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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 NADH-dependent 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.

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H + ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.

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 NADH-dependent 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.