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Largus T. Angenent - One of the best experts on this subject based on the ideXlab platform.
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Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation
Biotechnology for Biofuels, 2017Co-Authors: Sylvia Gildemyn, Bastian Molitor, Joseph G. Usack, Mytien Nguyen, Korneel Rabaey, Largus T. AngenentAbstract:Background The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures. Results Here, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n -caproic acid, which were up to 40 mM day^−1 (4.64 g L^−1 day^−1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n -caproic acid production. In addition, n -caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions. Conclusions Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.
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Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation
Biotechnology for biofuels, 2017Co-Authors: Sylvia Gildemyn, Bastian Molitor, Joseph G. Usack, Mytien Nguyen, Korneel Rabaey, Largus T. AngenentAbstract:Background The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures.
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a narrow ph range supports butanol hexanol and octanol production from syngas in a continuous co culture of Clostridium ljungdahlii and Clostridium kluyveri with in line product extraction
Frontiers in Microbiology, 2016Co-Authors: Hanno Richter, Bastian Molitor, Martijn Diender, Diana Machado De Sousa, Largus T. AngenentAbstract:Carboxydotrophic bacteria (CTB) have received attention due to their ability to synthesize commodity chemicals from producer gas and synthesis gas (syngas). CTB have an important advantage of a high product selectivity compared to chemical catalysts. However, the product spectrum of wild-type CTB is narrow. Our objective was to investigate whether a strategy of combining two wild-type bacterial strains into a single, continuously fed bioprocessing step would be promising to broaden the product spectrum. Here, we have operated a syngas-fermentation process with Clostridium ljungdahlii and Clostridium kluyveri with in-line product extraction through gas stripping and product condensing within the syngas recirculation line. The main products from C. ljungdahlii fermentation at a pH of 6.0 were ethanol and acetate at net volumetric production rates of 65.5 and 431 mmol C•L-1•d-1, respectively. An estimated 2/3 of total ethanol produced was utilized by C. kluyveri to chain elongate with the reverse β-oxidation pathway, resulting in n-butyrate and n-caproate at net rates of 129 and 70 mmol C•L-1•d-1, respectively. C. ljungdahlii likely reduced the produced carboxylates to their corresponding alcohols with the reductive power from syngas. This resulted in the longer-chain alcohols n-butanol, n-hexanol, and n-octanol at net volumetric production rates of 39.2, 31.7, and 0.045 mmol C•L-1•d-1, respectively. The continuous production of the longer-chain alcohols occurred only within a narrow pH spectrum of 5.7-6.4 due to the pH discrepancy between the two strains. Regardless whether other wild-type strains could overcome this pH discrepancy, the specificity (mol carbon in product per mol carbon in all other liquid products) for each longer-chain alcohol may never be high in a single bioprocessing step. This, because two bioprocesses compete for intermediates (i.e., carboxylates): 1) chain elongation; and 2) biological reduction. This innate competition resulted in a mixture of n-butanol and n-hexanol with traces of n-octanol.
Korneel Rabaey - One of the best experts on this subject based on the ideXlab platform.
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Biochar and activated carbon enhance ethanol conversion and selectivity to caproic acid by Clostridium kluyveri
Bioresource technology, 2020Co-Authors: Stef Ghysels, Korneel Rabaey, Sara Buffel, Frederik Ronsse, Ramon GaniguéAbstract:Abstract Syngas from biomass or steel mills can be fermented into a dilute stream of ethanol and acetic acid, which requires energy intensive distillation for product recovery. This can be circumvented by selective secondary fermentation of the syngas fermentation effluent to caproic acid as easier recoverable platform chemical with Clostridium kluyveri. Here, we explore the impact of biochar and activated carbon on this process. Changes during the fermentation with biochar or activated carbon were monitored, different doses were tested and the recyclability of biochar and activated carbon was assessed. Biochar decreased the lag phase and increased the caproic acid production rate (up to 0.50 g · L - 1 · h - 1 ). Upon recycling for subsequent fermentation, biochar retained this property largely. Activated carbon addition, especially at high dose, could potentially increase the conversion and selectivity towards caproic acid to 14.15 g · L - 1 (control: 11.01 g · L - 1 ) and 92% (control: 84%), respectively.
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Ethanol:propionate ratio drives product selectivity in odd-chain elongation with Clostridium kluyveri and mixed communities.
Bioresource technology, 2020Co-Authors: Pieter Candry, Korneel Rabaey, Barbara Ulcar, Camille Petrognani, Ramon GaniguéAbstract:Microbial production of valerate, a five-carbon carboxylate, can occur from propionate and ethanol through a process called odd-chain elongation. The generation of even-chain compounds in this process lowers product selectivity, forming a key challenge. This study investigated factors determining product selectivity during odd-chain elongation in an odd-chain elongating mixed community and the pure culture Clostridium kluyveri DSM555. Incubations at different ratios of ethanol:propionate showed that increasing ratios (from 0.5 to 7) lowered product specificity, as evidenced by a decrease in the odd:even product ratio from 5.5 to 1.5 for C. kluyveri and from 15 to 0.8 for the mixed community. The consistency of these observations with literature data suggests that control of ethanol:propionate ratio offers a robust tool for process control in odd-chain elongation, while the flexible metabolism can also have implications for efficient use of ethanol during even-chain elongation processes.
