【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。更多还原

【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.更多还原