for the remaining 14 years. This spacecraft does not require any physics breakthroughs or major advances in technology. The fission power system can be engineered and built by drawing upon known technologies developed for related systems over the past 40 years. The tug system would eventually reach 1000 a.u in 33 years, and would have adequate power to relay large amounts of data throughout its journey. {copyright} {ital 1999 American Institute of Physics.}

  9. Cellular metabolism of unnatural sialic acid precursors.

    PubMed

    Pham, Nam D; Fermaintt, Charles S; Rodriguez, Andrea C; McCombs, Janet E; Nischan, Nicole; Kohler, Jennifer J

    2015-10-01

    Carbohydrates, in addition to their metabolic functions, serve important roles as receptors, ligands, and structural molecules for diverse biological processes. Insight into carbohydrate biology and mechanisms has been aided by metabolic oligosaccharide engineering (MOE). In MOE, unnatural carbohydrate analogs with novel functional groups are incorporated into cellular glycoconjugates and used to probe biological systems. While MOE has expanded knowledge of carbohydrate biology, limited metabolism of unnatural carbohydrate analogs restricts its use. Here we assess metabolism of SiaDAz, a diazirine-modified analog of sialic acid, and its cell-permeable precursor, Ac4ManNDAz. We show that the efficiency of Ac4ManNDAz and SiaDAz metabolism depends on cell type. Our results indicate that different cell lines can have different metabolic roadblocks in the synthesis of cell surface SiaDAz. These findings point to roles for promiscuous intracellular esterases, kinases, and phosphatases during unnatural sugar metabolism and provide guidance for ways to improve MOE. PMID:25957566

  10. Fractionation of glycoside precursors of aroma in grapes and wine.

    PubMed

    Mateo, J J; Gentilini, N; Huerta, T; Jiménez, M; Di Stefano, R

    1997-08-22

    The glycosides in mono-, di- and trihydroxylated terpene and norisoprenoid alcohols and also those in the related shikimate pathway have been isolated on C18 reversed-phase cartridges and then fractionated into classes of different polarity at increasing percentages of methanol. The benzyl alcohol glycosides are the most polar, while those of terpene monohydroxylated alcohols and geranic acid are the least polar. The terpene diols, linalool furanoid and pyranoid oxides and also norisoprenoid precursors show intermediate polarity and separate into well defined fractions according to their polarity. PMID:9299735

  11. Selective methanol or formate production during continuous CO₂ fermentation by the acetogen biocatalysts engineered via integration of synthetic pathways using Tn7-tool.

    PubMed

    Tyurin, Michael; Kiriukhin, Michael

    2013-09-01

    Methanol-resistant mutant acetogen Clostridium sp. MT1424 originally producing only 365 mM acetate from CO₂/CO was engineered to eliminate acetate production and spore formation using Cre-lox66/lox71-system to power subsequent methanol production via expressing synthetic methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, three copies of each, assembled in cluster and integrated to chromosome using Tn7-based approach. Production of 2.2 M methanol was steady (p < 0.005) in single step fermentations of 20 % CO₂ + 80 % H₂ blend (v/v) 25 day runs each in five independent repeats. If the integrated cluster comprised only three copies of formate dehydrogenase the respective recombinants produced 95 mM formate (p < 0.005) under the same conditions. For commercialization, the suggested source of inorganic carbon would be CO₂ waste of IGCC power plant. Hydrogen may be produced in situ via powered by solar panels electrolysis. PMID:23519429

  12. Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits1

    PubMed Central

    Lewinsohn, Efraim; Schalechet, Fernond; Wilkinson, Jack; Matsui, Kenji; Tadmor, Yaakov; Nam, Kyoung-Hee; Amar, Orit; Lastochkin, Elena; Larkov, Olga; Ravid, Uzi; Hiatt, William; Gepstein, Shimon; Pichersky, Eran

    2001-01-01

    The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as γ- and α-tocopherols, lycopene, β-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering. PMID:11706204

  13. Premarital precursors of marital infidelity.

    PubMed

    Allen, Elizabeth S; Rhoades, Galena Kline; Stanley, Scott M; Markman, Howard J; Williams, Tamara; Melton, Jessica; Clements, Mari L

    2008-06-01

    Premarital precursors of infidelity were evaluated in a sample of 72 couples (N = 144) who were taking part in a longitudinal study of marriage. Premarital self-report and observational data were compared for couples who experienced infidelity and those who did not experience infidelity in the first years of marriage. Couples in which the male engaged in marital infidelity were characterized, premaritally, by significantly lower male sexual satisfaction, lower male positive communication, and higher female invalidation, whereas couples in which the female went on to engage in infidelity were characterized, premaritally, by significantly lower levels of female positive communication, higher levels of male and female negative communication, and higher levels of male and female invalidation. Implications of the findings for future research on the prediction and prevention of infidelity are discussed. PMID:18605124

  14. Precursors for Carbon Nitride Synthesis

    SciTech Connect

    Prashantha, M.; Gopal, E. S. R.; Ramesh, K.

    2011-07-15

    Nano structured carbon nitride films were prepared by pyrolysis assisted chemical vapour deposition. Pyrrole (C{sub 4}H{sub 5}N), Pyrrolidine (C{sub 4}H{sub 9}N), Azabenzimidazole (C{sub 6}H{sub 5}N{sub 3}) and Triazine (C{sub 6}H{sub 15}N{sub 3}) were used as precursors. The vibrational modes observed for C-N and C = N from FTIR spectra confirms the bonding of nitrogen with carbon. XPS core level spectra of C 1s and N 1s also show the formation of bonding between carbon and nitrogen atoms. The nitrogen content in the prepared samples was found to be around 25 atomic %.

  15. Activation mechanism of thiol protease precursor from broiler chicken specific Staphylococcus aureus strain CH-91.

    PubMed

    Wladyka, Benedykt; Dubin, Grzegorz; Dubin, Adam

    2011-01-10

    Staphylococcus aureus strain CH-91 isolated from chicken dermatitis lesions produces large quantities of thiol protease implicated in disease formation. Observed overproduction requires efficient activation of the protease precursor which mechanism is studied here in detail. Wild type and mutant precursor forms are expressed in E. coli to test different hypotheses on the activation process. It is demonstrated that wild type precursor undergoes rapid autocatalytic processing whereas proteolytically inactive catalytic triad cysteine mutant (C(249)A) of the precursor is stable, but can be processed by minute quantities of active protease. It is concluded that limited intramolecular proteolysis is mainly responsible for efficient activation but, a positive feedback loop also contributes to the process. Both activation pathways allow efficient production of mature extracellular thiol protease, a putative virulence factor specific for avian strains of S. aureus. PMID:20598816

  16. Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities

    SciTech Connect

    Patnaik, R.; Spitzer, R.G.; Liao, J.C.

    1995-05-20

    The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during the condensation reaction between phosphenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. In the present study the authors confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters, In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown hare are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.

  17. Vitamins and aging: pathways to NAD+ synthesis.

    PubMed

    Denu, John M

    2007-05-01

    Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction. PMID:17482537

  18. Metabolic engineering of Corynebacterium glutamicum for methionine production by removing feedback inhibition and increasing NADPH level.

    PubMed

    Li, Ying; Cong, Hua; Liu, Bingnan; Song, Jinzhu; Sun, Xueying; Zhang, Junzheng; Yang, Qian

    2016-09-01

    Relieving the feedback inhibition of key enzymes in a metabolic pathway is frequently the first step of producer-strain construction by genetic engineering. However, the strict feedback regulation exercised by microorganisms in methionine biosynthesis often makes it difficult to produce methionine at a high level. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for methionine production. First, the metD gene encoding the methionine uptake system was deleted to achieve extracellular accumulation of methionine. Then, random mutagenesis was performed to remove feedback inhibition by metabolic end-products. The resulting strain C. glutamicum ENM-16 was further engineered to block or decrease competitive branch pathways by deleting the thrB gene and changing the start codon of the dapA gene, followed by point mutations of lysC (C932T) and pyc (G1A, C1372T) to increase methionine precursor supply. To enrich the NADPH pool, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway were mutated to reduce their sensitivity to inhibition by intracellular metabolites. The resultant strain C. glutamicum LY-5 produced 6.85 ± 0.23 g methionine l(-1) with substrate-specific yield (Y P/S) of 0.08 mol per mol of glucose after 72 h fed-batch fermentation. The strategies described here will be useful for construction of methionine engineering strains. PMID:27255137

  19. Functional analyses and treatment of precursor behavior.

    PubMed

    Najdowski, Adel C; Wallace, Michele D; Ellsworth, Carrie L; MacAleese, Alicia N; Cleveland, Jackie M

    2008-01-01

    Functional analysis has been demonstrated to be an effective method to identify environmental variables that maintain problem behavior. However, there are cases when conducting functional analyses of severe problem behavior may be contraindicated. The current study applied functional analysis procedures to a class of behavior that preceded severe problem behavior (precursor behavior) and evaluated treatments based on the outcomes of the functional analyses of precursor behavior. Responding for all participants was differentiated during the functional analyses, and individualized treatments eliminated precursor behavior. These results suggest that functional analysis of precursor behavior may offer an alternative, indirect method to assess the operant function of severe problem behavior. PMID:18468282

  20. 1994 Accident sequence precursor program results

    SciTech Connect

    Belles, R.J.; Cletcher, J.W.; Copinger, D.A.

    1996-01-01

    The Accident Sequence Precursor (ASP) Program involves the systematic review and evaluation of operational events that have occurred at light-water reactors to identify and categorize precursors to potential severe core damage accident sequences. The results of the ASP Program are published in an annual report. The most recent report, which contains the analyses of the precursors for 1994, is NUREG/CR-4674, Vols. 21 and 22, Precursors to Potential Severe Core Damage Accidents: 1994, A Status Report, published in December 1995. This article provides an overview of the ASP review and evaluation process and a summary of the results for 1994. 12 refs., 2 figs., 4 tabs.

  1. 1995 Accident Sequence Precursor (ASP) Program results

    SciTech Connect

    Muhlheim, M.D.; Belles, R.J.; Cletcher, J.W.; Copinger, D.A.; O`Reilly, P.D.; Dolan, B.W.; Minarick, J.W.

    1997-01-01

    The Accident Sequence Precursor (ASP) Program involves the systematic review and evaluation of operational events that have occurred at light-water reactors to identify and categorize precursors to potential severe core damage accident sequences. The results of the ASP Program are published in an annual report. The most recent report, which contains the precursors for 1995, is NUREG/CR-4674, Volume 23, Precursors to Potential Severe Core Damage Accidents: 1995, A Status Report, published in April 1997. This article provides an overview of the ASP review and evaluation process and a summary of the results for 1995.

  2. Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

    PubMed

    Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo; Zhou, Kang; Phon, Too Heng; Nielsen, Michael Lynge; Lantz, Anna Eliasson; Kielland-Brandt, Morten C; Stephanopoulos, Gregory

    2013-07-01

    Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C₆ glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol. PMID:23636690

  3. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.

    PubMed

    Yang, Chen; Gao, Xiang; Jiang, Yu; Sun, Bingbing; Gao, Fang; Yang, Sheng

    2016-09-01

    Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The (13)C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0g/L isoprene with a yield of 0.267g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms. PMID:27174717

  4. Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production.

