Lee D. Mermelstein

Cloning of an NADH‐Dependent Butanol Dehydrogenase Gene from Clostridium acetobutylicuma

The acetone-butanol fermentation of C. acetobutylicum is characterized by the unique shift from acid to solvent production. The mechanism of the solventogenic switch involves the induction of several enzymes, including NADH-dependent butanol dehydrogenase (BDH) at the onset of solventogenesis. This enzyme is responsible for the final conversion of butyraldehyde to butanol, and is distinct from the NADPH-dependent alcohol dehydrogenase (ADH) also present in the organism. To characterize the genetic control of this gene, we have cloned and expressed it in E. coli. A lambda EMBL3 phage library of C. acetobutylicum DNA was screened via plaque hybridization using a [32P]-radiolabeled, 32-fold degenerate, 62-mer oligonucleotide probe. The probe was designed by reverse translation of the NH2-terminal amino acid sequence of purified BDH II. Southern blot experiments indicate that the phage insert was of clostridial origin and had no homology with the previously cloned NADPH-dependent ADH. Subcloning of DNA from purified positive plaques has localized the gene to a 3.5-kb EcoRI fragment from which the enzyme is well expressed. The sequence of the 25 NH2-terminal amino acids for the cloned enzyme purified from E. coli was determined and found to be identical to that for the clostridial NADH-dependent BDH II. Maxicell analysis of [35S]-radiolabeled plasmid-encoded proteins identified a species encoded by the clostridial insert with the expected Mr of 42 kD.

Amplification of Homologous Fermentative Genes in Clostridium Acetobutylicum ATCC 824

Clostridium acetobutylicum, a natural producer of butanol, acetone and ethanol, is of interest as a potential commercial source of these solvents. The biochemistry by which C. acetobutylicum initially produces acetate and butyrate and subsequently the above solvents, in batch culture, is well understood. The genetics of this anaerobic, highly regulated, branched, primary metabolism is, however, poorly understood despite the fact that many of the genes responsible for the formation of the various acids and solvents have recently been cloned [1–5].