The Experts below are selected from a list of 3015 Experts worldwide ranked by ideXlab platform
Lee R Lynd - One of the best experts on this subject based on the ideXlab platform.
-
Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.
Applied and environmental microbiology, 2020Co-Authors: Evert K. Holwerda, Lee R Lynd, Shuen Hon, Jilai Zhou, David M. Stevenson, Daniel Amador-noguez, Johannes P. Van DijkenAbstract:ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum. In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi → 2 pyruvate + 5 NTP + Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi → ADP + PPi. Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids. IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.
-
The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum.
Metabolic engineering, 2017Co-Authors: Shuen Hon, Adam M. Guss, Daniel G Olson, Sean Jeanloup Murphy, Anthony A Lanahan, Tianyong Zheng, Evert K. Holwerda, Marybeth Maloney, Beth Papanek, Lee R LyndAbstract:Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.
-
Expression of adhA from different organisms in Clostridium thermocellum
BMC, 2017Co-Authors: Tianyong Zheng, Lee R Lynd, Jingxuan Cui, Hye Ri Bae, Daniel G OlsonAbstract:Abstract Background Clostridium thermocellum is a cellulolytic anaerobic thermophile that is a promising candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels such as ethanol. It was previously shown that expressing Thermoanaerobacterium saccharolyticum adhA in C. thermocellum increases ethanol yield.In this study, we investigated expression of adhA genes from different organisms in Clostridium thermocellum. Methods Based on sequence identity to T. saccharolyticum adhA, we chose adhA genes from 10 other organisms: Clostridium botulinum, Methanocaldococcus bathoardescens, Thermoanaerobacterium ethanolicus, Thermoanaerobacter mathranii, Thermococcus strain AN1, Thermoanaerobacterium thermosaccharolyticum, Caldicellulosiruptor saccharolyticus, Fervidobacterium nodosum, Marinitoga piezophila, and Thermotoga petrophila. All 11 adhA genes (including T. saccharolyticum adhA) were expressed in C. thermocellum and fermentation end products were analyzed. Results All 11 adhA genes increased C. thermocellum ethanol yield compared to the empty-vector control. C. botulinum and T. ethanolicus adhA genes generated significantly higher ethanol yield than T. saccharolyticum adhA. Conclusion Our results indicated that expressing adhA is an effective method of increasing ethanol yield in wild-type C. thermocellum, and that this appears to be a general property of adhA genes
-
Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase
BMC, 2017Co-Authors: Liang Tian, Adam M. Guss, Daniel G Olson, Shuen Hon, Skyler J. Perot, Jilai Zhou, Xiaoyu Liang, Jason T. Bouvier, Lee R LyndAbstract:Abstract Background Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. Result Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. Conclusion The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield
-
Elimination of formate production in Clostridium thermocellum
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Thomas Rydzak, Lee R Lynd, Adam M. GussAbstract:The ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield is far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H_2, lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, genes encoding pyruvate:formate lyase ( pflB ) and PFL-activating enzyme ( pflA ) were deleted. Formate production in the resulting Δ pfl strain was eliminated and acetate production decreased by 50 % on both complex and defined medium. The growth rate of the Δ pfl strain decreased by 2.9-fold on defined medium and biphasic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δ pfl growth rate to 80 % of the parent strain. The role of pfl in metabolic engineering strategies and C_1 metabolism is discussed.
Daniel G Olson - One of the best experts on this subject based on the ideXlab platform.
-
metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production
Biotechnology for Biofuels, 2020Co-Authors: Daniel G Olson, Sean Jeanloup Murphy, Evert K. Holwerda, Marybeth Maloney, David M. Stevenson, Natalie M RuppertsbergerAbstract:Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.
-
The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum.
Metabolic engineering, 2017Co-Authors: Shuen Hon, Adam M. Guss, Daniel G Olson, Sean Jeanloup Murphy, Anthony A Lanahan, Tianyong Zheng, Evert K. Holwerda, Marybeth Maloney, Beth Papanek, Lee R LyndAbstract:Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.
