The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADHdependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.Clostridium acetobutylicum is a gram-positive spore-forming anaerobic bacterium capable of converting different sugars and polysaccharides to organic acids (acetate and butyrate) and solvents (acetone, butanol, and ethanol). Acetone is produced from acetoacetyl coenzyme A (acetoacetyl-CoA) in a two-step process that involves a CoA transferase and an acetoacetate decarboxylase. The CoA transferase consists of two different subunits encoded by the ctfA and ctfB genes, which are part of the sol operon (13). The acetoacetate decarboxylase gene (adc), adjacent but not part of the sol operon, is transcribed in the opposite direction from its own promoter. Both the sol operon and the adc gene are located on the pSOL1 megaplasmid (9). Ethanol and butanol are produced from acetyl-CoA and butyryl-CoA, respectively, in two reductive steps catalyzed by aldehyde dehydrogenases and alcohol dehydrogenases (ADHs). The aad gene of C. acetobutylicum ATCC 824 (referred to as adhE in strain DSM 792) is part of the sol operon, and it encodes a bifunctional aldehyde/alcohol dehydrogenase (AADH) (13,29). This enzyme is mainly involved in butanol formation. Two NADH-dependent butanol dehydrogenases (BDH) (BDH I and BDH II) have been purified, and their genes (bdhA and bdhB) have been cloned (43). These genes are adjacent on the chromosome but are each transcribed by their own promoters.In continuous culture, C. acetobutylicum can be maintained in three different stable metabolic states (15): acidogenic (production of acetic and butyric acids) when grown at neutral pH on glucose, solventogenic (production of acetone, butanol, and ethanol) when grown at low pH on glucose, and alcohologenic (formation of butanol and ethanol but not acetone) when grown at neutral pH under conditions of high NAD(P)H availability. When solventogenesis is induced by lowering the pH of a continuous culture, transcription ...