γ-谷氨酰激酶基因敲除对产L-精氨酸钝齿棒杆菌8-193生理代谢的影响

Effect of gamma-glutamyl kinase gene knock-out on metabolism in L-arginine-producing strain Corynebacterium crenatum 8-193

【目的】为了阻断L-精氨酸合成的前体物L-谷氨酸的分支代谢途径,增加L-精氨酸合成的代谢流,构建钝齿棒杆菌8-193(Corynebacterium crenatum 8-193)γ-谷氨酰激酶(EC:2.7.2.11,γ-glutamyl kinase)基因proB敲除的菌株,并研究proB基因敲除对菌株生理特性的影响。【方法】运用PCR技术分别扩增proB基因的上游和下游序列,构建带有内部缺失的proB基因的敲除载体。经过两次同源重组,敲除C.crenatum 8-193的proB基因,构建菌株8-193-ΔproB,并用带有proB基因的表达载体对8-193-ΔproB进行互补验证。通过摇瓶发酵研究8-193-ΔproB的生理特性。【结果】PCR验证、γ-谷氨酰激酶酶活测定和营养缺陷型鉴定表明,获得了proB基因缺陷的菌株。摇瓶发酵结果表明,与出发菌株相比,8-193-ΔproB生物量降低9.6%,L-精氨酸产量提高13.6%。副产物中谷氨酸族和天冬氨酸族氨基酸含量升高;α-酮戊二酸、磷酸烯醇式丙酮酸和琥珀酸含量降低。proB基因敲除后,菌株的磷酸烯醇式丙酮酸羧化酶和丙酮酸羧化酶活性提高。【结论】对谷氨酸分支代谢途径的阻断可以改善8-193菌株的葡萄糖利用和精氨酸合成能力。

[Objective] In order to optimize precursor supply for L-arginine biosynthesis,we constructed a Corynebacterium crenatum 8-193 mutant with gamma-glutamyl kinase gene（proB） in-frame deletion.The effects of proB knock-out on physiological characteristics of the mutant were investigated.[Methods] The upstream and downstream fragments of proB were cloned from C.crenatum 8-193 chromosome and ligated to integration vector.The mutant C.crenatum 8-193-ΔproB was obtained by homologous recombination.The mutant phenotype can be reversed by complementation with proB gene from the expression vector.The physiological characteristics of the mutant were investigated by measurement of the activities of phosphoenolpyruvate carboxylase（PEPCx） and pyruvate carboxylase（PYC）.[Results] The proB gene in-frame deletion was screened and confirmed by PCR,gamma-glutamyl kinase determination and complementation.The mutant lost the ability of growth on minimal medium without proline addition.The proB knock-out mutant resulted a decrease of cell mass by 9.6% and an increase of L-arginine accumulation by 13.6% compared with that of the parent strain.The analysis of by-products of fermentation broth showed that the concentrations of glutamate-related and aspartate-related amino acids increased,and the concentrations of α-ketoglutaric acid,PEP and succinic acid decreased.The specific activities of PEPCx and PYC increased in 8-193-ΔproB.[Conclusion] The proB gene knock-out of the strain 8-193 blocked branch catabolism of L-glutamate and improved efficiency of the glucose utilization and L-arginine accumulation.