代谢工程改造大肠杆菌合成L-组氨酸

Metabolic engineering of Escherichia coli for increased synthesis of L-histidine

该研究主要通过多种代谢工程策略对大肠杆菌进行改造从而增强菌株L-组氨酸的合成。首先,用人工的开放阅读框(hisL′-λattB′-trpE)替换大肠杆菌(Escherichia coli,E.coli)BL21(DE3)L-组氨酸操纵子的前导区,同时表达来源于E.coli的ATP转磷酸核糖基酶突变体基因hisGE271K后,菌株L-组氨酸产量达到872.50 mg/L;其次,通过比较来自谷氨酸棒状杆菌(Corynebacterium glutamicum,C.glutamicum)ATCC130132、粘质沙雷氏菌(Serratia marcescens,S.marcescens)ZJZ626和E.coli BL21(DE3)的11种hisG在不同抑制物浓度下的酶活力以及过表达11种hisG基因时L-组氨酸的产量,筛选出最优hisG基因,L-组氨酸产量提高至1480.42 mg/L;然后,通过CRISPR/Cas9技术将最优hisG整合至基因组;进一步比较不同来源的Prs对菌株L-组氨酸产量的影响,从中筛选出较优Prs进行基因组整合,结果显示,摇瓶发酵72 h后,菌株L-组氨酸产量提高至3898.06 mg/L,96 h时产量提高至5574.63 mg/L。该研究为将来L-组氨酸的工业化生产奠定了良好的基础。

In this study,several metabolic engineering strategies were used to improve the synthesis of L-histidine in Escherichia coli.Firstly,the precursor region of L-histidine operon in E.coli was replaced by an artificial open reading frame(hisL′-λattB′-trpE),and the titer of L-histidine was increased to 872.50 mg/L after the expression of hisGE271K.Secondly,the enzyme activities was compared under different concentrations of inhibitors,and L-histidine titer was analyzed when 11 kinds of hisG from Corynebacterium glutamicum ATCC 130132,Serratia marcescens ZJZ626 and E.coli BL21(DE3)were expressed.The optimal hisG was selected and L-histidine titer was increased to 1480.42 mg/L.Next,CRISPR/Cas9 was used to integrate the optimal hisG in the genome.Finally,after comparing the effects of two different Prs on L-histidine production,the better one was selected and integrated in the genome,resulting in the L-histidine titer of 3898.06 mg/L in 72 h and 5574.63 mg/L in 96 h.This work laid a good foundation for the industrial production of L-histidine in the future.