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Wolfgang Buckel - One of the best experts on this subject based on the ideXlab platform.
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Reduction of ferredoxin or oxygen by flavin‐based electron bifurcation in Megasphaera elsdenii
FEBS Journal, 2015Co-Authors: Nilanjan Pal Chowdhury, Jörg Kahnt, Wolfgang BuckelAbstract:Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to Butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421–432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + Butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or Butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2O2, whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed Butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles’ heel of anaerobes when exposed to air.
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effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase electron transferring flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
Journal of Bacteriology, 2013Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
Journal of Bacteriology, 2013Co-Authors: E A Aboulnaga, Wolfgang Buckel, Olaf Pinkenburg, Johannes Schiffels, Ahmed A. El-refai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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Crystal structure of the complex between 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and CoA
Archives of Microbiology, 2012Co-Authors: Sofia Macieira, Wolfgang Buckel, Jin Zhang, Albrecht MesserschmidtAbstract:Clostridium aminobutyricum ferments 4-aminobutyrate (γ-aminobutyrate, GABA) to ammonia, acetate and butyrate via 4-hydroxybutyrate that is activated to the CoA-thioester catalyzed by 4-hydroxybutyrate CoA-transferase. Then, 4-hydroxyButyryl-CoA is dehydrated to crotonyl-CoA, which disproportionates to Butyryl-CoA and acetyl-CoA. Cocrystallization of the CoA-transferase with the alternate substrate Butyryl-CoA yielded crystals with non-covalently bound CoA and two water molecules at the active site. Most likely, Butyryl-CoA reacted with the active site Glu238 to CoA and the mixed anhydride, which slowly hydrolyzed during crystallization. The structure of the CoA is similar but less stretched than that of the CoA-moiety of the covalent enzyme-CoA-thioester in 4-hydroxybutyrate CoA-transferase from Shewanella oneidensis . In contrast to the structures of the apo-enzyme and enzyme-CoA-thioester, the structure described here has a closed conformation, probably caused by a flip of the active site loop (residues 215–219). During turnover, the closed conformation may protect the anhydride intermediate from hydrolysis and CoA from dissociation from the enzyme. Hence, one catalytic cycle changes conformation of the enzyme four times: free enzyme—open conformation, CoA+ anhydride 1—closed, enzyme-CoA-thioester—open, CoA + anhydride-2—closed, free enzyme—open.
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The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Henning Seedorf, Wolfgang Buckel, Julia Hinderberger, W. Florian Fricke, Birgit Veith, Holger Brüggemann, Heiko Liesegang, Axel Strittmatter, Marcus Miethke, Fuli LiAbstract:Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic Butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to Butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions.
Thorsten Selmer - One of the best experts on this subject based on the ideXlab platform.
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effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase electron transferring flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
Journal of Bacteriology, 2013Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
Journal of Bacteriology, 2013Co-Authors: E A Aboulnaga, Wolfgang Buckel, Olaf Pinkenburg, Johannes Schiffels, Ahmed A. El-refai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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acryloyl coa reductase from clostridium propionicum an enzyme complex of propionyl coa dehydrogenase and electron transferring flavoprotein
FEBS Journal, 2003Co-Authors: Marc Hetzel, Thorsten Selmer, Matthias Brock, BERNARD THOMAS GOLDING, Antonio J Pierik, Wolfgang BuckelAbstract:Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow–greenish enzyme complex [(α2βγ)4; molecular mass 600 ± 50 kDa] composed of a propionyl-CoA dehydrogenase (α2, 2 × 40 kDa) and an electron-transferring flavoprotein (ETF; β, 38 kDa; γ, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per α2βγ subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 ± 1 µm; kcat = 4.5 s−1 at 100 µm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 µm; kcat = 29 s−1 at 300 µm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 µm; kcat = 2.0 s−1) or Butyryl-CoA (Km = 100 µm; kcat = 3.5 s−1) as electron donor and 200 µm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of Butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the β and γ subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.
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an enzyme complex of propionyl coa dehydrogenase and electron transferring flavoprotein
2003Co-Authors: Marc Hetzel, Thorsten Selmer, Matthias Brock, BERNARD THOMAS GOLDING, Antonio J Pierik, Wolfgang BuckelAbstract:dimeric propionyl-CoA dehydrogenase subunit is similar to those of Butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the b and c subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloylCoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.
E A Aboulnaga - One of the best experts on this subject based on the ideXlab platform.
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effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase electron transferring flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
Journal of Bacteriology, 2013Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
Journal of Bacteriology, 2013Co-Authors: E A Aboulnaga, Wolfgang Buckel, Olaf Pinkenburg, Johannes Schiffels, Ahmed A. El-refai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxyButyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the Butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and Butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating Butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The Butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide Butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating Butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
Eleftherios T. Papoutsakis - One of the best experts on this subject based on the ideXlab platform.
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aldehyde alcohol dehydrogenase and or thiolase overexpression coupled with coa transferase downregulation lead to higher alcohol titers and selectivity in clostridium acetobutylicum fermentations
Biotechnology and Bioengineering, 2009Co-Authors: Ryan Sillers, Eleftherios T. Papoutsakis, Mohab A AlhinaiAbstract:Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acet- one, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently devel- oped ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alco- hol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and etha- nol production from Butyryl-CoA and acetyl-CoA, respec- tively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of Butyryl-CoA. In order to increase then the flux towards Butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to Butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpres- sion in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular Butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. Biotechnol. Bioeng. 2009;102: 38-49.
