ppc基因敲除对大肠杆菌L-色氨酸发酵的影响

目的：减少大肠杆菌L-色氧酸前体物质磷酸烯醇式丙酮酸向草酰乙酸的代谢流，提高其L-色氨酸的产量。方法：以大肠杆菌TRTH0709为出发菌株，利用Red重组敲除技术敲除磷酸烯醇式丙酮酸羧化酶（Ppc）编码基因PPc，并经测序和酶活性检测确证；对出发菌株和基因敲除菌株进行L-色氖酸发酵，对比分析发酵结果。结果：测序和酶活性检测结果表明ppc基因被成功敲除。发酵结果表明，与出发菌株相比，基因敲除菌株TRTH0709△ppc生长速度减慢，最终生物量减少32％，L-色氯酸产量降低27％，但糖酸转化率提高6％；向发酵培养基中添加1％琥珀酸后，TRTH0709△ppc的生长速率和产酸量有所提高，但仍与出发菌株有一定差距。结论：虽然ppc基因敲除对菌体生长和产酸量影响较大，但能有效提高其糖酸转化率；选育Ppc弱化的突变株以达到减弱代谢流且不影响菌体生长，以及增加，L-色氨酸积累的目的，将是本研究今后的主要方向。

Effects of Gene ppc Disruption on L-Tryptophan Fermentation by Escherichia coli

Objective： To reduce the bypass flow of phosphoenolpyruvate and improve the L-tryptophan production of genetically engineered Escherichia coli. Methods： E.coli TRTH0709 was used as an original strain, and the gene ppc coding phosphoenolpyruvate carboxylase （Ppc） was disrupted by Red recombination technology. Results： The ppc mutant was screened and confirmed by result of sequencing and enzymatic activity detection. Fermentation assay showed that significant influence on cell growth was observed. The final biomass reduced by 32% and resulted in a 27% decrease in L-tryptophan production compared to that of parental strain. Addition approximate 1% of succinate in the fermentation medium can improve the biomass and L-tryptophan production, but still has certain gap compared with that of the original strain. Conclusion： Although disruption of ppc reduced biomass and L-tryptophan production, yield of L-tryptophan is effectively improved, indicating that it is useful to save raw materials and energy through further improving its growth rate and L-tryptophan production by metabolic engineering method. Breeding strains of attenuated Ppc is the main direction to improve growth and L-tryptophan production.