Calyculin A生合成機構の解析

• Project Area: Biosynthetic machinery: Deciphering and regulating the system for bioactive metabolite diversification
• Project/Area Number: 25108705
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: The University of Tokyo
• Principal Investigator: 脇本 敏幸 東京大学, 薬学研究科(研究院), 准教授 (70363900)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000); Fiscal Year 2014: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000); Fiscal Year 2013: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
• Keywords: カリクリンA / 海綿 / 生合成 / Calyculin A / ポリケタイド / メタゲノム

Outline of Annual Research Achievements

本研究では伊豆半島産チョコガタイシカイメンに含まれる細胞毒性物質カリクリンAの生合成経路の解明を目指した。カリクリンAはタンパク質脱リン酸化酵素1および2Aを特異的に阻害し、各種がん細胞に対してpMオーダーで増殖阻害活性を示す。我々はカリクリンAの生合成遺伝子クラスターの探索を進め、チョコガタイシカイメンよりメタゲノムライブラリーを作成、ポリケタイド合成酵素遺伝子に保存性の高い配列を足掛かりとしてスクリーニングを行った。その結果、全長150 kbpに及ぶ長大な生合成遺伝子クラスターの取得に至った。予想通り、本遺伝子クラスターはⅠ型ポリケタイド合成酵素と非リボソーム型ペプチド合成酵素をコードしており、それらのモジュールやドメインの構成はカリクリンAの構造と良い一致を示した。さらに、いくつかの推定修飾酵素について機能解析を試みた。遺伝子クラスター上流には３つのリン酸基転移酵素がコードされており、それらを大腸菌において異種発現し、酵素反応を試みた。その結果、３つのうち１つ（CalQ）がカリクリンAを基質として受け入れ、二リン酸化体を生成することを明らかにした。このことには海綿メタゲノムより同定した遺伝子クラスターがカリクリンA生合成遺伝子であることを支持している。カリクリンAの二リン酸体の生物活性を調べたところ、細胞毒性は1,000倍、PP2A阻害活性は20倍と著しく減弱しており、カリクリンAのプロドラッグ体であることが示唆された。したがって、カリクリン生合成における最終産物はカリクリンAではなく、本研究で見出されたホスホカリクリンAであることが明らかとなった。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont (2014)
  • Author(s): Wakimoto, T., Egami, Y., Nakashima, Y., Wakimoto,Y., Mori, T., Awakawa,T., Ito, T., Kenmoku, H., Asakawa, Y., Piel, J., Abe, I.
  • Journal Title: Nature Chem. Biol. Volume: 10 Issue: 8 Pages: 648-655
  • DOI: 10.1038/nchembio.1573
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid (2014)
  • Author(s): Matsuda, Y., Wakimoto, T., Mori, T., Awakawa, T., Abe, I.
  • Journal Title: J. Am. Chem. Soc. Volume: 136 Issue: 43 Pages: 15326-15336
  • DOI: 10.1021/ja508127q
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis (2014)
  • Author(s): Awakawa, T., Zhang, L., Wakimoto, T., Hoshino, S., Mori, T., Ito, T., Ishikawa, J., Tanner, M. E., Abe, I.
  • Journal Title: J. Am. Chem. Soc. Volume: 136 Issue: 28 Pages: 9910-9913
  • DOI: 10.1021/ja505224r
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx (2014)
  • Author(s): Y. Egami, T. Wakimoto, I. Abe
  • Journal Title: Bioorg. Med. Chem. Lett. Volume: 24 Issue: 22 Pages: 5150-5153
  • DOI: 10.1016/j.bmcl.2014.10.002
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Arcyriaflavin E, a new cytotoxic indolocarbazole alkaloid isolated by combined-culture of mycolic acid-containing bacteria and Streptomyces cinnamoneus NBRC 13823. (2014)
  • Author(s): S. Hoshino, L. Zhang, T. Awakawa, T. Wakimoto, H. Onaka, I. Abe
  • Journal Title: J. Antibiot. Volume: 67 Issue: 5 Pages: 1-3
  • DOI: 10.1038/ja.2014.147
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Three acyltetronic acid derivatives: noncanonical cryptic polyketides from Aspergillus niger identified by genome mining (2014)
  • Author(s): Yang, X.-L., Awakawa, T., Wakimoto, T., Abe, I.
