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

作　　者：陆敏涛[1,2] 赵智[1] 张英姿[1] 王宇[1] 丁久元[1] 

机构地区：[1]中国科学院微生物研究所,北京100101 [2]中国科学院研究生院,北京100049

出　　处：《微生物学报》2010年第10期1334-1340,共7页Acta Microbiologica Sinica

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。[ 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-Appc 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.

关 键 词：北京棒杆菌 磷酸烯醇式丙酮酸羧化酶 L-色氨酸 基因敲除 

分 类 号：Q93[生物学—微生物学]