    PubMed

    Chen, Binbin; Lee, Dong-Yup; Chang, Matthew Wook

    2015-09-01

    Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae. PMID:26164646

  5. Synaptogenesis: Modulation by Availability of Membrane Phospholipid Precursors.

    PubMed

    Cansev, Mehmet

    2016-09-01

    Phospholipids are the main constituents of brain membranes. Formation of new membranes requires that uridine, the omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA), and choline, the three circulating precursors of major phospholipids, interact via the Kennedy pathway. Supplementation of laboratory rodents with uridine, DHA and choline enhances the amount of brain membranes as well as synaptic proteins and increases the number of dendritic spines, the essential cytological precursor of new synapses. Hence, the newly formed membranes are utilized for synaptogenesis which underlies increased synaptic functioning evidenced by enhanced neurotransmission and cognition. In addition, this supplementation ameliorates the degeneration in a rat model of Parkinson's disease and mouse models of Alzheimer's disease (AD) when used in combination with several vitamins and cofactors. Hence, accumulating evidence shows that increasing the availability of phospholipid precursors, vitamins and cofactors to the brain through dietary supplementation enhances the formation of new synapses and provides protection under neurodegenerative conditions. The combination has been tested in clinical trials and a medication has been marketed for early-stage AD patients. PMID:27250850

  6. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

    PubMed

    Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

    2016-06-23

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids. PMID:27285777

  7. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway

    PubMed Central

    2016-01-01

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC=C + NC=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids. PMID:27285777

  8. Leading time domain seismic precursors

    NASA Astrophysics Data System (ADS)

    Boucouvalas, A. C.; Gkasios, M.; Keskebes, A.; Tselikas, N. T.

    2014-08-01

    The problem of predicting the occurrence of earthquakes is threefold. On one hand it is necessary to predict the date and magnitude of an earthquake, and on the other hand the location of the epicenter. In this work after a brief review of the state of earthquake prediction research, we report on a new leading time precursor for determining time onset of earthquake occurrence. We report the linking between earthquakes of the past with those which happen in the future via Fibonacci, Dual and Lucas numbers (FDL) numbers. We demonstrate it here with two example seed earthquakes at least 100 years old. Using this leading indicator method we can predict significant earthquake events >6.5R, with good accuracy approximately +- 1 day somewhere in the world. From a single seed we produce at least 100 trials simultaneously of which 50% are correct to +- 1day. The indicator is based on Fibonacci, Dual and Lucas numbers (FDL). This result hints that the log periodic FDL numbers are at the root of the understanding of the earthquake mechanism. The theory is based on the assumption that each occurred earthquake discontinuity can be thought of as a generating source of FDL time series. (The mechanism could well be linked to planetary orbits). When future dates are derived from clustering and convergence from previous strong earthquake dates at an FDL time distance, then we have a high probability for an earthquake to occur on that date. We set up a real time system which generates FDL time series from each previous significant earthquake (>7R) and we produce a year to year calendar of high probability earthquake dates. We have tested this over a number of years with considerable success. We have applied this technique for strong (>7R) earthquakes across the globe as well as on a restricted region such as the Greek geographic region where the magnitude is small (>4R-6.5R). In both cases the success of the method is impressive. It is our belief that supplementing this method with

  9. Precursors to potential severe core damage accidents: 1994, a status report. Volume 22: Appendix I

    SciTech Connect

    Belles, R.J.; Cletcher, J.W.; Copinger, D.A.; Vanden Heuvel, L.N.; Dolan, B.W.; Minarick, J.W. |

    1995-12-01

    Nine operational events that affected eleven commercial light-water reactors (LWRs) during 1994 and that are considered to be precursors to potential severe core damage are described. All these events had conditional probabilities of subsequent severe core damage greater than or equal to 1.0 {times} 10{sup {minus}6}. These events were identified by computer-screening the 1994 licensee event reports from commercial LWRs to identify those that could be potential precursors. Candidate precursors were then selected and evaluated in a process similar to that used in previous assessments. Selected events underwent engineering evaluation that identified, analyzed, and documented the precursors. Other events designated by the Nuclear Regulatory Commission (NRC) also underwent a similar evaluation. Finally, documented precursors were submitted for review by licensees and NRC headquarters and regional offices to ensure that the plant design and its response to the precursor were correctly characterized. This study is a continuation of earlier work, which evaluated 1969--1981 and 1984--1993 events. The report discusses the general rationale for this study, the selection and documentation of events as precursors, and the estimation of conditional probabilities of subsequent severe core damage for events. This document is bound in two volumes: Vol. 21 contains the main report and Appendices A--H; Vol. 22 contains Appendix 1.

  10. Precursors to potential severe core damage accidents: 1997 -- A status report. Volume 26

    SciTech Connect

    Belles, R.J.; Cletcher, J.W.; Copinger, D.A.; Muhlheim, M.D.; Dolan, B.W.; Minarick, J.W.

    1998-11-01

    This report describes the five operational events in 1997 that affected five commercial light-water reactors (LWRs) and that are considered to be precursors to potential severe core damage accidents. All these events had conditional probabilities of subsequent severe core damage greater than or equal to 1.0 {times} 10{sup {minus}6}. These events were identified by first computer-screening the 1997 licensee event reports from commercial LWRs to identify those events that could be precursors. Candidate precursors were selected and evaluated in a process similar to that used in previous assessments. Selected events underwent engineering evaluation that identified, analyzed, and documented the precursors. Other events designated by the Nuclear Regulatory Commission (NRC) also underwent a similar evaluation. Finally, documented precursors were submitted for review by licensees and NRC headquarters to ensure that the plant design and its response to the precursor were correctly characterized. This study is a continuation of earlier work, which evaluated 1969--1996 events. The report discusses the general rationale for this study, the selection and documentation of events as precursors, and the estimation of conditional probabilities of subsequent severe core damage for the events.

  11. Precursors to potential severe core damage accidents: 1995 A status report

    SciTech Connect

    Belles, R.J.; Cletcher, J.W.; Copinger, D.A.

    1997-04-01

    Ten operational events that affected 10 commercial light-water reactors during 1995 and that are considered to be precursors to potential severe core damage are described. All these events had conditional probabilities of subsequent severe core damage greater than or equal to 1.0 x 10{sup {minus}6}. These events were identified by first computer-screening the 1995 licensee event reports from commercial light-water reactors to identify those events that could potentially be precursors. Candidate precursors were selected and evaluated in a process similar to that used in previous assessments. Selected events underwent engineering evaluation that identified, analyzed, and documented the precursors. Other events designated by the Nuclear Regulatory Commission (NRC) also underwent a similar evaluation. Finally, documented precursors were submitted for review by licensees and NRC headquarters and regional offices to ensure the plant design and its response to the precursor were correctly characterized. This study is a continuation of earlier work, which evaluated 1969-1981 and 1984-1994 events. The report discusses the general rationale for this study, the selection and documentation of events as precursors, and the estimation of conditional probabilities of subsequent severe core damage for the events.

  12. Precursors to potential severe core damage accidents. A status report, 1982--1983

    SciTech Connect

    Forester, J.A.; Mitchell, D.B.; Whitehead, D.W.

    1997-04-01

    This study is a continuation of earlier work that evaluated 1969-1981 and 1984-1994 events affecting commercial light-water reactors. One-hundred nine operational events that affected 51 reactors during 1982 and 1983 and that are considered to be precursors to potential severe core damage are described. All these events had conditional probabilities of subsequent severe core damage greater than or equal to 1.0 x 10{sup {minus}6}. These events were identified by first computer screening the 1982-83 licensee event reports from commercial light-water reactors to select events that could be precursors to core damage. Candidates underwent engineering evaluation that identified, analyzed, and documented the precursors. This report discusses the general rationale for the study, the selection and documentation of events as precursors, and the estimation of conditional probabilities of subsequent severe core damage for the events.

  13. Nicotiana benthamiana as a Production Platform for Artemisinin Precursors

    PubMed Central

    van Herpen, Teun W. J. M.; Cankar, Katarina; Nogueira, Marilise; Bosch, Dirk; Bouwmeester, Harro J.; Beekwilder, Jules

    2010-01-01

    Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg−1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed. PMID:21151979

  14. Cloning and optimization of a nisin biosynthesis pathway for bacteriocin harvest.

    PubMed

    Kong, Wentao; Lu, Ting

    2014-07-18

    Nisin is an important antimicrobial peptide that has enormous applications in biotechnology. Despite many encouraging efforts, its overproduction has been a long-standing challenge due to the complexity of the underlying pathway and the difficulty in genetic modification of lactic acid bacteria. Here, we cloned an entire nisin biosynthesis pathway from a nisin-producing strain (Lactococcus lactis K29) into a plasmid and transplanted the plasmid into a nisin deficient strain Lactococcus lactis MG1363, resulting in successful heterologous expression of bioactive recombinant nisin. To increase nisin harvest, we also overexpressed nisA, a gene responsible for nisin precursor production, with a set of constitutive promoters. To further optimize nisin yield, we minimized the metabolic cost of the engineered strains by integrating nisA overexpression cassettes and the recombinant pathway into a single circuit. With our rational construction and optimization, our engineered optimized strain is able to produce bioactive nisin with a yield of 1098 IU/mL, which is more than six times higher than that of the original strain. PMID:24847677

  15. The Interrelationships of Mathematical Precursors in Kindergarten

    ERIC Educational Resources Information Center

    Cirino, Paul T.

    2011-01-01

    This study evaluated the interrelations among cognitive precursors across quantitative, linguistic, and spatial attention domains that have been implicated for math achievement in young children. The dimensionality of the quantity precursors was evaluated in 286 kindergarteners via latent variable techniques, and the contribution of precursors…

  16. Precursors in gas-liquid mixtures

    NASA Astrophysics Data System (ADS)

    Gasenko, V. G.; Gorelik, R. S.; Nakoryakov, V. E.; Timkin, L. S.

    2013-10-01

    Two types of precursors propagating at the speed of sound in a pure liquid have been revealed in the experiments on the evolution of pressure pulses in a gas-liquid mixture; at the same time, the main pressure pulse propagates at a low equilibrium speed of sound and its evolution is described by the Burgers-Korteweg-de Vries equation. The first high-frequency precursor is a complete analog of a classical Sommerfeld precursor, because the resonance dispersion equation for a bubble mixture coincides with that for insulators in the Lorentz model, and oscillates at a frequency close to the "plasma frequency." The second low-frequency precursor has been revealed in this work. The frequency of the low-frequency precursor is close to the resonance frequency of pulsations of bubbles, which is almost an order of magnitude lower than the frequency of the high-frequency precursor. The low-frequency precursor has a much larger amplitude of pulsations and smaller damping and is not described within the homogeneous model of the gas-liquid mixture. The observed phenomenon of low-frequency precursors has been explained within a simple heterogeneous model of a bubble liquid.

  17. Precursors and adjuncts of a lunar base

    NASA Technical Reports Server (NTRS)

    Burke, J. D.

    1988-01-01

    The automated, teleoperated, robotic and human-tended subsystems which will precede and accompany a lunar base program are discussed. The information about lunar conditions that can be provided by such precursors and adjuncts is addressed. The use of precursors and adjuncts for communications and navigation, for safety and survival, for lunar archives, and for entertainment and leisure is examined.

