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Screening of ADH gene-specific amplicons from metagenome for enzyme molecular evolution and biocatalysis

Research Project

Project/Area Number 22360349
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biofunction/Bioprocess
Research InstitutionToyama Prefectural University

Principal Investigator

ITOH Nobuya  富山県立大学, 工学部, 教授 (90213066)

Co-Investigator(Kenkyū-buntansha) 牧野 祥嗣  富山県立大学, 工学部, 教授 (20347355)
Project Period (FY) 2010-04-01 – 2014-03-31
Project Status Completed (Fiscal Year 2013)
Budget Amount *help
¥19,110,000 (Direct Cost: ¥14,700,000、Indirect Cost: ¥4,410,000)
Fiscal Year 2013: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
Fiscal Year 2012: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2011: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2010: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000)
Keywords生体触媒 / メタゲノム / スクリーニング / 光学活性アルコール / アルコール脱水素酵素 / 進化分子工学 / 進化分子アルゴリズム / 遺伝子ライブラリー / 進化アルゴリズム / 有用生体触媒
Research Abstract

We have developed an efficient PCR-based amplification of diverse alcohol dehydrogenase (adh) genes from soil and compost metagenomes to aid in the synthesis of optically pure alcohols. Forty lsadh-related genes (Hladh) and 30 par-homologous genes (Hpar), were obtained by screening of gene-specific amplicons from metagenome (S-GAM) from the metagenomic library. The amino acids sequences coded by the Hladh genes obtained by this approach were varied (8-99%). The recombinant enzymes obtained were characterized in detail. The results indicated that the enzymes are superior to the known ones to produce chiral alcohols. Thus S-GAM technique offers great biotechnological potential for biocatalysis and enzyme engineering.

Report

(5 results)
  • 2013 Annual Research Report   Final Research Report ( PDF )
  • 2012 Annual Research Report
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • Research Products

    (60 results)

All 2014 2013 2012 2011 2010 Other

All Journal Article (20 results) (of which Peer Reviewed: 9 results) Presentation (24 results) (of which Invited: 1 results) Book (8 results) Remarks (4 results) Patent(Industrial Property Rights) (4 results)

  • [Journal Article] Use of anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols2014

    • Author(s)
      N.Itoh
    • Journal Title

      Appl. Microbiol. Biotechnol

      Volume: 98 Pages: 3889-3904

    • Related Report
      2013 Final Research Report
  • [Journal Article] PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis2014

    • Author(s)
      N. Itoh, K..Isotani, Y. Makino, M. Kato, K. Kitayama, T. Ishimota
    • Journal Title

      Enzyme Microb. Technol.

      Volume: 55 Pages: 140-150

    • Related Report
      2013 Final Research Report
  • [Journal Article] Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure2014

    • Author(s)
      Y. Makino, N. Itoh
    • Journal Title

      Appl. Microbiol. Biotechnol

      Volume: 98 Pages: 4437-4443

    • Related Report
      2013 Final Research Report
  • [Journal Article] Use of anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols.2014

    • Author(s)
      N. Itoh
    • Journal Title

      Appl. Microbiol. Biotechnol.

      Volume: 98 Issue: 9 Pages: 3889-3904

    • DOI

      10.1007/s00253-014-5619-5

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.2014

    • Author(s)
      N. Itoh,K. Isotani, Y. Makino, M. Kato, K. Kitayama, T. Ishimota
    • Journal Title

      Enzyme Microb. Technol.

      Volume: 55 Pages: 140-150

    • DOI

      10.1016/j.enzmictec.2013.10.012

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure.2014

    • Author(s)
      Y. Makino, N. Itoh.
    • Journal Title

      Appl. Microbiol. Biotechnol.

      Volume: 未定 Issue: 10 Pages: 4437-4443

    • DOI

      10.1007/s00253-013-5406-8

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 91742013

    • Author(s)
      K. Isotani, J. Kurokawa, S. Nomoto, T. Negishi, M. Matsuda, N. Itoh
    • Journal Title

      Appl. Environ. Microbiol.

      Volume: 79 Pages: 1387-1384

    • Related Report
      2013 Final Research Report 2012 Annual Research Report
  • [Journal Article] Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis : application of engineered enzymes in a 2-propanol-water medium. Appl2012

    • Author(s)
      N. Itoh, K. Isotani, M. Nakamura, K. Inoue, Y. Isogai, Y. Makino
    • Journal Title

      Microbiol. Biotechnol.

