海洋微生物多环大环内酰胺的特殊代谢途径和生物合成机制

Polycyclic tetramate macrolactams (PTMs) are a growing class of natural products with complex ring systems and diverse biological activities, such as antimicrobial and antitumor activities. Most reported PTMs are derived from marine microorganisms. On the basis of our demonstration of the compact biosynthetic pathway of ikarugamycin, a PTM family natural product, and the mechanistic elucidation of formation of its inner five-membered ring, this project will focus on the following research aspects: (1) to discover novel PTMs from marine microorganisms; (2) to activate the silent PTM biosynthetic gene clusters in marine microbial genomes by manipulating transcriptional regulation and heterologous expression; (3) to unveil the distinct ring-formation mechanisms in PTMs with diverse ring systems by in vivo genetic knockouts and in vitro biochemical assays, especially the metabolic pathway and enzymatic mechanism for the formation of stereochemically distinct 5/5 or 5/6 ring systems by different oxidoreductases from a common intermediate; (4) to expand the structural diversity of PTMs by metabolic engineering and combinatorial biosynthesis, exploiting diverse tailoring enzymes for further modifications, such as hydroxylation, epoxidation and methylation, and to evaluate their antimicrobial and antitumor activities. This project will pave the way to discover PTM drug leads from marine microorganisms.

大环内酰胺类化合物（PTMs）是一类含复杂多环结构的天然产物，具有抗菌和抗肿瘤等多种生物活性，大多数PTMs源自海洋微生物。本项目拟在前期阐明PTM化合物斑鸠霉素生物合成途径的简洁性及内部五元环形成机理的基础上，展开下列研究：（1）从海洋微生物中发现新PTMs；（2）利用转录调控和异源表达方式激活PTM沉默基因簇；（3）结合体内敲除和体外生化手段揭示PTM不同多元环的成环机制，特别是在不同的氧化还原酶的介导下，分化形成立体构型多样的的5/5环或5/6环的代谢途径和酶学机理；（4）充分利用多样化的后修饰酶（羟基化、环氧化、甲基化、卤化等），采用代谢工程和组合生物合成技术，构建PTM化合物实体库并筛选抗菌和抗肿瘤活性。本项目将为充分利用海洋微生物资源，从中发现PTM类化合物，揭示特殊代谢途径和生物合成机制，拓展结构多样性，为发现PTM类先导化合物提供理论和物质基础。

大环内酰胺类化合物（PTMs）是一类含复杂多环结构的天然产物，具有抗菌、抗肿瘤等多种生物活性，大多数PTMs源自海洋微生物。本项目在前期阐明PTMs化合物斑鸠霉素生物合成途径简洁性及内部五元环形成机理的基础上展开研究，取得以下创新性成果：（1）通过筛选PTMs产生菌、激活PTM基因簇及组合生物合成的方法发掘了新的PTMs结构衍生物；（2）通过5（或6）元内环环化酶晶体结构指导研究了酶的体外功能、揭示了催化形成内环的酶反应机制。（3）结合体内敲除和体外生化手段揭示了形成5/5（或5/6）环的代谢途径和生化特性；（4）获得后修饰P450氧化酶IkaD的晶体结构及晶体结构底物复合物，推导了IkaD多位点催化酶学机理。（5）筛选出有显著肿瘤生长抑制活性的PTMs先导化合物。已发表包括Nat Commun、Angew Chem Int Ed Engl、Chem Sci，Org Lett等19篇SCI学术论文，奠定了进一步研究PTMs类化合物的理论与物质基础。