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

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.

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。

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，其旋光异构体(R)-乙偶姻、(S)-乙偶姻不仅具有普通乙偶姻消旋体的基本功能，而且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应用。由于乙偶姻属于有机溶剂，对生产菌种有毒性，从而导致发酵水平和生产强度较低。该项目首次研究了大肠杆菌对(R)-乙偶姻胁迫响应的分子机制并取得创新性成果，通过预测和筛选了11个可能与大肠杆菌耐受性相关的功能基因，利用基因过量表达和敲除实验对这些基因进行了考察和鉴定，结果表明tsf、fabD基因过量表达及yibT基因敲除均能够提高(R)-乙偶姻胁迫抗性，即通过调控细胞膜长链脂肪酸的合成可以提高细胞抗性，最终提高了(R)-乙偶姻的发酵浓度。利用合成生物学等多途径策略对菌种进行了构建和优化，获得高产(R)-乙偶姻的细胞工厂。开展了廉价生物质原料的生物炼制研究，对复合酶解工艺和发酵罐补料发酵工艺进行优化，利用非粮木薯粉和棉籽蛋白粉作为原料时，(R)-乙偶姻浓度为86.04 g/L，这是目前国内外报道利用大肠杆菌发酵制备高光学纯(R)-乙偶姻（光学纯度≥98%）的最高值。进一步研究了(R)-乙偶姻的下游产物，其中利用(R)-乙偶姻发酵液上清进行转化反应，四甲基吡嗪浓度为56.72 g/L，转化率85.30%，这是目前国内外报道利用生物质原料制备四甲基吡嗪最高浓度。