| Project/Area Number |
10556024
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
OIKAWA Hideaki Hokkaido Univ.Grad.School of Agriculture, Associate Prof., 大学院・農学研究科, 助教授 (00185175)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Hideaki Kirin Brewery, Institute of Exploring Medicine, Chief Researcher, 医薬探索研究所, 主任研究員
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1998: ¥3,500,000 (Direct Cost: ¥3,500,000)
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| Keywords | tautomycin / tautomycetin / protein phosphatase / inhibitor |
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| Research Abstract |
Aphidicolin(ACL), an antivirus agent from Phoma betae shows a various biological activity such as antitumor, phytotoxic and specific inhibition of DNA polymerase α. Although ACL can be obtained by traditional fermentation method of the fungus, its productivity and longer fermentation period hamper its large-scale production. Recent progress of prompted us to explore identification of entire biosynthetic genes responsible for ACL biosynthesis, and heterologous expression of the genes.
In order to identify the gene catalyzes cyclization of universal diterpene precusor GGDP, homology based PCR was conducted. RT-PCR with mRNA from P.betae and degenerate primers based on the conserved amino acid sequences of other diterpene cyclases allowed us to amplify 1100-bp band that showed a significant similarity to fungal ent-kaurene synthases. The nucleotide sequence of the full-length cDNA was determined by 5'-and 3'-RACE by using gene-specific primers. This contained the predicted 2997-dp open reading frame, encoding a product of 998 amino acids which was named aphidicolan-16 β-ol synthase(ACS). A full-length cDNA was ligated into a pGEX 4T-3 vector and the glutathione S-transferase(GST)-ACS fusion protein was expressed in Escherichia coli JM109. The hexane extracts of the reaction mixture obtained by incubation of GGDP with rACS afforded three products. These products of the rACS were identified, by comparison of retention time and mass spctra to those of synthetic standards, as aphidicolan-16 β-ol, aphidicol-16-ene and aphidicol-15-ene. Since all products are found in the mycelial extracts of P.betae, it suggests that these products are produced by a single enzyme.
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