  18. Anorectic activity of prostaglandin precursors.

    PubMed Central

    Doggett, N S; Jawaharlal, K

    1977-01-01

    1 Intraperitoneal and intragastric (i.g.) administration of prostaglandin precursors arachidonic (2 mg, 15 mg/kg, i.p; 30 mg/kg i.g.), linolenic (100 mg/kg i.p.; 200 mg/kg, i.g.) and linoleic (15, 100 mg/kg, i.p.; 100 mg/kg, i.g.) acids to 22 h food-deprived rats inhibits food intake. 2 This anorexia is similar to that induced by prostaglandin F2alpha (1 mg/kg, i.p.). 3 At anorectic doses these fatty acids do not cause pyrexia, in fact arachidonic acid causes hypothermia. 4 Prior treatment with indomethacin (15 mg/kg) and paracetamol (50 mg/kg) specifically reverses the anorexia and the behavioural satiety induced by the three fatty acids, while not affecting prostaglandin F2alpha-induced suppression of food intake. 5 Results of the present experiments suggest that both physiological and pharmacological modification of appetite could be brought about through an effect on prostaglandin generating systems. PMID:890209

  19. Precursor films in wetting phenomena.

    PubMed

    Popescu, M N; Oshanin, G; Dietrich, S; Cazabat, A-M

    2012-06-20

    The spontaneous spreading of non-volatile liquid droplets on solid substrates poses a classic problem in the context of wetting phenomena. It is well known that the spreading of a macroscopic droplet is in many cases accompanied by a thin film of macroscopic lateral extent, the so-called precursor film, which emanates from the three-phase contact line region and spreads ahead of the latter with a much higher speed. Such films have been usually associated with liquid-on-solid systems, but in the last decade similar films have been reported to occur in solid-on-solid systems. While the situations in which the thickness of such films is of mesoscopic size are fairly well understood, an intriguing and yet to be fully understood aspect is the spreading of microscopic, i.e. molecularly thin, films. Here we review the available experimental observations of such films in various liquid-on-solid and solid-on-solid systems, as well as the corresponding theoretical models and studies aimed at understanding their formation and spreading dynamics. Recent developments and perspectives for future research are discussed. PMID:22627067

  20. Classification of precursors in nanoscale droplets

    NASA Astrophysics Data System (ADS)

    Isele-Holder, Rolf E.; Ismail, Ahmed E.

    2016-04-01

    Molecular precursors, ultrathin films that precede spreading droplets, are still far from being understood, despite intensive study. The inherent microscopic length scales make small-scale experimental techniques and molecular simulation ideal methods to study this phenomenon. Previous work on molecular precursors using nanoscale droplets, however, consistently suffers from incorrect measurement of the dimensions of the precursor film. An alternative method to accurately characterize the precursor film is presented here. In contrast to previous measures, this method (i) allows for easy detection and characterization of precursors and (ii) yields wetting dynamics that agree with experimental observations. Finally, we briefly comment on previous studies whose conclusions may merit reconsideration in light of the present work.

  1. The Interrelationships of Mathematical Precursors in Kindergarten

    PubMed Central

    Cirino, Paul T.

    2011-01-01

    This study evaluated the interrelations among cognitive precursors across quantitative, linguistic, and spatial attention domains that have been implicated for math achievement in young children. The dimensionality of the quantity precursors was evaluated in 286 Kindergarteners via latent variable techniques, and the contribution of precursors from each domain was established for small sums addition. Results showed a five factor structure for the quantity precursors with the major distinction between nonsymbolic and symbolic tasks. The overall model demonstrated good fit, and strong predictive power (R2 = 55%) for addition number combinations. Linguistic and spatial attention domains showed indirect relationships with outcomes, with their effects mediated by symbolic quantity measures. These results have implications for the measurement of mathematical precursors, and yield promise for predicting future math performance. PMID:21194711

  2. Method of texturing a superconductive oxide precursor

    DOEpatents

    DeMoranville, Kenneth L.; Li, Qi; Antaya, Peter D.; Christopherson, Craig J.; Riley, Jr., Gilbert N.; Seuntjens, Jeffrey M.

    1999-01-01

    A method of forming a textured superconductor wire includes constraining an elongated superconductor precursor between two constraining elongated members placed in contact therewith on opposite sides of the superconductor precursor, and passing the superconductor precursor with the two constraining members through flat rolls to form the textured superconductor wire. The method includes selecting desired cross-sectional shape and size constraining members to control the width of the formed superconductor wire. A textured superconductor wire formed by the method of the invention has regular-shaped, curved sides and is free of flashing. A rolling assembly for single-pass rolling of the elongated precursor superconductor includes two rolls, two constraining members, and a fixture for feeding the precursor superconductor and the constraining members between the rolls. In alternate embodiments of the invention, the rolls can have machined regions which will contact only the elongated constraining members and affect the lateral deformation and movement of those members during the rolling process.

  3. Engineering of Tomato Glandular Trichomes for the Production of Specialized Metabolites.

    PubMed

    Kortbeek, R W J; Xu, J; Ramirez, A; Spyropoulou, E; Diergaarde, P; Otten-Bruggeman, I; de Both, M; Nagel, R; Schmidt, A; Schuurink, R C; Bleeker, P M

    2016-01-01

    Glandular trichomes are specialized tissues on the epidermis of many plant species. On tomato they synthesize, store, and emit a variety of metabolites such as terpenoids, which play a role in the interaction with insects. Glandular trichomes are excellent tissues for studying the biosynthesis of specialized plant metabolites and are especially suitable targets for metabolic engineering. Here we describe the strategy for engineering tomato glandular trichomes, first with a transient expression system to provide proof of trichome specificity of selected promoters. Using microparticle bombardment, the trichome specificity of a terpene-synthase promoter could be validated in a relatively fast way. Second, we describe a method for stable expression of genes of interest in trichomes. Trichome-specific expression of another terpene-synthase promoter driving the yellow-fluorescence protein-gene is presented. Finally, we describe a case of the overexpression of farnesyl diphosphate synthase (FPS), specifically in tomato glandular trichomes, providing an important precursor in the biosynthetic pathway of sesquiterpenoids. FPS was targeted to the plastid aiming to engineer sesquiterpenoid production, but interestingly leading to a loss of monoterpenoid production in the transgenic tomato trichomes. With this example we show that trichomes are amenable to engineering though, even with knowledge of a biochemical pathway, the result of such engineering can be unexpected. PMID:27480691

  4. Engineering the central pathways in Lactococcus lactis: functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions.

    PubMed

    Papagianni, Maria; Avramidis, Nicholaos

    2012-08-10

    The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277 mM. The high maximum specific uptake rate of glucose of 1.8 mMs(-1)gCDW(-1) at 277 mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk 13-pkaC-aox1-transformant strain's fermentation at 277 mM glucose gave a final biomass concentration of 7.5 g/l and nisin activity of 14,000 IU/ml which is, compared to the parental strain's production levels at its optimal 55 mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin. PMID:22759530

  5. Methylerythritol 4-phosphate (MEP) pathway metabolic regulation.

    PubMed

    Banerjee, A; Sharkey, T D

    2014-08-01

    Covering: up to February 2014. The methylerythritol 4-phosphate (MEP) pathway is the recently discovered source of isoprenoid precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The precursors lead to the formation of various isoprenoids having diverse roles in different biological processes. Some isoprenoids have important commercial uses. Isoprene, which is made in surprising abundance by some trees, plays a significant role in atmospheric chemistry. The genetic regulation of this pathway has been discussed but information about metabolic regulation is just now becoming available. This review covers metabolic regulation of the MEP pathway starting from the inputs of carbon, ATP, and reducing power. A number of different regulatory mechanisms involving intermediate metabolites and/or enzymes are discussed. Some recent data indicate that methylerythritol cyclodiphosphate (MEcDP), the fifth intermediate of this pathway, is a key metabolite. It has been found to play diverse roles in regulation within the pathway as well as coordinating other biological processes by acting as a stress regulator in bacteria and possibly a retrograde signal from plastids to the nucleus in plants. In this review we focus on the role of the MEP pathway in photosynthetic leaves during isoprene emission and more generally the metabolic regulation of the MEP pathway in both plants and bacteria. PMID:24921065

  6. Metabolic Engineering VII Conference

    SciTech Connect

    Kevin Korpics

    2012-12-04

    The aims of this Metabolic Engineering conference are to provide a forum for academic and industrial researchers in the field; to bring together the different scientific disciplines that contribute to the design, analysis and optimization of metabolic pathways; and to explore the role of Metabolic Engineering in the areas of health and sustainability. Presentations, both written and oral, panel discussions, and workshops will focus on both applications and techniques used for pathway engineering. Various applications including bioenergy, industrial chemicals and materials, drug targets, health, agriculture, and nutrition will be discussed. Workshops focused on technology development for mathematical and experimental techniques important for metabolic engineering applications will be held for more in depth discussion. This 2008 meeting will celebrate our conference tradition of high quality and relevance to both industrial and academic participants, with topics ranging from the frontiers of fundamental science to the practical aspects of metabolic engineering.

  7. The Significance of Different Diacylgycerol Synthesis Pathways on Plant Oil Composition and Bioengineering

    PubMed Central

    Bates, Philip D.; Browse, John

    2012-01-01

    The unique properties of vegetable oils from different plants utilized for food, industrial feedstocks, and fuel is dependent on the fatty acid (FA) composition of triacylglycerol (TAG). Plants can use two main pathways to produce diacylglycerol (DAG), the immediate precursor molecule to TAG synthesis: (1) De novo DAG synthesis, and (2) conversion of the membrane lipid phosphatidylcholine (PC) to DAG. The FA esterified to PC are also the substrate for FA modification (e.g., desaturation, hydroxylation, etc.), such that the FA composition of PC-derived DAG can be substantially different than that of de novo DAG. Since DAG provides two of the three FA in TAG, the relative flux of TAG synthesis from de novo DAG or PC-derived DAG can greatly affect the final oil FA composition. Here we review how the fluxes through these two alternate pathways of DAG/TAG synthesis are determined and present evidence that suggests which pathway is utilized in different plants. Additionally, we present examples of how the endogenous DAG synthesis pathway in a transgenic host plant can produce bottlenecks for engineering of plant oil FA composition, and discuss alternative strategies to overcome these bottlenecks to produce crop plants with designer vegetable oil compositions. PMID:22783267

  8. Synthesis and structures of metal chalcogenide precursors

    NASA Technical Reports Server (NTRS)

    Hepp, Aloysius F.; Duraj, Stan A.; Eckles, William E.; Andras, Maria T.

    1990-01-01

    The reactivity of early transition metal sandwich complexes with sulfur-rich molecules such as dithiocarboxylic acids was studied. Researchers recently initiated work on precursors to CuInSe2 and related chalcopyrite semiconductors. Th every high radiation tolerance and the high absorption coefficient of CuInSe2 makes this material extremely attractive for lightweight space solar cells. Their general approach in early transition metal chemistry, the reaction of low-valent metal complexes or metal powders with sulfur and selenium rich compounds, was extended to the synthesis of chalcopyrite precursors. Here, the researchers describe synthesis, structures, and and routes to single molecule precursors to metal chalcogenides.