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Enrichment and characterisation of ethanol chain elongating communities from natural and engineered environments.
Scientific reports, 2020Co-Authors: Pieter Candry, Korneel Rabaey, Shengle Huang, José M. Carvajal-arroyo, Ramon GaniguéAbstract:Chain elongation is a microbial process in which an electron donor, such as ethanol, is used to elongate short chain carboxylic acids, such as acetic acid, to medium chain carboxylic acids. This metabolism has been extensively investigated, but the spread and differentiation of chain elongators in the environment remains unexplored. Here, chain elongating communities were enriched from several inocula (3 anaerobic digesters, 2 animal faeces and 1 caproic acid producing environment) using ethanol and acetic acid as substrates at pH 7 and 5.5. This approach showed that (i) the inoculum’s origin determines the pH where native chain elongators can grow; (ii) pH affects caproic acid production, with average caproic acid concentrations of 6.4 ± 1.6 g·L−1 at pH 7, versus 2.3 ± 1.8 g·L−1 at pH 5.5; however (iii) pH does not affect growth rates significantly; (iv) all communities contained a close relative of the known chain elongator Clostridium kluyveri; and (v) low pH selects for communities more enriched in this Clostridium kluyveri-relative (57.6 ± 23.2% at pH 7, 96.9 ± 1.2% at pH 5.5). These observations show that ethanol-consuming chain elongators can be found in several natural and engineered environments, but are not the same everywhere, emphasising the need for careful inoculum selection during process development.
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A novel high-throughput method for kinetic characterisation of anaerobic bioproduction strains, applied to Clostridium kluyveri.
Scientific reports, 2018Co-Authors: Pieter Candry, Ramon Ganigué, Timothy Van Daele, Kyrina Denis, Youri Amerlinck, Stephen Andersen, Jan Arends, Ingmar Nopens, Korneel RabaeyAbstract:Hexanoic acid (HA), also called caproic acid, can be used as an antimicrobial agent and as a precursor to various chemicals, such as fuels, solvents and fragrances. HA can be produced from ethanol and acetate by the mesophilic anaerobic bacterium Clostridium kluyveri, via two successive elongation steps over butyrate. A high-throughput anaerobic growth curve technique was coupled to a data analysis framework to assess growth kinetics for a range of substrate and product concentrations. Using this method, growth rates and several kinetic parameters were determined for C. kluyveri. A maximum growth rate (µmax) of 0.24 ± 0.01 h-1 was found, with a half-saturation index for acetic acid (KS,AA) of 3.8 ± 0.9 mM. Inhibition by butyric acid occurred at of 124.7 ± 5.7 mM (KI,BA), while the final product, HA, linearly inhibited growth with complete inhibition above 91.3 ± 10.8 mM (KHA of 10.9*10-3 ± 1.3*10-3 mM-1) at pH = 7, indicating that the hexanoate anion also exerts toxicity. These parameters were used to create a dynamic mass-balance model for bioproduction of HA. By coupling data collection and analysis to this modelling framework, we have produced a powerful tool to assess the kinetics of anaerobic micro-organisms, demonstrated here with C. kluyveri, in order further explore the potential of micro-organisms for chemicals production.
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Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation
Biotechnology for Biofuels, 2017Co-Authors: Sylvia Gildemyn, Bastian Molitor, Joseph G. Usack, Mytien Nguyen, Korneel Rabaey, Largus T. AngenentAbstract:Background The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures. Results Here, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n -caproic acid, which were up to 40 mM day^−1 (4.64 g L^−1 day^−1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n -caproic acid production. In addition, n -caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions. Conclusions Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.
Richard W. Pickersgill - One of the best experts on this subject based on the ideXlab platform.
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structure of a trimeric bacterial microcompartment shell protein etub associated with ethanol utilization in Clostridium kluyveri
Biochemical Journal, 2009Co-Authors: Dana Heldt, Stefanie Frank, Arefeh Seyedarabi, Dimitrios Ladikis, Martin J. Warren, Joshua B Parsons, Richard W. PickersgillAbstract:It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.
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Structure of a trimeric bacterial microcompartment shell protein, EtuB, associated with ethanol utilisation in Clostridium kluyveri
Biochemical Journal, 2009Co-Authors: Dana Heldt, Stefanie Frank, Arefeh Seyedarabi, Martin J. Warren, Joshua B Parsons, Dimitrios Ladakis, Richard W. PickersgillAbstract:It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins (EtuA and EtuB) are found encoded on the genome clustered with the genes for ethanol utilisation. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates, and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.
