scholarly journals Energy Conservation Associated with Ethanol Formation from H2and CO2in Clostridium autoethanogenum Involving Electron Bifurcation

2015 ◽  
Vol 197 (18) ◽  
pp. 2965-2980 ◽  
Author(s):  
Johanna Mock ◽  
Yanning Zheng ◽  
Alexander P. Mueller ◽  
San Ly ◽  
Loan Tran ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Acetyl Coa ◽  
Content Type ◽  
Substrate Level Phosphorylation ◽  
Clostridium Ljungdahlii ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Substrate Level ◽  
Clostridium Autoethanogenum ◽  
Specific Activities

ABSTRACTMost acetogens can reduce CO2with H2to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such asClostridium autoethanogenum,Clostridium ljungdahlii, andClostridium ragsdalei, also form large amounts of ethanol from CO2and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grownC. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD+oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2and H2.IMPORTANCEEthanol formation from syngas (H2, CO, and CO2) and from H2and CO2that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

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 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 Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Varsha Patil ◽  
Vikas Jain
Keyword(s):  
Oxidative Phosphorylation ◽  
Atp Synthase ◽  
Drug Targets ◽  
Energy State ◽  
Β Subunit ◽  
Wild Type ◽  
Content Type ◽  
Substrate Level Phosphorylation ◽  
Substrate Level ◽  
Physiological Differences

ABSTRACT Mycobacterium tuberculosis, a bacterium that causes tuberculosis, poses a serious threat, especially due to the emergence of drug-resistant strains. M. tuberculosis and other mycobacterial species, such as M. smegmatis, are known to generate an inadequate amount of energy by substrate-level phosphorylation and mandatorily require oxidative phosphorylation (OXPHOS) for their growth and metabolism. Hence, antibacterial drugs, such as bedaquiline, targeting the multisubunit ATP synthase complex, which is required for OXPHOS, have been developed with the aim of eliminating pathogenic mycobacteria. Here, we explored the influence of suboptimal OXPHOS on the physiology and metabolism of M. smegmatis. M. smegmatis harbors two identical copies of atpD, which codes for the β subunit of ATP synthase. We show that upon deletion of one copy of atpD (M. smegmatis ΔatpD), M. smegmatis synthesizes smaller amounts of ATP and enters into an energy-compromised state. The mutant displays remarkable phenotypic and physiological differences from the wild type, such as respiratory slowdown, reduced biofilm formation, lesser amounts of cell envelope polar lipids, and increased antibiotic sensitivity compared to the wild type. Additionally, M. smegmatis ΔatpD overexpresses genes belonging to the dormancy operon, the β-oxidation pathway, and the glyoxylate shunt, suggesting that the mutant adapts to a low energy state by switching to alternative pathways to produce energy. Interestingly, M. smegmatis ΔatpD shows significant phenotypic, metabolic, and physiological similarities with bedaquiline-treated wild-type M. smegmatis. We believe that the identification and characterization of key metabolic pathways functioning during an energy-compromised state will enhance our understanding of bacterial adaptation and survival and will open newer avenues in the form of drug targets that may be used in the treatment of mycobacterial infections. IMPORTANCE M. smegmatis generates an inadequate amount of energy by substrate-level phosphorylation and mandatorily requires oxidative phosphorylation (OXPHOS) for its growth and metabolism. Here, we explored the influence of suboptimal OXPHOS on M. smegmatis physiology and metabolism. M. smegmatis harbors two identical copies of the atpD gene, which codes for the ATP synthase β subunit. Here, we carried out the deletion of only one copy of atpD in M. smegmatis to understand the bacterial survival response in an energy-deprived state. M. smegmatis ΔatpD shows remarkable phenotypic, metabolic, and physiological differences from the wild type. Our study thus establishes M. smegmatis ΔatpD as an energy-compromised mycobacterial strain, highlights the importance of ATP synthase in mycobacterial physiology, and further paves the way for the identification of novel antimycobacterial drug targets.

scholarly journals Distribution, Diversity, and Activities of Sulfur Dioxygenases in Heterotrophic Bacteria

2014 ◽  
Vol 80 (5) ◽  
pp. 1799-1806 ◽  
Author(s):  
Honglei Liu ◽  
Yufeng Xin ◽  
Luying Xun
Keyword(s):  
Elemental Sulfur ◽  
Heterotrophic Bacteria ◽  
Sulfur Oxidation ◽  
Wide Distribution ◽  
Content Type ◽  
Ethylmalonic Encephalopathy ◽  
Cell Extracts ◽  
Gene Coding ◽  
Chemolithotrophic Bacteria ◽  
Specific Activities

