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Scientific research on elucidation of terpene-cyclic reactions in the biosynthesis of bioactive microbial terpenoids.

Research Project

Project/Area Number 13306009
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field Bioproduction chemistry/Bioorganic chemistry
Research InstitutionYamagata University

Principal Investigator

SASSA Takeshi  Yamagata University, Dept.of Bioresource Engineering, Professor, 農学部, 教授 (80023456)

Co-Investigator(Kenkyū-buntansha) OIKAWA Hideaki  Hokkaido University, Division of Chemistry, Professor, 大学院・理学研究科, 教授 (00185175)
KATO Nobuo  Osaka University, The Institute of Scientific and Industrial Research, Professor, 産業科学研究所, 教授 (50150537)
HOSHINO Tsutomu  Niigata University, Dept.of Applied Biological Chemistry, Professor, 農学部, 教授 (30165542)
TOYOMASU Tomonobu  Yamagata University, Assistant Prof., 農学部, 助教授 (60272085)
DAIRI Tohru  Toyama Prefectural University, the Biotechnology Research Center, Assistant Prof., 生物工学研究センター, 助教授 (70264679)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥56,290,000 (Direct Cost: ¥43,300,000、Indirect Cost: ¥12,990,000)
Fiscal Year 2003: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
Fiscal Year 2002: ¥16,380,000 (Direct Cost: ¥12,600,000、Indirect Cost: ¥3,780,000)
Fiscal Year 2001: ¥28,080,000 (Direct Cost: ¥21,600,000、Indirect Cost: ¥6,480,000)
Keywordsditerpene / terpene cyclase / biosynthesis / cyclization mechanism / biosynthetic intermediate / tetraterpene / squarene / フォマクチン / エリナシン / サイアタジエン / アフィジコリン / テルペンテシン / フシコクシン / 環化酵素遺伝子 / ヤマブシタケ / テルペンテトリエン / アフィジコリンの環化機構 / スクアレン-ホペン環化酵素 / 非天然型トリテルペン
Research Abstract

(1)We isolated (+)-cyatha-3,12-diene from an erinacine-producing fungus, and confirmed its stereostructure by semi-synthesis from erinacine P. The cyathane hydrocarbon could be obtained from GGDP by the cell-free system. From a fusicoccin-mass-producing fungus we isolated (+)-δ-araneosene as the presumed bicyclic hydrocarbon intermediate. (+)-Fusicocca-3(16),10(14)-diene was also isolated and its identification suggested the presence of a new fusicoccenyl cation in the biosynthetic pathway. (2)We identified two gene clusters responsible for diterpene biosynthesis by chromosome waling from GGDP synthase genes from Phomopsis amygdali. The gene clusters led us to successful isolation and characterization of two cDNAs encoding diterpene cyclases. (3)The gene cluster for aphidicolin biosynthesis has been identified by PCR-based genome walking using reported sequence of cDNA coding the diterpene cyclase. Based on non-enzymatic cyclization under various conditions and molecular orbital calculations, cyclization mechanism for construction of aphidicolane skeleton was proposed. (4)We have cloned two diterpene cyclase genes essential for biosynthesis of terpentecin. By using recombinant enzymes, it was revealed that one enzyme converted GGDP into a cyclized intermediate (terpentedienol diphosphate), and then it was transformed into terpentetriene by the other enzyme. (5)By using the site-specific mutants and the substrate analogs, we clearly demonstrated that the steric bulk size of active site residues has an important role in determining the folding conformation of squalene substrate during the polycyclization cascade.

Report

(4 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • Research Products

    (48 results)

All Other

All Publications (48 results)

