Elucidation of the molecular mechanism that regulates the diversity of the tetracyclic diterpene skeleton produced by Scoparia dulcis

• Project/Area Number: 18K06729
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 47050:Environmental and natural pharmaceutical resources-related
• Research Institution: University of Toyama
• Principal Investigator: Yamamura Yoshimi 富山大学, 学術研究部薬学・和漢系, 助教 (30464027)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 植物二次代謝 / ジテルペン / 環化酵素 / チトクロームP450 / 有用物質生産 / P450 / ジャスモン酸 / RNA-Seq / エリシター / 二次代謝

Outline of Final Research Achievements

It was clarified that the Tyr528 clearly contributes to the stability of the carbocation intermediate in the novel cyclization enzyme SdCPS2 (syn-copalyl diphosphate synthase). Next, we showed that SdKSL1 is a novel cyclizing enzyme that biosynthesizes scopadula-13-α-ol, which has a scopadulane skeleton characteristic of SDB. Finally, we found seven P450 candidate genes involved in the desired hydroxylation of scopadula-13-α-ol. This information obtained in this study greatly contributes to the elucidation of the mechanism of skeletal diversity of labdane-related diterpenes produced by plants.

Academic Significance and Societal Importance of the Research Achievements

本研究の学術的な特色は、大腸菌のもつ一次代謝産物（IPP）を利用して、大腸菌内にジテルペン生合成酵素群を共導入させ、目的ジテルペンのin vivo生合成システムを構築し、目的の酵素の機能を解析する、という点にある。この手法は基質の生合成すなわち新たな標準物質の供給も可能にするものである。この手法により、これまで解析が困難と思われた酵素反応を大腸菌内で行い、培養液の成分を分析することで、その酵素の機能を特定することができる。このような手法を介して植物の持つ機能を全面的に利用することができれば、新たな有用物質生産や新規創薬シーズの開発への波及効果が期待できる。

Research Products

• [Journal Article] Divergent Evolution of the Diterpene Biosynthesis Pathway in Tea Plants (Camellia sinensis) Caused by Single Amino Acid Variation of ent-Kaurene Synthase (2020)
  • Author(s): Yang Mei、Liu Guanhua、Yamamura Yoshimi、Chen Feng、Fu Jianyu
  • Journal Title: Journal of Agricultural and Food Chemistry Volume: 68 Issue: 37 Pages: 9930-9939
  • DOI: 10.1021/acs.jafc.0c03488
• [Journal Article] Functional characterization of NADPH-cytochrome P450 reductase and cinnamic acid 4-hydroxylase encoding genes from Scoparia dulcis L. (2020)
  • Author(s): Yamamura Yoshimi、Mabuchi Ayaka
  • Journal Title: Botanical Studies Volume: 61 Issue: 1 Pages: 6-6
  • DOI: 10.1186/s40529-020-00284-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Identification of the P450 gene involved in scopadulane-type diterpene biosynthesis in Scoparia dulcis (2021)
  • Author(s): Yamada A, Yoshimi Yamamura Y, Lee JB.
  • Organizer: 第6回富山-バーゼルジョイントシンポジウム
  • Int'l Joint Research
• [Presentation] Identification of key genes involved in scopadulane-type diterpene biosynthesis in Scoparia dulcis (2020)
  • Author(s): Yamamura Y and Lee JB
  • Organizer: Plant Biology 2020 World Wide Summit
  • Int'l Joint Research
• [Presentation] Isolation and functional characterization of UDP-glucosyltransferases involved in the benzoxazinoid biosynthesis in Scoparia dulcis (2020)
  • Author(s): Kasai Y, Lee JB, Yamamura Y
  • Organizer: Plant Biology 2020 World Wide Summit
  • Int'l Joint Research
• [Presentation] In vivo functional characterization of eight new terpene synthase genes from medicinal plant Scoparia dulcis (2020)
  • Author(s): Yamamoto R, Yamamura Y, Lee JB
  • Organizer: Plant Biology 2020 World Wide Summit
  • Int'l Joint Research
• [Presentation] 薬用植物スコパリアのBenzoxazinoid生合成における糖転移酵素の解析 (2020)
  • Author(s): 笠井優理亜, 倉岡孝好, 吾郷良輔, 李 貞範, 山村良美
  • Organizer: 第61回日本植物生理学会年会
• [Presentation] 薬用植物スコパリア由来のテルペン合成酵素遺伝子群のin vivoにおける機能解析 (2019)
  • Author(s): 石田和也, 山本 涼, 山村良美, 黒崎文也, 李 貞範
  • Organizer: 第60回日本植物生理学会年会