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Engineered biosynthesis of new natural products for innovative drug discovery

Planned

Project AreaCreation of Complex Functional Molecules by Rational Redesign of Biosynthetic Machineries
Project/Area Number 16H06449
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Science and Engineering
Research InstitutionUniversity of Shizuoka

Principal Investigator

渡辺 賢二  静岡県立大学, 薬学部, 教授 (50360938)

Co-Investigator(Kenkyū-buntansha) 植木 尚子  岡山大学, 資源植物科学研究所, 准教授 (50622023)
Project Period (FY) 2016-06-30 – 2021-03-31
Project Status Granted (Fiscal Year 2020)
Budget Amount *help
¥72,020,000 (Direct Cost: ¥55,400,000、Indirect Cost: ¥16,620,000)
Fiscal Year 2020: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000)
Fiscal Year 2019: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000)
Fiscal Year 2018: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000)
Fiscal Year 2017: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000)
Fiscal Year 2016: ¥15,860,000 (Direct Cost: ¥12,200,000、Indirect Cost: ¥3,660,000)
Keywords生合成 / リデザイン / 天然物 / 担子菌 / 抗生物質 / 生合成リデザイン / テルペノイド / 天然物化学 / キノコ / 酵素 / 遺伝子 / 微量天然物 / 異種発現 / 生合 / 反応機構 / 糸状菌 / Aspoquinolone / ゲノム情報 / 物質生産
Outline of Annual Research Achievements

担子菌ウシグソヒトヨタケCoprinopsis cinereaにおける内因性遺伝子の強制発現
C. cinereaを宿主とした異種遺伝子発現を行う準備段階として、まずはC. cinerea自身の遺伝子を強制発現させることを試みた。既に我々は本遺伝子についてcDNAを取得した後にクローニングし、出芽酵母を用いて異種発現させることでその産物がオルセリン酸 (1)であることを報告している。そこでCC1G_05377の上流に強制発現が可能と考えられるb-tublin、EF3、DED19の各プロモーターを配置したプラスミドを構築し、これをC. cinerea 326株に導入した。PCRによって遺伝子導入が確認された変異体をMYG培地にて振盪培養し、その代謝産物をLC-MSにて解析したところ、それぞれ最大で約10倍(pb-tublin)、約100倍(pEF3)、約250倍(pDED1)と野生株よりも生産性の向上したクローンを得ることに成功した。
続いてcop6遺伝子(CC1G_03563)に着目した。本遺伝子はセスキテルペン合成酵素Cop6をコードしており、FPPよりa-cupreneneを生合成することが報告されている。C. cinerea由来のa-cuprenene関連化合物としてはlagopodin類が知られており、加えてlagopodin類がさらに代謝されて生成したと考えられるhytoyol類の構造がつい最近、報告された。しかし、lagopodinおよびhytoyol生合成におけるcop6遺伝子の関与について直接的な証拠はこれまでに得られていなかった。そこでcop6遺伝子についてDED1プロモーターによる強制発現を試みた。得られた形質転換体は、野生株と比較してlagopodin類が約2-3倍、hytoyol類の生産性が約15倍に増加したクローンを見出すことに成功した。

Current Status of Research Progress
Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

Lagopodin類の生合成経路に関して、α-cupreneneが順次酸化されることで生合成されると予想されているものの、これまでに実験的に証明はされていない。そこで、本研究ではlagopodin類の生合成経路の解明を目指した。
研究対象として、lagopodin類を生産し、遺伝子の操作系も確立されているC. cinereaを用いた。C. cinereaのゲノム情報を参照すると、テルペン環化酵素をコードするcop6に隣接して酸化酵素であるシトクロムP450をコードする遺伝子が2つ(cox1、cox2)存在していることが確認された。そこで、本研究ではcop6、cox1およびcox2遺伝子を出芽酵母Saccharomyces cerevisiaeで異種種宿主発現することや、C. cinereaのこれら遺伝子を破壊することで、lagopodin類の生合成経路について解析した。S. cerevisiaeを宿主としてcop6等を発現させるためのベクターを作製し、形質転換した後、それらの生産物を確認したところ、cop6導入株ではα-cupreneneの生産が観測された。さらに、cop6、cox1およびcox2の共発現株では2の生産が確認されたものの、lagopodin類の産生は確認されなかった。
続いて、C. cinereaのcop6遺伝子等の破壊株を用いてlagopodin類が生産されるか調べたところ、cop6破壊株ではlagopodinの生産は確認されなくなったものの、cox1やcox2破壊株ではlagopodin類の生産は依然として観測された。そのため、α-cupreneneや2をlagopodin類へと酸化する酵素がcox1およびcox2以外にもC. cinereaにコードされていることが示唆された。