  9. A retrosynthetic biology approach to metabolic pathway design for therapeutic production

    PubMed Central

    2011-01-01

    Background Synthetic biology is used to develop cell factories for production of chemicals by constructively importing heterologous pathways into industrial microorganisms. In this work we present a retrosynthetic approach to the production of therapeutics with the goal of developing an in situ drug delivery device in host cells. Retrosynthesis, a concept originally proposed for synthetic chemistry, iteratively applies reversed chemical transformations (reversed enzyme-catalyzed reactions in the metabolic space) starting from a target product to reach precursors that are endogenous to the chassis. So far, a wider adoption of retrosynthesis into the manufacturing pipeline has been hindered by the complexity of enumerating all feasible biosynthetic pathways for a given compound. Results In our method, we efficiently address the complexity problem by coding substrates, products and reactions into molecular signatures. Metabolic maps are represented using hypergraphs and the complexity is controlled by varying the specificity of the molecular signature. Furthermore, our method enables candidate pathways to be ranked to determine which ones are best to engineer. The proposed ranking function can integrate data from different sources such as host compatibility for inserted genes, the estimation of steady-state fluxes from the genome-wide reconstruction of the organism's metabolism, or the estimation of metabolite toxicity from experimental assays. We use several machine-learning tools in order to estimate enzyme activity and reaction efficiency at each step of the identified pathways. Examples of production in bacteria and yeast for two antibiotics and for one antitumor agent, as well as for several essential metabolites are outlined. Conclusions We present here a unified framework that integrates diverse techniques involved in the design of heterologous biosynthetic pathways through a retrosynthetic approach in the reaction signature space. Our engineering methodology

  10. Neovascularization in Tissue Engineering

    PubMed Central

    Chung, Jennifer C.-Y.; Shum-Tim, Dominique

    2012-01-01

    A prerequisite for successful tissue engineering is adequate vascularization that would allow tissue engineering constructs to survive and grow. Angiogenic growth factors, alone and in combination, have been used to achieve this, and gene therapy has been used as a tool to enable sustained release of these angiogenic proteins. Cell-based therapy using endothelial cells and their precursors presents an alternative approach to tackling this challenge. These studies have occurred on a background of advancements in scaffold design and assays for assessing neovascularization. Finally, several studies have already attempted to translate research in neovascularization to clinical use in the blossoming field of therapeutic angiogenesis. PMID:24710553

  11. Sulfur Oxidation and Contrail Precursor Chemistry

    NASA Technical Reports Server (NTRS)

    DeWitt, Kenneth J.

    2003-01-01

    Sulfuric acid (H2SO4), formed in commercial aircraft operations via fuel-S (goes to) SO2 (goes to) SO3 (goes to) H2SO4 plays an important role in the formation of contrails. It is believed that the first step occurs inside the combustor, the second step in the engine exit nozzle, and the third step in the exhaust plume. Thus, measurements of the sulfur oxidation rates are critical to the understanding of contrail formation. Field measurements of contrails formed behind commercial aircraft indicate that significantly greater conversion of fuel-bound sulfur to sulfate aerosol occurs than can be explained by our current knowledge of contrail physics and chemistry. The conversion of sulfur from S(IV) to S(VI) oxidation state, required for sulfate aerosol formation, is thermodynamically favored for the conditions that exist within jet engines but is kinetically disfavored. The principal reaction pathway is O+SO2+M (goes to) SO3+M. The rates of this reaction have never been measured in the temperature and pressure regimes available to aircraft operation. In the first year (FY02) of this project, we performed a series of experiments to elucidate the rate information for the O+SO2+M (goes to) SO3+M reaction. The work performed is described following the proposed work plan. Because we used the H2/O2 system for an O-atom source and rate coefficients were obtained via computer simulation, construction of a reaction mechanism and either recalculation or estimation of thermodynamic properties of H(x)SO(y) species are described first.

  12. A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factories

    PubMed Central

    Fisher, Amanda K.; Freedman, Benjamin G.; Bevan, David R.; Senger, Ryan S.

    2014-01-01

    Microbial cell factories (MCFs) are of considerable interest to convert low value renewable substrates to biofuels and high value chemicals. This review highlights the progress of computational models for the rational design of an MCF to produce a target bio-commodity. In particular, the rational design of an MCF involves: (i) product selection, (ii) de novo biosynthetic pathway identification (i.e., rational, heterologous, or artificial), (iii) MCF chassis selection, (iv) enzyme engineering of promiscuity to enable the formation of new products, and (v) metabolic engineering to ensure optimal use of the pathway by the MCF host. Computational tools such as (i) de novo biosynthetic pathway builders, (ii) docking, (iii) molecular dynamics (MD) and steered MD (SMD), and (iv) genome-scale metabolic flux modeling all play critical roles in the rational design of an MCF. Genome-scale metabolic flux models are of considerable use to the design process since they can reveal metabolic capabilities of MCF hosts. These can be used for host selection as well as optimizing precursors and cofactors of artificial de novo biosynthetic pathways. In addition, recent advances in genome-scale modeling have enabled the derivation of metabolic engineering strategies, which can be implemented using the genomic tools reviewed here as well. PMID:25379147

  13. AIP Career Pathways

    NASA Astrophysics Data System (ADS)

    Palchak, Amanda

    2012-02-01

    American Institute of Physics (AIP) Career Pathways is a new project funded by the National Science Foundation. One of the goals of AIP Career Pathways is to prepare students to compete for Science, Technology, Engineering, and Mathematics (STEM) careers with a bachelor's degree in physics. In order to do so, I reviewed and compiled useful resources on finding a STEM career with a bachelor's degree in physics. These resources not only supply the job seeker with job postings in STEM careers but also provide them with information on resumes, interviewing skills, and networking. Recently at the 2011 Industrial Physics Forum, I interviewed companies in the private sector to obtain a unique perspective on what types of skills potential employers expect an applicant to posses with a bachelor's degree in physics. Ultimately, these components will be used as supplements at student career workshops held at annual Society of Physics Students Zone Meetings.

  14. A biosynthetic pathway for anandamide

    PubMed Central

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Osei-Hyiaman, Douglas; Razdan, Raj; Gong, Qian; Chan, Andrew C.; Zhou, Zhifeng; Huang, Bill X.; Kim, Hee-Yong; Kunos, George

    2006-01-01

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released “on demand” by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE–PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE–PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target. PMID:16938887

  15. Biochemical Removal of HAP Precursors From Coal

    SciTech Connect

    Olson, G.; Tucker, L.; Richards, J.

    1997-07-01

    This project addresses DOE`s interest in advanced concepts for controlling emissions of air toxics from coal-fired utility boilers. We are determining the feasibility of developing a biochemical process for the precombustion removal of substantial percentages of 13 inorganic hazardous air pollutant (HAP) precursors from coal. These HAP precursors are Sb, As, Be, Cd, Cr, Cl, Co, F, Pb, Hg, Mn, Ni, and Se. Although rapid physical coal cleaning is done routinely in preparation plants, biochemical processes for removal of HAP precursors from coal potentially offer advantages of deeper cleaning, more specificity, and less coal loss. Compared to chemical processes for coal cleaning, biochemical processes potentially offer lower costs and milder process conditions. Pyrite oxidizing bacteria, most notably Thiobacillusferrooxidans, are being evaluated in this project for their ability to remove HAP precursors from U.S. coals.

  16. Progress in molecular precursors for electronic materials

    SciTech Connect

    Buhro, W.E.

    1996-09-01

    Molecular-precursor chemistry provides an essential underpinning to all electronic-materials technologies, including photovoltaics and related areas of direct interest to the DOE. Materials synthesis and processing is a rapidly developing field in which advances in molecular precursors are playing a major role. This article surveys selected recent research examples that define the exciting current directions in molecular-precursor science. These directions include growth of increasingly complex structures and stoichiometries, surface-selective growth, kinetic growth of metastable materials, growth of size-controlled quantum dots and quantum-dot arrays, and growth at progressively lower temperatures. Continued progress in molecular-precursor chemistry will afford precise control over the crystal structures, nanostructures, and microstructures of electronic materials.

  17. Explorations Precursor Robotic Missions (xPRM)

    NASA Video Gallery

    Jay Jenkins delivers a presentation from the Exploration Precursor Robotic Missions (xPRM) study team on May 25, 2010, at the NASA Exploration Enterprise Workshop held in Galveston, TX. The purpose...

  18. AN INACTIVE PRECURSOR OF STREPTOCOCCAL PROTEINASE

    PubMed Central

    Elliott, Stuart D.; Dole, Vincent P.

    1947-01-01

    1. Streptococcal proteinase is derived from an inactive precursor found in culture filtrates of proteinase-producing streptococci. 2. The precursor can be converted into the proteinase by low concentrations of trypsin but not by chymotrypsin. 3. In cultures grown in suitable media the conversion of precursor to proteinase is effected autocatalytically. This reaction occurs under reducing conditions and is initiated by active proteinase present in low concentrations with the precursor. 4. The autocatalytic reaction is suppressed or retarded by conditions which decrease the activity of the proteinase, e.g. by growing cultures at 22°C. instead of at 37°C. or by growing them under markedly aerobic conditions. It is also retarded in the presence of casein. PMID:19871616

  19. [Structure and functions of bacterial proteinase precursors].

    PubMed

    Serkina, A V; Shevelev, A B; Chestukhina, G G

    2001-01-01

    The data on the precursors of bacterial proteases were generalized. The structure and special features of processing of the precursors of bacillary subtilisins, the alpha-lytic protease from Lysobacter enzymogenes and the related chymotrypsin-like proteases from Streptomyces griseus, and the metalloproteases from bacilli and Pseudomonas aeruginosa were discussed. The approaches to producing the precursors and the protease propeptides and to in vitro characterizing them were particularly analyzed. The following physiological functions of the propeptides within the protease precursors were considered probable: (a) inhibition of the proteases to protect the host cells from the proteolytic damage; (b) participation in the folding of the mature enzyme; and (c) providing for the protease interaction with the bacterial cell surveillance mechanisms, including protease translocation through the cell wall. PMID:11641907

  20. Theoretical study on the initial reaction mechanisms of ansa-metallocene zirconium precursor on hydroxylated Si(1 0 0) surface.

    PubMed

    Zhou, Guangfen; Ren, Jie; Zhang, Shaowen

    2016-05-01

    The initial reaction mechanisms for depositing ZrO2 thin films using ansa-metallocene zirconium (Cp2CMe2)ZrMe2 precursor were studied by density functional theory (DFT) calculations. The (Cp2CMe2)ZrMe2 precursor could be absorbed on the hydroxylated Si(1 0 0) surface via physisorption. Possible reaction pathways of (Cp2CMe2)ZrMe2 were proposed. For each reaction, the activation energies and reaction energies were compared, and stationary points along the reaction pathways were shown. In addition, the influence of dispersion effects on the reactions was evaluated by non-local dispersion corrected DFT calculations. PMID:27138945

  1. Adult neurogenesis in the crayfish brain: proliferation, migration and possible origin of precursor cells

    PubMed Central

    Zhang, Y.; Allodi, S.; Sandeman, D.C.; Beltz, B.S.

