title: 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 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 (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
authors.affiliation: Aachen University of Applied Sciences, Campus Juelich, Department of Chemistry and Biotechnology, Juelich, Germany.
authors.forename: El-Hussiny
authors.initials: el-H
authors.lastname: Aboulnaga
authors.forename: Olaf
authors.initials: O
authors.lastname: Pinkenburg
authors.forename: Johannes
authors.initials: J
authors.lastname: Schiffels
authors.forename: Ahmed
authors.initials: A
authors.lastname: El-Refai
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Thorsten
authors.initials: T
authors.lastname: Selmer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism*; D044926:Butyryl-CoA Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D044927:Electron-Transferring Flavoproteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D010100:Oxygen; D011994:Recombinant Proteins
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D044927:Electron-Transferring Flavoproteins; D011994:Recombinant Proteins; D044926:Butyryl-CoA Dehydrogenase; D010100:Oxygen