      Volume: 93 Pages: 1075-1085

    • Related Report
      2013 Final Research Report
  • [Journal Article] メタゲノムスクリーニング と進化分子工学的手法の融合による有用生 体触媒の創製2012

    • Author(s)
      伊藤 伸哉
    • Journal Title

      日本応用酵素協会誌

      Volume: 46 Pages: 31-40

    • Related Report
      2013 Final Research Report
  • [Journal Article] Production of (R)-3-quinuclidinol by E. coli biocatalysts possessing NADH-d ependent 3- q uinuclidinone r eductase (QNR or bacC) from Microbacterium luteolum and Leifsonia alcohol dehydrogenase (LSADH)2012

    • Author(s)
      K. Isotani, J. Kurokawa, N. Itoh
    • Journal Title

      Int. J. Mol. Sci.

      Volume: 13 Pages: 13542-13553

    • Related Report
      2013 Final Research Report
  • [Journal Article] Efficient biocatalysis for the production of enantiopure (S)-epoxides using styrene monooxygenase(SMO) and Leifsonia alcohol dehydrogenase (LSADH) system2012

    • Author(s)
      H. Toda, R. Imae, N. Itoh
    • Journal Title

      Tetrahedron : Asymmetry

      Volume: 23 Pages: 1542-1549

    • Related Report
      2013 Final Research Report
  • [Journal Article] Production of (R)-3-quinuclidinol by E. coli biocatalysts possessing NADH-dependent 3-quinuclidinone reductase (QNR or bacC) from Microbacterium luteolum and Leifsonia alcohol dehydrogenase (LSADH)2012

    • Author(s)
      K. Isotani, J. Kurokawa, N. Itoh
    • Journal Title

      Int. J. Mol. Sci.

      Volume: 13 Issue: 10 Pages: 13542-13553

    • DOI

      10.3390/ijms131013542

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Efficient biocatalysis for the production of enantiopure (S)-epoxides using styrene monooxygenase (SMO) and Leifsonia alcohol dehydrogenase (LSADH) system2012

    • Author(s)
      H. Toda, R. Imae, N. Itoh
    • Journal Title

      Tetrahedron: Asymmetry

      Volume: 23 Issue: 22-23 Pages: 1542-1549

    • DOI

      10.1016/j.tetasy.2012.09.017

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis : application of engineered enzymes in a 2-propanol-water medium2012

    • Author(s)
      N.Itoh, K.Isotani, M.Nakamura, K.Inoue, Y.Isogai, Y.Makino
    • Journal Title

      Applied Microbiology and Biotechnology

      Volume: 93 Issue: 3 Pages: 1075-1085

    • DOI

      10.1007/s00253-011-3447-4

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Isolation and characterization of styrene metabolism genes from styrene-assimilating soil bacteria Rhodococcus sp.ST-5 and ST-102012

    • Author(s)
      H.Toda, N.Itoh
    • Journal Title

      Journal of Bioscience and Bioengineering

      Volume: 113 Issue: 1 Pages: 12-19

    • DOI

      10.1016/j.jbiosc.2011.08.028

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] メタゲノムスクリーニングと進化分子工学的手法の融合による有用生体触媒の創製2012

    • Author(s)
      伊藤伸哉
    • Journal Title

      日本応用酵素協会誌

      Volume: 第46号 Pages: 31-40

    • Related Report
      2011 Annual Research Report
  • [Journal Article] Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrate2010

    • Author(s)
      H. Asako, M. Shimizu, Y. Makino, N. Itoh
    • Journal Title

      Tetrahedron Lett

      Volume: 51 Pages: 2664-2666

    • Related Report
      2013 Final Research Report
  • [Journal Article] Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum : A useful enzymatic regeneration system of NAD+ from NADH2010

    • Author(s)
      J. Kurokawa, M. Asano, S. Nomoto, Y. Makino, N. Itoh
    • Journal Title

      J. Biosci. Bioeng.

      Volume: 109 Pages: 218-223

    • Related Report
      2013 Final Research Report
  • [Journal Article] Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrat2010

    • Author(s)
      H.Asako, M.Shimizu, Y.Makino, N.Itoh
    • Journal Title

      Tetrahedron Lett.

      Volume: 51 Pages: 2664-2666

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum : A useful enzymatic regeneration system of NAD^+ from NADH2010

    • Author(s)
      J.Kurokawa, M.Asano, S.Nomoto, Y.Makino, N.Itoh
    • Journal Title

      J.Biosci.Bioeng.