    2015-01-01

    The birth of new neurons and their incorporation into functional circuits in the adult brain is a characteristic of many vertebrate and invertebrate organisms, including decapod crustaceans. Precursor cells maintaining life-long proliferation in the brains of crayfish (Procambarus clarkii, Cherax destructor) and clawed lobsters (Homarus americanus) reside within a specialized niche on the ventral surface of the brain; their daughters migrate to two proliferation zones along a stream formed by processes of the niche precursors. Here they divide again, finally producing interneurons in the olfactory pathway. The present studies in P. clarkii explore (1) differential proliferative activity among the niche precursor cells with growth and aging, (2) morphological characteristics of cells in the niche and migratory streams, and (3) aspects of the cell cycle in this lineage. Morphologically symmetrical divisions of neuronal precursor cells were observed in the niche near where the migratory streams emerge, as well as in the streams and proliferation zones. The nuclei of migrating cells elongate and undergo shape changes consistent with nucleokinetic movement. LIS1, a highly conserved dynein-binding protein, is expressed in cells in the migratory stream and neurogenic niche, implicating this protein in the translocation of crustacean brain neuronal precursor cells. Symmetrical divisions of the niche precursors and migration of both daughters raised the question of how the niche precursor pool is replenished. We present here preliminary evidence for an association between vascular cells and the niche precursors, which may relate to the life-long growth and maintenance of the crustacean neurogenic niche. PMID:19294644

  2. Adult neurogenesis in the crayfish brain: proliferation, migration, and possible origin of precursor cells.

    PubMed

    Zhang, Yi; Allodi, Silvana; Sandeman, David C; Beltz, Barbara S

    2009-06-01

    The birth of new neurons and their incorporation into functional circuits in the adult brain is a characteristic of many vertebrate and invertebrate organisms, including decapod crustaceans. Precursor cells maintaining life-long proliferation in the brains of crayfish (Procambarus clarkii, Cherax destructor) and clawed lobsters (Homarus americanus) reside within a specialized niche on the ventral surface of the brain; their daughters migrate to two proliferation zones along a stream formed by processes of the niche precursors. Here they divide again, finally producing interneurons in the olfactory pathway. The present studies in P. clarkii explore (1) differential proliferative activity among the niche precursor cells with growth and aging, (2) morphological characteristics of cells in the niche and migratory streams, and (3) aspects of the cell cycle in this lineage. Morphologically symmetrical divisions of neuronal precursor cells were observed in the niche near where the migratory streams emerge, as well as in the streams and proliferation zones. The nuclei of migrating cells elongate and undergo shape changes consistent with nucleokinetic movement. LIS1, a highly conserved dynein-binding protein, is expressed in cells in the migratory stream and neurogenic niche, implicating this protein in the translocation of crustacean brain neuronal precursor cells. Symmetrical divisions of the niche precursors and migration of both daughters raised the question of how the niche precursor pool is replenished. We present here preliminary evidence for an association between vascular cells and the niche precursors, which may relate to the life-long growth and maintenance of the crustacean neurogenic niche. PMID:19294644

  3. Summer 2014 Pathways Report

    NASA Technical Reports Server (NTRS)

    Hand, Zachary

    2014-01-01

    Over the summer I had the exciting opportunity to work for NASA at the Kennedy Space Center as a Mission Assurance Engineering intern. When I was offered a position in mission assurance for the Safety and Mission Assurance directorate's Launch Services Division, I didn't really know what I would be doing, but I knew it would be an excellent opportunity to learn and grow professionally. In this report I will provide some background information on the Launch Services Division, as well as detail my duties and accomplishments during my time as an intern. Additionally, I will relate the significance of my work experience to my current academic work and future career goals. This report contains background information on Mission Assurance Engineering, a description of my duties and accomplishments over the summer of 2014, and relates the significance of my work experience to my school work and future career goals. It is a required document for the Pathways program.

  4. Hydridosiloxanes as precursors to ceramic products

    DOEpatents

    Blum, Yigal D.; Johnson, Sylvia M.; Gusman, Michael I.

    1997-01-01

    A method is provided for preparing ceramic precursors from hydridosiloxane starting materials and then pyrolyzing these precursors to give rise to silicious ceramic materials. Si--H bonds present in the hydridosiloxane starting materials are catalytically activated, and the activated hydrogen atoms may then be replaced with nonhydrogen substituents. These preceramic materials are pyrolyzed in a selected atmosphere to give the desired ceramic product. Ceramic products which may be prepared by this technique include silica, silicon oxynitride, silicon carbide, metal silicates, and mullite.

  5. Hydridosiloxanes as precursors to ceramic products

    DOEpatents

    Blum, Y.D.; Johnson, S.M.; Gusman, M.I.

    1997-06-03

    A method is provided for preparing ceramic precursors from hydridosiloxane starting materials and then pyrolyzing these precursors to give rise to silicious ceramic materials. Si-H bonds present in the hydridosiloxane starting materials are catalytically activated, and the activated hydrogen atoms may then be replaced with nonhydrogen substituents. These preceramic materials are pyrolyzed in a selected atmosphere to give the desired ceramic product. Ceramic products which may be prepared by this technique include silica, silicon oxynitride, silicon carbide, metal silicates, and mullite.

  6. Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae.

    PubMed

    Song, Ji-Yoon; Park, Joon-Song; Kang, Chang Duk; Cho, Hwa-Young; Yang, Dongsik; Lee, Seunghyun; Cho, Kwang Myung

    2016-05-01

    Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142g/L with production yield of 0.89g/g and productivity of 3.55gL(-1)h(-1) under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast. PMID:26384570

  7. Pathways of Transport Protein Evolution: Recent Advances

    PubMed Central

    Lam, Vincent H.; Lee, Jong-Hoon; Silverio, Abe; Chan, Henry; Gomolplitinant, Kenny M.; Povolotsky, Tatyana L.; Orlova, Ekaterina; Sun, Eric I.; Welliver, Carl H.; Saier, Milton H.

    2014-01-01

    We herein report recent advances in our understanding of transport protein evolution. The Drug-Metabolite Transporter (DMT) superfamily (TC# 2.A.7) arose from a 2TMS precursor to give 4TMS proteins which then added one and duplicated to give 10. The proposed pathway is 2 –> 4 –> 5 –> 10. This superfamily provides a rare example where all proposed topological intermediates in this evolutionary pathway have been identified in current protein databases. Another family, the Oligopeptide Transporter (OPT) family (TC# 2.A.67), also started with a 2 TMS peptide precursor, but it followed the pathway: Only 16 and 17 TMS OPT family members have been identified in current databases. The TRIC family of K+ channels, characterized in animals, arose via the pathway: where the seventh TMS was added c-terminally to the 6 TMS precursor that resulted from a 3 TMS duplication. Surprisingly, animal TRIC channels proved to have numerous 7 TMS homologues in prokaryotes, none of which had been identified previously. We found that two families of integral membrane proteins gave rise to multiple current topological types. Members of the SdpC killer factor immunity protein family, SdpI (TC# 9.A.32) probably arose via the pathway: while members of the Heme Handling Protein (HHP) Family (TC# 9.B.14) arose via the pathway: Predictions are also made for an evolutionary pathway giving rise to the seven topological types of P-type ATPases so far identified in the P-ATPase superfamily. Finally, the ubiquitous CDF carriers (TC# 1.A.4) of 6TMSs probably gave rise to CRAC channels of 4TMSs by loss of the first two TMSs an unusual example of retroevolution. PMID:21194372

  8. Combining Chemoinformatics with Bioinformatics: In Silico Prediction of Bacterial Flavor-Forming Pathways by a Chemical Systems Biology Approach “Reverse Pathway Engineering”

    PubMed Central

    Liu, Mengjin; Bienfait, Bruno; Sacher, Oliver; Gasteiger, Johann; Siezen, Roland J.; Nauta, Arjen; Geurts, Jan M. W.

    2014-01-01

    The incompleteness of genome-scale metabolic models is a major bottleneck for systems biology approaches, which are based on large numbers of metabolites as identified and quantified by metabolomics. Many of the revealed secondary metabolites and/or their derivatives, such as flavor compounds, are non-essential in metabolism, and many of their synthesis pathways are unknown. In this study, we describe a novel approach, Reverse Pathway Engineering (RPE), which combines chemoinformatics and bioinformatics analyses, to predict the “missing links” between compounds of interest and their possible metabolic precursors by providing plausible chemical and/or enzymatic reactions. We demonstrate the added-value of the approach by using flavor-forming pathways in lactic acid bacteria (LAB) as an example. Established metabolic routes leading to the formation of flavor compounds from leucine were successfully replicated. Novel reactions involved in flavor formation, i.e. the conversion of alpha-hydroxy-isocaproate to 3-methylbutanoic acid and the synthesis of dimethyl sulfide, as well as the involved enzymes were successfully predicted. These new insights into the flavor-formation mechanisms in LAB can have a significant impact on improving the control of aroma formation in fermented food products. Since the input reaction databases and compounds are highly flexible, the RPE approach can be easily extended to a broad spectrum of applications, amongst others health/disease biomarker discovery as well as synthetic biology. PMID:24416282

  9. Improving peppermint essential oil yield and composition by metabolic engineering

    PubMed Central

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R.; Turner, Glenn W.; Davis, Edward M.; Lange, Iris; Baker, Raymond C.; Boydston, Rick A.; Croteau, Rodney B.

    2011-01-01

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost. PMID:21963983

  10. Improving peppermint essential oil yield and composition by metabolic engineering.

    PubMed

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R; Turner, Glenn W; Davis, Edward M; Lange, Iris; Baker, Raymond C; Boydston, Rick A; Croteau, Rodney B

    2011-10-11

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost. PMID:21963983

  11. Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway.

    PubMed

    Hirokawa, Yasutaka; Maki, Yuki; Tatsuke, Tsuneyuki; Hanai, Taizo

    2016-03-01

    Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B12-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B12, suggesting that the intrinsic pseudovitamin B12 served as a substitute of coenzyme B12. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study. PMID:26769097

  12. The role of amorphous precursors in the crystallization of La and Nd carbonates

    NASA Astrophysics Data System (ADS)

    Vallina, Beatriz; Rodriguez-Blanco, Juan Diego; Brown, Andrew P.; Blanco, Jesus A.; Benning, Liane G.

    2015-07-01

    Crystalline La and Nd carbonates can be formed from poorly-ordered nanoparticulate precursors, termed amorphous lanthanum carbonate (ALC) and amorphous neodymium carbonate (ANC). When reacted in air or in aqueous solutions these precursors show highly variable lifetimes and crystallization pathways. We have characterized these precursors and the crystallization pathways and products with solid-state, spectroscopic and microscopic techniques to explain the differences in crystallization mechanisms between the La and Nd systems. ALC and ANC consist of highly hydrated, 10-20 nm spherical nanoparticles with a general formula of REE2(CO3)3.5H2O (REE = La, Nd). The stabilities differ by ~2 orders of magnitude, with ANC being far more stable than ALC. This difference is due to the Nd3+ ion having a far higher hydration energy compared to the La3+ ion. This, together with temperature and reaction times, leads to clear differences not only in the kinetics and mechanisms of crystallization of the amorphous precursor La- and Nd-carbonate phases but also in the resulting crystallite sizes and morphologies of the end products. All crystalline La and Nd carbonates developed spherulitic morphologies when crystallization occurred from hydrous phases in solution at temperatures above 60 °C (La system) and 95 °C (Nd system). We suggest that spherulitic growth occurs due to a rapid breakdown of the amorphous precursors and a concurrent rapid increase in supersaturation levels in the aqueous solution. The kinetic data show that the crystallization pathway for both La and Nd carbonate systems is dependent on the reaction temperature and the ionic potential of the REE3+ ion.Crystalline La and Nd carbonates can be formed from poorly-ordered nanoparticulate precursors, termed amorphous lanthanum carbonate (ALC) and amorphous neodymium carbonate (ANC). When reacted in air or in aqueous solutions these precursors show highly variable lifetimes and crystallization pathways. We have

  13. Natural Modulators of Amyloid-Beta Precursor Protein Processing

    PubMed Central

    Zhang, Can; Tanzi, Rudolph E.

