磷酸烯醇式丙酮酸羧化酶基因的敲除对北京棒杆菌PD-67 的生理代谢的影响

Effect of phosphoenolpyruvate carboxylase gene knock-out on metabolism in Corynebacterium pekinense PD-67

摘要:

摘要：【目的】为了优化L-色氨酸合成的前体供应，构建北京棒杆菌PD-67（Corynebacterium pekinense PD-67）磷酸烯醇式丙酮酸羧化酶（EC:4.1.1.31，phosphoenolpyruvate carboxylase，PEPCx）基因ppc敲除的菌株，并研究ppc基因敲除对菌株生理特性的影响。【方法】运用PCR技术扩增ppc基因的上游和下游序列，构建带有目标基因内部缺失的基因整合载体。通过同源重组技术将C. pekinense PD-67的ppc基因敲除，构建ppc基因缺陷突变株C. pekinense PD-67-Δppc。通过摇瓶发酵研究突变株的生理特性，并测定突变株丙酮酸激酶和丙酮酸羧化酶的活性。【结果】PCR验证和PEPCx活性分析结果表明，筛选到ppc缺陷的突变株。摇瓶发酵结果表明，与出发菌株相比，突变株的生长速率下降，生物量降低20%，L-色氨酸积累降低62%，丙酮酸激酶活力提高，而丙酮酸羧化酶活力下降。【结论】C. pekinense PD-67的ppc基因敲除以后，对菌株的代谢影响较大。仅通过阻断PEPCx催化的回补途径，减少磷酸烯醇式丙酮酸的分支代谢，不能提高该菌株L-色氨酸的积累。

Abstract:

Abstract:[Objective] In order to optimize precursor supply for L-tryptophan biosynthesis, a Corynebacterium pekinense PD-67 mutant with phosphoenolpyruvate carboxylase gene(ppc) in-frame deletion was constructed. The effect of ppc knock-out on physiological characteristics of the mutant was investigated. [Methods] The upstream and downstream fragments of ppc were cloned from C. pekinense PD-67 chromosome and ligated to integration vector. The mutant C. pekinense PD-67-Δppc was screened by homologous recombination. The physiological characteristics of the mutant were investigated by fermentation experiments and measurement of pyruvate carboxylase (PCx) and pyruvate kinase (PK). [Results] The mutant with ppc gene in-frame deletion was screened and confirmed by PCR check and phosphoenolpyruvate carboxylase determination. The mutant exhibited slow growth and less cell mass, 80% as much as the parent strain. The ppc knock-out resulted in decrease of L-tryptophan accumulation and overproduction of pyruvate-related amino acids, which accompanied by increase of PK activity and the decrease of PCx activity, in C. pekinense PD-67. [Conclusion] The knock-out of ppc gene affected the metabolism of the strain to some extent. Only by blocking the anaplerotic pathway PEPCx participated was insufficient to increase the accumulation of L-tryptophan in C. pekinense PD-67.