  • [Publications] H.Kenmoku: "Isolation of (-)-cyatha-3,12-diene, a common biosynthetic intermediate of cyathane diterpenoids from an erinacine-producing basidiomycete, Hericium erinaceum, and its formation in a cell-free system."Tetrahedron Lett.. 42. 7439-7442 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Sato: "Functional analyses of Tyr420 and Leu607 of Alicyclobacillus acidocaldarius squalene-hopenecyclase. Neoachillapentaene, a novel triterpene with the 1,5,6-trimethylcyclohexene moiety produced through a folding of the constrained boat structure."Biosci.Biotechnol.Biochem.. 66(8). 1660-1670 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hoshino: "Importance of the methyl group at C(10) of squalene for hopene biosythesis and novel carbocyclic skeletons with 6/5 + 5/5 + (6) ring system(s)."Organic Lett.. 4(15). 2553-2556 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Oikawa: "Proposed mechanism for the reaction catalyzed by a diterpene cyclase, aphidicolan-16β-ol synthase : Experimental results on biomimetic cyclization pathway by ab initio calculations."J.Am.Chem.Soc.. 124(31). 9145-9153 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku: "Erinecine Q, a new erinacine from Hericium erinaceum, and its biosynthetic route to erinacine C in the basidiomycete."Biosci.Biotechnol.Biochem.. 66(3). 571-575 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hoshino: "Squalene-hopene cyclase : catalytic mechanism and substrate recognition."Chem.Commun.. (4). 291-301 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 豊増知伸: "微生物テルペノイド生合成研究の新展開-環化酵素遺伝子-"日本農芸化学会誌. 76(12). 1186-1205 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Kawasaki: "A relationship between the mevalonate pathway and isoprenoid production in Actinomycetes."J.Antibiot.. 56(12). 957-966 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 及川 英秋: "生物活性ジテルペンの酵素的生産を目指して"化学と生物. 41(12). 807-814 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Toyomasu: "Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α."Biosci.Biotechnol.Biochem.. 68(1). 146-152 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Sato: "Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius."Biosci.Biotechnol.Biochem.. 68(3). 728-738 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hoshino: "Squalene-hopene cyclase : final deprotonation reaction, conformational analysis for the cyclization of (3R, S)-2.3-oxidosqualene and further evidence for the requirement of isopropylidene moiety both for initiation of the polycyclization cascade and for the formation of 5-membered E-ring."Org. & Biomol.Chem.. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku: "Erinacol (cyatha-3,12-dien-14β-ol) and 11-O-acetylcyathin A_3, new cyathane metabolites from an erinacine Q-producing Hericium erinaceum."Biosci.Biotechnol.Biochem.. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Sassa: "Fusicocca-3(16),10(14)-diene, and β- and δ-araneosenes, new fusicoccin biosynthesis-relating diterpene hydrocarbons from Phomopsis amygdali."Biosci.Biotechnol.Biochem.. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku: "Identification of (+)-phyllocladene, (-)-sandaracopimaradiene, and (+)-kaurene, as new fungal metabolites from the fusicoccin-producing Phomopsis amygdali F6."Biosci.Biotechnol.Biochem.. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku et al.: "Isolation of (-)-cyatha-3,12-diene, a common biosynthetic intermediate of cyathane diterpenoids, from an erinacine-producing basidiomycete, Hericium erinaceum, and its formation in a cell-free system."Tetrahedron Lett.. 42. 7439-7442 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Sato et al.: "Functional analyses of Tyr420 and Leu607 of Alicyclobacillus acidocaldarius squalene-hopene cyclase. Neoachillapentaene, a novel triterpene with the 1, 5, 6-trimethylcyclohexene moiety produced through a folding of the constrained boat structure."Biosci.Biotechnol.Biochem.. 66. 1660-1670 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hoshino et al.: "Importance of the methyl group at C(10) of squalene for hopene biosythesis and novel carbocyclic skeletons with 6/5 + 5/5 + (6) ring system(s)."Organic Lett.. 4. 2553-2556 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Oikawa et al.: "Proposed mechanism for the reaction catalyzed by a diterpene cyclase, aphidicolan-16 β -ol synthase : Experimental results on biomimetic cyclization pathway by ab initio calculations."J.Am.Chem.Soc.. 123. 9145-9153 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku et al.: "Erinecine Q, a new erinacine from Hericium erinaceum, and its biosynthetic route to erinacine C in the basidiomycete."Biosci Biotechnol Biochem.. 63. 571-575 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hoshino et al.: "Squalene-hopene cyclase : catalytic mechanism and substrate recognition."Chem.Commun.. 291-301 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Toyornasu et al.: "New approach of studies on microbial terpenoid biosynthesis-cyclase genes-"Nippon Nogeikagaku Kaishi. 76. 1186-1205 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Kawasaki et al.: "A relationship between the mevalonate pathway and isoprenoid production in Actinomycetes."J.Antibiot.. 