PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis

Itoh, Nobuhide; Isotani, Kentaro; Makino, Yoshihide; Kato, Masaki; Kitayama, Kouta; Ishimota, Tuyoshi

doi:10.1016/j.enzmictec.2013.10.012

Cited by 23 publications

(17 citation statements)

References 36 publications

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“…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.…”

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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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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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“…The targeting of specific classes of enzymes can be directly incorporated into PCR‐based analyses of metagenomic DNA (Fig. ), via highly conserved domains in particular enzymes . In these strategies primer design represents the critical step and can introduce intrinsic bias through a marked influence on the types and relative novelty of genes that may be amplified.…”

Section: Functional Metagenomics: Methodologies and Challengesmentioning

confidence: 99%

From lignocellulosic metagenomes to lignocellulolytic genes: trends, challenges and future prospects

Batista-García

Sánchez-Carbente

Talia

et al. 2016

Biofuels Bioprod Bioref

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Lignocellulose is the most abundant biomass on Earth with immense potential to act as a primary resource for the production of a range of compounds currently obtained from fossil fuel sources. However, lignocellulosic feedstocks remain largely underexploited due to the complex mixture of recalcitrant polymers present, whose structural features hinder access to the utilizable monosaccharide reservoir within cellulose. Various fungi and bacteria have been identified that can enzymatically decompose lignocellulose to its monomeric compounds for use as carbon sources. The investigation of such lignocellulolytic organisms has proven very useful in gaining primary insights into degradation processes and key microbial enzymes, but the established limitations of culture‐based approaches suggest that we have yet to understand the full range of lignocellulolytic mechanisms, likely expressed within natural systems. In this review, we focus on metagenomic approaches to study lignocellulose degradation from structural and functional perspectives, which may provide novel insights into this process in order to rationally design methods for the extraction of compounds from biomass that could enhance biorefinery efficiencies. © 2016 Society of Chemical Industry and John Wiley & Sons, Ltd

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“…; Itoh et al. ). The expression level of recombinant enzymes was extremely increased compared to which in natural cells.…”

Section: Introductionmentioning

confidence: 96%

RNA‐seq transcriptome analysis of a Pseudomonas strain with diversified catalytic properties growth under different culture medium

et al. 2016

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Biocatalysis is an emerging strategy for the production of enantio‐pure organic molecules. However, lacking of commercially available enzymes restricts the widespread application of biocatalysis. In this study, we report a Pseudomonas strain which exhibited versatile oxidation activity to synthesize chiral sulfoxides when growing under M9‐toluene medium and reduction activity to synthesize chiral alcohols when on Luria‐Bertani (LB) medium, respectively. Further comparative transcriptome analysis on samples from these two cultural conditions has identified 1038 differentially expressed genes (DEG). Gene Ontology (GO) enrichment and KEGG pathways analysis demonstrate significant changes in protein synthesis, energy metabolism, and biosynthesis of metabolites when cells cultured under different conditions. We have identified eight candidate enzymes from this bacterial which may have the potential to be used for synthesis of chiral alcohol and sulfoxide chemicals. This work provides insights into the mechanism of diversity in catalytic properties of this Pseudomonas strain growth with different cultural conditions, as well as candidate enzymes for further biocatalysis of enantiomerically pure molecules and pharmaceuticals.

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PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis

Cited by 23 publications

References 36 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

From lignocellulosic metagenomes to lignocellulolytic genes: trends, challenges and future prospects

RNA‐seq transcriptome analysis of a Pseudomonas strain with diversified catalytic properties growth under different culture medium

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