A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase

Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhE(Meta). The AdhE(Meta) protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhE(Meta) did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhE Meta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhE(Meta) is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhE Meta did, however, confer one phenotype to E. coli. AdhE(-) mutants of E. coli fail to ferment glucose and introduction of adhE Meta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions