I型聚酮合酶中酰基转移酶的分子作用机制

Many valuable microbial medicines are biosynthesized by type I polyketide synthase (PKS). Acyltransferase (AT) domain plays an essential role to type I PKS via transfer acyl units into acyl carrier proteins (ACP) of PKS. Since incorporation of acyl units into PKS machineries is the first and rate-limiting step of biosynthesis, and determine the sturcture of polyketide,unveiling mechanism of AT under molecular level has great value. In this project, we chose four ATs of FK506 PKS from an industrial producing strain, Streptomyces sp.YN06, as a model to reveal molecular mechanism of AT. This project focus on main contents as following: (i)catalytic mechanism of AT, including substrate specificity, substrate competition, product inhibition, and kinetic assay in the transfer pathway of acyl units; (ii) relationship between structure and function, especially the effects of acyl-specific motif of AT and configuration of intermediates, (iii) evolutionary analysis of AT from different prokaryotes.Our research not only reveal the catalytic mechanism of AT under molecular level, but also may help us to improve the activities and substrate specificity by engineering of AT, resulting in an high efficiency biosynthetic pathway of microbial medicine.

许多重要的微生物药物由I型聚酮合酶（PKS）合成，酰基转移酶（AT）是其不可或缺的功能域，它负责将酰基底物转移到PKS酰基载体蛋白ACP上，该过程是I型PKS药物合成的首步决速反应，也决定了药物的分子结构，因此深入揭示AT的分子机制具有重要的科学意义和应用价值。本项目以FK506工业菌（链霉菌YN06）的PKS中4个AT（转移不同酰基）为模式研究对象，通过研究AT催化酰基转移过程中底物选择性、底物竞争性、产物抑制效应，阐明AT的催化机理；通过研究AT的酰基特异性模序、催化过程系列复合物晶体构象变化，解析其结构与功能的关系；结合其它原核生物I型PKS进行AT进化分析，研究酰基特异性模序与酰基底物选择性、催化效率之间的关系，阐释原核生物AT的分子进化。同时，基于机制研究改造AT结构，提高其催化活性和底物选择性，并强化合成通路中的前体供应，从而为建立I型PKS微生物药物高效生物合成通路奠定基础。

本项目以他克莫司（FK506）产生菌筑波链霉菌为研究对象，解析了I型聚酮合酶（PKS）次级代谢产物合成途径中酰基转移酶（AT）识别与转运酰基的底物选择性机制，阐述了AT上载酰基的特异性模序结构，揭示了聚酮合酶合成天然产物的生物多样性奥秘；基于上述机制，我们发现通过对AT结构域关键位点进行点突变改变酰基的选择性，从而将所需酰基引入到主链中，产生了新颖人工天然产物。 上述AT底物选择性的分子机制研究，揭示了聚酮天然化合物生物多样性的奥秘，是聚酮药物生产菌高产改造和人工设计新衍生物的重要理论基础。. 本项目已发表SCI论文6篇，尚有1篇正在修回，授权发明专利3项，培养博士生2名、硕士生2名。