Development of a heterologous expression method for exploitation of large biosynthetic gene clusters over 200 kb.

• Project/Area Number: 18K14349
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 37020:Chemistry and chemical methodology of biomolecules-related
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: Hashimoto Takuya 国立研究開発法人産業技術総合研究所, 生命工学領域, 研究員 (10783714)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: 二次代謝 / 放線菌 / ポリケタイド / 大環状マクロライド / 異種発現 / 細菌人工染色体（BAC） / 生合成 / 細菌人工染色体 / 合成生物学 / 生合成遺伝子クラスター / 異種生産 / 二次代謝産物 / BAC library / 生物有機化学 / 応用微生物

Outline of Final Research Achievements

Production of secondary metabolites using biosynthetic gene clusters over 200 kb had not been reported previously. Establishment of a method for heterologous expression of biosynthetic gene cluster over 200 kb will enable us to exploit biosynthetic gene clusters without limitation of size. To this end, we demonstrated heterologous expression of the biosynthetic gene cluster of quinolidomicin A1 derived from Micromonospora chalcea AK-AN57, which is the largest macrolide from terrestrial origins. Its biosynthetic gene cluster spanning 215 kb was cloned and co-expressed with two positive transcriptional regulators in Streptomyces lividans TK23ΔredDX. As a result, production of quinolidomicinA1 in the heterologous host was accomplished. This is the first example of the heterologous expression of the biosynthetic gene cluster over 200 kb.

Academic Significance and Societal Importance of the Research Achievements

これまで生合成遺伝子を利用した異種発現による化合物生産の例は限られており150 kb を超える例はほとんどない。本研究の結果、200 kbを超える生合成遺伝子クラスターを取得し転写因子と共発現することで生合成経路の異種宿主内での再構築に成功した。200 kb を超える遺伝子クラスターを利用して化合物生産を達成した初めての例である。本研究と同様の手法を利用することで、非リボソームペプチドやポリケタイドの生合成に関わる巨大生合成遺伝子クラスターを利用した有用物質の探索や生産につながると期待される。

Research Products

• [Journal Article] Biosynthesis of Quinolidomicin, the Largest Known Macrolide of Terrestrial Origin: Identification and Heterologous Expression of a Biosynthetic Gene Cluster over 200 kb (2018)
  • Author(s): Hashimoto Takuya、Hashimoto Junko、Kozone Ikuko、Amagai Keita、Kawahara Teppei、Takahashi Shunji、Ikeda Haruo、Shin-ya Kazuo
  • Journal Title: Organic Letters Volume: 20 Issue: 24 Pages: 7996-7999
  • DOI: 10.1021/acs.orglett.8b03570
  • Peer Reviewed / Open Access
• [Presentation] BACを利用した200 kb を超えるquinoliidomicin生合成遺伝子クラスターの異種発現 (2020)
  • Author(s): 橋本 拓哉、橋本 絢子、小曽根 郁子、酒井 紀子、高橋 俊二、池田 治生、新家 一男
  • Organizer: 日本農芸化学会 2020 大会
• [Presentation] Biosynthesis of quinolidomicin: Identification and heterologous expression of the biosynthetic gene cluster over 200 kb (2018)
  • Author(s): Hashimoto Takuya、Amagai Keita、Kawahara Teppei、Hashimoto Junko、Kozone Ikuko、Takahashi Shunji、Ikeda Haruo、Shin-ya Kazuo
  • Organizer: 3rd European Conference on Natural Products
  • Int'l Joint Research