磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响

【目的】L-缬氨酸生物合成的前体物质是丙酮酸。为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1(Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-Δpepc,并研究pepc敲除后菌株生理特性的改变。【方法】运用交叉PCR方法得到pepc基因内部缺失的同源片段Δpepc,并构建敲除质粒pK18mobsacB-Δpepc。利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-Δpepc。采用摇瓶发酵对C.glutamicum V1-Δpepc进行发酵特性的研究。对谷氨酸棒杆菌模式菌株C.glutamicum ATCC 13032、出发菌株C.glutamicum V1和敲除菌株C.glu-tamicum V1-Δpepc的丙酮酸激酶(Pyruvate kinase,PK)、丙酮酸脱氢酶(Pyruvate dehydro-genase,PDH)、丙酮酸羧化酶(Pyruvate carboxylase,PC)分别进行测定和分析。【结果】PCR验证以及PEPC酶活测定都表明筛选到pepc缺陷的突变菌株C.glutamicum V1-Δpepc,摇瓶发酵结果表明,突变菌株C.glutamicum V1-Δpepc不再积累L-缬氨酸而是积累L-精氨酸达到7.48 g/L。酶活测定结果表明出发菌株的PDH和PC酶活均低于模式菌株C.glu-tamicum ATCC13032和重组菌株C.glutamicum V1-Δpepc,出发菌株的PK与PEPC酶活与模式菌株没有较大的差异。【结论】研究表明,通过切断PEPC参与的三羧酸循环的回补途径,增加磷酸烯醇式丙酮酸向丙酮酸的流向使丙酮酸向TCA循环的流量增加,精氨酸的累积量提高。同时,以丙酮酸为前体的L-缬氨酸和丙氨酸的积累量降低。

Effect of phosphoenolpyruvate carboxylase gene knock-out on physiological metabolism in Corynebacterium glutamicum V1

[Objective] In order to optimize precursor supply for L-valine biosynthesis,a Corynebacterium glutamicum V1 mutant with phosphoenolpyruvate carboxylase gene(pepc) in-frame deletion was constructed through crossover PCR and homologous recombination.The effect of pepc knock-out on physiological characteristics of the mutant was investigated.[Methods] The upstream and downstream fragments of pepc were cloned from C.glutamicum V1 chromosome and ligated to integration vector.The mutant C.glutamicum V1-Δpepc was screened by homologous recombination.The physiological characteristics of the mutant were investigated by fermentation experiments and enzymes activity measurement of pyruvate carboxylase(PC),pyruvate dehydrogenase(PDH) and pyruvate kinase(PK).The mutant with pepc gene in-frame deletion was screened and confirmed by PCR and phosphoenolpyruvate carboxylase activity determination.[Results] The pepc knock-out resulted in L-argine accumulation to 7.48 g/L and no accumulation of L-valine,which accompanied by increase of PDH activity and PC activity in C.glutamicum V1-Δpepc.The knock-out of pepc gene affected the metabolism of the strain to some extent.[Conclusion] Blocking the anaplerotic pathway PEPC participated increased TCA cycle,leading to the increase of L-argine and decrease of amino acids with pyruvic acid as precursor,such as L-valine and alanine.