Strategy for Future Research Activity

今回の研究成果によって、キノコを用いリード化合物の探索および大量生産という2大課題を同時に達成できる高い技術に手が届いたと言えるであろう。キノコ類に関しては、単離されてきた天然物の化学構造を眺めてみると糸状菌由来天然物と類似した分子も見受けられるが、その生合成起源および生合成経路が不明瞭な分子も少なくない。このような分子は糸状菌とは異なる生合成遺伝子・酵素から生合成されていると予想できる。本法論を用いて未開拓のキノコを調査することで数多くの新規成分の発見が期待できる。

Report

(3 results)
  • 2018 Annual Research Report
  • 2017 Annual Research Report
  • 2016 Annual Research Report

Research Products

(41 results)

All 2019 2018 2017 2016 Other

All Int'l Joint Research Journal Article Presentation Book Remarks Patent(Industrial Property Rights)

  • [Int'l Joint Research] University of California Los Angeles(米国)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] Leibniz Institute(ドイツ)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] カリフォルニア大ロサンゼルス校(米国)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] University of California, Los Angeles(USA)

    • Related Report
      2016 Annual Research Report
  • [Int'l Joint Research]

    • Related Report
      2016 Annual Research Report
  • [Journal Article] Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions.2019

    • Author(s)
      Masuya Takahiro、Tsunematsu Yuta、Hirayama Yuichiro、Sato Michio、Noguchi Hiroshi、Nakazawa Takehito、Watanabe Kenji
    • Journal Title

      Organic and Biomolecular Chemistry

      Volume: 17 Pages: 234-239

    • DOI

      10.1039/c8ob02814a

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Enzymatic one-step ring contraction for quinolone biosynthesis2018

    • Author(s)
      Kishimoto Shinji、Hara Kodai、Hashimoto Hiroshi、Hirayama Yuichiro、Champagne Pier Alexandre、Houk Kendall N.、Tang Yi、Watanabe Kenji
    • Journal Title

      Nature Commun

      Volume: 9 Pages: 1-1

    • DOI

      10.1038/s41467-018-05221-5

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Genome Mining and Assembly-Line Biosynthesis of the UCS1025A Pyrrolizidinone Family of Fungal Alkaloids2018

    • Author(s)
      Li Li、Tang Man-Cheng、Tang Shoubin、Gao Shushan、Soliman Sameh、Hang Leibniz、Xu Wei、Ye Tao、Watanabe Kenji、Tang Yi
    • Journal Title

      Journal of the American Chemical Society

      Volume: 140 Pages: 2067-2071

    • DOI

      10.1021/jacs.8b00056

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Enzymatic Amide Tailoring Promotes Retro-Aldol Amino Acid Conversion To Form the Antifungal Agent Aspirochlorine2018

    • Author(s)
      Tsunematsu Yuta、Maeda Naoya、Yokoyama Mamoru、Chankhamjon Pranatchareeya、Watanabe Kenji、Scherlach Kirstin、Hertweck Christian
    • Journal Title

      Angewandte Chemie International Edition

      Volume: 57 Pages: 14051-14054

    • DOI

      10.1002/anie.201806740

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Polyketide synthase-nonribosomal peptide synthetase hybrid enzymes of fungi2018

    • Author(s)
      Kishimoto, S., Hirayama, Y., Watanabe, K.
    • Journal Title

      The Mycota XV: Physiology and Genetics

      Volume: 368 Pages: 1-1

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Integration of Chemical, Genetic, and Bioinformatic Approaches Delineates Fungal Polyketide-Peptide Hybrid Biosynthesis2017

    • Author(s)
      Yokoyama M, Hirayama Y, Yamamoto T, Kishimoto S, Tsunematsu Y, Watanabe K
    • Journal Title

      Org Lett

      Volume: 19 Pages: 2002-2005

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis2017

    • Author(s)
      1.Ohashi, M., Liu, F., Hai, Y., Chen, M., Tang, M.-C., Yang, Z., Sato, M., Watanabe, K., Houk, K. N., Tang, Y.
    • Journal Title

      Nature

      Volume: 549 Pages: 502-506

    • DOI

      10.1038/nature23882

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Oxidative Cyclization in Natural Product Biosynthesis2017

