Studies on the substrate range of Clostridium kluyveri; the use of propanol and succinate

Kenealy, William R.; Waselefsky, David M.

doi:10.1007/bf00408056

Cited by 116 publications

(106 citation statements)

References 18 publications

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“…A pathway was proposed, one in which succinate is first activated and then reduced by a two-step reaction to give 4-hydroxybutyrate, which is then further metabolized to crotonyl-coenzyme A (CoA) ( Fig. 1) (27). In a previous study, we discussed enzymes involved in the anaerobic breakdown of succinate by C. kluyveri, specifically, a succinyl-CoA:CoA transferase, a CoA-and NADP ϩ -dependent succinate-semialdehyde dehydrogenase, and a 4-hydroxybutyrate dehydrogenase (49).…”

mentioning

confidence: 99%

Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri

Söhling¹,

Gottschalk²

1996

J Bacteriol

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The gram-positive anaerobic bacterium Clostridium kluyveri (3) ferments ethanol and acetate to butyrate, caproate, and molecular hydrogen (7). ATP, required for growth, is gained by substrate-level phosphorylation from acetyl phosphate, and the quantity is proportional to the amount of hydrogen produced (46, 50). Investigations of additional metabolic abilities revealed that this organism can utilize crotonate, vinylacetate, and 4-hydroxybutyrate as substrates (4, 5) and is able to ferment the unusual substrate combination of succinate plus ethanol (27). A pathway was proposed, one in which succinate is first activated and then reduced by a two-step reaction to give 4-hydroxybutyrate, which is then further metabolized to crotonyl-coenzyme A (CoA) ( Fig. 1) (27). In a previous study, we discussed enzymes involved in the anaerobic breakdown of succinate by C. kluyveri, specifically, a succinyl-CoA:CoA transferase, a CoA-and NADP ϩ -dependent succinate-semialdehyde dehydrogenase, and a 4-hydroxybutyrate dehydrogenase (49). Wolff et al. (55) independently confirmed these data by 13 C-nuclear magnetic resonance studies as well as enzymatic investigations on the dehydrogenases. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes the last step of the succinatespecific pathway, the dehydration and isomerization of 4-hydroxybutyryl-CoA to crotonyl-CoA, was recently identified, purified, and characterized (45). We present here some molecular aspects of this pathway, including the cloning, sequencing, and heterologous expression of a C. kluyveri DNA region which encodes a succinyl-CoA:CoA transferase, the succinatesemialdehyde dehydrogenase, and the 4-hydroxybutyrate dehydrogenase. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. C. kluyveri (DSM 555) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Escherichia coli JM109 (58) and the pBluescript SK vector (Stratagene, San Diego, Calif.) were from the laboratory collection. C. kluyveri cells used for DNA isolation were grown at 37ЊC under strictly anaerobic conditions on ethanol (300 mM) and succinate (100 mM) as previously described (49). Cells for RNA preparation were cultured in the same medium except that succinate was replaced by acetate (100 mM). E. coli cultures were routinely grown at 30ЊC in Luria-Bertani (LB) medium (42) on a rotary shaker. Tetrazolium indicator plates (6) containing 4 g of 4-hydroxybutyrate per liter were employed for the screening procedure (oxidation of 4-hydroxybutyrate). Utilization of 4-hydroxybutyrate (4 g/liter) as a carbon source for recombinant E. coli clones was investigated in M9 medium (42), supplemented with a small amount of yeast extract (0.2 g/liter), MgSO 4 (2 mM), and CaCl 2 (0.1 mM). Ampicillin (75 mg/liter) was added to the media for E. coli as a selection marker, when needed.Nucleic acids isolation and recombinant DNA techniques. Chromosomal DNA from C. kluyveri was isolated by the method of Saito and Miura (41). Total RNA from C. kluyveri...

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confidence: 99%

Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri

Söhling¹,

Gottschalk²

1996

J Bacteriol

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“…C, kluyveri is also able to grow on crotonate, vinylacetate and 4-hydroxybutyrate as sole carbon and energy sources [4, 51. More recently it was shown that C. kluyveri can ferment a substrate combination of ethanol and succinate, producing acetate, butyrate and caproate [6]. A pathway was proposed, in which succinate is activated to succinyl-CoA, then metabolized to 4-hydroxybutyrate via two reduction steps analogous to acetaldehyde and alcohol dehydrogenase /6].…”

mentioning

confidence: 99%

Purification and characterization of a coenzyme‐A‐dependent succinate‐semialdehyde dehydrogenase from Clostridium kluyveri

Söhling

Gottschalk

1993

European Journal of Biochemistry

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Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinylCoA : 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; SDSRAGE 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 optimum was 7.0 for the reduction of succinyl-CoA, and 8.5 for the reverse reaction. K,,, values were determined for both the forward and. reverse directions. The kinetic data suggest a ping-pong mechanism.Clostridium kluyveri is an organism with an unusual fermentative metabolism; ethanol and acetate are oxidized to butyrate and caproate plus molecular hydrogen [l]. The amount of H, produced is related to the ATP which becomes available to the organism for growth and other expenditures 12, 31. C, kluyveri is also able to grow on crotonate, vinylacetate and 4-hydroxybutyrate as sole carbon and energy sources [4, 51. More recently it was shown that C. kluyveri can ferment a substrate combination of ethanol and succinate, producing acetate, butyrate and caproate [6]. A pathway was proposed, in which succinate is activated to succinyl-CoA, then metabolized to 4-hydroxybutyrate via two reduction steps analogous to acetaldehyde and alcohol dehydrogenase /6]. Experimental evidence of the enzymes required has not been presented. We report here on the detection of a succinyl-CoA : CoA transferase, a 4-hydroxybutyrate dehydrogenase [7] and a succinate-semialdehyde dehydrogenase in ethanolhccinate-grown cells of C. kluyveri and on the punfication and characterization of succinate-semialdehyde dehydrogenase. (0.05 %), para-amino benzoic acid (0.05 mg/l), biotin (0.01 mgA) and with the trace element solution (10 mVI) described by Kenealy and Zeikus [9]. The medium was prepared strictly anaerobically using an atmosphere of N,/CO, (80 : 20). After sterilization, the medium was reduced with cysteine/HCl and Na,S 3 9 H,O (0.025% of each). The cells (450 g) were harvested anaerobically, frozen in liquid nitrogen and stored at -2OOC until used. MATERIALS AND METHODS Organism and growth conditions MaterialsQ-Sepharose fast flow was obtained from Pharmacia. Reactive-Red-120-agarose (type 3000), succinate semialdehyde and the coenzyme-A derivatives were obtained from Sigma. 4-Hydroxybutyrate (sodium salt) and 2-amino-2-methyl-l,3-propanediol were from Aldrich, coenzyme A and other biochemicals from Boehringer.Succinyl-CoA was prepared from succinate anhydride and coenzyme A according to Simon and Shemin [lo]. The reaction was followed by the increasing absorbance at 235 nm, due to the formation of the thioester bond [ll]. To remove the remaining coenzyme A, a few crystals of 53'-dithi...

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“…When propanol is used as a substrate, propionate and valerate are produced instead of butyrate and caproate (Kenealy and Waselefsky, 1985). The fermentation of C. tyrobutyricum is similar to that of C. kluyveri.…”

Section: Butyrate and Caproatementioning

confidence: 97%

“…A special case is C. kluyveri, which can metabolize ethanol (or propanol), in the absence or presence of acetate (or succinate), to butyrate and caproate (Kenealy and Waselefsky, 1985). The fermentation pathway performed by saccharolytic clostridia is exemplified by the clostridial-type strain C. butyricum.…”

Section: The Saccharolytic Clostridiamentioning

confidence: 99%

Acetogenic and Acid-Producing Clostridia

1989

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Studies on the substrate range of Clostridium kluyveri; the use of propanol and succinate

Cited by 116 publications

References 18 publications

Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri

Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri

Purification and characterization of a coenzyme‐A‐dependent succinate‐semialdehyde dehydrogenase from Clostridium kluyveri

Acetogenic and Acid-Producing Clostridia

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