淫羊藿苷合成中关键异戊烯基转移酶的挖掘及其调控研究

Icariin, the major bioactive compound in Herba epimedii, is one of the most important prenylated flavonoids. It shows great potential to be applied in food and drug industry. Benefiting from the development of systematic biology, the biosynthesis of prenylated flavonoids in microorganisms has become a new trend. However, site determined transferase activity, the order of the reactions and the regulation of prenyltransferase for icariin biosynthesis in microbes remain puzzled, which turns to be a great obstacle. In the current project, the new prenyltranferases are cloned from Epimedium via RNA-seq methods and heterologously expressed in Saccharomyces cerevisiae. The enzymes are purified and assayed, and the site determined transferase activity as well as the reaction orders are figured out with the method of LCMS and NMR. A biosynthetic pathway of prenylated naringenin is introduced into S. cerevisiae, the regulatory mechanisms of prenyltransferase are further studied by the alteration of genes involved in the mevalonate pathway. The results of this project will provide theoretical and technical references for the biosynthesis and metabolism of complex pentenylated flavonoids in microorganisms. The constructed yeast with a high yield of isoprene donors will serve as a platform strain for further study of prenylated nature products.

淫羊藿苷是一种重要的异戊烯基黄酮类化合物，是传统中药淫羊藿的主要活性成分，在食品和医药领域具有广阔的应用前景。随着合成生物学的飞速发展，利用微生物合成淫羊藿苷成为趋势。然而，淫羊藿苷合成中关键异戊烯基转移酶修饰作用的位点特异性与反应顺序性研究不充分，以及在微生物中催化异戊烯基化修饰的调控机制不明晰，严重制约了微生物法生产淫羊藿苷的发展。本项目利用酿酒酵母异源表达挖掘获得的淫羊藿异戊烯基转移酶，验证酶基因的功能。通过产物结构鉴定，解析该酶催化异戊烯基化修饰的位点特异性与反应顺序性。最后，构建酿酒酵母异戊烯基柚皮素合成途径，并强化甲羟戊酸途径，阐明甲羟戊酸途径对异戊烯基化修饰的协同调控机制。本项目的研究成果，将为涉及异戊烯基化修饰的复杂黄酮化合物的微生物合成及代谢提供理论与技术参考，获得的高效积累异戊二烯前体的酿酒酵母将作为异戊烯化修饰的其他苯丙素类天然产物的生物合成研究平台菌株。

淫羊藿苷是食品和医药领域应用前景广阔的活性黄酮化合物，利用微生物法合成是未来制备该化合物的绿色新技术，然而其合成途径中关键异戊烯基转移酶的催化与调控机制尚不明晰。本项目从朝鲜淫羊藿转录组测序数据，挖掘潜在的途径关键异戊烯基转移酶基因，利用酿酒酵母异源表达，通过底物饲喂结合代谢工程改造实验，实现关键异戊烯基转移酶基因鉴定及调控机制解析。通过项目研究，鉴定淫羊藿来源新的异戊烯基转移酶基因2个、3-O-糖基转移酶基因3个、UDP-鼠李糖合酶基因1个、3-黄酮醇合酶基因1个、黄烷酮-3-羟化酶基因1个；揭示糖基化修饰对异戊烯基转移酶催化的阻遏作用，证明充足的DMAPP前体供给对淫羊藿苷合成的促进作用，发现异戊烯基转移酶准确细胞器定位对其催化活性的重要影响；在此基础上，构建了3个高效积累异戊二烯前体的酿酒酵母底盘，以及3个不同UDP糖基前体供给优化的酿酒酵母底盘。本研究的发现将促进淫羊藿苷的合成生物学研究，完善酿酒酵母合成淫羊藿苷的代谢工程改造策略，获得的多个高催化活性途径基因以及前体供给强化的底盘酵母均可用于复杂黄酮的合成。