Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes

Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh ) genes from metagenomes based on theLeifsonia speciesadh gene (lsadh ), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs oflsadh , and PCR was used to amplify nearly full-lengthadh genes from metagenomic DNAs. Alladh preparations were fused withlsadh at the terminal region and used to constructEscherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified asadh positive (∼60%). Finally, 40adh genes,Hladh -001 toHladh -040 (forh omologousL eifsoniaadh ), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. TheHladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols.