操纵子前导区的修饰及PRPP合成途径的加强对大肠杆菌积累L-组氨酸的影响

Effects of Modification in Operon-leader Region and Strengthening in PRPP Synthesis Pathway on L-histidine Accumulation by Escherichia coli

目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。

Objective： Based on Escherichia coli MG1655-ΔtktA,histidine-operon leader region was replaced by strong promoter T5,6-phosphoglucose dehydrogenase（zwf）,6-phosphogluconate dehydrogenase（gnd） and phosphoribosylpyrophosphate synthetase（prs） were overexpressed.Effects of the above modifications on L-histidine accumulation were investigated.Methods： In strain with tktA interrupted,leader region in histidine operon was replaced by T5 promoter by Red recombination system from bacteriophage λ.By cloning technology,zwf and gnd were coexpressed on pSTV28 and prs was expressed on pQE30.Effects of the above modifications on L-histidine accumulation were compared by fermentation in flasks.Results： Quantified by HPLC,trace L-histidine was accumulated in fermentation mediums for those strains with leader region replaced by promoter T5——MG1655-ΔtktA-PT5,MG1655-ΔtktA-PT5（prs-pQE30）,MG1655-ΔtktA-PT5（zwf-gnd-pSTV28）,and MG1655-ΔtktA-PT5（prs-pQE30,zwf-gnd-pSTV28） with 60.12 mg/L,66.47 mg/L,89.69 mg/L and 111.56 mg/L,respectively.Conclusion： Modification of leader region in operon strengthened the ability to synthesize L-histidine.Afterward,elevation in oxidative pentose phosphate pathway level and PRPP synthetase activity resulted in increased accumulation.