  • Journal Title: ChemBioChem Volume: 15 Issue: 11 Pages: 1578-1583
  • DOI: 10.1002/cbic.201402172
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Lipodiscamides A-C, new cytotoxic lipopeptides from the marine sponge Discodermia kiiensis (2014)
  • Author(s): K. C. Tan, T. Wakimoto, I. Abe
  • Journal Title: Org. Lett. Volume: 16 Issue: 12 Pages: 3256-3259
  • DOI: 10.1021/ol501271v
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] An environmental bacterial taxon with a large and distinct metabolic repertoire (2014)
  • Author(s): Wilson, M. C., Mori, T., Ruckert, C., Uria, A. R., Helf, M. J., Takada, K., Gernert, C., Steffens, U. A. E., Heycke, N., Schmitt, S., Rinke, C., Helfrich, E. J. N., Brachmann, A. O., Gurgui, C., Wakimoto. T., Kracht, M., Crusemann, M., Hentschel, U., Abe, I., Matsunaga, S., Kalinowski, J., Takeyama, H., Piel, J.
  • Journal Title: Nature Volume: 506 Issue: 7486 Pages: 58-62
  • DOI: 10.1038/nature12959
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Revised structure of cyclolithistide A, a cyclic depsipeptide from the marine sponge Discodermia japonica (2014)
  • Author(s): H. Tajima, T. Wakimoto, K. Takada, Y. Ise, I. Abe
  • Journal Title: J. Nat. Prod. Volume: 77 Issue: 1 Pages: 154-158
  • DOI: 10.1021/np400668k
  • Peer Reviewed
• [Journal Article] Enantioselective Synthesis of SB-203207 (2014)
  • Author(s): Yasuo Hirooka, Kazutada Ikeuchi, Yuichiro Kawamoto, Yusuke Akao, Takumi Furuta, Tomohiro Asakawa, Makoto Inai, Toshiyuki Wakimoto, Tohru Fukuyama and Toshiyuki Kan
  • Journal Title: Org. Lett. Volume: 16 Issue: 6 Pages: 1646-1646
  • DOI: 10.1021/ol5002973
  • Peer Reviewed
• [Journal Article] Spiro-ring formation is catalyzed by a multifunctional dioxygenase in austinol biosynthesis (2013)
  • Author(s): Y. Matsuda, T. Awakawa, T. Wakimoto, and I. Abe
  • Journal Title: J. Am. Chem. Soc. Volume: 135 Issue: 30 Pages: 10962-10965
  • DOI: 10.1021/ja405518u
  • Peer Reviewed
• [Journal Article] Pyranonigrin E : a novel PKS-NRPS hybrid metabolite from Aspergillus niger ide ntified by genome mining (2013)
  • Author(s): Takayoshi Awakawa., Xiao-Long Xia o-Long Yang, Toshiyuki Wakimoto., Ikuro Abe
  • Journal Title: Chembiochem Volume: 14 Issue: 16 Pages: 2095-2099
  • DOI: 10.1002/cbic.201300430
  • Peer Reviewed
• [Journal Article] Cycloforskamide, a cytotoxic macrocyclic peptide from the sea slug Pleurobranchus forskalii (2013)
  • Author(s): K. C. Tan, T. Wakimoto, K. Takada, T. Ohtsuki, N. Uchiyama, Y. Goda, I. Abe
  • Journal Title: Toxicon Volume: 72 Pages: 1-4
  • DOI: 10.1016/j.toxicon.2013.05.021
  • Peer Reviewed
• [Journal Article] Indole-porphyrin hybrids produced by metagenomics (2013)
  • Author(s): Yang, X. -L., Wakimoto, T., Takeshige, Y., He, R., Egami, Y., Abe, I.
