title: Cloning and Expression of a Clostridium acetobutylicum Alcohol Dehydrogenase Gene in Escherichia coli.
abstract: An alcohol dehydrogenase (ADH) gene from Clostridium acetobutylicum was cloned on a recombinant plasmid, pCADH100. Escherichia coli HB101, and an allyl alcohol-resistant mutant, HB101-adh1, containing this plasmid were unable to grow aerobically or anaerobically on agar media containing sublethal concentrations of allyl alcohol. E. coli HB101 and HB101-adh1 transformed with the plasmid pCADH100 produced increased levels of ethanol when grown anaerobically under alkaline conditions in the absence of nitrate. Cell extracts from aerobically and anaerobically grown E. coli HB101(pCADH100) and HB101-adhl(pCADH100) cells exhibited increased levels of NADP-dependent ADH activity with either ethanol or butanol as the substrate. The inability of E. coli HB101(pCADH100) to grow in the presence of allyl alcohol correlated with the appearance of an NADP-dependent ADH activity band on nondenaturing polyacrylamide gel electrophoresis with either ethanol or butanol as the substrate. The position of the cloned NADP-dependent ADH activity bands in E. coli HB101(pCADH100) cell extracts with either ethanol or butanol as the substrate coincided with the position of a single NADP-dependent ADH activity band in extracts of C. acetobutylicum cells. E. coli HB101(pCADH100) cell extracts prepared from both aerobically and anaerobically grown cells exhibited an additional protein band with an apparent M(r) of approximately 33,000 on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis which was absent in cell extracts of E. coli HB101. A protein band with a similar apparent M(r) was observed in cell extracts of C. acetobutylicum, and in vitro transcription and translation experiments with pCADH100 produced a major protein product with a similar apparent M(r).
authors.affiliation: Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Youngleson
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Santangelo
authors.forename: David T
authors.initials: DT
authors.lastname: Jones
authors.forename: David R
authors.initials: DR
authors.lastname: Woods