MOLECULAR CHARACTERIZATION OF AN ALDEHYDE ALCOHOL DEHYDROGENASE GENE FROM CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824/

A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was id entified immediately upstream of the previously cloned ctfA (J.W. Cary , D.J. Petersen, E.T. Papoutsakis, and G N. Bennett, Appl. Environ. Mi crobiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 an d sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer ex tension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD sho ws homology to aldehyde dehydrogenases of bacterial, fungal, mammalian , and plant origin, while the C-terminal section shows homology to alc ohol dehydrogenases of bacterial (which includes three clostridial alc ohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunct ional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a similar to 96-kDa band in denaturing protein gels, provides elevate d activities of NADH-dependent butanol dehydrogenase, NAD-dependent ac etaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open readin g frame that could potentially encode a 36,704-Da protein was identifi ed upstream of aad.