초록
<P>The butyrogenic genes from <I>Clostridium difficile</I> DSM 1296<SUP>T</SUP> have been cloned and expressed in <I>Escherichia coli</I>. 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 <I>E. coli</I> and kinetically characterized <I>in vitro</I>. 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. <I>In vitro</I> and <I>in vivo</I> studies demonstrated that <I>C. difficile</I> 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<SUP>+</SUP>-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from <I>C. difficile</I> 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 (<U><I>R</I></U><I>hodobacter</I> <U>n</U>itrogen <U>f</U>ixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.</P>