雷公藤甲素生物合成二萜合酶基因克隆及功能研究

Triptolide is an important abietane-type diterpene. Recently, it has aroused scientists' interest because of its unique chemical configuration and significant pharmacological activities, but the research of the key enzyme genes involved in the biosynthesis which determines its chemical structure has not been carried out yet. Based on the study of Tripterygium wilfordii transcriptome by the high throughput sequencing, the project will construct the new Digital Gene Expression (DGE) on suspension cell of Tripterygium wilfordii induced by Methyl Jasmonate (MeJA), in order to screen diterpene synthase gene fragments. The full-length cDNAs of candidate diterpene synthases (TwCPS, TwASL) will be cloned by RACE, which are used for construction of prokaryotic expression vector and the next expression of heterologous proteins, and these candidate diterpene synthases could be preliminary identified by enzymatic reaction in vitro. On this basis, for the diterpene synthase genes can't be accurately identified, engineering bacteria will be established to enrich fermentation for further structural analysis by spectral meansand to know the specific functions of TwCPS, TwASL and chemical structures of the final catalytic product (triptolide precursor). In this study, the full-length cDNAs of diterpene synthases involved in triptolide biosynthesis will be cloned and identified accurately.Then the structure of triptolide precursor will be resolved, which will reveal the starting key step of triptolide biosynthesis from the linear GGPP into the abietane-type nucleus. These results will lay the foundation for study on the downstream pathway of triptolide biosynthesis and its producing by means of synthetic biology.

雷公藤甲素是一类重要的松香烷型二萜，由于其独特构型和显著活性而备受关注，但决定其化学构型的生物合成关键酶基因研究却尚未开展。本项目拟在雷公藤转录组研究的基础上，构建基于茉莉酸甲酯诱导的雷公藤悬浮细胞数字基因表达谱，筛选出雷公藤甲素生物合成二萜合酶基因片段；克隆候选二萜合酶全长cDNA（TwCPS、TwASL），并进行异源蛋白表达，通过体外酶促反应初步鉴定其功能；在此基础上，对于无法准确鉴定的二萜合酶基因，构建工程菌，富集发酵产物，采用光谱等手段对产物进一步结构解析，明确TwCPS、TwASL特异性功能及其催化终产物（雷公藤甲素二萜烯中间体）的化学结构。本项目将获得雷公藤甲素生物合成二萜合酶全长基因，通过基因功能鉴定和解析雷公藤甲素二萜烯中间体的化学结构，揭示雷公藤甲素生物合成从线性GGPP环化成松香烷型母核的生物学过程和起始关键步骤，为其生物合成下游途径的阐释和合成生物学研究奠定基础。

雷公藤甲素是一类重要的松香烷型二萜，由于其独特构型和显著活性而备受关注，但决定其化学构型的生物合成二萜合酶基因研究却尚未开展。本项目以决定雷公藤甲素母核构型的关键二萜合酶基因为研究目标，运用转录组测序、基因全长克隆及基因体内外功能鉴定等方法对雷公藤二萜合酶基因进行研究，成功鉴定出雷公藤甲素生物合成二萜合酶基因功能及其二萜烯类中间体结构。取得的研究结果如下：1.建立了稳定高效的雷公藤悬浮细胞培养体系；2.获得了一个全面的雷公藤转录组数据；3.筛选并克隆鉴定了7条雷公藤二萜合酶基因，通过体外功能研究获得了催化生成中间体Miltiradiene的二萜合酶TwTPS27v2；4. 通过基因枪介导转化的体内功能研究发现Miltiradiene为雷公藤甲素生物合成的母核前体化合物。研究成果揭示了雷公藤甲素生物合成从线性 GGPP环化成独特松香烷型母核的生物学过程和起始关键步骤，对于阐释雷公藤甲素生物合成的分子机制具有重要意义，并为雷公藤甲素合成生物学生产奠定基础；在本项目的资助下，以第一或通讯作者发表学术论文22篇，其中SCI收录17篇，国内核心期刊7篇。申请国家发明专利3项（已受理）。项目负责人以第四完成人获得中国药学会科学技术奖一等奖，并获中国药学会-赛诺菲青年生物药物奖、以岭生物医药青年奖和中华中医药学会中青年创新人才等奖项。参加了5次国际学术会议。培养研究生8名。