-
engineering electron metabolism to increase ethanol production in Clostridium thermocellum
Metabolic Engineering, 2017Co-Authors: Daniel G Olson, Sean Jeanloup Murphy, Liang Tian, Shuen Hon, Anthony A LanahanAbstract:The NfnAB (NADH-dependent reduced ferredoxin: NADP+ oxidoreductase) and Rnf (ion-translocating reduced ferredoxin: NAD+ oxidoreductase) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P)+. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes had little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.
-
Expression of adhA from different organisms in Clostridium thermocellum
BMC, 2017Co-Authors: Tianyong Zheng, Lee R Lynd, Jingxuan Cui, Hye Ri Bae, Daniel G OlsonAbstract:Abstract Background Clostridium thermocellum is a cellulolytic anaerobic thermophile that is a promising candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels such as ethanol. It was previously shown that expressing Thermoanaerobacterium saccharolyticum adhA in C. thermocellum increases ethanol yield.In this study, we investigated expression of adhA genes from different organisms in Clostridium thermocellum. Methods Based on sequence identity to T. saccharolyticum adhA, we chose adhA genes from 10 other organisms: Clostridium botulinum, Methanocaldococcus bathoardescens, Thermoanaerobacterium ethanolicus, Thermoanaerobacter mathranii, Thermococcus strain AN1, Thermoanaerobacterium thermosaccharolyticum, Caldicellulosiruptor saccharolyticus, Fervidobacterium nodosum, Marinitoga piezophila, and Thermotoga petrophila. All 11 adhA genes (including T. saccharolyticum adhA) were expressed in C. thermocellum and fermentation end products were analyzed. Results All 11 adhA genes increased C. thermocellum ethanol yield compared to the empty-vector control. C. botulinum and T. ethanolicus adhA genes generated significantly higher ethanol yield than T. saccharolyticum adhA. Conclusion Our results indicated that expressing adhA is an effective method of increasing ethanol yield in wild-type C. thermocellum, and that this appears to be a general property of adhA genes
-
Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase
BMC, 2017Co-Authors: Liang Tian, Adam M. Guss, Daniel G Olson, Shuen Hon, Skyler J. Perot, Jilai Zhou, Xiaoyu Liang, Jason T. Bouvier, Lee R LyndAbstract:Abstract Background Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. Result Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. Conclusion The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield
Adam M. Guss - One of the best experts on this subject based on the ideXlab platform.
-
Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics
BMC, 2018Co-Authors: Jason M. Whitham, Adam M. Guss, Thomas Rydzak, Miguel Rodriguez, Ji-won Moon, Nancy L. Engle, Dawn M. Klingeman, Malaney M. Abel, Timothy J. Tschaplinski, Steven D. BrownAbstract:Abstract Background Clostridium (RuminiClostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools. Results Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h−1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow). Conclusions Clostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values
-
The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum.
Metabolic engineering, 2017Co-Authors: Shuen Hon, Adam M. Guss, Daniel G Olson, Sean Jeanloup Murphy, Anthony A Lanahan, Tianyong Zheng, Evert K. Holwerda, Marybeth Maloney, Beth Papanek, Lee R LyndAbstract:Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.
-
Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase
BMC, 2017Co-Authors: Liang Tian, Adam M. Guss, Daniel G Olson, Shuen Hon, Skyler J. Perot, Jilai Zhou, Xiaoyu Liang, Jason T. Bouvier, Lee R LyndAbstract:Abstract Background Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. Result Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. Conclusion The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield
-
Elimination of formate production in Clostridium thermocellum
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Thomas Rydzak, Lee R Lynd, Adam M. GussAbstract:The ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield is far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H_2, lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, genes encoding pyruvate:formate lyase ( pflB ) and PFL-activating enzyme ( pflA ) were deleted. Formate production in the resulting Δ pfl strain was eliminated and acetate production decreased by 50 % on both complex and defined medium. The growth rate of the Δ pfl strain decreased by 2.9-fold on defined medium and biphasic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δ pfl growth rate to 80 % of the parent strain. The role of pfl in metabolic engineering strategies and C_1 metabolism is discussed.
