真菌Bipolaris sorokiniana 11134中seco-sativene型倍半萜罕见桥环及裂环结构的生物合成机制解析

Terpenoids is a rich source for the discovery of novel natural product drug leads. Terpene synthases catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse terpene metabolites. Understanding the mechanism how the complicate structures have been synthesized of terpene synthase is crucial for further natural product leads compounds discovery. In our previous study, we have identified a serial unique seco-sativene sesquiterpenoids with neuroprotection activity. The reactions correlated to the formation of the unique 5/6 or 5/7 bridged rings and seco-ring based on the cleavage on C14-C15 bond are the most interesting issues in the biosynthetic pathway of this type of compounds. In this proposal, we will apply bioinformatics firstly to deduce the potential terpene synthase and oxygenase responsible for these reactions. We will develop two platforms for identify the responsible enzyme and as well as characterize their functions: i) The deletion potential genes in wild-type strains and detect the intermediates; ii) develop a rapid screening system for sesquiterpene synthase and oxyganse using the reliable Aspergillus nidulans, Saccharomyces cerevisiae, and Escherichia coli expression system. Further, to identify the cyclized products, a S. cerevisiae system will be applied to heterologously express the sesquiterpene synthase and the enzyme function will be characterized using in vitro enzymatic reactions. At the end, in vivo analyses of the sesquiterpene synthase with isotopically labeled precursors will allow the elucidation of its cyclization mechanism. A good understanding of key reactions and mechanisms of seco-sativene sesquiterpenoids will provide important guidance for further new drug leads discovery based on combinatorial biosynthesis approach.

萜类化合物是重要的天然产物药物来源之一，其骨架结构的多样性源于萜合酶复杂的催化功能。深入研究萜合酶的催化环合机制，对揭开萜类复杂结构“生物合成机器”的运作规律有重要作用。申请者在前期工作基础中，从一株植物病原真菌Bipolaris sorokiniana 11134中发现了系列骨架独特和潜在成药价值的seco-sativene倍半萜新化合物，其骨架中罕见的桥环及C14-C15键断裂形成裂环，与其相关的酶催化机制尚不清楚。本项目拟利用生物信息学分析定位潜在基因簇；采用本源和异源两种策略进行鉴定负责催化桥环结构的合酶和负责氧化裂环的P450酶的功能：i）在野生型菌株中通过敲除和回补实验，ii）构建多种异源表达平台结合体内异源表达和体外酶催化反应；最后，通过稳定同位素标记实验解析合酶的环化机制。本项目通过深入解析桥环和裂环的合成机制，对更多萜类药物先导物的研究具有重要的意义。

本项目主要研究了一类seco-sativene类倍半萜化合物的生物合成研究，这类分子具有多种生物活性，在我们前期研究中发现其还具有潜在的神经保护活性。通过全基因组测序、生物信息学分析和异源表征，成功定位了一条可能的生物合成基因簇。经对基因簇中关键基因的表征，中间体化合物的结构鉴定，推测了其生物合成过程，进一步通过底物喂养实验，验证了生物合成的过程，验证了参与形成其骨架中罕见的桥环及C14-C15键断裂的关键基因。进一步通过质谱MS/MS分子网络快速定位技术，从基因簇异源表征产物中发现了一系列新的seco-sativene倍半萜化合物。该研究拓宽了我们对倍半萜结构中桥环及C14-C15键断裂的认识，并成功激活获得了一系列新颖的seco-sativene骨架化合物，为开发及合成生物学创制具有丰富活性的该类倍半萜奠定了理论基础。

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