title: PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.
abstract: The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. Electronic address: 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, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Isotani
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Makino
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kato
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Kouta
authors.initials: K
authors.lastname: Kitayama
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Tuyoshi
authors.initials: T
authors.lastname: Ishimota
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D055162:Biocatalysis; D019117:Burkholderia / Q000235:genetics; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D000426:Alcohol Dehydrogenase
keywords: Alcohol dehydrogenase gene; Biocatalysis; Chiral alcohol; Pseudomonas; Pseudomonas vector pNUKT; Soil metagenome