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

本研究主要对真菌重要二萜类植物激素赤霉素（giberellin）、DNA聚合酶α特异性抑制剂aphidicolon和II^+- atp酶激活剂fusicoccin的生物合成中间体环烃进行了鉴定，并对其环化酶进行了cDNA克隆和功能分析。利用RT-PCR技术从藤黑赤霉素中分离到编码ent-kaurene合成酶的cDNA。其重组蛋白在大肠杆菌中表达，将GGDP转化为赤霉素生物合成中的环烃中间体-叔丁烯。通过对产生阿蚜霉素的Phoma betae菌丝体中环烃的分析，鉴定出阿蚜霉素生物合成中间体阿蚜霉素-8β-醇、茎-13-烯及其相关化合物。利用RT-PCR技术从该真菌中克隆出了aphidicolan-8β-ol合成酶的cDNA。在大肠杆菌中表达的重组蛋白将GGDP转化为蚜虫-8β-醇。通过对产生梭菌素J及其相关化合物的Phomopsis amygdali F6培养菌丝中环烃的分析，分离出(+)- fusicocca-2,10 (14)- dicine作为梭菌素生物合成中间体的重要候选物。通过立体控制合成确定了其立体结构。该化合物是由杏仁核F6制备的无细胞体系由GGDP形成的。合成的氘标记的(+)-fusicocca- 2,10(14) -二烯被代谢为(+)-fusicocca- 2,10(14) -二烯-8β-醇，也被鉴定为该真菌的天然产物。进一步合成氘标记的(+)- fusicocca-2,10 (14) -dien-8β-ol本身在真菌中转化为fusicocin J。fusicocca- 2,10(14) -二烯合成酶cDNA的克隆正在进行中。

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.