    2013-01-01

    Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the primary cause of dementia, with no cure currently available. The pathogenesis of AD is believed to be primarily driven by Aβ, the principal component of senile plaques. Aβ is an ~4 kDa peptide generated from the amyloid-β precursor protein (APP) through proteolytic secretases. Natural products, particularly those utilized in traditional Chinese medicine (TCM), have a long history alleviating common clinical disorders, including dementia. However, the cell/molecular pathways mediated by these natural products are largely unknown until recently when the underlying molecular mechanisms of the disorders begin to be elucidated. Here, the mechanisms with which natural products modulate the pathogenesis of AD are discussed, in particular, by focusing on their roles in the processing of APP. PMID:22998566

  14. Hydrogen platelets in crystalline silicon - Precursors for the "smart cut"

    SciTech Connect

    Reboredo, Fernando A; Pantelides, Sokrates T

    1999-01-01

    High concentrations of H in crystalline Si are known to induce planar defects (platelets), primarily in the (100) and the (Ill)planes. These platelets are precursors for the so-called "smart cut" technique for fabricating Silicon-On-insulator (SOI) structures. The atomic-scale mechanisms for the nucleation and growth of the H-induced platelets and many other experimental observations have remained elusive. We review recent extensive first-principles calculations in terms of which a comprehensive picture of platelet nucleation and growth has emerged. The theory naturally accounts for the observed preference for (111) and (100) planes, for H release and trapping of H-2 molecules, and other observations. The platelets are arrays of second-neighbor hydrogenated vacancies that do not bind to each other, but their formation is due to energetically preferred reaction pathways.

  15. Accident Precursor Analysis and Management: Reducing Technological Risk Through Diligence

    NASA Technical Reports Server (NTRS)

    Phimister, James R. (Editor); Bier, Vicki M. (Editor); Kunreuther, Howard C. (Editor)

    2004-01-01

    Almost every year there is at least one technological disaster that highlights the challenge of managing technological risk. On February 1, 2003, the space shuttle Columbia and her crew were lost during reentry into the atmosphere. In the summer of 2003, there was a blackout that left millions of people in the northeast United States without electricity. Forensic analyses, congressional hearings, investigations by scientific boards and panels, and journalistic and academic research have yielded a wealth of information about the events that led up to each disaster, and questions have arisen. Why were the events that led to the accident not recognized as harbingers? Why were risk-reducing steps not taken? This line of questioning is based on the assumption that signals before an accident can and should be recognized. To examine the validity of this assumption, the National Academy of Engineering (NAE) undertook the Accident Precursors Project in February 2003. The project was overseen by a committee of experts from the safety and risk-sciences communities. Rather than examining a single accident or incident, the committee decided to investigate how different organizations anticipate and assess the likelihood of accidents from accident precursors. The project culminated in a workshop held in Washington, D.C., in July 2003. This report includes the papers presented at the workshop, as well as findings and recommendations based on the workshop results and committee discussions. The papers describe precursor strategies in aviation, the chemical industry, health care, nuclear power and security operations. In addition to current practices, they also address some areas for future research.

  16. Radon earthquake precursor: A short review

    NASA Astrophysics Data System (ADS)

    Woith, H.

    2015-05-01

    More than 100 publications reporting radon anomalies to precede earthquakes were evaluated. A clear apparent negative correlation between the number of reported anomalies and the published length of the timeseries is evident. 19% of all timeseries are longer than 5 years, characterized by a precursor rate of less than one precursor per year, the extreme case being 1 anomaly in 18 years of monitoring. Contrary, precursor rates between 1 and more than 10 precursors per year stem from published timeseries shorter than 3 years. Nearly 50% of the timeseries contain exactly one radon anomaly, independent of the length of the observation interval. Generally, the number of anomalies is about 5 times higher at sites where radon is measured in soil air as compared to radon in groundwater. In conclusion: (i) significant radon anomalies exist, and (ii) seismo-tectonically induced radon anomalies probably exist. But, radon anomalies of non-tectonic origin also exist and may look strikingly similar to tectonic ones. Thus, presumably only a fraction of all reported radon precursors are real in the sense that they are physically related to the preparation process of an impending earthquake.

  17. Precursors of storage proteins in Lupinus angustifolius.

    PubMed Central

    Gayler, K R; Boadle, B G; Snook, M; Johnson, E D

    1984-01-01

    The proteins that are synthesized during differentiation and development in the cotyledons of Lupinus angustifolius L. were characterized both in situ and after purification. The proteins present in situ were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and subjected to 'Western'-blot analysis to identify immunologically related polypeptides. The major storage proteins of the lupin, conglutins alpha and beta, were both present in juvenile tissue only as higher Mr precursors. For conglutin beta, a family of at least three polypeptides of Mr 66 000-72 000 accumulated during the earliest phases of protein synthesis in the developing cotyledon (20-28 days after flowering). Later in development each of these polypeptides disappeared and there was the concurrent appearance in the cotyledon of the lower-Mr fragments characteristic of mature conglutin beta. For conglutin alpha, an equivalent family of precursor polypeptides of Mr 60 000-83 000 was detected. Multiple internal sites for proteolytic cleavage of all these precursors appeared to be present. However, processing of the precursors was sufficiently slow to allow them to accumulate to over 50% of total soluble protein in juvenile tissue. The precursors were purified by column chromatography under non-dissociating conditions and shown by ultracentrifugation to be multimeric proteins with Mr values in the range 150 000-200 000. Images Fig. 1. Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:6548131

  18. Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis

    PubMed Central

    2013-01-01

    Background L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. Results Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L-1 of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. Conclusions This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of “L” isomers precursors in the culture medium. We have engineered K. lactis

  19. A Purification Process for Heparin and Precursor Polysaccharides Using the pH Responsive Behavior of Chitosan

    PubMed Central

    Bhaskar, Ujjwal; Hickey, Anne M.; Li, Guoyun; Mundra, Ruchir V.; Zhang, Fuming; Fu, Li; Cai, Chao; Ou, Zhimin; Dordick, Jonathan S.; Linhardt, Robert J.

    2015-01-01

    The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical Ndeacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatographymass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition. PMID:26147064

  20. A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan.

    PubMed

    Bhaskar, Ujjwal; Hickey, Anne M; Li, Guoyun; Mundra, Ruchir V; Zhang, Fuming; Fu, Li; Cai, Chao; Ou, Zhimin; Dordick, Jonathan S; Linhardt, Robert J

    2015-01-01

    The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical N-deacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatography-mass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition. PMID:26147064

  1. MOVPE growth of Ga(AsBi)/GaAs using different metalorganic precursors

    NASA Astrophysics Data System (ADS)

    Nattermann, L.; Ludewig, P.; Meckbach, L.; Ringler, B.; Keiper, D.; von Hänisch, C.; Stolz, W.; Volz, K.

    2015-09-01

    In this paper different metalorganic Bismuth- and Gallium-precursors such as Triisopropylbismuth, Tritertiarybutylbismuth, Trimethylbismuth, Triethylgallium and Tritertiarybutylgallium, were investigated to grow Ga(AsBi) by metal organic vapour phase epitaxy (MOVPE). Use of different precursors which dissociate at different temperatures and have various decomposition pathways can lead to an understanding of the complex growth chemistry at low temperatures, possibly enabling the growth of Ga(AsBi) at very low growth temperatures in order to further increase the Bi fraction. The growth conditions were varied over a wide range in order to investigate the Bi-incorporation and growth rate comprehensively. Using different methods like high resolution X-ray diffraction, scanning electron microscopy and secondary ion mass spectroscopy, we found that especially in the low temperature regime not only the decomposition of the precursors is important but also the interaction between the different molecules and processes at the surface, like surface coverage and pyrolysis of the precursors, needs to be taken into account. Furthermore, it is demonstrated that the growth of Ga(AsBi) with the novel precursors is possible and results in high quality Ga(AsBi) samples.

  2. Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1

    PubMed Central

    Whitmer, Serap; Canel, Camilo; Hallard, Didier; Gonçalves, Cecilia; Verpoorte, Robert

    1998-01-01

    We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures. PMID:9490777

  3. P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex

    PubMed Central

    Wehrendt, Diana P.; Carmona, Fernando; González Wusener, Ana E.; González, Ángela; Martínez, Juan M. Lázaro; Arregui, Carlos O.

    2016-01-01

    It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface. PMID:27254316

  4. Sentinel-5 Precursor Payload Data Ground Segment

    NASA Astrophysics Data System (ADS)

    Kiemle, S.; Knispel, R.; Schwinger, M.; Weiland, N.

    2012-11-01

    The Payload Data Ground Segment (PDGS) for the Sentinel-5 Precursor mission operating the TROPOMI instrument is currently being developed at DLR Oberpfaffenhofen. The PDGS covers the functions of payload data acquisition, level 0 to level 2 near real-time and offline processing, re-processing, short- and long-term archiving, product quality and service monitoring. A major challenge in the PDGS development is the handling of high data rates for processing, transmission and archiving, and the integration of several different processing systems.This contribution describes the Sentinel-5 Precursor PDGS concept and architecture, gives an overview on the system requirements and presents the current PDGS development status. The ESA Sentinel-5 Precursor to be launched in March 2015 is an important mission for the continuous operational remote sensing of the atmosphere filling the gap between the ENVISAT era and the GMES Sentinel 5 mission.

  5. Sequestration and transport of lignin monomeric precursors.

    PubMed

    Liu, Chang-Jun; Miao, Yu-Chen; Zhang, Ke-Wei

    2011-01-01

    Lignin is the second most abundant terrestrial biopolymer after cellulose. It is essential for the viability of vascular plants. Lignin precursors, the monolignols, are synthesized within the cytosol of the cell. Thereafter, these monomeric precursors are exported into the cell wall, where they are polymerized and integrated into the wall matrix. Accordingly, transport of monolignols across cell membranes is a critical step affecting deposition of lignin in the secondarily thickened cell wall. While the biosynthesis of monolignols is relatively well understood, our knowledge of sequestration and transport of these monomers is sketchy. In this article, we review different hypotheses on monolignol transport and summarize the recent progresses toward the understanding of the molecular mechanisms underlying monolignol sequestration and transport across membranes. Deciphering molecular mechanisms for lignin precursor transport will support a better biotechnological solution to manipulate plant lignification for more efficient agricultural and industrial applications of cell wall biomass. PMID:21245806

  6. Sequestration and Transport of Lignin Monomeric Precursors

    SciTech Connect

    Liu, C.J.; Miao, Y.-C.; Zhang, K.-W.

    2011-01-18

    Lignin is the second most abundant terrestrial biopolymer after cellulose. It is essential for the viability of vascular plants. Lignin precursors, the monolignols, are synthesized within the cytosol of the cell. Thereafter, these monomeric precursors are exported into the cell wall, where they are polymerized and integrated into the wall matrix. Accordingly, transport of monolignols across cell membranes is a critical step affecting deposition of lignin in the secondarily thickened cell wall. While the biosynthesis of monolignols is relatively well understood, our knowledge of sequestration and transport of these monomers is sketchy. In this article, we review different hypotheses on monolignol transport and summarize the recent progresses toward the understanding of the molecular mechanisms underlying monolignol sequestration and transport across membranes. Deciphering molecular mechanisms for lignin precursor transport will support a better biotechnological solution to manipulate plant lignification for more efficient agricultural and industrial applications of cell wall biomass.

  7. Nozzle designs with pitch precursor ablatives

    NASA Technical Reports Server (NTRS)

    Blevins, H. R.; Bedard, R. J.

    1976-01-01

    Recent developments in carbon phenolic ablatives for solid rocket motor nozzles have yielded a pitch precursor carbon fiber offering significant raw material availability and cost saving advantages as compared to conventional rayon precursor material. This paper discusses the results of an experimental program conducted to assess the thermal performance and characterize the thermal properties of pitch precursor carbon phenolic ablatives. The end result of this program is the complete thermal characterization of pitch fabric, pitch mat, hybrid pitch/rayon fabric and pitch mat molding compound. With these properties determined an analytic capability now exists for predicting the thermal performance of these materials in rocket nozzle liner applications. Further planned efforts to verify material performance and analytical prediction procedures through actual rocket motor firings are also discussed.

