模块化工程改造大肠杆菌生产L-色氨酸

Modular engineering of Escherichia coli for high-level production of L-tryptophan

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein,trpR)、替换L-色氨酸弱化子(trpL)、引入抗反馈调节的aroGfbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid,SA)途径至分支酸(chorismic acid,CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为L-色氨酸的规模化生产奠定了良好的基础。

As an essential amino acid,L-tryptophan is widely used in food,feed and medicine sectors.Nowadays,microbial L-tryptophan production suffers from low productivity and yield.Here we construct a chassis E.coli TRP3 producing 11.80 g/L L-tryptophan,which was generated by knocking out the L-tryptophan operon repressor protein(trpR)and the L-tryptophan attenuator(trpL),and introducing the feedback-resistant mutant aroGfbr.On this basis,the L-tryptophan biosynthesis pathway was divided into three modules,including the central metabolic pathway module,the shikimic acid pathway to chorismate module and the chorismate to tryptophan module.Then we used promoter engineering approach to balance the three modules and obtained an engineered E.coli TRP9.After fed-batch cultures in a 5 L fermentor,tryptophan titer reached to 36.08 g/L,with a yield of 18.55%,which reached 81.7%of the maximum theoretical yield.The tryptophan producing strain with high yield laid a good foundation for large-scale production of tryptophan.