scholarly journals The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene,adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

2015 ◽  
Vol 197 (8) ◽  
pp. 1386-1393 ◽  
Author(s):  
Jonathan Lo ◽  
Tianyong Zheng ◽  
Shuen Hon ◽  
Daniel G. Olson ◽  
Lee R. Lynd
Keyword(s):  
Ethanol Production ◽  
Aldehyde Dehydrogenase ◽  
Clostridium Thermocellum ◽  
Deletion Strain ◽  
Aldh Activity ◽  
Content Type ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dehydrogenase Gene

ABSTRACTThermoanaerobacterium saccharolyticumandClostridium thermocellumare anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene,adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of theadhEgene inC. thermocellumandT. saccharolyticum. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In bothadhEdeletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. InT. saccharolyticum, theadhEdeletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in theT. saccharolyticumparent strain but did not increase activity in theadhEdeletion strain, suggesting that ALDH activity was inhibited. InC. thermocellum, theadhEdeletion strain lost >90% of ALDH and ADH activity in cell extracts. TheC. thermocellumadhEdeletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase.IMPORTANCEThermoanaerobacterium saccharolyticumandClostridium thermocellumare bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation ofadhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In strains withoutadhE, we note changes in biochemical activity, product formation, and growth.

scholarly journals Deletion ofnfnABin Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

2015 ◽  
Vol 197 (18) ◽  
pp. 2920-2929 ◽  
Author(s):  
Jonathan Lo ◽  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
Natalie Ruppertsberger ◽  
Shital A. Tripathi ◽  
...  
Keyword(s):  
Ethanol Yield ◽  
Oxidoreductase Activity ◽  
Fermentation Products ◽  
Content Type ◽  
Ferredoxin Oxidoreductase ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Adh Activity ◽  
Reversible Transfer

ABSTRACTNfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP+. The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains ofThermoanaerobacterium saccharolyticumengineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts ofT. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. ThenfnABgene was deleted in different strains ofT. saccharolyticumto determine its effect on end product formation. In wild-typeT. saccharolyticum, deletion ofnfnABresulted in a 46% increase in H2formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss ofnfnABcaused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnABstrain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnABstrain with unchanged ethanol yield had NADH-linked ADH activity. Deletion ofnfnABcaused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lostnfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct fromnfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity.IMPORTANCEThermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)+. The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP+and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion ofnfnABand demonstrate a role for NfnAB in metabolism and ethanol formation inThermoanaerobacterium saccharolyticumand show that this may be an important feature among other thermophilic ethanologenic anaerobes.

scholarly journals Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA, Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

2016 ◽  
Vol 199 (3) ◽  
Author(s):  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
Sean J. Murphy ◽  
Xiongjun Shao ◽  
Liang Tian ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Single Gene ◽  
Maximum Yield ◽  
Primary Alcohol ◽  
Content Type ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dehydrogenase Gene ◽  
High Ethanol

ABSTRACT Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here, we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%. IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. Here, we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.

scholarly journals Ferredoxin:NAD+Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

2016 ◽  
Vol 82 (24) ◽  
pp. 7134-7141 ◽  
Author(s):  
Liang Tian ◽  
Jonathan Lo ◽  
Xiongjun Shao ◽  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Redox Balance ◽  
Wild Type ◽  
Anaerobic Microorganism ◽  
Content Type ◽  
Ethanol Formation ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Different Strains ◽  
Electron Carriers

ABSTRACTFerredoxin:NAD+oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway inThermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activityin vitrowith a ferredoxin-based FNOR assay. To determine its role in metabolism, thetsac_1705gene was deleted in different strains ofT. saccharolyticum. In wild-typeT. saccharolyticum, deletion oftsac_1705resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR inT.saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 inClostridium thermocellumand found improvements in both the titer and the yield of ethanol.IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD+, a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms.

scholarly journals Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

2015 ◽  
Vol 197 (15) ◽  
pp. 2610-2619 ◽  
Author(s):  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
Liang Tian ◽  
Yannick J. Bomble ◽  
Michael E. Himmel ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Ethanol Production ◽  
Aldehyde Dehydrogenase ◽  
Clostridium Thermocellum ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type ◽  
Cofactor Specificity ◽  
Theoretical Maximum ◽  
Adh Activity

ABSTRACTClostridium thermocellumandThermoanaerobacteriumsaccharolyticumare thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains ofT. saccharolyticumproduce ethanol with a yield of 90% of the theoretical maximum, engineered strains ofC. thermocellumproduce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in theiradhEgenes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, theadhEgenes from six strains ofC. thermocellumandT. saccharolyticumwere cloned and expressed inEscherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains ofT. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain ofC. thermocellumhas acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced intoC. thermocellumandT. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level.IMPORTANCEThis work describes the characterization of the AdhE enzyme from different strains ofC. thermocellumandT. saccharolyticum.C. thermocellumandT. saccharolyticumare thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production byC. thermocellumandT. saccharolyticum.

