大肠杆菌ptsHI-crr基因的敲除及其对苯丙氨酸生产的影响

Knockout of ptsG Gene of Escherichia coli w3110 and Its Impact on Production of Phenylalanine

L-苯丙氨酸(L-phenylalanine)是重要的食品和医药中间体。利用大肠杆菌发酵葡萄糖生产苯丙氨酸时,对葡萄糖转运起重要作用的磷酸烯醇丙酮酸糖磷酸转移酶系统(PTS)对苯丙氨酸产量有很大影响。由ptsHI-crr操纵子编码的磷酸组氨酸载体蛋白(HPr),酶I(EI)和酶IIAGlc是PTS的必要组分,通过敲除ptsHI-crr得到PTS缺陷菌株,可以使葡萄糖代谢更多地流向苯丙氨酸生物合成。采用Red同源重组技术将大肠杆菌染色体上的ptsHI-crr基因替换为四环素抗性基因,得到PTS缺陷菌株。该菌株在以葡萄糖为惟一碳源的培养基中摇瓶培养,菌密度为对照菌株的2.7倍,苯丙氨酸产量为对照菌株的6.3倍。

L-phenylalanine is an important intermediate in food and pharmaceutical industry.The fermentive method is often used in L-phenylalanine production.In Escherichia coli,phosphoenolpyruvate sugar phosphotransferase system（PTS）which is important in glucose uptake has a great negative impact on phenylalanine production.In PTS,the enzyme I,enzyme IIAGlc and phosphohistidine carrier protein（HPr）were essential components.They were all encoded by genes ptsHI-crr.In the PTS defective strain which can be obtained by knocking out genes ptsHI-crr,the flow of glucose metabolism is changed to synthesis more valuable metabolism products such as phenylalanine.The Red homologous recombination technology was used to produce PTS defective strain.With this method,genes ptsHI-crr in E.coli w3110 chromosome was replaced by tetracycline resistance gene.When glucose was used as the sole carbon source in the culture medium,the biomass of PTS defective strain was 2.7 times of parent strain.When the stain was used to produce L-phenylalanine,the output of L-phenylalanine is 6.3 times of parent strain.