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赤霉素(植物激素)、除草剂(α聚合酶特异性抑制剂)和褐变素(Ⅱ~+-ATPase激活剂)的生物合成中间体环烃进行了鉴定，并对其环化酶进行了克隆和功能分析。利用RT-PCR技术从藤库里赤霉菌中分离到Ent-Kaurene合成酶基因，重组蛋白在大肠杆菌中表达，将GGDP转化为赤霉素生物合成的环状碳氢化合物中间体Ent-Kaurene。通过对甜菜夜蛾菌丝体中环状碳氢化合物的分析，鉴定出其生物合成中间体APhidicolan-8β-ol、茎-13-烯及其相关化合物。用RT-PCR方法从该真菌中克隆了APHIPDICORAN-8β-ol合成酶基因，重组蛋白在大肠杆菌中表达后转化为APHIPICORAN-8β-OL。通过对产生褐霉素J及其相关新化合物的杏树拟南星F6菌丝体中环状烃的分析，分离得到一种重要的褐霉素-2，10(14)-二环己二烯。它的立体结构是由其立体控制合成确定的。这种碳氢化合物是由GGDP通过由杏仁侧耳F6制备的无细胞系统形成的。合成的氚标记的fusicocca-2，10(14)-diene明显代谢为fusicocca-2，10(14)-dien-8β-ol，该产物也被鉴定为该真菌的天然产物。此外，在真菌中，合成的标记()-fusicocca-2，10(14)-dien-8β-ol本身被转化为fusicocin J。Fusicocca-2，10(14)-二烯合酶基因的克隆正在进行中。

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.