α-酮戊二酸（α-ketoglutarate，α-KG）作为一种二元短链羧酸，是谷氨酸、琥珀酸等化合物的重要前体，在转氨基等反应中也起着重要作用。磷酸烯醇式丙酮酸羧化酶是谷氨酸棒杆菌中催化合成TCA循环中重要代谢物草酰乙酸的关键酶，本文以谷氨酸棒状杆菌GDK-10为出发菌株，通过同源重组技术对磷酸烯醇式丙酮酸羧化酶编码基因pepc进行以下改造：将GDK-10的Ppepc启动子替换为强启动子Ptuf，获得菌株GDK-11；构建pepc双拷贝菌株GDK-12；对GDK-10基因组pepc进行定点突变(N917G)，获得菌株GDK-13。实时荧光定量PCR检测表明，菌株GDK-11和GDK-12的pepc表达量是GDK-10的47.05倍和2.03倍。发酵结果表明，菌株GDK-11和GDK-12的产酸量相对于出发菌株分别下降45.50%和13.90%，但GDK-12的单位菌体产量较GDK-10提高38.59%。菌株GDK-13产酸量无明显变化，但其单位菌体产量和糖酸转化率分别提高21.14%和8.97%。可见，适量过量表达pepc和将GDK-10基因组pepc替换为解除天冬氨酸反馈抑制的pepc (N917G)均可提高α-KG的单位菌体产量且后者可显著提高糖酸转化率。本研究结果可对α-KG的基因工程改造奠定基础。

Alpha-ketoglutarate (α-KG), a dicarboxylic acid, is one of the key intermediates in amino acid metabolism and serves as the precursor of glutamic acid, succinic acid, etc. In the pathways ofα-KG fermentation for Corynebacterium glutamicum, phosphoenolpyruvate carboxylase is a key enzyme to synthesize oxaloacetate. In this study, we overexpressed gene pepc using homologous recombination with C. glutamicum GDK-10 as the parent strain. Then we obtained series of recombinant strains: GDK-11(Ppepc::Ptuf), GDK-12(△gdh::pepc) and GDK-13(pepc(N917G)). RT-qPCR was employed to evaluate the transcript quantification of the target gene. The expression of pepc gene in GDK-11 and GDK-12 was about 47.50 and 2.03 times, respectively of that in strain GDK-10. The fermentation experiments showed that the yield in strain GDK-11 and GDK-12 decreased by 45.50% and 13.90%. But the YP/S in strain GDK-12 increased by 38.59%. The α-KG concentration of strain GDK-13 did not significantly increased, but the YP/S and the yield of glucose to α-KG increased by 21.14% and 8.97% respectively. These results demonstrate that properly enhancing pepc transcription and strain GDK-12(△gdh::) and GDK-13 (pepc(N917G)) can increase the YP/S, and the point mutations (N917G) into the pepc gene can increase conversion rate of glucose obviously. The results obtained may be useful for the further genetic modification.

天津市教委科研究计划项目（项目编号：20120630）