Cloning of an NADH-dependent butanol dehydrogenase gene from Clostridium acetobutylicum

Abstract

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.