      Volume: 109 Pages: 218-223

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Presentation] PCR 法により取得したメタゲノム由来新規単鎖アルコール脱水素酵素遺伝子とその翻訳産物の諸性質2014

    • Author(s)
      黒川純司, 狩谷理美, 礒谷健太郎, 伊藤伸哉
    • Organizer
      2014年度日本農芸化学会大会
    • Place of Presentation
      東京
    • Related Report
      2013 Final Research Report
  • [Presentation] S749由来アルコール脱水素酵素(LSADH) のメタゲノムスクリーニングと解析2013

    • Author(s)
      狩谷理美,黒川純司,伊藤伸哉, Lesifonia sp
    • Organizer
      2013年 度日本農芸化学会大会
    • Place of Presentation
      仙台
    • Related Report
      2013 Final Research Report
  • [Presentation] メタゲノムを用いた酸化還元酵素遺伝子の探索とその応用2013

    • Author(s)
      礒谷健太郎, 石母田剛, 加藤真輝, 黒川 純司, 牧野祥嗣, 伊藤伸哉
    • Organizer
      2013年度日本農芸化学会大会
    • Place of Presentation
      仙台
    • Related Report
      2013 Final Research Report 2012 Annual Research Report
  • [Presentation] メタゲノムスクリーニングと 進化分子工学による有用生体触媒の創製2013

    • Author(s)
      伊藤伸哉
    • Organizer
      応用酵素協会成果発表会
    • Place of Presentation
      大阪
    • Related Report
      2013 Final Research Report
  • [Presentation] メタゲノム由来フェニル アセトアルデヒド還元酵素(PAR)相同遺伝子 の配列情報と酵素の改変2013

    • Author(s)
      礒谷健太郎・黒川純司・竹内奈美・牧野 祥嗣, 伊藤伸哉
    • Organizer
      第65回日本生物工学会大会
    • Place of Presentation
      広島
    • Related Report
      2013 Final Research Report
  • [Presentation] メタゲ ノムスクリーニングによる新規短鎖アルコ ール脱水素酵素遺伝子の取得とその解析2013

    • Author(s)
      狩谷理美, 黒川純司, 伊藤伸哉
    • Organizer
      第17回生体触媒化学シンポジウム
    • Place of Presentation
      岡山
    • Related Report
      2013 Final Research Report
  • [Presentation] メタゲノム由来フェニルアセトアルデヒド還元酵素(PAR)相同遺伝子の配列情報と酵素機能の改変2013

    • Author(s)
      風間美輝, 礒谷健太郎, 黒川純司, 竹内奈美, 牧野祥嗣, 伊藤伸哉
    • Organizer
      第17回生体触媒化学シンポジウム
    • Place of Presentation
      岡山
    • Related Report
      2013 Final Research Report
  • [Presentation] Lesifonia sp. S749由来アルコール脱水素酵素(LSADH)のメタゲノムスクリーニングと解析2013

    • Author(s)
      狩谷 理美、黒川純司、伊藤伸哉
    • Organizer
      2013年度日本農芸化学会大会
    • Place of Presentation
      仙台
    • Related Report
      2012 Annual Research Report
  • [Presentation] メタゲノムを用いた酸化還元酵素遺伝子の探索とその応用2012

    • Author(s)
      礒谷・石母田・加藤・黒川・牧野・伊藤
    • Organizer
      日本農芸化学会2012年度大会
    • Place of Presentation
      京都女子大学
    • Year and Date
      2012-03-24
    • Related Report
      2011 Annual Research Report
  • [Presentation] フェニルアセトアルデヒド還元酵素(PAR)相同遺伝子のメタゲノムスクリーニングとその解析2012

    • Author(s)
      伊藤・礒谷・竹内・牧野・大段・黒川
    • Organizer
      日本農芸化学会2012年度大会
    • Place of Presentation
      京都女子大学
    • Year and Date
      2012-03-24
    • Related Report
      2011 Annual Research Report
  • [Presentation] Development of novel biocatalysts for producing chiral compounds2012

    • Author(s)
      N. Itoh
    • Organizer
      Seminar on Green Sustainable Chemistry in Tottori
    • Place of Presentation
      Tottori, Japan
    • Related Report
      2013 Final Research Report
  • [Presentation] フェニルアセトアルデヒド還元酵素(PAR)相同遺伝子のメタゲノムスクリーニングとその解析2012

