石杉碱甲合成途径中细胞色素单加氧酶P450功能的研究

Huperzine A (HupA), a lycopodium alkaloid isolated initially from traditional Chinese medicinal plant Qian Ceng Ta (Huperzia serrata Thunb. Ex Murray), has shown to be a powerful and selective inhibitor of acetylcholinesterase, and has been used for alzheimer’s disease as a promising drug since 1990s in China. Due to the medicinal importance of H. serrata and its economic value of bioactive ingredients, like HupA, this plant has been extensively harvested and is in danger of extinction in China. Currently, synthetic biology was developed to successfully produce the targeted specialized metabolites in heterologous hosts. Hence, elucidating the HupA biosynthesis pathway, and then producing HupA in heterologous hosts will be an alternatively efficiency method to over the issue. Cytochrome P450s (P450) were proposed to participated in the formation, oxidation and modification of HupA skeleton. Thus, it is pivotal to characterize P450s involved in HupA biosynthesis..Previously, global transcriptomics and metabolomics analysis of different tissues in H. serrata were, respectively, carried out. Accordingly, the main purpose of this program is to characterize the genes of P450 involved in HupA biosynthesis by using activity-based metabolic profiling approach and agro-infiltration based tobacco transformants. After the characterization, tobacco chassis producing intermediates of HupA could be developed by plant synthetic biology. The results of this project could be used for elucidation of HupA biosynthetic pathway and for large-scale HupA production in heterologous hosts in future.

石杉碱甲是治疗阿尔兹海默病的高效乙酰胆碱酯酶抑制剂，在国内已临床使用多年。由于该药物的市场需求和难以工业化合成，蛇足石杉仍是石杉碱甲的唯一来源，导致该资源日渐匮乏濒危。解析石杉碱甲生物合成途径，利用合成生物学的策略在异源宿主中合成石杉碱甲是解决这一问题的有效途径。细胞色素单加氧酶P450在石杉碱甲合成过程中具有重要作用：可能参与石杉碱甲骨架的形成、氧化和修饰等过程。因此，石杉碱甲合成途径中P450功能的研究具有重要意义。.本项目基于前期蛇足石杉不同组织的转录组学和代谢组学分析，筛选参与石杉碱甲合成的P450基因，利用基于活性的代谢图谱策略和烟草瞬时表达系统，鉴定候选P450的功能，同步获得合成蛇足石杉中间体的烟草底盘。该项目的实施为后续石杉碱甲合成途径的解析和利用合成生物学规模化、工程化异源合成石杉碱甲提供研究基础。

石杉碱甲是具有重要药理活性的药用化合物，其合成途径仍不清晰。根据前人研究，P450可能参与了石杉碱甲的生物合成。本课题结合转录组和代谢组对蛇足石杉和龙骨马尾杉中参与石杉碱甲骨架修饰的P450、P450还原酶CPR和参与关键前体4PAA合成的PKS基因进行了系统挖掘和鉴定。体外功能表征结果显示，1) 除HsSLS2可以催化loganin生成secologanin外，其余P450均为未检测到催化活性；2) 筛选到的两个P450 还原酶（HsCPR1和HsCPR2）可以与CrCYP72A1结合，催化loganin生成secologanin，CrCYP72A1-HsCPR1 组合的微粒体催化活性高于 CrCYP72A1-HsCPR2 微粒体催化活性。3) 筛选到10个候选PKS，其中AaPYKS、HsPKS1、HsPKS4及PgPKS1均可以催化Δ1-哌啶生成4PAA；4）在体外一锅法可以实现LDC、CAO 和PKS三步酶的偶联反应后，我们将LDC、CAO 和PKS 基因依次导入到酿酒酵母基因组，实现了石榴碱在酿酒酵母的异源合成。同时将这三个基因构建农杆菌表达载体，通过瞬时侵染的策略，在烟草中成功检测到4PAA的生成。成功构建的产4PAA的酵母和烟草底盘，为后续的石杉碱甲合成途径解析奠定了基础。.综上，本研究结合转录组和代谢组方法，筛选和鉴定参与石杉碱甲合成相关的P450、CPR和PKS等候选基因。最后在酵母和烟草中分别构建了合成关键前体4PAA和石榴碱的底盘，为后续石杉碱甲或石松生物碱合成途径的解析奠定了基础。