The Effect of Enhanced Activity of Phosphoenolpyruvate Carboxylase on the Accumulation of α-Ketoglutarate by Corynebacterium glutamicum
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Abstract:
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.