-
elimination of hydrogenase active site assembly blocks h2 production and increases ethanol yield in Clostridium thermocellum
Biotechnology for Biofuels, 2015Co-Authors: Daniel G Olson, Lee R Lynd, Tianyong Zheng, Ranjita Biswas, Adam M. GussAbstract:Background The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol.
Evert K. Holwerda - One of the best experts on this subject based on the ideXlab platform.
-
Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.
Applied and environmental microbiology, 2020Co-Authors: Evert K. Holwerda, Lee R Lynd, Shuen Hon, Jilai Zhou, David M. Stevenson, Daniel Amador-noguez, Johannes P. Van DijkenAbstract:ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum. In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi → 2 pyruvate + 5 NTP + Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi → ADP + PPi. Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids. IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.
-
metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production
Biotechnology for Biofuels, 2020Co-Authors: Daniel G Olson, Sean Jeanloup Murphy, Evert K. Holwerda, Marybeth Maloney, David M. Stevenson, Natalie M RuppertsbergerAbstract:Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.
-
The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum.
Metabolic engineering, 2017Co-Authors: Shuen Hon, Adam M. Guss, Daniel G Olson, Sean Jeanloup Murphy, Anthony A Lanahan, Tianyong Zheng, Evert K. Holwerda, Marybeth Maloney, Beth Papanek, Lee R LyndAbstract:Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.
-
simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum
Biotechnology for Biofuels, 2016Co-Authors: Daniel G Olson, Liang Tian, Evert K. Holwerda, Beth Papanek, Thomas Rydzak, Tianyong ZhengAbstract:Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed.
-
simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum
Biotechnology for Biofuels, 2016Co-Authors: Daniel G Olson, Liang Tian, Evert K. Holwerda, Beth Papanek, Thomas Rydzak, Tianyong ZhengAbstract:Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Here, we started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. The resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. A two step selection method successfully improved the ethanol yield and the titer. This evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.
Liang Tian - One of the best experts on this subject based on the ideXlab platform.
-
engineering electron metabolism to increase ethanol production in Clostridium thermocellum
Metabolic Engineering, 2017Co-Authors: Daniel G Olson, Sean Jeanloup Murphy, Liang Tian, Shuen Hon, Anthony A LanahanAbstract:The NfnAB (NADH-dependent reduced ferredoxin: NADP+ oxidoreductase) and Rnf (ion-translocating reduced ferredoxin: NAD+ oxidoreductase) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P)+. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes had little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.
-
Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase
BMC, 2017Co-Authors: Liang Tian, Adam M. Guss, Daniel G Olson, Shuen Hon, Skyler J. Perot, Jilai Zhou, Xiaoyu Liang, Jason T. Bouvier, Lee R LyndAbstract:Abstract Background Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. Result Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. Conclusion The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield
-
glycolysis without pyruvate kinase in Clostridium thermocellum
Metabolic Engineering, 2017Co-Authors: Daniel G Olson, Liang Tian, Jingxuan Cui, Marybeth Maloney, Jilai Zhou, Manuel Horl, Tobias Fuhrer, Daniel Amadornoguez, Uwe SauerAbstract:The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33±2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. This provides the first direct evidence of the in-vivo function of the malate shunt.
-
simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum
Biotechnology for Biofuels, 2016Co-Authors: Daniel G Olson, Liang Tian, Evert K. Holwerda, Beth Papanek, Thomas Rydzak, Tianyong ZhengAbstract:Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed.
-
simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum
Biotechnology for Biofuels, 2016Co-Authors: Daniel G Olson, Liang Tian, Evert K. Holwerda, Beth Papanek, Thomas Rydzak, Tianyong ZhengAbstract:Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Here, we started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. The resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. A two step selection method successfully improved the ethanol yield and the titer. This evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.