雷公藤倍半萜生物碱母核形成的关键酶基因挖掘及其功能研究

雷公藤倍半萜生物碱具有抗风湿、抗肿瘤和杀虫等活性，极具药用潜力和市场前景，然而，雷公藤倍半萜生物碱的生物合成途径解析和母核形成机制研究尚未开展。在前期研究中，我们发现了一条雷公藤倍半萜合酶基因，其催化产物结构与雷公藤倍半萜生物碱母核近似。基于此，我们提出假说，雷公藤倍半萜生物碱的母核也是由倍半萜合酶催化形成的。本项目拟在前期研究的基础上，对雷公藤倍半萜合酶基因进行筛选和克隆，通过体外酶促、异源表达和RNA干扰等方法鉴定其功能，利用分子对接和定点突变等手段揭示雷公藤倍半萜生物碱母核形成的分子机制，并对酿酒酵母进行特异性改造，实现雷公藤倍半萜生物碱关键前体的高效异源合成。本项目将获得催化雷公藤倍半萜生物碱母核形成的关键酶基因，阐明其催化过程的分子机制，并构建合成雷公藤倍半萜生物碱关键前体的细胞工厂，为进一步解析雷公藤倍半萜生物碱的合成途径、实现其生物合成奠定基础。

The sesquiterpene alkaloids in Tripterygium wilfordii are a class of compounds with great medicinal potential and market prospect due to their variety of biological activities, such as immunosuppression, anti-tumor, insecticidal and so on. However, the biosynthetic pathway of sesquiterpene alkaloids in T. wilfordii and the mechanism of mother nucleus formation are still unknown. In a previous study, we found a sesquiterpene synthase gene from T. wilfordii, whose product structure is similar to the mother nucleus of sesquiterpene alkaloids in T. wilfordii. According to this, we hypothesized that the mother nucleus of sesquiterpenoid alkaloids in T. wilfordii was also catalyzed by sesquiterpene synthase. On the basis of the previous study, this project intends to screen and clone the sesquiterpene synthase genes from T. wilfordii, and characterize their functions by the enzymatic reaction, yeast heterologous expression and RNA interference. Molecular docking and site-directed mutagenesis will be used to reveal the molecular mechanism of the formation of sesquiterpene alkaloids mother nucleus. The optimization of Saccharomyces cerevisiae for the sesquiterpene biosynthesis will be carried out to improve the production of the key precursor of sesquiterpene alkaloids in T. wilfordii. This project will obtain the key enzyme that catalyzes the formation of the mother nucleus of sesquiterpenoid alkaloids in T. wilfordii, clarify its catalytic mechanism, and construct an engineered yeast with high-yield precursors of T. wilfordii sesquiterpene alkaloids, which will lay a foundation for the further study of the biosynthetic pathway and biosynthesis of sesquiterpenoid alkaloids in T. wilfordii.