2000 Fiscal Year Final Research Report Summary
cDNA cloning and its development of diterpene cyclases forming its cyclic carbon skeleton from bioactive diterpene producing microorganisms
| Project/Area Number |
11460053
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B).
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Bioproduction chemistry/Bioorganic chemistry
|
|---|
| Research Institution | Yamagata University |
|---|
Principal Investigator |
SASSA Takeshi Yamagata Univ. Bioresoures Engineering, Professor, 農学部, 教授 (80023456)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MITSUHASHI Wataru Yamagata Univ. Bioresoures Engineering, Associate Professor, 農学部, 助教授 (50192761)
TOYOMASU Tomonobu Yamagata Univ. Bioresoures Engineering Associate Professor, 農学部, 助教授 (60272085)
KATO Nobuo Kyushu Univ. Institute of advanced material study Associate Professor, 機能物質科学研究所, 助教授 (50150537)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Keywords | Gibberella fujikuroi / Phoma betae / ent-kaurene synthase / aphidicolan-8β-ol synthase / gibberellin / aphidicolin / fusicoccin / terpene biosynthesis |
|---|
| Research Abstract |
In this study the identification of biosynthetic intermediacy cyclic-hydrocarbons of fungal important diterpenes such as gibberellin (phytohormone), aphidicolon (specific inhibitor for DNA polymerase α) and fusicoccin (activator for II^+-ATPase), and cDNA cloning and functional analysis of their cyclases were carried out cheifly. The cDNA encoding ent-kaurene synthase was isolated from Gibberella fujikuroi by RT-PCR.Its recombinant protein which was expressed in Escherichia coli converted GGDP to ent-kaurene which is the cyclic hydrocarbon intermediate in gibberellin biosynthesis. As a result of analysis of cyclic hydrocarbons in the cultural mycelia of Phoma betae producing aphidicolin, aphidicolan-8β-ol (a biosynthetic intermediate of aphidicolin), stemar-13-ene and their related compounds were identified. The cDNA encoding aphidicolan-8β-ol synthase was cloned from the fungus by RT-PCR.Its recombinant protein which was expressed in E.coli converted GGDP to aphidicolan-8β-ol. By the analysis of cyclic hydrocarbons in the cultural mycelia of Phomopsis amygdali F6 in which fusicoccin J and its novel related compounds are found to be produced, (+)- fusicocca-2,10 (14)-dicne was isolated as an important candidate of fusicoccin-bisosynthetic intermediates. Its stereostructure was determined by its stereocontrolled synthesis. This hydrocarbon was formed from GGDP by a cell free system prepared from P.amygdali F6. Synthetic deuterium-labeled (+)-fusicocca-2, 10 (14) -diene was clearly metabolized to (+) -fusicocca-2, 10 (14) -dien-8β-ol which was also identified as a natural product from this fungus. Further synthetic deuterium-labeled (+)- fusicocca-2,10 (14) -dien-8β-ol itself was converted into fusicocin J in the fungus. The cloning of cDNA encoding fusicocca-2, 10 (14) -diene synthase is being progresssed.
|