ABSTRACTSulfur oxidation by chemolithotrophic bacteria is well known; however, sulfur oxidation by heterotrophic bacteria is often ignored. Sulfur dioxygenases (SDOs) (EC 1.13.11.18) were originally found in the cell extracts of some chemolithotrophic bacteria as glutathione (GSH)-dependent sulfur dioxygenases. GSH spontaneously reacts with elemental sulfur to generate glutathione persulfide (GSSH), and SDOs oxidize GSSH to sulfite and GSH. However, SDOs have not been characterized for bacteria, including chemolithotrophs. The gene coding for human SDO (human ETHE1 [hETHE1]) in mitochondria was discovered because its mutations lead to a hereditary human disease, ethylmalonic encephalopathy. Using sequence analysis and activity assays, we discovered three subgroups of bacterial SDOs in the proteobacteria and cyanobacteria. Ten selected SDO genes were cloned and expressed inEscherichia coli, and the recombinant proteins were purified. The SDOs used Fe2+for catalysis and displayed considerable variations in specific activities. The wide distribution of SDO genes reveals the likely source of the hETHE1 gene and highlights the potential of sulfur oxidation by heterotrophic bacteria.

scholarly journals Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

2011 ◽  
Vol 77 (23) ◽  
pp. 8234-8240 ◽  
Author(s):  
Grigoriy E. Pinchuk ◽  
Oleg V. Geydebrekht ◽  
Eric A. Hill ◽  
Jennifer L. Reed ◽  
Allan E. Konopka ◽  
...  
Keyword(s):  
Electron Acceptor ◽  
Shewanella Oneidensis ◽  
Anaerobic Growth ◽  
Lactate Metabolism ◽  
Content Type ◽  
Substrate Level Phosphorylation ◽  
Formate Oxidation ◽  
Pyruvate Fermentation ◽  
Wide Range ◽  
Substrate Level

ABSTRACTShewanella oneidensisMR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O2, lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions,S. oneidensisMR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O2limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation byS. oneidensisMR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independentd-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.

scholarly journals Olsenella scatoligenes sp. nov., a 3-methylindole- (skatole) and 4-methylphenol- (p-cresol) producing bacterium isolated from pig faeces

2015 ◽  
Vol 65 (Pt_4) ◽  
pp. 1227-1233 ◽  
Author(s):  
Xiaoqiong Li ◽  
Rikke Lassen Jensen ◽  
Ole Højberg ◽  
Nuria Canibe ◽  
Bent Borg Jensen
Keyword(s):  
Acetic Acid ◽  
Type Species ◽  
Phylogenetic Analyses ◽  
Boar Taint ◽  
Rrna Gene ◽  
Strictly Anaerobic ◽  
Content Type ◽  
Link Type ◽  
Cell Extracts ◽  
Hydroxyphenylacetic Acid

Strain SK9K4T, which is a strictly anaerobic, non-motile, non-sporulating, Gram-stain-positive, saccharolytic coccobacillus, was isolated from pig faeces. SK9K4T metabolized indol-3-acetic acid to 3-methylindole (skatole), which is the main contributor to boar taint; it also produced 4-methylphenol (p-cresol) from p-hydroxyphenylacetic acid. Phylogenetic analyses, based on 16S rRNA gene sequences, revealed that the isolate represented a new lineage within the genus Olsenella of the family Atopobiaceae . Strain SK9K4T was most closely related to the type strains of the three species of the genus Olsenella with validly published names; Olsenella profusa DSM 13989T (93.6 %), Olsenella uli DSM 7084T (93.5 %) and Olsenella umbonata DSM 22620T (92.7 %). DNA–DNA relatedness values of strain SK9K4T with O. profusa , O. uli and O. umbonata were 28.3 %, 69.1 % and 27.2 %, respectively. The genomic DNA G+C content was 62.1 mol% and the major cellular fatty acids (constituting >10 % of the total) were C14 : 0 and C18 : 1ω9c. The major end product of glucose fermentation was lactic acid, with minor amounts of acetic acid and formic acid; no H2 was produced. Discrepancies in the fatty acid profiles, the MALDI-TOF mass spectra of cell extracts and the physiological and biochemical characteristics differentiated strain SK9K4T from other species of the genus Olsenella and indicate that the isolate represents a novel species within this genus. The name Olsenella scatoligenes sp. nov., is proposed and the type strain is SK9K4T ( = JCM 19907T = DSM 28304T).

scholarly journals Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP

2020 ◽  
Vol 287 (1934) ◽  
pp. 20201538
Author(s):  
Euki Yazaki ◽  
Keitaro Kume ◽  
Takashi Shiratori ◽  
Yana Eglit ◽  
Goro Tanifuji ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Metabolic Pathways ◽  
Large Scale ◽  
Phylogenetic Position ◽  
Acetyl Coa ◽  
Transcriptomic Data ◽  
Substrate Level Phosphorylation ◽  
Substrate Level ◽  
Phylogenomic Analyses

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

scholarly journals Uncoupling of Substrate-Level Phosphorylation in Escherichia coli during Glucose-Limited Growth

2012 ◽  
Vol 78 (19) ◽  
pp. 6908-6913 ◽  
Author(s):  
Poonam Sharma ◽  
Klaas J. Hellingwerf ◽  
Maarten J. Teixeira de Mattos ◽  
Martijn Bekker
Keyword(s):  
Escherichia Coli ◽  
Proton Translocation ◽  
Growth Conditions ◽  
Physiological Data ◽  
Synthesis Rate ◽  
Limited Growth ◽  
Content Type ◽  
E Coli ◽  
Substrate Level Phosphorylation ◽  
Substrate Level

ABSTRACTThe respiratory chain ofEscherichia colicontains three different cytochrome oxidases. Whereas the cytochromebooxidase and the cytochromebd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochromebd-II oxidase. Recently, however, this view was challenged by anin vitrobiochemical analysis that showed that the activity of cytochromebd-II oxidase does contribute to proton translocation with an H+/e−stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growingE. colistrains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochromebd-II oxidase, compatible with an H+/e−ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation inE. coliunder energy-limited growth conditions.

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 Functional Expression of theClostridium ljungdahliiAcetyl-Coenzyme A Synthase inClostridium acetobutylicumas Demonstrated by a NovelIn VivoCO Exchange Activity En Route to Heterologous Installation of a Functional Wood-Ljungdahl Pathway

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Alan G. Fast ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Clostridium Acetobutylicum ◽  
Coenzyme A ◽  
Functional Expression ◽  
Recombinant Enzymes ◽  
Carbonyl Carbon ◽  
Acetyl Coa ◽  
Content Type ◽  
Clostridium Ljungdahlii ◽  
Exchange Activity

ABSTRACTEngineering the Wood-Ljungdahl pathway (WLP) in the established industrial organismClostridium acetobutylicumwould allow for the conversion of carbohydrates into butanol, acetone, and other metabolites at higher yields than are currently possible, while minimizing CO2and H2release. To this effect, we expressed 11Clostridium ljungdahliicore genes coding for enzymes and accessory proteins of the WLP inClostridium acetobutylicum. The engineered WLP inC. acetobutylicumshowed functionality of the eastern branch of the pathway based on the formation of labeled 5,10-methylenetetrahydrofolate from13C-labeled formate, as well as functionality of the western branch as evidenced by the formation of CO from CO2. However, the lack of labeling in acetate and butyrate pools indicated that the connection between the two branches is not functional. The focus of our investigation then centered on the functional expression of the acetyl-coenzyme A (CoA) synthase (ACS), which forms a complex with the CO dehydrogenase (CODH) and serves to link the two branches of the WLP. The CODH/ACS complex catalyzes the reduction of CO2to CO and the condensation of CO with a methyl group to form acetyl-CoA, respectively. Here, we show the simultaneous activities of the two recombinant enzymes. We demonstratein vivothe classicalin vitroACS carbonyl carbon exchange assay, whereby the carbonyl carbon of acetyl-CoA is exchanged with the CO carbon. Our data suggest that the low heterologous expression of ACS may limit the functionality of the heterologous WLP inC. acetobutylicum.IMPORTANCEThe bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) fromC. ljungdahliiwas heterologously expressed in the obligate heterotrophC. acetobutylicum. The functional activity of the CODH was confirmed through both the oxidation and reduction of CO, as had previously been shown for the heterologous CODH fromClostridium carboxidivorans. Significantly, a novelin vivoassay for ACS exchange activity using13C-tracers was developed and used to confirm functional ACS expression.

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