Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production

Mutant M5 of Clostridium acetobutylicum ATCC 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase, and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase activities, was transformed with plasmid pCAAD, which carries the gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol, 176:871-885, 1994). In batch fermentation studies, aad expression restored butanol formation (84 mM) in mutant M5 without any acetone formation or any significant increase in ethanol production. The corresponding protein (AAD) appeared as a ca. 96-kDa band in a denaturing protein gel. Expression of AAD in M5 resulted in restoration of BYDH activity and small increases in the activities of acetaldehyde dehydrogenase, butanol dehydrogenase, and ethanol dehydrogenase. These findings suggest that BYDH activity in C. acetobutylicum ATCC 824 resides largely in AAD, and that AAD's primary role is in the formation of butanol rather than of ethanol.