    • Author(s)
      礒谷健太郎, 黒川純司, 竹内奈美, 牧野 祥嗣, 伊藤伸哉
    • Organizer
      第16回生体触媒化学シンポジウム
    • Place of Presentation
      富山
    • Related Report
      2013 Final Research Report
  • [Presentation] Efficient synthesis of optically pure (S)-epoxides using Rhodococcus styrene monbooxygenase (SMO) and Leifsonina alcohol dehydrogenase (LSDH) system2012

    • Author(s)
      N. Itoh
    • Organizer
      Enzyme Engineering XXII
    • Place of Presentation
      Toyama, Japan
    • Related Report
      2013 Final Research Report
  • [Presentation] Efficient synthesis of enantiopure (S)-epoxides by styrene monooxygenase of Rhodococcous sp. ST-102012

    • Author(s)
      N. Itoh, R. Imae, H. Toda
    • Organizer
      12 th Japan-Korea-China Joint Symposium on Enzyme Engineering
    • Place of Presentation
      富山
    • Related Report
      2012 Annual Research Report
  • [Presentation] フェニルアセトアルデヒド還元酵素(PAR)相同遺伝子のメタゲノムスクリーニングとその解析2012

    • Author(s)
      礒谷健太郎、黒川純司、竹内奈美、牧野祥嗣、伊藤伸哉
    • Organizer
      第16回生体触媒化学シンポジウム
    • Place of Presentation
      富山
    • Related Report
      2012 Annual Research Report
  • [Presentation] 3-キヌクリジノン還元酵素遺伝子のクローニングと(R)-3-キヌクリジノール生産2011

    • Author(s)
      礒谷・黒川・伊藤
    • Organizer
      第4回北陸合同バイオシンポジウム
    • Place of Presentation
      宇奈月国際会議場
    • Year and Date
      2011-11-11
    • Related Report
      2011 Annual Research Report
  • [Presentation] Development of an efficient method for producing optically pure alcohols using an asymmetric hydrogen-transfer biocatalysis2010

    • Author(s)
      N.Itoh, Y.Makino
    • Organizer
      Pacifichem2010
    • Place of Presentation
      Hawaii, USA
    • Year and Date
      2010-12-19
    • Related Report
      2010 Annual Research Report
  • [Presentation] メタゲノムを用いた酸化還元酵素遺伝子の探索とその応用2010

    • Author(s)
      黒川、礒谷、石母田、加藤、牧野、伊藤
    • Organizer
      第62回日本生物工学会
    • Place of Presentation
      宮崎市
    • Year and Date
      2010-10-28
    • Related Report
      2010 Annual Research Report
  • [Presentation] Development of an efficient method for producing optically pure alcoholsusing an asymmetric hydrogen-transfer biocatalysis2010

    • Author(s)
      N. Itoh, Y. Makino
    • Organizer
      Pacifichem2010
    • Place of Presentation
      Hawaii, USA
    • Related Report
      2013 Final Research Report
  • [Presentation] メタゲノム由来フェニルアセトアルデヒド還元酵素(PAR)相同遺伝子の配列情報と酵素の改変

    • Author(s)
      礒谷健太郎、黒川純司、竹内奈美、牧野祥嗣、伊藤伸哉
    • Organizer
      第65回日本生物工学会大会
    • Place of Presentation
      広島
    • Related Report
      2013 Annual Research Report
  • [Presentation] メタゲノムスクリーニングと進化分子工学による有用生体触媒の創製

    • Author(s)
      伊藤伸哉
    • Organizer
      応用酵素協会成果発表会
    • Place of Presentation
      大阪
    • Related Report
      2013 Annual Research Report
  • [Presentation] メタゲノム由来フェニルアセトアルデヒド還元酵素(PAR)相同遺伝子の配列情報と酵素機能の改変

    • Author(s)
      風間美輝、礒谷健太郎、黒川純司、竹内奈美、牧野祥嗣、伊藤伸哉
    • Organizer
      第17回生体触媒化学シンポジウム
    • Place of Presentation
      岡山
    • Related Report
      2013 Annual Research Report
  • [Presentation] PCR法により取得したメタゲノム由来新規短鎖アルコール脱水素酵素遺伝子とその翻訳産物の諸性質

    • Author(s)
      黒川 純司、狩谷 理美、礒谷 健太郎、伊藤 伸哉
    • Organizer
      2014年度日本農芸化学会
    • Place of Presentation
      東京
    • Related Report
      2013 Annual Research Report
  • [Presentation] Development of novel biocatalysts for producing chiral compounds

    • Author(s)
      N. Itoh
    • Organizer
      Seminar on Green Sustainable Chemistry in Tottori, 2012.12 (Tottori, Japan)
    • Place of Presentation
      鳥取
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Book] Protein engineering: development of novel enzymes for the improved reduction of C= O double bonds2014

