cDNA cloning and its development of diterpene cyclases forming its cyclic carbon skeleton from bioactive diterpene producing microorganisms

本研究对真菌重要二萜类化合物如赤霉素（植物激素）、蚜虫素（DNA聚合酶α的特异性抑制剂）和梭孢菌素（II^+-ATP酶的激活剂）等生物合成中间环烃进行了鉴定，并对其环化酶进行了cDNA克隆和功能分析。利用RT-PCR方法从藤仓赤霉菌中克隆了内贝壳杉烯合成酶基因，并在大肠杆菌中表达了该基因的重组蛋白，该蛋白可将GGDP转化为赤霉素合成的环烃中间体内贝壳杉烯。通过对产蚜虫霉素的甜菜茎点霉菌丝体中环烃类化合物的分析，鉴定出了蚜虫霉素的生物合成中间体8β-醇（aphidicolan-8β-ol）、stemar-13-ene及其相关化合物。利用RT-PCR技术从该真菌中克隆了aphidicolan-8β-ol合成酶的cDNA，并在大肠杆菌中表达了其重组蛋白，该重组蛋白可转化GGDP为aphidicolan-8β-ol。通过对产生梭孢菌素J及其相关化合物的扁桃拟茎点霉（Phomopsis amygdali）F6培养菌丝体中的环烃类化合物的分析，分离到了（+）-梭孢菌素-2，10（14）-二炔，它是梭孢菌素-双合成中间体的重要候选者。其立体结构由立体控制合成确定。该烃由GGDP通过由苦杏仁P.amygdali F6制备的无细胞系统形成。合成的氘标记的（+）-fusicocca-2，10（14）-diene被明显代谢为（+）-fusicocca-2，10（14）-dien-8β-ol，其也被鉴定为该真菌的天然产物。进一步合成的氘标记的（+）- fusicocca-2，10（14）-dien-8β-ol本身在真菌中转化为fusicocin J。目前正在进行梭壳-2，10（14）-二烯合酶cDNA的克隆。

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.