    • Author(s)
      Tang Man-Cheng、Zou Yi、Watanabe Kenji、Walsh Christopher T.、Tang Yi
    • Journal Title

      Chemical Reviews

      Volume: 117 Pages: 5226-5333

    • DOI

      10.1021/acs.chemrev.6b00478

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Elucidation of Biosynthetic Pathways of Natural Products2017

    • Author(s)
      Kishimoto, S., Tsunematsu, Y., Sato, M., Watanabe, K.
    • Journal Title

      The Chemical Record

      Volume: 印刷中 Pages: 1095-1108

    • DOI

      10.1002/tcr.201700015

    • Related Report
      2017 Annual Research Report 2016 Annual Research Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Integration of chemical, genetic and bioinformatic approaches delineates fungal polyketide-peptide hybrid biosynthesis.2017

    • Author(s)
      Yokoyama, M., Hirayama, Y., Yamamoto, T., Kishimoto, S., Tsunematsu, Y., Watanabe, K.
    • Journal Title

      Org. Lett.

      Volume: 印刷中

    • DOI

      10.1021/acs.orglett.7b00559

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Biochemical characterization of an eukaryotic decalin-forming Diels-Alderase.2016

    • Author(s)
      Li, L., Yu, P., Tang, M. C., Zou, Y., Gao, S.-S., Hung, Y.-S., Zhao, M., Watanabe, K., Houk, K., Tang, Y.
    • Journal Title

      J. Am. Chem. Soc.

      Volume: 138 Pages: 15837-15840

    • DOI

      10.1021/jacs.6b10452

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Regioselective dichlorination of a non-activated aliphatic carbon and phenol bismethylation by a multifunctional fungal flavoenzyme.2016

    • Author(s)
      Chankhamjon, P., Tsunematsu, Y., Ishida-Ito, M., Sasa, Y., Meyer, F., Boettger-Schmidt, D., Urbansky, B., Menzel, K. D., Scherlach, K., Watanabe, K., Hertweck, C.
    • Journal Title

      Angew. Chem. Int. Ed.

      Volume: 55 Pages: 11955-11959

    • DOI

      10.1002/anie.201604516

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] OxidativetranstocisIsomerization of Olefins in Polyketide Biosynthesis2016

    • Author(s)
      Tsuyoshi Yamamoto, Yuta Tsunematsu, Kodai Hara, Tomohiro Suzuki, Shinji Kishimoto, Hirokazu Kawaguchi, Hiroshi Noguchi, Hiroshi Hashimoto, Yi Tang, Kinya Hotta, & Kenji Watanabe
    • Journal Title

      Angewandte Chemie International Edition

      Volume: 印刷中 Pages: 6207-6210

    • DOI

      10.1002/anie.201600940

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Simple Screening for High-Colibactin Producers from Clinical Samples Using Activity- Based Fluorescent Probe2018

    • Author(s)
      Kenji Watanabe
    • Organizer
      U.S.-Japan Cooperative Medical Sciences Program (USJCMSP)
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] ENZYMATIC ONE-STEP RING CONTRACTION FOR QUINOLONE BIOSYNTHESIS2018

    • Author(s)
      Kenji Watanabe
    • Organizer
      2nd China-Japan Joint Symposium on Natural Product Biosynthesis
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Simple Screening for High-Colibactin Producers from Clinical Samples Using Activity- Based Fluorescent Probe2018

    • Author(s)
      Kenji Watanabe
    • Organizer
      30th International Symposium on the Chemistry of Natural Products
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Simple Screening for High-Colibactin Producers from Clinical Samples Using Activity- Based Fluorescent Probe2018

    • Author(s)
      Kenji Watanabe
    • Organizer
      6th Biennial International Conference on New Developments in Drug Discovery from Natural Products and Traditional Medicines
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Crystallographic characterization of a [4+2]-cyclase binding site and mutational alteration of stereoselectivity2018

    • Author(s)
      Kenji Watanabe
    • Organizer
      The 2nd International Symposium on Organic Reaction Mechanism
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Genetic indoctrination of Escherichia coli for finding genotoxins “colibactin” facilitating inflammation-induced colorectal cancer2017

    • Author(s)
      Kenji Watanabe
    • Organizer
      US-Japan Cooperative Medical Sciences Program, 19th International Conference on Emerging Infectious Diseases in the Pacific Rim
    • Place of Presentation
      Seoul, South Korea
    • Year and Date
      2017-02-07
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Disfavored exo-adduct formation by a modified octalin-forming Diels-Alderase2017

