基于模块化聚酮合成酶系统进行顺,顺-己二烯二酸的新型组合生物合成研究

Combinatorial biosynthesis is a method of editing, modification, combination of existing biosynthetic modules to produce various analogues of antibiotics or drugs. The new generation of combinatorial biosynthesis is a kind of "Legoization" of biomodule for de novo synthesis of chemicals, which is based on retrosynthesis analysis of chemistry and traditional combinatorial biosynthesis. This study focuses on the de novo synthesis of cis,cis-muconic acid (ccMA), an important platform chemical with two cis conjugated double bonds, based on the modular polyketide synthase (PKS) system and the techniques of novel combinatorial biology. After retrosynthesis analysis, ccMA will be divided into three two-carbon unit, and PKS domains and modules with specific substrate-specificity, stereoselectivity and catalytic activity will be selected, based on the correspondence of chemical structure and catalytic domains of various PKS modules. And a series of non-natural chimeric PKS will be constructed by multi-modular assembly via different linkers in DNA level, and expressed in Saccharomyces cerevisiae to produce ccMA from inexpensive glucose. Based on the modular PKS system in our work, dicarboxylic acid containing cis conjugated double bonds will be biosynthesized in vivo and de novo, expanding the varieties of chemicals that modular PKS can synthesize, as well as providing a new strategy for production of chemicals containing conjugated double bond or/and cis-trans isomer. In addition, our study will provide more experiences and general rules for this emerging biosynthesis method for retrosynthesis of non-natural products based on modular PKS, and also develop an innovative production method with high stereoselectivity and high yield for ccMA, which result in the positive theoretical value and promising application prospects.

新型组合生物学借鉴了化学的逆合成分析思路及组合生物学技术，是一种“乐高式”的化学品从头合成方法。本研究拟基于模块化的生物聚酮合成酶（PKS）系统对含顺式共轭双键的重要平台化合物顺,顺-己二烯二酸（ccMA）进行“乐高式”的新型组合生物合成研究。对ccMA结构进行逆合成分析，将其拆分成三个二碳单元；据每个单元的化学结构，筛选具有特定底物特异性、催化活性和立体选择性的PKS结构域和模块，在基因层面以不同的连接序列进行模块化组装得到一系列非天然PKS嵌合酶，在酿酒酵母中高效表达并利用廉价葡萄糖从头合成ccMA。本研究基于PKS系统在体内从头合成含共轭双键及顺式结构的二元羧酸，将扩展PKS系统合成的化学品种类，为基于模块化PKS的非天然产物的新型组合生物合成研究提供更多科研经验和普适性规律，也为ccMA提供一种高立体选择性、高产率的合成新方法，具有重要的理论价值和实际应用前景。

聚酮合成酶PKS是一类模块化的复杂多酶体系，能参与多种具有重要药理活性的天然产物及其衍生物。对聚酮合成酶及其产物的生物合成途径和关键酶/模块的解析，有助于开发具有更好药理活性的天然药物和设计新型生物催化剂。本项目发现了一类尚未报道的聚酮化合物Varicidin A，是一类具有稀有顺式-十氢萘环结构的新型抗真菌剂，并通过组合生物学和合成生物学方法构建工程菌获得了产量提高的Varicidin A 产物。同时通过对其生物合成途径的关键酶解析，发现了一种新型的能催化立体选择性的狄尔斯-阿尔德反应（DA反应）的酶PvhB，是目前报道的唯一能催化独特的Disfavored DA反应的强大的酶，以合成稀有的一般酶催化难以合成的顺式-十氢萘环结构。本项目的研究对于扩展基于聚酮合成酶的组合生物合成的化合物种类，开发设计新型的生物催化剂具有重要的理论和应用价值。