高产光学纯（R）-乙偶姻工程菌株的构建与发酵工艺优化

Construction of High Yield Optically Pure(R)Acetoin Engineering Strain and Optimization of Fermentation Process

本研究拟在大肠杆菌中构建光学纯(R)-乙偶姻的合成途径和利用辅酶工程调控NADH/NAD+氧化还原平衡,并对工程菌发酵(R)-乙偶姻进行优化。将来源于Enterobacter cloacae的α-乙酰乳酸合成酶基因budB、α-乙酰乳酸脱羧酶基因budA和来源于Lactobacillus brevis的NADH氧化酶基因noxE进行密码子优化后组成基因簇,构建表达质粒并导入大肠杆菌,进一步优化工程菌的培养基成分和发酵条件,提高(R)-乙偶姻的合成能力。结果表明:获得专一性合成高光学纯(R)-乙偶姻的大肠杆菌工程菌株GXASR,对其发酵条件进行系统优化后,摇瓶发酵的(R)-乙偶姻产量为36.82g/L,光学纯度达99.1%,发酵罐补料发酵的(R)-乙偶姻产量达到67.65g/L。在大肠杆菌细胞中过表达外源基因簇budB-budA-noxE能够高效合成光学纯(R)-乙偶姻,经发酵优化后,工程菌的(R)-乙偶姻产量、生产强度和得率均显著提高,为代谢工程改造大肠杆菌生产高光学纯(R)-乙偶姻提供了理论基础。

The synthetic pathway of optically pure(R)acetoin was constructed and coenzyme engineering was applied to regulate NADH/NAD+redox equilibrium in Escherichiacoliwhich was used to optimize the fermentation conditions.The gene cluster was constructed by codon-optimizing theα?acetolactate synthase gene budB,α?acetolactate decarboxylase gene budA from Enterobacter cloacae,and NADH oxidase gene noxE fromLactobacillus brevis.The expression plasmids were constructed and introduced into Ec.oli.The medium components and fermentation conditions of engineered strain were optimized to improve the synthesis ability of(R)acetoin.The results showed that an engineered strain of E.coli with high specific optical purity(R)acetoin GXASR was obtained.After systematic optimization of fermentation conditions,the(R)acetoin yield of shake flask fermentation was 36.82 g/L,the optical purity reached 99.1%.And the yield of(R)acetoin by fed-batch fermentation reached 67.65 g/L.(R)acetoin could be synthesized efficiently by overexpressing the exogenous gene cluster budB-budA-noxEin Ec.oli cells.After optimization of fermentation,the(R)acetoin yield,production intensity and receiving rate were significantly increased.It provided a theoretical basis for metabolic engineering transformation of Ec.oli to produce high optically pure(R)acetoin.