RRC ID 34390
著者 Itoh N, Kariya S, Kurokawa J.
タイトル Efficient PCR-based amplification of diverse alcohol dehydrogenase genes from metagenomes for improving biocatalysis: screening of gene-specific amplicons from metagenomes.
ジャーナル Appl Environ Microbiol
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.
巻・号 80(20)
ページ 6280-9
公開日 2014-10-1
DOI 10.1128/AEM.01529-14
PII AEM.01529-14
PMID 25085492
PMC PMC4178635
MeSH Actinomycetales / genetics Alcohol Dehydrogenase / genetics* Alcohol Dehydrogenase / metabolism* Alcohols / metabolism Amino Acid Sequence Biotechnology / methods Catalysis Cloning, Molecular DNA Primers / genetics Escherichia coli / genetics Escherichia coli / metabolism Gene Library Metagenome* Molecular Sequence Data Phylogeny Polymerase Chain Reaction Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Recombinant Proteins / metabolism Sequence Homology, Amino Acid
IF 4.016
引用数 11
WOS 分野 BIOTECHNOLOGY & APPLIED MICROBIOLOGY MICROBIOLOGY
リソース情報
一般微生物 JCM 15132