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Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.
Biotechnology and Bioengineering, 2009Co-Authors: Ryan Sillers, Mohab A. Al-hinai, Eleftherios T. PapoutsakisAbstract:Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acet- one, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently devel- oped ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alco- hol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and etha- nol production from Butyryl-CoA and acetyl-CoA, respec- tively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of Butyryl-CoA. In order to increase then the flux towards Butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to Butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpres- sion in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular Butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. Biotechnol. Bioeng. 2009;102: 38-49.
Kevin A. Reynolds - One of the best experts on this subject based on the ideXlab platform.
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Precursor supply for polyketide biosynthesis: the role of crotonyl-CoA reductase.
Metabolic Engineering, 2020Co-Authors: Kevin A. ReynoldsAbstract:Abstract Crotonyl-CoA reductase (CCR), which catalyzes the reduction of crotonyl-CoA to Butyryl-CoA, is common to most streptomycetes and appears to be inducible by either lysine or its catabolites in Streptomyces cinnamonensis grown in chemically defined medium. A major role of CCR in providing Butyryl-CoA from acetate for monensin A biosynthesis has been demonstrated by the observation of a change in the monensin A/monensin B ratio in the parent C730.1 strain (50/50) and a ccr (encoding CCR) disruptant (12:88) of S. cinnamonensis in a complex medium. Both strains produce significantly higher monensin A/monensin B ratios in a chemically defined medium containing valine as a major carbon source than in either complex medium or chemically defined medium containing alternate amino acids. This observation demonstrates that under certain growth conditions valine catabolism may have a more significant role than CCR in providing Butyryl-CoA. Such a process most likely involves an isomerization of the valine catabolite isoButyryl-CoA, catalyzed by the coenzyme B12-dependent isoButyryl-CoA mutase. Monensin labeling experiments using dual 13C-labeled acetate in the ccr-disrupted S. cinnamonensis indicate the presence of an additional coenzyme B12-dependent mutase linking branched and straight-chain C4 compounds by a new pathway.
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Multiple pathways for acetate assimilation in Streptomyces cinnamonensis.
Journal of Industrial Microbiology & Biotechnology, 2005Co-Authors: Konstantin Akopiants, Galina Florova, Chaoxuan Li, Kevin A. ReynoldsAbstract:In most bacteria acetate assimilation is accomplished via the glyoxylate pathway. Isocitrate lyase (ICL) and malate synthase (MS) are two key enzymes of this pathway, which results in the net generation of one molecule of succinyl-CoA from two acetyl-CoA molecules. Genetic and biochemical data have shown that genes encoding these key enzymes are present in streptomycetes, yet there has been no clear demonstration of the importance of these genes to acetate assimilation. In fact, for Streptomyces collinus an alternative Butyryl-CoA pathway has been shown to be critical for growth on acetate as a sole carbon source. Crotonyl-CoA reductase (CCR) is a key enzyme in this pathway and catalyzes the last step of the conversion of 2-acetyl-CoA molecules to Butyryl-CoA. In Streptomyces cinnamonensis C730.1, it has been shown that CCR and this Butyryl-CoA pathway provide the majority of methylmalonyl-CoA and ethylmalonyl-CoA for monensin A biosynthesis in an oil-based fermentation medium. We have cloned a MS homologue gene from this strain. Reverse transcription and direct enzyme assays demonstrated that neither this nor other MS genes were expressed during fermentation in an oil-based fermentation of either the C730.1 or L1 strain (a ccr mutant). Similarly, no ICL activity could be detected. The C730.1 but not the L1 strain was able to grow on acetate as a sole carbon source. The Streptomyces coelicolor aceA and aceB2 genes encoding ICL and MS were cloned into a Streptomyces expression plasmid (a derivative of pSET152) to create pExIM1. Enzyme assays and transcript analyses demonstrated expression of both of these proteins in C730.1/pExIM1 and L1/pExIM1 grown in an oil-based fermentation and tryptic soy broth media. Nonetheless, L1/pExIM1, like L1, was unable to grow on acetate as a sole carbon source, and was unable to efficiently generate precursors for monensin A biosynthesis in an oil-based fermentation, indicating that the additional presence of these two enzyme activities does not permit a functional glyoxylate cycle to occur. UV mutagenesis of S. cinnamonensis L1 and L1/pExIM1 led to mutants which were able to grow efficiently on acetate despite a block in the Butyryl-CoA pathway. Analysis of enzyme activity and monensin production from these mutants in an oil-based fermentation demonstrated that neither the glyoxylate cycle nor the Butyryl-CoA pathway function, suggesting the possibility of alternative pathways of acetate assimilation.
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In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.
Journal of Bacteriology, 1997Co-Authors: Kimberlee K. Wallace, Hamish A. I. Mcarthur, S. Lobo, Kevin A. ReynoldsAbstract:A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isoButyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while Butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that Butyryl-CoA and isoButyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from Butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isoButyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or Butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.
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In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes
Fems Microbiology Letters, 1995Co-Authors: Kimberlee K. Wallace, Bitao Zhao, Hamish A. I. Mcarthur, Kevin A. ReynoldsAbstract:The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylButyryl-CoA and isoButyryl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using Butyryl-CoA as a starter unit. Isomerization of the valine catabolite isoButyryl-CoA was shown to be only a minor source of this Butyryl-CoA.