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 23 Issue: 13 Pages: 2810-2813
  • DOI: 10.1016/j.bmcl.2013.04.076
  • Peer Reviewed
• [Journal Article] Induced production of novel prenyldepside and coumarins in endophytic fungi Pestalotiopsis acaciae (2013)
  • Author(s): X.-L. Yang, T. Awakawa, T. Wakimoto, I. Abe
  • Journal Title: Tetrahedron Lett. Volume: 54 Pages: 5814-5817
  • Peer Reviewed
• [Journal Article] Induced production of the novel glycolipid ustilagic acid C in the plant pathogen Ustilago maydis (2013)
  • Author(s): X.-L. Yang, T. Awakawa, T. Wakimoto, I. Abe
  • Journal Title: Tetrahedron Lett. Volume: 54 Pages: 3655-3657
  • Peer Reviewed
• [Journal Article] Induced biosyntheses of a novel butyrophenone and two aromatic polyketides in the plant pathogen Stagonospora nodorum (2013)
  • Author(s): X.-L. Yang, T. Awakawa, T. Wakimoto, I. Abe
  • Journal Title: Nat. Prod. Bioprospect. Volume: 3 Pages: 141-144
  • Peer Reviewed
• [Journal Article] Epigenetic modifier-induced biosynthesis of novel fusaric acid derivatives in endophytic fungi from Datura stramonium L. (2013)
  • Author(s): H.-J. Chen, T. Awakawa, J.-Y. Sun, T. Wakimoto, I. Abe
  • Journal Title: Nat. Prod. Bioprospect. Volume: 3 Pages: 20-23
  • Peer Reviewed
• [Presentation] The significance of unstable marine natural products (2015)
  • Author(s): Toshiyuki Wakimoto
  • Organizer: Marine Biotechnology Seminar
  • Place of Presentation: Adelaide, Australia
  • Year and Date: 2015-02-09
  • Invited
• [Presentation] 海綿由来生物活性物質の生合成に関する研究 (2014)
  • Author(s): 脇本敏幸
  • Organizer: 日本生薬学会第61年会
  • Place of Presentation: 福岡県福岡市
  • Year and Date: 2014-09-14
  • Invited
• [Presentation] 多様な天然物を生産する海綿共生バクテリア (2014)
  • Author(s): 脇本敏幸
  • Organizer: 静岡大学農学部公開講演会
  • Place of Presentation: 静岡県静岡市
  • Year and Date: 2014-07-14
  • Invited
• [Presentation] A bacterial symbiont of Japanese marine sponge Discodermia calyx, produces biologically active metabolites (2013)
  • Author(s): T. Wakimoto, Y. Egami, Y. Nakashima, I. Abe
  • Organizer: 9th World Sponge Conference
  • Place of Presentation: Perth, Australia
  • Invited
• [Presentation] A bacterial symbiont of Japanese marine sponge Discodermia calyx, produces biologically active metabolites (2013)
  • Author(s): T. Wakimoto, Y. Egami, Y. Nakashima, I. Abe
  • Organizer: The 14th International Symposium of Marine Natural Products
  • Place of Presentation: La Toja, Spain
  • Invited
• [Presentation] 海洋生物に由来する医薬品資源の開拓と利用を目指して
  • Author(s): 脇本敏幸
  • Organizer: 第34回和漢医薬学総合研究所 特別セミナー
  • Place of Presentation: 富山県富山市
  • Invited
• [Presentation] 海洋生物に由来する医薬品資源の開拓と利用を目指して
  • Author(s): 脇本敏幸
  • Organizer: 福井県立大学 生物資源学特別セミナー
  • Place of Presentation: 福井県福井市
  • Invited
• [Presentation] 海綿共生微生物を起源とする生理活性物質の生産機構解析
  • Author(s): 脇本敏幸
  • Organizer: 第1回薬学の有機化学を考えるin北海道
  • Place of Presentation: 北海道札幌市
  • Invited
• [Book] Handbook of Cytotoxic cyclic peptides from the marine sponges (2014)
  • Author(s): T. Wakimoto, K. C. Tan, H. Tajima, I. Abe
  • Total Pages: 800
  • Publisher: Springer
• [Remarks] カイメンを気づかう共生バクテリア ―チョコガタイシカイメンの毒性物質はバクテリアが生産していた―
  • URL: http://www.u-tokyo.ac.jp/public/public01_260630_02_j.html
• [Remarks] 海綿動物の化学防御を担う共生細菌
  • URL: http://first.lifesciencedb.jp/archives/9108