目前，从野生中药植物狼毒大戟中发现的蓖麻烯型二萜包括巴豆二萜烷和巨大戟烷两种亚型。针对其中先导化合物巨大戟醇（巨大戟烷亚型）和候选药物prostratin（巴豆二萜烷亚型）含量甚微并且化学合成收率低的问题，本项目拟开展其生物合成方面的研究。申请人在前期研究中，克隆了蓖麻烯合成酶（CS）等蓖麻烯型二萜生物合成相关酶，并构建了产生蓖麻烯的工程菌；采用长距离PCR技术证明狼毒大戟基因组含有蓖麻烯型二萜的生物合成基因簇；确定该基因组为388.95 Mb。以此为依据和基础，拟通过建立和筛选狼毒大戟细菌人工染色体的方法，① 鉴定蓖麻烯型二萜的生物合成基因簇，② 阐明从蓖麻烯向巴豆二萜烷和巨大戟烷等复杂多环骨架转化的关键步骤，③ 揭示隐秘的从巴豆二萜烷亚型成分到瑞香烷亚型成分的代谢途径，④ 并构建产生重要中间体的工程菌，为实现合成生物学技术对prostratin等化合物的规模化生产奠定基础。

The main bioactive constituents of TCM herb Euphorbia fischeriana are casbene-type diterpenoids including tigliane and ingenane subfamilies. Among these compounds, prostratin (tigliane-type) was approved by FDA as an anti-AIDS drug candidate in clinical trial, and ingenol derivatives possess the same core structure as the anticancer drug ingenolmebutate. To address the problems of low contents in planta and low synthetic yields of these compounds, the applicant will study their biosynthesis. The applicant cloned a casbene synthase (CS), two cytochrome P450 CYP726A enzymes and a short-chain alcohol dehydrogenase, identified the function of CS and constructed engineering strains of producing casbene; documented that the genome of E.fischeriana possesses the biosynthetic gene cluster of casbene-type diterpenoids and its size is 388.95 Mb. Based on these results, the applicant will ① construct and screen Bacterial ArtificialChromosome of E.fischeriana to identify the target cluster, ② clarify key biosynthetic steps involving conversion of casbene to complex cyclic skeletons (e.g. tigliane and ingenane), ③reveal cryptic biosynthetic pathway from tigliane-type diterpenoids to daphnane-type diterpenoids and ④ construct engineering strains of producing key biosynthetic intermediates to pave the way for establishment of synthetic biology systems for industrially producing these comounds.