Rudolf K Thauer - One of the best experts on this subject based on the ideXlab platform.
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NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri
Journal of Bacteriology, 2010Co-Authors: Shuning Wang, Johanna Moll, Haiyan Huang, Rudolf K ThauerAbstract:It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP+ with reduced ferredoxin (Fdred) and the endergonic reduction of NADP+ with NADH in a reversible reaction: Fdred2− + NADH + 2 NADP+ + H+ = Fdox + NAD+ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.
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coupled ferredoxin and crotonyl coenzyme a coa reduction with nadh catalyzed by the butyryl coa dehydrogenase etf complex from Clostridium kluyveri
Journal of Bacteriology, 2008Co-Authors: Fuli Li, Julia Hinderberger, Henning Seedorf, Wolfgang Buckel, Jin Zhang, Rudolf K ThauerAbstract:Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.
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Coupled ferredoxin- and crotonyl-CoA reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri
2008Co-Authors: H. Hinderberger, Henning Seedorf, Wolfgang Buckel, Jin Zhang, Rudolf K ThauerAbstract:Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper
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Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri
Journal of Bacteriology, 2007Co-Authors: Fuli Li, Julia Hinderberger, Henning Seedorf, Wolfgang Buckel, Jin Zhang, Rudolf K ThauerAbstract:Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH (E0′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.
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Re-citrate synthase from Clostridium kluyveri is phylogenetically related to homocitrate synthase and isopropylmalate synthase rather than to Si-citrate synthase.
Journal of bacteriology, 2007Co-Authors: Christoph H. Hagemeier, Henning Seedorf, Gerhard Gottschalk, Rudolf K ThauerAbstract:The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.
Gerhard Gottschalk - One of the best experts on this subject based on the ideXlab platform.
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Re-citrate synthase from Clostridium kluyveri is phylogenetically related to homocitrate synthase and isopropylmalate synthase rather than to Si-citrate synthase.
Journal of bacteriology, 2007Co-Authors: Christoph H. Hagemeier, Henning Seedorf, Gerhard Gottschalk, Rudolf K ThauerAbstract:The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.
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Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri.
Journal of bacteriology, 1996Co-Authors: B Söhling, Gerhard GottschalkAbstract:A region of genomic DNA from Clostridium kluyveri was cloned in Escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene. The gene region (6,575 bp) contained several open reading frames which encoded the coenzyme A (CoA)- and NADP+-dependent succinate-semialdehyde dehydrogenase (sucD), the 4-hydroxybutyrate dehydrogenase (4hbD), and a succinyl-CoA;CoA transferase (cat1), as analyzed by heterologous expression in E. coli. An open reading frame encoding a putative membrane protein (orfY) and the 5' region of a gene encoding a sigma 54-homologous sigma factor (sigL) were identified as well. Transcription was investigated by Northern (RNA) blot analysis. Protein sequence comparisons of SucD and 4HbD revealed similarities to the adhE (aad) gene products from E. coli and Clostridium acetobutylicum and to enzymes of the novel class (III) of alcohol dehydrogenases. A comparison of CoA-dependent aldehyde dehydrogenases is presented.
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succinate ethanol fermentation in Clostridium kluyveri purification and characterisation of 4 hydroxybutyryl coa dehydratase vinylacetyl coa 3 2 isomerase
Archives of Microbiology, 1994Co-Authors: Uwe Scherf, Gerhard Gottschalk, Brigitte Söhling, Dietmar Linder, Wolfgang BuckelAbstract:Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.
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Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ^3-Δ^2-isomerase
Archives of Microbiology, 1994Co-Authors: Uwe Scherf, Gerhard Gottschalk, Brigitte Söhling, Dietmar Linder, Wolfgang BuckelAbstract:Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum . The dehydratase was revealed as being a homotetramer ( m =59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum . Moreover, the N -terminal sequences of the dehydratases from both organisms were found to be 63% identical.
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Purification and characterization of a coenzyme-A-dependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri.
European Journal of Biochemistry, 1993Co-Authors: Brigitte Söhling, Gerhard GottschalkAbstract:Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinyl-CoA:CoA transferase (0.28 U/mg), a coenzyme-A-dependent succinate-semialdehyde dehydrogenase (0.73 U/mg) and a NAD+-dependent 4-hydroxybutyrate dehydrogenase (0.25 U/mg). The semialdehyde dehydrogenase, which catalyzed the NADPH-dependent reduction of succinyl-CoA to succinate semialdehyde, was purified 59-fold to homogeneity. A molecular mass of 115000 Da was determined for the native enzyme; SDS/PAGE revealed one protein band at 55000, indicating that the active form is a dimer. The enzyme was highly specific for succinyl-CoA and succinate semialdehyde. The pH optium was 7.0 for the reduction of succinyl-CoA, and 8.5 for the reverse reaction Km values were determined for both the forward and reverse directions. The kinetic data suggest a ping-pong mechanism.