Gene Cloning and Expression of<i>Leifsonia</i>Alcohol Dehydrogenase (LSADH) Involved in Asymmetric Hydrogen-Transfer Bioreduction to Produce (<i>R</i>)-Form Chiral Alcohols

Inoue, Kousuke; Makino, Yoshihide; Dairi, Tohru; Itoh, Nobuhide

doi:10.1271/bbb.70.418

Cited by 44 publications

(22 citation statements)

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“…However, PAR and LSADH do not fully possess the required substrate specificity or stereospecificity; for example, LSADH does not accept methyl benzoylformate, 2-acetylpyridine, or 3-quinuclidinone as a substrate (26,27). Thus, we sought to clone genes encoding enzymes with properties distinct from those of LSADH.…”

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confidence: 99%

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

Kariya

Kurokawa

2014

Appl Environ Microbiol

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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 (ϳ60%). 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 antiPrelog chiral alcohols. Metagenomics is an emerging and powerful tool for the isolation of genes, the enzyme products of which have industrial applications (1-6). Screening of metagenomic libraries to find novel enzymes or enzymes homologous to those described previously has been used successfully to isolate lipases (7, 8), amylases (9), amidases (10), oxidoreductases (11), dehydratases (12), cytochrome P450 (13), styrene monooxygenase (14), and other enzymes (1-6). However, most previous approaches featured metagenomic DNA extraction and Escherichia coli library construction, followed by sequence-or function/molecule-based screens of the library. Such approaches are very time-consuming and inefficient, especially in terms of detection; much of the DNA sequenced and analyzed is irrelevant, and target genes may be expressed ambiguously in E. coli host cells. PCR amplification of truncated genes from metagenomes would facilitate the identification of genes encoding superior enzymes and yield homologous gene sets that could be used for DNA shuffling (15). Although previous studies based on PCR-mediated methods that utilize primers designed from inner conserved sequences have been conducted for biocatalysts, including lipase (8), cytochrome P450 (13), 2,5-diketo-D-gluconic acid reductase (16), Pseudomonas alcohol dehydrogenase (ADH) (17), and other biocatalysts (3, 4, 6), the methods are not very efficient in many cases and often fail to produce complete functional genes.Enantioselective organic synthesis is useful for producing chiral synthones for the preparation of fine chemicals, including pharmaceuticals and agricultural chemicals. The asymmetric reduction of ketones is one of the most promising approaches, because no substrate is lost, in contrast to when racemic separation is performed. Chiral metal complexes, such as BINAP-Ru, have been used successfully as chemocatalysts in a number of cases of enantioselective synthe...

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

Kariya

Kurokawa

2014

Appl Environ Microbiol

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“…The two enzymes have similar biochemical properties, (R)-specificity, and NAD-dependency for their reactions. The similarities of the deduced amino acid sequences of bacterial (R)-specific alcohol dehydrogenases isolated from Leifsonia sp., 8,12) Lactobacillus kefir, 6,11) and Lactobacillus brevis 5) were about 41.3%, 42.2%, and 38.9% respectively. The substrate specificities of the enzymes resembled those of AFPDH, even though they are tetrameric enzymes.…”

Section: Discussionmentioning

confidence: 97%

“…4) On the other hand, only a few enzymes follow the antiPrelog rule. 5) Several NADH-or NADPH-dependent (R)-specific alcohol dehydrogenases, from bacteria and NADPH-dependent ones from yeast have been purified, [5][6][7][8][9][10] and the genes of these enzymes have been cloned and sequenced, 5,11,12) but there have been no reports on the DNA sequence of an NADH-dependent highly enantioselective (R)-specific alcohol dehydrogenase from yeast.…”

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Cloning and Overexpression of an NADH-Dependent Alcohol Dehydrogenase Gene fromCandida marisInvolved in (R)-Selective Reduction of 5-Acetylfuro[2,3-c]pyridine

Kawano

Yano

Hasegawa

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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“…ChKRED20, predicted ketone reductase in this work, KC342020; LSADH, alcohol dehydrogenase from Leifsonia sp. S749, BAD99642 [31]; LK-ADH, R-specific alcohol dehydrogenase from Lactobacillus kefir, AAP94029 [28]; LB-RADH, R-specific alcohol dehydrogenase from Lactobacillus brevis, AJ544275 [32]; TtADH, alcohol dehydrogenase from Thermus thermophilus, 2D1Y [36]; CPADH, (S)-1-phenyl-1,2-ethanediol dehydrogenase from Candida parapsilosis, DQ675534 [29]. Gray shading indicates residues highly conserved in the SDR family.…”

Section: Discussionmentioning

confidence: 99%

“…S749 appeared to be most closely related to ChKRED20 with 51% identity. Furthermore, it has been reported that it follows the anti-Prelog's rule [31]. All other SDRs shared <40% identity with ChKRED20 ( Table 2).…”

Section: Sequence Analysis Of Chkred20mentioning

confidence: 91%

Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

Liu

Tang

Pei

et al. 2014

Journal of Molecular Catalysis B: Enzymatic

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Gene Cloning and Expression ofLeifsoniaAlcohol Dehydrogenase (LSADH) Involved in Asymmetric Hydrogen-Transfer Bioreduction to Produce (R)-Form Chiral Alcohols

Cited by 44 publications

References 28 publications

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

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

Cloning and Overexpression of an NADH-Dependent Alcohol Dehydrogenase Gene fromCandida marisInvolved in (R)-Selective Reduction of 5-Acetylfuro[2,3-c]pyridine

Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

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