Large-scale Production of Antivirus Agent Based on Engineered Biosynthesis

基于工程生物合成的抗病毒剂规模化生产

• 批准号: 10556024
• 负责人: OIKAWA Hideaki
• 金额: $ 4.99万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10556024/
• 关键词: tautomycin; tautomycetin; protein phosphatase; inhibitor

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.

Aphidicolin（ACL）是一种来源于Phoma betae的抗病毒药物，具有抗肿瘤、植物毒性和特异性抑制DNA聚合酶α等多种生物活性。传统的真菌发酵方法虽然可以获得ACL，但其产量高，发酵周期长，阻碍了其大规模生产。近年来的研究进展促使我们探索ACL生物合成的全基因鉴定和异源表达，为了鉴定催化通用二萜前体GGDP环化的基因，我们进行了基于同源性的PCR。RT-PCR与P.betae的mRNA和简并引物的基础上保守的氨基酸序列的其他二萜环化酶允许我们扩增1100 bp的带，表现出显着的相似性，真菌ent-kaurene环化酶。利用基因特异性引物，通过5 '-和3'-RACE确定全长cDNA的核苷酸序列。该基因含有预测的2997-dp开放阅读框架，编码998个氨基酸的产物，命名为蚜虫烷-16 β-醇合酶（ACS）。将全长cDNA连接到pGEX 4 T-3载体中，并在大肠杆菌JM 109中表达谷胱甘肽S-转移酶（GST）-ACS融合蛋白。通过将GGDP与rACS孵育获得的反应混合物的己烷提取物得到三种产物。通过与合成标准品的保留时间和质量谱的比较，将这些rACS产物鉴定为aphidicolan-16 β-ol、aphidicol-16-ene和aphidicol-15-ene。由于所有的产物都存在于甜菜夜蛾的菌丝体提取物中，这表明这些产物是由单一的酶产生的。

项目成果

H.Oikawa et al.: "Diversity of Diterpene Hydrocarbons in Fungus Phoma betae"Tetrahedron Lett.. 42(3). 2329-2332 (2001)

H.Oikawa 等人：“真菌Phoma betae 中二萜烃的多样性”Tetrahedron Lett.. 42(3)。

H.Oikawa: "Biosynthesis of Diterpenoid Aphidicolin : Isolation of Intermediates from P-450 Inhibitor Treated Mycelia of Phoma betae"Tetrahedron. 55. 7541-7554 (1999)

H.Oikawa：“二萜类 Aphidicolin 的生物合成：从 P-450 抑制剂处理的短柄霉菌丝体中分离中间体”四面体。

H.Toshima et al.: "Total Synthesis of Both Enantiomers of Copalol via Optical Resolution of a General Synthetic intermediate"Tetrahedron. 56(43). 8443-8450 (2000)

H.Toshima 等人：“通过通用合成中间体的光学拆分来全合成柯巴洛的两种对映体”四面体。

H.Toshima: "Total Synthesis of Both Enantiomers of Copalol via Optical Resolution of a General Synthetic intermediate"Tetrahedron. 56・43. 8443-8450 (2000)

H. Toshima：“通过一般合成中间体的光学拆分来完全合成柯巴洛的两种对映体”Tetrahedron 56・43（2000）。

H.Oikawa: "Diversity of Diterpene Hydrocarbons in Fungus Phoma betae"Tetrahedron Lett.. 42・3. 2329-2332 (2001)

H.Oikawa：“真菌中的二萜烃的多样性”Tetrahedron Lett.. 42・3（2001）