贯叶金丝桃素分子中多异戊烯基取代双环结构的生物合成研究

贯叶金丝桃素是多环多异戊烯基取代酰基间苯三酚（PPAPs）类复杂结构天然产物的代表，具有抗抑郁等多种显著活性。然而，该化合物特征结构的生物合成机制至今不明。推测其结构中不同类型单、偕双、侧链异戊烯基取代以及双环结构的形成由系列异戊烯基转移酶（PT）介导完成。本项目前期已完成贯叶金丝桃中部分PT基因的筛选及功能研究，拟在此基础上进一步结合转录组学、蛋白组学、生物信息学和现代分子酶学技术，发现并功能鉴定贯叶金丝桃素多异戊烯基取代双环结构生物合成关键酶基因，开展深入的酶催化机制研究，并通过工程酵母系统进行生物合成途径的体内重构和验证，以解析该生物合成过程的分子机制。本研究将发现新颖类型的PT基因和酶学机制，阐明贯叶金丝桃素多异戊烯基取代双环结构的生物合成机制，为PPAPs类复杂结构天然产物的生物合成研究提供启示，并为通过生物合成方式高效合成该类型药用先导化合物奠定基础，具重要理论及实际应用意义。

Hyperforin, which possesses multiple prenyl substitutions and highly oxygenated double-ring skeleton, is a representative of polycyclic polyprenylated acylphloroglucinols (PPAPs) and is well known for its significant antidepressant activity. However, the biosynthesis mechanisms determined its characteristic multi-prenylated double-ring structure has not been elucidated yet. It is hypothesized that the monoprenylation, gem-diprenylation and side-chain prenylation substitutions and the formation of double-ring system are conducted by series of prenyltransferases. In our previous investigations, some of the prenyltransferase genes have been screened out and functionally identified from Hypericum perforatum. This project is proposed based on these accumulations, combining with the transcriptome, proteomics, bioinformatics and modern molecular enzymology techniques, to explore and functionally identify the key enzymes involved in the formation of multi-prenylated double ring in hyperforin. Besides, in-depth mechanistic investigations will be performed to clarify the catalysis mechanism underlying these reactions. Moreover, the biosynthesis pathway of hyperforin will also be artificially reconstructed and verified in engineered yeast expression system to illuminate the molecular mechanisms of the biosynthesis pathways. The implementation of this project will discover the novel prenyltransferases and catalysis mechanisms that determined the biosynthesis of the multi-prenylated double-ring moiety in hyperforin. The proposed results will also provide useful hints to the biosynthesis investigations of natural PPAPs with complex structures and make a solid foundation for the efficient biosynthetic production of these leading compounds, with both theoretical and practical significance.