    • Author(s)
      渡辺賢二
    • Organizer
      China/Japan Symposium on Natural Product Biosynthesis
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Disfavored exo-adduct generation by a modified octalin-forming Diels-Alderase2017

    • Author(s)
      渡辺賢二
    • Organizer
      Society for Industrial Microbiology
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Pseurotin biosynthesis in fungus2017

    • Author(s)
      渡辺賢二
    • Organizer
      9th US-Japan Seminar: The international Conference of Natural Product Biosynthesis
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Novel Genotoxins “Colibactin” Produced by E.coli and colon carcinogenesis (In Vitro, In Vivo Experiments)2017

    • Author(s)
      渡辺賢二
    • Organizer
      The 12th international conference and 5th Asian congress on environmental mutagens with the 33rd annual meeting of KSOT/KEMS
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Genetic indoctrination of Escherichia coli for finding genotoxins colibactin facilitating inflammation-induced colorectal cancer2017

    • Author(s)
      渡辺賢二
    • Organizer
      US-Japan Cooperative Medical Sciences Program Cancer Panel Meeting
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 実用的物質生産系構築にむけたゲノム情報に基づく 新規生合成システムのリデザイ2017

    • Author(s)
      渡辺賢二
    • Organizer
      生物合成系の再設計による複雑骨格機能分子の革新的創成化学(新学術領域)第一回公開シンポジウム
    • Place of Presentation
      東京大学, 東京
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Oxidative trans-to-cis isomerization of olefin in polyketide biosynthesis2016

    • Author(s)
      Kenji Watanabe
    • Organizer
      The 13th International Symposium on the Genetics of Industrial Microorganisms
    • Place of Presentation
      Wuhan, People’s Republic of China
    • Year and Date
      2016-10-16
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Book] 天然物の化学Ⅱ(科学のとびら64)2018

    • Author(s)
      上村大輔, 渡辺賢二
    • Total Pages
      192
    • Publisher
      東京化学同人
    • ISBN
      9784807915057
    • Related Report
      2018 Annual Research Report
  • [Book] 天然物生合成におけるS-アデノシルメチオニン依存性酵素により触媒されるペリ環状反応2018

    • Author(s)
      大橋雅生, 渡辺賢二
    • Total Pages
      6
    • Publisher
      現代化学
    • Related Report
      2018 Annual Research Report
  • [Book] Polyketide synthase and nonribosomal peptide synthetase hybrid enzymes of fungi. The Mycota XV: Physiology and Genetics2018

    • Author(s)
      Kishimoto, S., Hirayama, Y., Watanabe, K.
    • Total Pages
      7
    • Publisher
      Springer
    • Related Report
      2017 Annual Research Report
  • [Book] 現代化学2017

    • Author(s)
      大橋雅生, 渡辺賢二
    • Total Pages
      6
    • Publisher
      東京化学同人
    • Related Report
      2017 Annual Research Report
  • [Book] BIO Clinica2017

    • Author(s)
      渡辺賢二
    • Total Pages
      4
    • Publisher
      北隆館/ニュー・サイエンス社
    • Related Report
      2017 Annual Research Report
  • [Remarks] 渡辺賢二 研究室

    • URL

      http://sweb.u-shizuoka-ken.ac.jp/~kenji55-lab/

    • Related Report
      2018 Annual Research Report
  • [Remarks] 研究内容

    • URL

      http://sweb.u-shizuoka-ken.ac.jp/~kenji55-lab/

    • Related Report
      2017 Annual Research Report
  • [Remarks] WATANABE RESEARCH GROUP

    • URL

      http://sweb.u-shizuoka-ken.ac.jp/~kenji55-lab/

    • Related Report
      2016 Annual Research Report
  • [Patent(Industrial Property Rights)] コリバクチン産生菌検出用プライマーセット及び検出用キット2019

    • Inventor(s)
      渡辺賢二
    • Industrial Property Rights Holder
      渡辺賢二
    • Industrial Property Rights Type
      特許
    • Industrial Property Number
      2019-027695
    • Filing Date
      2019
    • Related Report
      2018 Annual Research Report
  • [Patent(Industrial Property Rights)] コリバクチンおよびコリバクチン産生菌の検出方法および検出プローブ2018

    • Inventor(s)
      渡辺賢二
    • Industrial Property Rights Holder
      渡辺賢二
    • Industrial Property Rights Type
      特許
    • Filing Date
      2018
    • Related Report
      2018 Annual Research Report
    • Overseas

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Published: 2016-07-04   Modified: 2020-08-26  

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