黄脂菌素生物合成甲基化后修饰的研究

Polycyclic xanthone antibiotics are a subgroup of the microbial nature products characterized by the unique xanthone ring nucleus and charming diverse biological activities. In our previous studies, the xantholipin biosynthetic gene cluster was cloned, and the biosynthetic pathway has been proposed based on both of the bioinformatics analysis of gene cluster and the genetic functional identifications of several key genes. This study aims to demonstrate the methylation modifications introduced by unique methyltransferase in xantholipin biosynthesis, by means of in vivo genetic complementation, feeding experiments, biochemical analysis and enzymatic assay. Inspired by the possible necessity of the methyl group for the halogenation and the formation of methylenedioxy bridge (ring G), we will illustrate the important effect of specific methyl group on the enzymatic recognition during halogenation and the formation of ring G, unveiling the vital role of methyl group as an important biochemical checkpoint in controlling xantholipin biosynthetic line. Meanwhile, we will explore the possible important role the methyl groups play in the enhancement of biological activity and the attenuation of cytotoxicity. The new roles outlined here will improve the understanding of the methylation modifications during natural product biosynthesis. Our study will set a solid stage for the diversifying of natural product biosynthetic pathways and the structural innovation for more effective derivatives.

聚环呫吨酮类抗生素是一类具有良好的生物活性和独特呫吨酮结构的微生物来源天然产物。本项目在前期鉴定了黄脂菌素生物合成基因簇、构建了黄脂菌素生物合成关键基因缺失突变株、分离鉴定了多个中间体的基础上，采用体内遗传回补、中间代谢物分析、体外酶学表征手段，解析黄脂菌素生物合成甲基化后修饰途径。以氯代修饰以及亚甲基双氧桥结构形成依赖于特定位点的甲基化为线索，通过系统研究黄脂菌素生物合成中氧甲基转移酶催化的甲基化对氯代酶和P450单加氧酶的底物识别方面的影响，揭示甲基化修饰在黄脂菌素生物合成途径中的作用。预期对一种新的甲基化介导黄脂菌素生物合成机制的解析，将增加对天然产物生物合成途径中广泛存在的甲基化作用的理解。为进一步运用合成生物学手段修饰聚环呫吨酮类化合物生物合成途径，拓展其结构多样性建立基础。

黄脂菌素具有高度氧化且呈角状折叠排列的六环骨架和核心呫吨酮环，具有抗细菌、抗真菌及抗肿瘤活性，属于聚环呫吨酮类化合物。黄脂菌素核心呫吨酮环形成的酶学机制的揭示，为同类化合物生物合成研究奠定了关键基础。然而，核心呫吨酮环形成之后多种后修饰机制尚未得到解析。本项目以解析黄脂菌素生物合成甲基化修饰为目标，以催化C13-OH和C17-OH甲基化建立蛋白活性表征体系，证实氧甲基转移酶XanM1-M3分别主要负责特定位点羟基的甲基化、同时具有功能交叉，体现出依赖于底物分子结构的区域选择性，表征了复杂甲基化修饰网络。构建了XanM1-M3与其相同进化分支同源蛋白的祖先蛋白，成功获得了一个同时具有XanM1-M3所有活性的氧甲基转移酶。由XanH催化的依赖于C17-OH甲基化的氯代修饰表征，再次证明了甲基化的重要性，为甲基化介导黄脂菌素生物合成提供重要理论依据。项目研究增加了对天然产物生物合成途径中广泛存在的甲基化作用的理解，为进一步运用合成生物学手段修饰聚环呫吨酮类化合物生物合成途径，拓展其结构多样性提供了新的思路和借鉴。