茯苓三萜类化合物合成中相关细胞色素P450基因的鉴定与功能研究

Triterpenoids are one of the main medicinal ingredients of Wolfiporia cocos (Schwein.) Ryvarden et Glib., which exhibit marked diuretic, anti-tumor, and other significant pharmacological activities, and thus possess huge potential in market demand. Submerged fermentation and biosynthesis in heterogeneous cell factories are potential approaches for producing a large quantity of Wolfiporia cocos triterpenoids. The previous finding revealed that lanosterol is biosynthesized through the MVA pathway in Wolfiporia cocos. However, the biosynthesis processes from lanosterol to triterpenoids remain largely unknown. Our previous results showed that the transcriptional levels of total 27 Cytochrome P450 (CYP) genes were up-regulated when the mycelium producing more triterpenoids, implying that these 27 CYPs are possibly involved in triterpenoids biosynthesis processes. In the present study, these 27 CYPs would be identified individually as functional CYP(s) by in vivo enzymatic activity assays in an engineered yeast strain, which permits the over-expression of tHMGR and UPC2-1. And thus functional CYP gene(s) would be determined. The process(es) catalyzed by CYP(s) would be further determined by in vitro enzymatic activity assays and the modification site(s) would be elucidated. Finally, over-expression and knock-down genetic manipulations in Wolfiporia cocos will be developed, and the biological role of CYP(s) would be determined. The information obtained from this study will provide a theoretical basis for biosynthesis of Wolfiporia cocos triterpenoids.

三萜类化合物是茯苓主要的药效成分之一，有利尿、抗肿瘤及其它重要的药效活性，市场需求潜力巨大。液体发酵与异源细胞生物合成是大量获取茯苓三萜类化合物潜在的方法。已有报道揭示茯苓经MVA途径合成羊毛甾醇，但羊毛甾醇到三萜类化合物的生物合成过程仍未知。项目组前期研究结果发现当茯苓菌丝产三萜含量增加时，27个细胞色素P450（CYPs）基因转录水平上调，暗示它们可能参与茯苓三萜类化合物的合成。本研究拟构建tHMGR与UPC2-1过表达酵母工程菌，应用该工程菌进行体内酶学活性分析分别鉴定27个CYPs是否参与羊毛甾醇到三萜类化合物的合成，明确发挥功能的CYP(s)；应用体外酶学活性分析解析CYP(s)催化的化学反应类型与反应过程；建立茯苓基因过表达与敲低遗传操作体系，明确CYP(s)的体内生物学功能。本研究结果将为茯苓三萜类化合物的生物合成提供理论基础。

项目执行期间共收集到20株茯苓菌株，应用ISSR-PCR技术理清了菌株之间的亲缘关系，结果表明20条ISSR引物可以将20种菌株有效区分，但体细胞不亲和性分析结果与分子遗传标记结果不完全吻合。应用液体深层发酵方式，分析了16种菌株产胞内多糖与三萜的潜能，就生产茯苓胞内多糖与胞内三萜而言，AS 5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株，Y 1，Jingzhou 28，Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。选择了产三萜含量较高的菌株Y 1与CGMCC 5.78进行后续的研究工作，应用农杆菌介导的基因转化方法，将鲨烯合成酶（sqs）基因成功转化到Y 1菌株，发现过表达sqs可显著提高Y 1菌株产胞内三萜的含量，该技术体系为将来在茯苓本体内应用分子生物学策略过表达功能性的细胞色素P450基因达到高产特异性的茯苓三萜提供理论基础。成功构建过表达tHMGR、过表达UPC2.1、同时过表达tHMGR与UPC2.1酵母工程菌，发现只过表达tHMGR较对照可提高羊毛甾醇含量约5倍，但其作为底盘细胞的性能不如BY-T3菌株。经过优化钓取基因的各个环节，如引物设计、反转录引物的选择、Gibson组装重组臂的选择、PCR反应程序等，熟化了实验室的分子构建系统，并应用该系统成功构建了含有147个细胞色素P450的过表达基因库。以BY-T3作为底盘细胞，对逐个细胞色素P450基因进行筛选，发现CYP22843酵母发酵产物较对照菌株多一个特异性的峰，通过HPLC-MS对CYP22843酵母发酵产物进行分子量鉴定，结果表明该特异性峰的相对分子量为473.0，初步判定CYP22843介导了羊毛甾醇的修饰，形成了茯苓三萜类化合物。