A novel bifunctional aldehyde/alcohol dehydrogenase mediating ethanol formation from acetyl-CoA in hyperthermophiles

2020 ◽  
Author(s):  
Qiang Wang ◽  
Chong Sha ◽  
Hongcheng Wang ◽  
Kesen Ma ◽  
Juergen Wiegel ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Fermentation ◽  
Cellulosic Ethanol ◽  
Decarboxylase Activity ◽  
Aldh Activity ◽  
Acetyl Coa ◽  
Ethanol Formation

Abstract Background: Hyperthermophilic fermentation at temperatures above 80 °C allows in situ product removal to mitigate the ethanol toxicity, and reduces microbial contamination without autoclaving/cooling of feedstock. Many species of Thermotoga grow at temperatures up to 90 °C, and have enzymes to degrade and utilize lignocelluloses, which provide advantages for achieving consolidated processes of cellulosic ethanol production. However, no CoA-dependent aldehyde dehydrogenase (CoA-Aldh) from any hyperthermophiles has been documented in literature so far. The pyruvate ferredoxin oxidoreductases from hyperthermophiles have pyruvate decarboxylase activity, which convert about 2% and 98% of pyruvate to acetaldehyde and acetyl-CoA (ac-CoA), respectively. Acetyl-CoA can be converted to acetic acid, if there is no CoA-Aldh to convert ac-CoA to acetaldehyde and further to ethanol. Therefore, the current study aimed to identify and characterize a CoA-Aldh activity that mediates ethanol fermentation in hyperthermophiles.Results: In Thermotoga neapolitana (Tne), a hyperthermophilic iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) was, for the first time, revealed to catalyze the ac-CoA reduction to form ethanol via an acetaldehyde intermediate, while the annotated aldh gene in Tne genome only encodes a CoA-independent Aldh that oxidizes aldehyde to acetic acid. Three other Tne alcohol dehydrogenases (Adh) exhibited specific physiological roles in ethanol formation and consumption: Fe-Adh2 mainly catalyzed the reduction of acetaldehyde to produce ethanol, and Fe-Adh1 showed significant activities only under extreme conditions, while Zn-Adh showed special activity in ethanol oxidation. In the in vitro formation of ethanol from ac-CoA, a strong synergy was observed between Fe-Adh1 and Fe-AAdh. The Fe-AAdh gene is highly conserved in Thermotoga spp. and in Pyrococus sp., which is probably responsible for ethanol metabolism in hyperthermophiles.Conclusions: Hyperthermophilic Thermotoga spp. are excellent candidates for biosynthesis of cellulosic ethanol fermentation strains. The finding of a novel hyperthermophilic CoA-Aldh activity of Tne Fe-AAdh revealed the existence of a hyperthermophilic fermentation pathway from ac-CoA to ethanol, which offers a basic frame for in vitro synthesis of a highly active AAdh for effective ethanol fermentation pathway in hyperthermophiles, which is a key element for the approach to the consolidated processes of cellulosic ethanol production.

scholarly journals In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13C and 2H Tracers

mSystems ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Tyler B. Jacobson ◽  
Travis K. Korosh ◽  
David M. Stevenson ◽  
Charles Foster ◽  
Costas Maranas ◽  
...  
Keyword(s):  
Free Energy ◽  
Thermodynamic Analysis ◽  
Clostridium Thermocellum ◽  
Metabolic Networks ◽  
Glycolytic Pathway ◽  
Cellulosic Biomass ◽  
Model Organisms ◽  
Content Type ◽  
Thermoanaerobacterium Saccharolyticum

ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2H and 13C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli. The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellum than in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. IMPORTANCE Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum, two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae, the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.

scholarly journals Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Evert K. Holwerda ◽  
Jilai Zhou ◽  
Shuen Hon ◽  
David M. Stevenson ◽  
Daniel Amador-Noguez ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pyruvate Kinase ◽  
Clostridium Thermocellum ◽  
Proton Pumping ◽  
Nucleoside Triphosphate ◽  
Flux Analysis ◽  
Wild Type ◽  
Content Type ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Chemostat Cultures

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.

scholarly journals The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum

2017 ◽  
Vol 42 ◽  
pp. 175-184 ◽  
Author(s):  
Shuen Hon ◽  
Daniel G. Olson ◽  
Evert K. Holwerda ◽  
Anthony A. Lanahan ◽  
Sean J.L. Murphy ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Clostridium Thermocellum ◽  
Thermoanaerobacterium Saccharolyticum
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