  8. A Revised Classification System and Recommendations From the Baltimore Consensus Meeting for Neoplastic Precursor Lesions in the Pancreas.

    PubMed

    Basturk, Olca; Hong, Seung-Mo; Wood, Laura D; Adsay, N Volkan; Albores-Saavedra, Jorge; Biankin, Andrew V; Brosens, Lodewijk A A; Fukushima, Noriyoshi; Goggins, Michael; Hruban, Ralph H; Kato, Yo; Klimstra, David S; Klöppel, Günter; Krasinskas, Alyssa; Longnecker, Daniel S; Matthaei, Hanno; Offerhaus, G Johan A; Shimizu, Michio; Takaori, Kyoichi; Terris, Benoit; Yachida, Shinichi; Esposito, Irene; Furukawa, Toru

    2015-12-01

    distinguish them from IPMN. (7) Human lesions resembling the acinar to ductal metaplasia and atypical flat lesions of genetically engineered mouse models exist and may reflect an alternate pathway of carcinogenesis; however, their biological significance requires further study. These revised recommendations are expected to improve our management and understanding of precursor lesions in the pancreas. PMID:26559377

  9. Precursors prior to type IIn supernova explosions are common: Precursor rates, properties, and correlations

    SciTech Connect

    Ofek, Eran O.; Steinbok, Aviram; Arcavi, Iair; Gal-Yam, Avishay; Tal, David; Ben-Ami, Sagi; Yaron, Ofer; Sullivan, Mark; Shaviv, Nir J.; Kulkarni, Shrinivas R.; Nugent, Peter E.; Kasliwal, Mansi M.; Cenko, S. Bradley; Silverman, Jeffrey M.

    2014-07-10

    There is a growing number of Type IIn supernovae (SNe) which present an outburst prior to their presumably final explosion. These precursors may affect the SN display, and are likely related to poorly charted phenomena in the final stages of stellar evolution. By coadding Palomar Transient Factory (PTF) images taken prior to the explosion, here we present a search for precursors in a sample of 16 Type IIn SNe. We find five SNe IIn that likely have at least one possible precursor event (PTF 10bjb, SN 2010mc, PTF 10weh, SN 2011ht, and PTF 12cxj), three of which are reported here for the first time. For each SN we calculate the control time. We find that precursor events among SNe IIn are common: at the one-sided 99% confidence level, >50% of SNe IIn have at least one pre-explosion outburst that is brighter than 3 × 10{sup 7} L{sub ☉} taking place up to 1/3 yr prior to the SN explosion. The average rate of such precursor events during the year prior to the SN explosion is likely ≳ 1 yr{sup –1}, and fainter precursors are possibly even more common. Ignoring the two weakest precursors in our sample, the precursors rate we find is still on the order of one per year. We also find possible correlations between the integrated luminosity of the precursor and the SN total radiated energy, peak luminosity, and rise time. These correlations are expected if the precursors are mass-ejection events, and the early-time light curve of these SNe is powered by interaction of the SN shock and ejecta with optically thick circumstellar material.

  10. Single Source Precursors for Thin Film Solar Cells

    NASA Technical Reports Server (NTRS)

    Banger, Kulbinder K.; Hollingsworth, Jennifer A.; Harris, Jerry D.; Cowen, Jonathan; Buhro, William E.; Hepp, Aloysius F.

    2002-01-01

    The development of thin film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. At NASA GRC we have focused on the development of new single source precursors (SSP) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD (chemical vapor deposition) process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV (photovoltaic) devices.

  11. Polymeric precursors for fibers and matrices

    NASA Technical Reports Server (NTRS)

    Hurwitz, Frances I.

    1992-01-01

    Candidate polymeric precursors for ceramic fiber and matrix processing are discussed, with a view to the advantages and disadvantages of this approach relative to existing alternatives. The properties of ceramic products thus derived are noted to strongly depend on the molecular weight and structure of the starting polymer; in particular, the ceramic's composition and morphology are dependent on the character and extent of crosslinking, as well as on the path of pyrolysis. While large and complex structural ceramic components may ultimately be obtainable by these means, the polymer-precursor method is still in its developmental infancy.

  12. Microbial engineering for the production of fatty acids and fatty acid derivatives

    DOEpatents

    Stephanopoulos, Gregory; Abidi, Syed Hussain Imam

    2014-07-01

    Some aspects of this invention relate to methods useful for the conversion of a carbon source to a biofuel or biofuel precursor using engineered microbes. Some aspects of this invention relate to the discovery of a key regulator of lipid metabolism in microbes. Some aspects of this invention relate to engineered microbes for biofuel or biofuel precursor production.

  13. Engine Test and Measurements

    NASA Technical Reports Server (NTRS)

    Wey, Chown Chou

    1999-01-01

    Although the importance of aerosols and their precursors are now well recognized, the characterization of current subsonic engines for these emissions is far from complete. Furthermore, since the relationship of engine operating parameters to aerosol emissions is not known, extrapolation to untested and unbuilt engines necessarily remains highly uncertain. 1997 NASA LaRC engine test, as well as the parallel 1997 NASA LaRC flight measurement, attempts to address both issues by expanding measurements of aerosols and aerosol precursors with fuels containing different levels of fuel sulfur content. The specific objective of the 1997 engine test is to obtain a database of sulfur oxides emissions as well as the non-volatile particulate emission properties as a function of fuel sulfur and engine operating conditions. Four diagnostic systems, extractive and non-intrusive (optical), will be assembled for the gaseous and particulate emissions characterization measurements study. NASA is responsible for the extractive gaseous emissions measurement system which contains an array of analyzers dedicated to examining the concentrations of specific gases (NO, NO(x), CO, CO2, O2, THC, SO2) and the smoke number. University of Missouri-Rolla uses the Mobile Aerosol Sampling System to measure aerosol/particulate total concentration, size distribution, volatility and hydration property. Air Force Research Laboratory uses the Chemical Ionization Mass Spectrometer to measure SO2, SO3/H2SO4, and HN03 Aerodyne Research, Inc. uses Infrared Tunable Diode Laser system to measure SO2, SO3, NO, H2O, and CO2.

  14. Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

    PubMed

    Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy

  15. Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis1[C][W][OPEN

    PubMed Central

    Wright, Louwrance P.; Rohwer, Johann M.; Ghirardo, Andrea; Hammerbacher, Almuth; Ortiz-Alcaide, Miriam; Raguschke, Bettina; Schnitzler, Jörg-Peter; Gershenzon, Jonathan; Phillips, Michael A.

    2014-01-01

    The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway. PMID:24987018

  16. A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop

    PubMed Central

    Fuentes, Paulina; Zhou, Fei; Erban, Alexander; Karcher, Daniel; Kopka, Joachim; Bock, Ralph

    2016-01-01

    Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. DOI: http://dx.doi.org/10.7554/eLife.13664.001 PMID:27296645

  17. THE CAROTENOID BIOSYNTHETIC PATHWAY: THINKING IN ALL DIMENSIONS

    PubMed Central

    Shumskaya, Maria; Wurtzel, Eleanore T.

    2013-01-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signalling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavour of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and predictability of metabolic engineering of carotenoids rests in the ability to target carotenoids for specific physiological purposes as well as to simultaneously modify carotenoids along with other desired traits. Here, we ask whether predictive metabolic engineering of the carotenoid pathway is indeed possible. Despite a long history of research on the pathway, at this point in time we can only describe the pathway as a parts list and have almost no knowledge of the location of the complete pathway, how it is assembled, and whether there exists any trafficking of the enzymes or the carotenoids themselves. We discuss the current state of knowledge regarding the “complete” pathway and make the argument that predictive metabolic engineering of the carotenoid pathway (and other pathways) will require investigation of the three dimensional state of the pathway as it may exist in plastids of different ultrastructures. Along with this message we point out the need to develop new types of visualization tools and resources that better reflect the dynamic nature of biosynthetic pathways. PMID:23683930

  18. The carotenoid biosynthetic pathway: thinking in all dimensions.

    PubMed

    Shumskaya, Maria; Wurtzel, Eleanore T

    2013-07-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signaling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavor of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and predictability of metabolic engineering of carotenoids rests in the ability to target carotenoids for specific physiological purposes as well as to simultaneously modify carotenoids along with other desired traits. Here, we ask whether predictive metabolic engineering of the carotenoid pathway is indeed possible. Despite a long history of research on the pathway, at this point in time we can only describe the pathway as a parts list and have almost no knowledge of the location of the complete pathway, how it is assembled, and whether there exists any trafficking of the enzymes or the carotenoids themselves. We discuss the current state of knowledge regarding the "complete" pathway and make the argument that predictive metabolic engineering of the carotenoid pathway (and other pathways) will require investigation of the three dimensional state of the pathway as it may exist in plastids of different ultrastructures. Along with this message we point out the need to develop new types of visualization tools and resources that better reflect the dynamic nature of biosynthetic pathways. PMID:23683930

  19. Superconductor precursor mixtures made by precipitation method

    DOEpatents

    Bunker, Bruce C.; Lamppa, Diana L.; Voigt, James A.

    1989-01-01

    Method and apparatus for preparing highly pure homogeneous precursor powder mixtures for metal oxide superconductive ceramics. The mixes are prepared by instantaneous precipitation from stoichiometric solutions of metal salts such as nitrates at controlled pH's within the 9 to 12 range, by addition of solutions of non-complexing pyrolyzable cations, such as alkyammonium and carbonate ions.

  20. Dynamic Yielding Behind Near-Steady Precursors

    NASA Astrophysics Data System (ADS)

    Sano, Yukio; Sano, Tomokazu

    2007-12-01

    In the precursor decay process in a lithium fluoride single crystal, plastic flow is revealed to occur in an evolving follower through an evaluated time variation in the dislocation density at its leading edge that is considerable higher than the initial density in the bulk. The occurrence of plastic flow in the follower suggests that the compressibility changes in an unsteady portion behind a near-steady precursor in a single plane wave, and its change causes the plane wave to be unstable and to separate into a near-steady precursor and a follower. The follower changes from wave C to waves I, II, R', and Rb in sequence. Here C is the contraction (compression) wave; I and II the degenerate contraction waves I and II; R' the subrarefaction wave; and Rb the rarefaction wave. Dynamic yield phenomena are illustrated by the schematics of three different types of dynamic relations between stress and strain, which are induced by a precursor C, a follower (i) C, (ii) I or II, or (iii) R' or Rb, and a plastic wave C behind the follower.

  1. SUMMER 1990 ATLANTA OZONE PRECURSOR STUDY

    EPA Science Inventory

    During the summer of 1990, the U.S. Environmental Protection Agency (EPA) conducted a major monitoring study in Atlanta, Georgia, addressing the measurement of ozone and its precursors. The study was undertaken to obtain an information base to support the development and implemen...

