title: Efficient PCR-based amplification of diverse alcohol dehydrogenase genes from metagenomes for improving biocatalysis: screening of gene-specific amplicons from metagenomes.
abstract: 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 the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh 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.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan nbito@pu-toyama.ac.jp.
authors.forename: Nobuya
authors.initials: N
authors.lastname: Itoh
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Kariya
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Kurokawa
mesh_terms: D000192:Actinomycetales / Q000235:genetics; D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000438:Alcohols; D017931:DNA Primers; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase
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