Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli

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 cooxidant 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 (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed. G enome sequencing of organisms provides information regarding the distribution of genes encoding biotechnologically important metabolic pathways. This is true for the clostridial butyrogenic pathway, which converts acetyl-conenzyme A (CoA)-the terminal oxidation product of glucose via glycolysis-to butyrate. Genes encoding enzymes from this pathway are widespread in genome-sequenced clostridia and related species (1-8). In spite of the central importance of butyrate-forming genes in these organisms, only individual enzymes from a comparably small selection of organisms have been purified and carefully studied in the past (9-15). In particular, Clostridium acetobutylicum enzymes were of great interest due to the organism's capability of producing acetone and butanol (16)(17)(18). Driven by the urgent need to replace oil-derived fossil fuels, genetic engineering of microbes for production of butan-1-ol, a promising alternative transportation fuel, is proceeding worldwide (16,(18)(19)(20)(21)(22)(23)(24).A long-known metabolic route such as the butyrate pathway (Fig. 1A) can suddenly gain fundamentally new meanings when hitherto-unknown properties of individual enzymes within this pathway become known. While for decades the role of butyrate formation was generally a...

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

“…2B). This optimal ratio agrees well with the molecular masses of the stable clostridial Bcd-Etf complexes of Clostridium kluyveri (3), Clostridium difficile (30), and C. tetanomorphum (31, 32) with compositions of Etf 1 -Bcd(dimer) or Etf 2 -Bcd(tetramer) (see also Fig. 5…”

“…2E). As demonstrated for other flavinbased electron bifurcating systems (5,6,30), ferredoxin reduction is almost 100% indicating a tight energetic coupling between ferredoxin and crotonyl-CoA reduction. Limiting amounts of crotonyl-CoA under these conditions resulted in the reduction of 1 mol of ferredoxin/mol of crotonyl-CoA; hence at equilibrium the completely oxidized Fd accepts two electrons, one by each [4Fe-4S] cluster.…”

Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis

“…As shown in Table 1, the K M of butyrate was 79.9 µM and was the lowest of all the SCCAs tested. This K M value was similar to those previously reported for Ptb from other organisms [20,31]. The shortest and longest substrates tested, acetyl CoA (C2) and hexanoyl CoA (C6), proved to be poor substrates for Ptb judging by their poor fit to the Michaelis-Menten equation and low catalytic efficiency ( k cat / K M ) suggesting that the carbon chain length was suboptimal for efficient binding to the active site.…”

Regulating the Intersection of Metabolism and Pathogenesis in Gram-positive Bacteria

“…If proline is limiting in the growth medium or if proline reductase or PrdR is inactive, alternative pathways for NAD + regeneration [e.g., glycine reductase, alcohol dehydrogenase, succinate to crotonyl CoA, crotonyl CoA to butyrate (183, 184)] are induced (Bouillaut et al, manuscript in preparation). These alternative pathways (Fig.…”