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

• Project/Area Number: 22360349
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Toyama 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: ¥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.

Research Products

• [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 Pages: 3889-3904
• [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
• [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: 98 Pages: 4437-4443
• [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
  • 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
  • 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
  • Peer Reviewed
• [Journal Article] Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 9174 (2013)
  • Author(s): K. Isotani, J. Kurokawa, S. Nomoto, T. Negishi, M. Matsuda, N. Itoh
  • Journal Title: Appl. Environ. Microbiol. Volume: 79 Pages: 1387-1384
• [Journal Article] Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis : application of engineered enzymes in a 2-propanol-water medium. Appl (2012)
  • Author(s): N. Itoh, K. Isotani, M. Nakamura, K. Inoue, Y. Isogai, Y. Makino
  • Journal Title: Microbiol. Biotechnol. Volume: 93 Pages: 1075-1085
• [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
• [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
• [Journal Article] Efficient biocatalysis for the production of enantiopure (S)-epoxides using styrene monooxygenase(SMO) and Leifsonia alcohol dehydrogenase (LSADH) system (2012)
  • Author(s): H. Toda, R. Imae, N. Itoh
  • Journal Title: Tetrahedron : Asymmetry Volume: 23 Pages: 1542-1549
• [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
  • Peer Reviewed
• [Journal Article] Efficient biocatalysis for the production of enantiopure (S)-epoxides using styrene monooxygenase (SMO) and Leifsonia alcohol dehydrogenase (LSADH) system (2012)
  • 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
  • Peer Reviewed
• [Journal Article] Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis : application of engineered enzymes in a 2-propanol-water medium (2012)
  • 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
  • Peer Reviewed
• [Journal Article] Isolation and characterization of styrene metabolism genes from styrene-assimilating soil bacteria Rhodococcus sp.ST-5 and ST-10 (2012)
  • 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
  • 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
• [Journal Article] Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrate (2010)
  • Author(s): H. Asako, M. Shimizu, Y. Makino, N. Itoh
  • Journal Title: Tetrahedron Lett Volume: 51 Pages: 2664-2666
• [Journal Article] Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum : A useful enzymatic regeneration system of NAD+ from NADH (2010)
  • Author(s): J. Kurokawa, M. Asano, S. Nomoto, Y. Makino, N. Itoh
  • Journal Title: J. Biosci. Bioeng. Volume: 109 Pages: 218-223
• [Journal Article] Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrat (2010)
  • Author(s): H.Asako, M.Shimizu, Y.Makino, N.Itoh
  • Journal Title: Tetrahedron Lett. Volume: 51 Pages: 2664-2666
  • Peer Reviewed
• [Journal Article] Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum : A useful enzymatic regeneration system of NAD^+ from NADH (2010)
  • Author(s): J.Kurokawa, M.Asano, S.Nomoto, Y.Makino, N.Itoh
  • Journal Title: J.Biosci.Bioeng. Volume: 109 Pages: 218-223
  • 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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [Presentation] Development of novel biocatalysts for producing chiral compounds (2012)
  • Author(s): N. Itoh
  • Organizer: Seminar on Green Sustainable Chemistry in Tottori
  • Place of Presentation: Tottori, Japan
• [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
• [Presentation] Efficient synthesis of optically pure (S)-epoxides using Rhodococcus styrene monbooxygenase (SMO) and Leifsonina alcohol dehydrogenase (LSDH) system (2012)
  • Author(s): N. Itoh
  • Organizer: Enzyme Engineering XXII
  • Place of Presentation: Toyama, Japan
• [Presentation] Efficient synthesis of enantiopure (S)-epoxides by styrene monooxygenase of Rhodococcous sp. ST-10 (2012)
  • Author(s): N. Itoh, R. Imae, H. Toda
  • Organizer: 12 th Japan-Korea-China Joint Symposium on Enzyme Engineering
  • Place of Presentation: 富山
• [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
• [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
• [Presentation] Development of an efficient method for producing optically pure alcohols using an asymmetric hydrogen-transfer biocatalysis (2010)
  • Author(s): N.Itoh, Y.Makino
  • Organizer: Pacifichem2010
  • Place of Presentation: Hawaii, USA
  • Year and Date: 2010-12-19
• [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
• [Presentation] Development of an efficient method for producing optically pure alcoholsusing an asymmetric hydrogen-transfer biocatalysis (2010)
  • Author(s): N. Itoh, Y. Makino
  • Organizer: Pacifichem2010
  • Place of Presentation: Hawaii, USA
• [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
• [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
• [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
• [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
• [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
  • Invited
• [Book] Protein engineering: development of novel enzymes for the improved reduction of C= O double bonds (2014)
  • Author(s): N. Itoh, Y. Makino
  • Publisher: E. Brenna (ed.) in Synthetic methods for biologically active molecules. (Wiley, Germany)
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [Remarks]
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [Remarks] 応用生物プロセス学研究室
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [Remarks]
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [Remarks]
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [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
• [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
• [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
• [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