萜类化合物是天然产物中化学结构多样性最丰富的家族,包括抗疟药物青蒿素及抗癌药物紫杉醇。Ophiobolins(Ophs）是一种5-8-5环二倍半萜，对阿霉素耐药乳腺癌有增敏作用，对脑胶质瘤、帕金森症等疾病有潜在疗效。阐明Ophs的生物合成机制对大量制备该化合物有重要意义。前期研究发现在红树林真菌焦曲霉094102中，Ophs的合成至少与五个基因簇相关，与通常一个基因簇负责一种化合物骨架合成有很大区别。本研究将通过CRISPR/CAS9基因敲除、蛋白表达与体外反应等方法，对参与Ophs生物合成的相关基因进行功能鉴定, 结合大肠杆菌底盘系统重建Ophs合成途径，解析来自不同基因簇的基因对Ophs合成的影响。目的是揭示一种多基因簇参与的萜类化合物生物合成模式，同时建立大肠杆菌底盘的Ophs合成体系，为生物合成法大量制备Ophs奠定基础。

Terpenoids are the most diverse and abundant natural products among which artemisinin and paclitaxel are well known antimalarial and anticancer drugs. Ophiobolins are tricyclic 5-8-5 ring sesterterpenes with potential therapeutic application in drug resistent breast cancer, glioma as well as Parkinson’s disease. Involvement of five gene clusters in ophiobolin synthesis was found in Aspergillus ustus 094102 isolated from mangrove rizhosphare, which is an unususal biosynthesis pathway. In this project, using a combined strategy of gene knockout by CRISPR/CAS9 system, gene expression and in vitro verification, together with pathway reconstruction in E. coli chassis, we will illuminate this unique multiple gene clusters involved terpene biosynthesis mechanism and symutanously, construct a heterologus ophiobolin biosynthesis system in E. coli, which will be of significant application in large amount production of ophiobolin compound.