禾谷镰刀菌二倍半萜类化合物Mangicols生物合成机理研究

The sesterterpenoids of filamentous fungi are a kind of terpenoid with unusual chemical structures, complex biosynthetic mechanisms, and extensive biological activities, which are attracting more attention for their theoretical research and potential application values. Bioinformatics data have shown that filamentous fungi are among the most important sources of sesterterpenoids; however, the biosynthetic mechanisms and the structures of sesterterpenoids of this microorganism were poorly studied. As a kind of sesterterpenoid, Mangicols (Mangicol A-G) was first isolated from the filamentous fungi Fusarium heterosporum CNC-477, and shows anti-inflammatory and antitumor activity. The characterized function of sesterterpene synthase FgMS, which catalyzes the biosynthesis of the complex core skeleton of Mangicols, makes it possible for us to mine the gene cluster and reveal the biosynthetic mechanism of Mangicols. In this project, we first propose using the well-established Targeted Synthetic Metabolism strategy to engineer the mevalonate pathway of Aspergillus oryzae to build a sesterterpene overproduction platform. Next, we can utilize this well-established sesterterpene overproduction platform to characterize the function of each enzyme in this gene cluster by promoter replacement, gene knockout, and complement and structural characterization of metabolic mediates. With the aid of these molecular biology and natural product chemistry studies, we can propose the biosynthetic mechanism of Mangicols and these results can be further verified by in vitro reconstitution of each enzyme in the gene cluster by feeding the appropriate substrate. This will efficiently and specifically provide a solid theoretical basis for the overproduction of anti-inflammatory sesterterpenoid Mangicol A, and also provide an ideal scheme for characterization of the mechanism and overproduction of sesterterpenoids from other filamentous fungi.

真菌来源的二倍半萜类化合物因有独特新颖的产物结构，复杂的产物合成机理以及广泛的生物活性而具有重要的研究价值以及潜在的应用价值。然而目前研究人员对真菌来源二倍半萜化合物生物合成机理的研究还非常不足。Mangicols是一类真菌来源具有抗炎症、抗肿瘤等生物活性的重要的二倍半萜化合物，本项目拟在申请人成功解析负责合成其核心骨架的二倍半萜合酶FgMS的工作基础上，解析其生物合成途径。首先利用“定向合成代谢”策略构建一个高效合成二倍半萜化合物的米曲霉平台；随后以此平台为基础，通过分子生物学及天然产物化学操作研究Mangicols生物合成基因簇中各个基因的功能；并结合基因簇中各个酶的体外酶促反应结果，解析其生物合成机理。为后续特异性高效合成具有显著抗炎症活性的Mangicol A奠定坚实的理论基础，同时也为其它真菌来源二倍半萜化合物生物合成途径的解析及高效合成提供一个理想的解决方案。

在前期研究中，我们从和禾谷镰刀菌体内鉴定了二倍半萜合酶FgMS及其产物mangicdiene。我们发现二倍半萜类化合物mangicdiene 具有mangicols类化合物的核心骨架。这类化合物是从红树林的异孢镰刀菌（Fusarium heterosporum CNC-477）中分离到的一类重要的二倍半萜化合物，因其复杂的结构和良好的抗炎活性和低细胞毒性，吸引了大量研究人员的目光。然而，目前该类化合物主要通过传统的分离方法获得，效率低成本高。迄今为止，该类化合物的生物合成途径还未被揭示。为此，本研究利用米曲霉（Aspergillus oryzae）异源表达平台对FgMS的生物合成基因簇进行挖掘，并阐明mangicols类化合物的生物合成途径。我们将FgMS基因簇中的基因在米曲霉中进行异源表达，构建了一系列突变株。我们在AO-mgcDE、AO-mgcDEF和AO-mgcCDE三个突变株中成功检测到了6个后修饰产物，其中化合物1-5均为magicols类新化合物，并且中间产物3表现出良好的抗炎活性。根据以上结果，我们首次阐明了mangicols化合物的生物合成途径。在该生物合成途径中涉及环氧化物水解酶mgcC、二倍半萜合酶mgcD以及两个P450酶（mgcE和mgcF）4个酶的参与。同时我们发现mgcE为多功能的P450酶，能连续催化多步反应。本研究的发现填补了mangicols化合物生物合成研究领域的空白。基于化合物Mangicol J的显著的抗炎活性，本研究还在A. oryzae宿主中进行了Mangicol J高效合成的代谢工程改造。为此，通过以质粒在基因组中随机插入的方式，过表达了整个MVA途径和限速酶tHMG1,以基因高表达位点整合型过表达的方式进行菌株构建，实现了27.39 mg/L mangicdiene和8.93 mg/L mangicol J的高效合成，较出发菌株分别提升了约41.5倍和111倍。