56. 957-966 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Oikawa et al.: "Production by enzymatic reaction of bioactive ditrepenes."Kagaku to Seibutsu. 41. 807-814 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Toyomasu et al.: "Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitior of DNA polymerase α."Biosci Biotechnol Biochem. 68. 146-152 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Sato et al.: "Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius."Biosci Biotechnol Biochem. 68. 728-738 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku et al.: "Erinacol (cyatha-3,12-dien-14 β -ol) and 11-O-acetylcyathin A_3, new cyathane metabolites from an erinacine Q-producing Hericium erinaceum."Biosci.Biothechnol.Biochem. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Sassa et al.: "Fusicocca-3(16), 10(14)-diene, and β -and δ -araneosenes, new fusicoccin biosynthesis-relating hydrocarbons from Phomopsis amygdali."Biosci.Biothechnol.Biochem. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] H.Kenmoku et al.: "Identification of (+)-phyllocladene, (-)-sandaracopimaradiene, and (+)-kaurene, as new fungal metabolites from the fusicoccin-producing Phomopsis amygdali F6."Biosci.Biothechnol.Biochem. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hoshino et al.: "Squalene-hopene cyclase : final deprotonation reaction, conformational analysis for the cyclization of (3R, S)-2,3-oxidosqualene and further evidence for the requirement of isopropylidene moiety both for initiation of the polycyclization cascade and for the formation of 5-membered E-ring."Org.& Biomol.Chem.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kenmoku, H.: "Erinacol(cyatha-3,12-dien-14β-ol)and 11-O-acetylcyathin A_3, new cyathane metabolites from an erinacine Q-producing Hericium erinaceum"Biosci.Biotechnol.Biochem.. 68(in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Sassa, T.: "Fusicocca-3(16),10(14)-diene, and β-and δ-araneosenes, new fusicoccin biosynthesis-relating diterpene hydrocarbons from Phomopsis amygdali"Biosci.Biotechnol.Biochem.. 68(in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Sato, T.: "Site-directed mutagenesis experiments on the putative deprotonation site of squalenehopene cyclase from Alicyclobacillus acidocaldarius"Biosci.Biotechnol.Biochem.. 68(3)(in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kawasaki, T.: "A relationship between the mevalonate pathway and isoprenoid production in Actinomycetes"J.Antibiot.. 56(12). 957-966 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Toyomasu, T.: "Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α"Biosci.Biotechnol.Biochem.. 68(1). 146-152 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] 及川 英秋: "生物活性ジテルペンの酵素的生産を目指して"化学と生物. 41(12). 807-814 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] T.Hoshino: "Importance of the methyl group at C(10) of squalene for hopene biosynthesis and novel carbocyclic skeletons with 6/5+5/5+(6) ring system(s)"Org. Lett.. 4. 2553-2556 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] T.Sato: "Functional analysis of tyr 420 and leu 607 of Alicyclobacillus acidocaldarius squalene-hopene cyclase"Biosci. Biotechnol. Biochem.. 1660-1670 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 星野 力: "スクアレン環化酵-環化機構と非天然型トリテルペンの創製-"日本農芸化学会誌. 76・12. 1187-1190 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 加藤 修雄: "特異な生理活性菌類ジテルペン配糖体-フシコクシン,エリナシン-における初期生合成経路の解明と応用"日本農芸化学会誌. 76・12. 1202-1205 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 及川 英秋: "環化酵素を鍵としたジテルペンの酵素的全合成"日本農芸化学会誌. 76・12. 1199-1201 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Y.Hamano: "Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic terpentecin"J. Biol. Chem.. 277. 37098-37104 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] H.Kenmoku: "Isolation of (-)-cyatha-3. 12-diene, a common biosynthetic intermediate of cyathane diterpenoides, from an erinacine-producing basidiomycete and its formation in a cell-free system"Tetrahedron Lett. 42. 7439-7442 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] H.Kenmoku: "Erinacine Q, a new erinacine from Hericium erinaceum and its biosynthetic route to erinacine C in the Bosidiomycete"Biosci. Biotechnol. Bioclem. 66・3. 571-575 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Sato: "Catalytic function of the residues of phenylalanine and tryosine conserved in sgualene-hopene cyclases"Beosci. Biotechnel, Bioclem. 65・10. 2233-2242 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Hoshino: "Engymic products of the 2.3-oxidosqualene an alog having an ethyl residue at 10-position. First trapping of the trimethyl cyclo hexanone ring by lanosterol systhase"Tetrahedron Lett. 42. 7319-7324 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Hoshino: "Squalene-hopene cyclase : catalytic mechanism and sabstrate reisgnition"J,Chem. Commun. ・4. 291-301 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T, Dairi: "Eubacterial diterpene cyclase genes essential for production of isoprenoid dntibiotic terpenticin"J,Bacteriology. 183・20. 6085-6094 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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