  2. Targeting bactoprenol-coupled cell envelope precursors.

    PubMed

    Ulm, Hannah; Schneider, Tanja

    2016-09-01

    Targeting the bactoprenol-coupled cell wall precursor lipid II is a validated antibacterial strategy. In this review, selected prototype lipid II-binding antibiotics of different chemical classes are discussed. Although these compounds attack the same molecular target, they trigger nuanced and diverse cellular effects. Consequently, the mechanisms of antibacterial resistance and the likelihood of resistance development may vary substantially. PMID:27495122

  3. Detection of Chemical Precursors of Explosives

    NASA Technical Reports Server (NTRS)

    Li, Jing

    2012-01-01

    Certain selected chemicals associated with terrorist activities are too unstable to be prepared in final form. These chemicals are often prepared as precursor components, to be combined at a time immediately preceding the detonation. One example is a liquid explosive, which usually requires an oxidizer, an energy source, and a chemical or physical mechanism to combine the other components. Detection of the oxidizer (e.g. H2O2) or the energy source (e.g., nitromethane) is often possible, but must be performed in a short time interval (e.g., 5 15 seconds) and in an environment with a very small concentration (e.g.,1 100 ppm), because the target chemical(s) is carried in a sealed container. These needs are met by this invention, which provides a system and associated method for detecting one or more chemical precursors (components) of a multi-component explosive compound. Different carbon nanotubes (CNTs) are loaded (by doping, impregnation, coating, or other functionalization process) for detecting of different chemical substances that are the chemical precursors, respectively, if these precursors are present in a gas to which the CNTs are exposed. After exposure to the gas, a measured electrical parameter (e.g. voltage or current that correlate to impedance, conductivity, capacitance, inductance, etc.) changes with time and concentration in a predictable manner if a selected chemical precursor is present, and will approach an asymptotic value promptly after exposure to the precursor. The measured voltage or current are compared with one or more sequences of their reference values for one or more known target precursor molecules, and a most probable concentration value is estimated for each one, two, or more target molecules. An error value is computed, based on differences of voltage or current for the measured and reference values, using the most probable concentration values. Where the error value is less than a threshold, the system concludes that the target

  4. Tissue engineered periodontal products.

    PubMed

    Bartold, P M; Gronthos, S; Ivanovski, S; Fisher, A; Hutmacher, D W

    2016-02-01

    Attainment of periodontal regeneration is a significant clinical goal in the management of advanced periodontal defects arising from periodontitis. Over the past 30 years numerous techniques and materials have been introduced and evaluated clinically and have included guided tissue regeneration, bone grafting materials, growth and other biological factors and gene therapy. With the exception of gene therapy, all have undergone evaluation in humans. All of the products have shown efficacy in promoting periodontal regeneration in animal models but the results in humans remain variable and equivocal concerning attaining complete biological regeneration of damaged periodontal structures. In the early 2000s, the concept of tissue engineering was proposed as a new paradigm for periodontal regeneration based on molecular and cell biology. At this time, tissue engineering was a new and emerging field. Now, 14 years later we revisit the concept of tissue engineering for the periodontium and assess how far we have come, where we are currently situated and what needs to be done in the future to make this concept a reality. In this review, we cover some of the precursor products, which led to our current position in periodontal tissue engineering. The basic concepts of tissue engineering with special emphasis on periodontal tissue engineering products is discussed including the use of mesenchymal stem cells in bioscaffolds and the emerging field of cell sheet technology. Finally, we look into the future to consider what CAD/CAM technology and nanotechnology will have to offer. PMID:25900048

  5. Precursors to potential severe core damage accidents: 1994, a status report. Volume 21: Main report and appendices A--H

    SciTech Connect

    Belles, R.J.; Cletcher, J.W.; Copinger, D.A.; Vanden Heuvel, L.N.; Dolan, B.W.; Minarick, J.W. |

    1995-12-01

    Nine operational events that affected eleven commercial light-water reactors (LWRs) during 1994 and that are considered to be precursors to potential severe core damage are described. All these events had conditional probabilities of subsequent severe core damage greater than or equal to 1.0 {times} 10{sup {minus}6}. These events were identified by computer-screening the 1994 licensee event reports from commercial LWRs to identify those that could be potential precursors. Candidate precursors were then selected and evaluated in a process similar to that used in previous assessments. Selected events underwent engineering evaluation that identified, analyzed, and documented the precursors. Other events designated by the Nuclear Regulatory Commission (NRC) also underwent a similar evaluation. Finally, documented precursors were submitted for review by licensees and NRC headquarters and regional offices to ensure that the plant design and its response to the precursor were correctly characterized. This study is a continuation of earlier work, which evaluated 1969--1981 and 1984--1993 events. The report discusses the general rationale for this study, the selection and documentation of events as precursors, and the estimation of conditional probabilities of subsequent severe core damage for events. This document is bound in two volumes: Vol. 21 contains the main report and Appendices A--H; Vol. 22 contains Appendix 1.

  6. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production.

    PubMed

    Kang, Aram; George, Kevin W; Wang, George; Baidoo, Edward; Keasling, Jay D; Lee, Taek Soon

    2016-03-01

    Branched C5 alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C5 alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene. PMID:26708516

  7. Engineering Cellular Metabolism.

    PubMed

    Nielsen, Jens; Keasling, Jay D

    2016-03-10

    Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation. PMID:26967285

  8. Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid.

    PubMed

    Farré, Gemma; Perez-Fons, Laura; Decourcelle, Mathilde; Breitenbach, Jürgen; Hem, Sonia; Zhu, Changfu; Capell, Teresa; Christou, Paul; Fraser, Paul D; Sandmann, Gerhard

    2016-08-01

    Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid. PMID:26931320

  9. The Peroxide Pathway

    NASA Technical Reports Server (NTRS)

    McNeal, Curtis I., Jr.; Anderson, William

    1999-01-01

    NASA's current focus on technology roadmaps as a tool for guiding investment decisions leads naturally to a discussion of NASA's roadmap for peroxide propulsion system development. NASA's new Second Generation Space Transportation System roadmap calls for an integrated Reusable Upper-Stage (RUS) engine technology demonstration in the FY03/FY04 time period. Preceding this integrated demonstration are several years of component developments and subsystem technology demonstrations. NASA and the Air Force took the first steps at developing focused upper stage technologies with the initiation of the Upper Stage Flight Experiment with Orbital Sciences in December 1997. A review of this program's peroxide propulsion development is a useful first step in establishing the peroxide propulsion pathway that could lead to a RUS demonstration in 2004.

  10. Isopropanol production with engineered Cupriavidus necator as bioproduction platform.

    PubMed

    Grousseau, Estelle; Lu, Jingnan; Gorret, Nathalie; Guillouet, Stéphane E; Sinskey, Anthony J

    2014-05-01

    Alleviating our society's dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO2. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a β-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l(-1) isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l(-1) of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g(-1) h(-1), yield 0.32 Cmole Cmole(-1)) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole(-1)) and the overall specific productivity (0.044 g g(-1) h(-1)) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures. PMID:24604499

  11. Isopropanol production with engineered Cupriavidus necator as bioproduction platform

    SciTech Connect

    Grousseau, E; Lu, JN; Gorret, N; Guillouet, SE; Sinskey, AJ

    2014-03-07

    Alleviating our society's dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO2. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a beta-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l(-1) isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l(-1) of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g(-1) h(-1), yield 0.32 Cmole Cmole(-1)) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole(-1)) and the overall specific productivity (0.044 g g(-1) h(-1)) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures.

  12. Layered Systems Engineering Engines

    NASA Technical Reports Server (NTRS)

    Breidenthal, Julian C.; Overman, Marvin J.

    2009-01-01

    A notation is described for depicting the relationships between multiple, contemporaneous systems engineering efforts undertaken within a multi-layer system-of-systems hierarchy. We combined the concepts of remoteness of activity from the end customer, depiction of activity on a timeline, and data flow to create a new kind of diagram which we call a "Layered Vee Diagram." This notation is an advance over previous notations because it is able to be simultaneously precise about activity, level of granularity, product exchanges, and timing; these advances provide systems engineering managers a significantly improved ability to express and understand the relationships between many systems engineering efforts. Using the new notation, we obtain a key insight into the relationship between project duration and the strategy selected for chaining the systems engineering effort between layers, as well as insights into the costs, opportunities, and risks associated with alternate chaining strategies.

  13. Fragmentation pathways of polymer ions.

    PubMed

    Wesdemiotis, Chrys; Solak, Nilüfer; Polce, Michael J; Dabney, David E; Chaicharoen, Kittisak; Katzenmeyer, Bryan C

    2011-01-01

    Tandem mass spectrometry (MS/MS) is increasingly applied to synthetic polymers to characterize chain-end or in-chain substituents, distinguish isobaric and isomeric species, and determine macromolecular connectivities and architectures. For confident structural assignments, the fragmentation mechanisms of polymer ions must be understood, as they provide guidelines on how to deduce the desired information from the fragments observed in MS/MS spectra. This article reviews the fragmentation pathways of synthetic polymer ions that have been energized to decompose via collisionally activated dissociation (CAD), the most widely used activation method in polymer analysis. The compounds discussed encompass polystyrenes, poly(2-vinyl pyridine), polyacrylates, poly(vinyl acetate), aliphatic polyester copolymers, polyethers, and poly(dimethylsiloxane). For a number of these polymers, several substitution patterns and architectures are considered, and questions regarding the ionization agent and internal energy of the dissociating precursor ions are also addressed. Competing and consecutive dissociations are evaluated in terms of the structural insight they provide about the macromolecular structure. The fragmentation pathways of the diverse array of polymer ions examined fall into three categories, viz. (1) charge-directed fragmentations, (2) charge-remote rearrangements, and (3) charge-remote fragmentations via radical intermediates. Charge-remote processes predominate. Depending on the ionizing agent and the functional groups in the polymer, the incipient fragments arising by pathways (1)-(3) may form ion-molecule complexes that survive long enough to permit inter-fragment hydrogen atom, proton, or hydride transfers. PMID:20623599

  14. Crystallization trends of precursor pyroxene in ordinary chondrites: Implications for igneous origin of precursor

    NASA Technical Reports Server (NTRS)

    Fujita, T.; Kitamura, M.

    1994-01-01

    Various observations suggest that the precursor of the fine fragments and the relict pyroxene was formed by an igneous process in a grandparent body of the chondrite. Furthermore, the fact that the precursors appear as fragments in the matrix as well as relicts in the chondrule suggests a shock origin for the chondrite by collision of two or more bodies, which had already to some extent undergone igneous differentiation.

  15. Biocommodity Engineering.

    PubMed

    Lynd; Wyman; Gerngross

    1999-10-01

    xylose and other nonglucose sugars, and "consolidated bioprocessing" in which cellulase production, cellulose hydrolysis, and fermentation of soluble carbohydrates to desired products occur in a single process step. With respect to product diversification, a distinction is made between replacement of a fossil resource-derived chemical with a biomass-derived chemical of identical composition and substitution of a biomass-derived chemical with equivalent functional characteristics but distinct composition. The substitution strategy involves larger transition issues but is seen as more promising in the long term. Metabolic engineering pursuant to the production of biocommodity products requires host organisms with properties such as the ability to use low-cost substrates, high product yield, competitive fitness, and robustness in industrial environments. In many cases, it is likely to be more successful to engineer a desired pathway into an organism having useful industrial properties rather than trying to engineer such often multi-gene properties into host organisms that do not have them naturally. Identification of host organisms with useful industrial properties and development of genetic systems for these organisms is a research challenge distinctive to biocommodity engineering. Chemical catalysis and separations technologies have important roles to play in downstream processing of biocommodity products and involve a distinctive set of challenges relative to petrochemical processing. At its current nascent state of development, the definition and advancement of the biocommodity field can benefit from integration at multiple levels. These include technical issues associated with integrating unit operations with each other, integrating production of individual products into a multi-product biorefinery, and integrating biorefineries into the broader resource, economic, and environmental systems in which they function. We anticipate that coproduction of multiple products