高产光学纯(R)-乙偶姻细胞工厂的构建及胁迫抗性分子机制研究

乙偶姻在食品、化工、医药、烟草、化妆品等行业具有广泛的用途。光学纯(R)-乙偶姻不仅具有普通乙偶姻的基本功能，而且因其独特的立体结构在不对称合成方面优势突出，在合成高附加值手性药物中间体、化学中间体、液晶材料等方面具有重要应用。虽然已有较多报道采用基因工程菌株生产(R)-乙偶姻，但尚未有涉及乙偶姻胁迫抗性的研究报道，也无法解决发酵后期产物乙偶姻的细胞毒性问题，从而导致目前的工程菌株普遍存在发酵水平较低，生产强度不高的问题，无法实现商业化生产。本项目在前期已构建的高产菌株基础上，重点研究菌株对(R)-乙偶姻胁迫抗性的分子机制，鉴别与乙偶姻胁迫响应关键因子；进一步对大肠杆菌工程菌株进行系统改造，有效提高菌株的胁迫抗性，克服细胞代谢网络的刚性，实现胞内碳架物质流量分布的重大改变，获得遗传性质稳定，胁迫抗性、发酵水平和转化率提高的高性能细胞工厂，为光学纯(R)-乙偶姻工业化生产奠定理论和技术基础。

Acetoin (3-hydroxy-2-butanone) is widely used in foods, cigarettes, cosmetics, detergents and chemical synthesis, which largely works as flavour and fragrance. Acetoin has a stereogenic center resulting R- and S-isomers, respectively. The pure stereoisomers have additional values as chiral starting materials for the synthesis of high-value added specialty chemicals as well as chiral pharmaceutical products. As the pure stereoisomers of acetoin could not be obtained by the chemical synthesis or ordinary fermentation process by native strains, increasing attention is being paid to biotechnological process by engineered strains. Although many efforts aiming to improve the production of action has been reported, there no investigation regarding the molecular mechanism of stress tolerance to acetoin on microbial cells was published. This is vital to further improve the production performance since acetoin is physiologically toxic to bacterial cells. In this proposal, based on our previously engineered strain for efficient production of R-acetoin, we will focus on exploration of molecular mechanism of stress tolerance to acetoin by E. coli. The key factors responsible for acetoin stress tolerance are expected to be identified and functionally verified. The synthetic pathway of acetoin will be further improved by knocking out branch pathway genes, screening more efficient key enzymes and by regulating the gene expression on a pathway level. Then the following systematic metabolic engineering strategies will be applied to fully optimize the cells, including optimization of the cofactor and conducting physiological engineering to improve the traits of better tolerance to high concentration of acetoin. The engineered microbial cell factory developed in this study will laid the fundamental for efficiently producing (R)-acetoin on an industrial scale in future.