Cloning and chatacterization of a zymomonas mobilis alcohol dehydrogenase gene, and development of plasmid vectors for zymomonas

Abstract

From the $\underline{Zymomonas}$ $\underline{mobilis}$, having potential value for ethanol fermentation, the structural gene encoding alcohol dehydrogenase (ADH; EC 1.1.1.1) responsible for the final step during alcoholic fermentation was cloned into $\underline{Escherichia}$ $\underline{coli}$ with plasmic pUC9 using allyl alcohol. Two recombinant plasmids were isolated from E.coli transformants showing ADH activity, which were sensitive to allyl alcohol, named pADS93 and pADL99. These plasmids was shown to share a common $\underline{Z.}$ $\underline{mobilis}$ chromosomal DNA of 2.6 kb. Electrophoretical analysis of total cell extrats on a nondenaturing polyacrylamide gel indicated that the two clones, $\underline{E.}$ $\underline{coli}$ (pADS93) and $\underline{E.}$ $\underline{coli}$ (pADL99), produced the identical enzyme displaying a same band of activity comigrating with one (ZADH-2) of the two $\underline{Z.}$ $\underline{mobilis}$ alcohol dehydrogenase isozymes (ZADH). In addition, the enzyme from crude extracts of $\underline{E.}$ $\underline{coli}$ (pADS93) was purified to compare with the ADH produced by $\underline{Z.}$ $\underline{mobilis}$. SDS-polyacrylamide gel electrophoresis of these final preparations revealed that ZADH-2 subunit purified from $\underline{E.}$ $\underline{coli}$ (pADS93) had a single band identical to the enzyme subunit from $\underline{Z.}$ $\underline{mobilis}$ at 40,000 dalton of molecular weight. Analytical gel filtration of Sephadex G-200 led to conclusion that this enzyme is a tetramer with molecular mass 170,000 dalton. Southern hybridization indicated that the structural gene (zadhII) for ZADH-2 was homologous between $\underline{Zymomonas}$ strains, though it did not have homology to that for other isozyme (ZADH 1). The complete nucleotide sequence of the zadhII was determined. The zadhII consists of an open reading frame, 1152 bp long, commencing from the ATG start codon encoding a polypeptide of 383 amino acid resi...