CLONING, SEQUENCING, AND EXPRESSION OF CLUSTERED GENES ENCODING BETA-HYDROXYBUTYRYL-COENZYME-A (COA) DEHYDROGENASE, CROTONASE, AND BUTYRYL-COA DEHYDROGENASE FROM CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824

The enzymes beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase (BHBD), crotonase, and butyryl-CoA dehydrogenase (BCD) from Clostridium aceto butylicum are responsible for the formation of butyryl-CoA from acetoa cetyl-CoA. These enzymes are essential to both acid formation and solv ent formation by clostridia. Clustered genes encoding BHBD, crotonase, BCD, and putative electron transfer flavoprotein alpha and beta subun its have been cloned and sequenced. The nucleotide sequence of the crt gene indicates that it encodes crotonase, a protein with 261 amino ac id residues and a calculated molecular mass of 28.2 kDa; the hbd gene encodes BHBD, with 282 residues and a molecular mass of 30.5 kDa. Thre e open reading frames (bcd, etfB, and etfA) are located between crt an d hbd. The nucleotide sequence of bcd indicates that it encodes BCD, w hich consists of 379 amino acid residues and has high levels of homolo gy with various acyl-CoA dehydrogenases. Open reading frames etfB and etfA, located downstream of bcd, encode 27.2- and 36.1-kDa proteins, r espectively, and show homology with the fixAB genes and the alpha and beta subunits of the electron transfer flavoprotein. These findings su ggest that BCD in clostridia might interact with the electron transfer flavoprotein in its redox function. Primer extension analysis identif ied a promoter consensus sequence upstream of the crt gene, suggesting that the clustered genes are transcribed as a transcriptional unit an d form a BCS (butyryl-CoA synthesis) operon. A DNA fragment containing the entire BCS operon was subcloned into an Escherichia coli-C. aceto butylicum shuttle vector. Enzyme activity assays showed that crotonase and BHBD were highly overproduced in cell extracts from E. coli harbo ring the subclone. In C. acetobutylicum harboring the subclone, the ac tivities of the enzymes crotonase, BHBD, and BCD were elevated.