    • Author(s)
      N. Itoh, Y. Makino
    • Publisher
      E. Brenna (ed.) in Synthetic methods for biologically active molecules. (Wiley, Germany)
    • Related Report
      2013 Annual Research Report
  • [Book] Protein engineering: development of novel enzymes for the improved reduction of C=O double bonds(In: E. Brenna (ed) Synthetic methods for biologically active molecules)2013

    • Author(s)
      N. Itoh, Y. Makino
    • Publisher
      Wiley-VCH
    • Related Report
      2013 Final Research Report
  • [Book] 進化分子工学的手法による極性有機溶媒耐性PAR の創製と生産性の向上伏見譲(監修進化分子工学)2013

    • Author(s)
      伊藤伸哉
    • Publisher
      NTS出版
    • Related Report
      2013 Final Research Report
  • [Book] 進化分子工学を利用した光学活性エポキシ化合物生産プロセスの開発(伏見譲監修進化分子工学)2013

    • Author(s)
      戸田弘, 伊藤伸哉
    • Publisher
      NTS出版
    • Related Report
      2013 Final Research Report
  • [Book] 進化分子工学的手法による極性有機溶媒耐性PARの創製と生産性の向上2013

    • Author(s)
      伊藤 伸哉
    • Publisher
      伏見 譲 監修 進化分子工学 エヌ・ティー・エス
    • Related Report
      2013 Annual Research Report
  • [Book] 進化分子工学を利用した光学活性エポキシ化合物生産プロセスの開発2013

    • Author(s)
      戸田弘、伊藤 伸哉
    • Publisher
      伏見 譲 監修 進化分子工学 エヌ・ティー・エス
    • Related Report
      2013 Annual Research Report
  • [Book] 酸化還元反応を利用する有用物質生産2010

    • Author(s)
      伊藤伸哉, エコバイオリファイナリー
    • Publisher
      シーエム シー出版
    • Related Report
      2013 Final Research Report
  • [Book] エコバイオリファイナリー,酸化還元反応を利用する有用物質生産2010

    • Author(s)
      伊藤伸哉
    • Total Pages
      10
    • Publisher
      シーエムシー出版
    • Related Report
      2010 Annual Research Report
  • [Remarks]

    • URL

      http://www.pu-toyama.ac.jp/BR/itoh/

    • Related Report
      2013 Final Research Report
  • [Remarks] 応用生物プロセス学研究室

    • URL

      http://www.pu-toyama.ac.jp/BR/itoh/

    • Related Report
      2012 Annual Research Report
  • [Remarks]

    • URL

      http://www.pu-toyama.ac.jp/BR/itoh/

    • Related Report
      2011 Annual Research Report
  • [Remarks]

    • URL

      http://www.pu-toyama.ac.jp/BR/itoh/

    • Related Report
      2010 Annual Research Report
  • [Patent(Industrial Property Rights)] 還元酵素遺伝子増幅用プライマーセット,および還元酵素遺伝子取得方法2013

    • Inventor(s)
      伊藤伸哉, 黒川純司, 朝子弘之
    • Industrial Property Rights Holder
      富山県, 住友化学
    • Industrial Property Rights Type
      特許
    • Filing Date
      2013
    • Related Report
      2013 Final Research Report
  • [Patent(Industrial Property Rights)] 還元酵素遺伝子増幅用プライマーセット、および還元酵素遺伝子取得方法2013

    • Inventor(s)
      伊藤、黒川、朝子
    • Industrial Property Rights Holder
      伊藤、黒川、朝子
    • Industrial Property Rights Type
      特許
    • Filing Date
      2013-03-01
    • Related Report
      2012 Annual Research Report
  • [Patent(Industrial Property Rights)] 変異型還元酵素及びその利用2012

    • Inventor(s)
      伊藤伸哉, 朝子弘之
    • Industrial Property Rights Holder
      富山県, 住友化学
    • Industrial Property Rights Type
      特許
    • Filing Date
      2012
    • Related Report
      2013 Final Research Report
  • [Patent(Industrial Property Rights)] 変異型還元酵素及びその利用2012

    • Inventor(s)
      伊藤伸哉, 朝子弘之
    • Industrial Property Rights Holder
      富山県住友化学
    • Industrial Property Number
      2012-039796
    • Filing Date
      2012
    • Related Report
      2011 Annual Research Report

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Published: 2010-08-23   Modified: 2019-07-29  

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