| Alpha-ketoglutarate(a-KG),a dicarboxylic acid,is one of the key intermediates in the chemical synthesis and amino acid metabolism and serves as the precursor of glutamic acid,succinic acid,etc.In this article,in order to construct L-2-aminobutyrate producing strains,a-KG producing strain C.glutamicum GDK-10 was used as parent strain under the guidance of metabolic engineering theory and molecular biology methods.In the pathways of a-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 a-KG concentration of strain GDK-13 did not significantly increased,but the YP/S and the yield of glucose to a-KG increased by 21.14%and 8.97%respectively.These results demonstrate that properly enhancing pepc transcription and strain GDK-12 and GDK-13 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.Succinic acid,acetic acid and lactic acid is the primary by-product in the fermentation of C.glutamicum GDK-10.It does harm to the synethsis and extracting of a-KG We obtained series of recombinant strains by homologous recombination:GDK-14(△ldh),GDK-15(△pqo),GDK-16(△ackA),GDK-17(△pqo△ackA)and GDKY-19(△aceA△pknG).The fermentation experiments showed that the yield in strain GDK-14 decreased by 8.54%.It demonstrates that the synethsis of a-KG can’t improved by knocking out the ldh gene.The a-KG concentration of strain GDK-15 is 47.88 g/L,increased by 8.99%.The a-KG concentration of strain GDK-16 and GDK-17 decreased 27.9%and 14.57%respectively,which compared with GDK-10.These results demonstrate that the deletion of ackA may affect the strain’s normal metabolism,causing growth deficieny and the decrease of the a-KG synethsis.The yield of a-KG has an impressive promotion by knocking out the pqo gene alone.The a-KG concentration of strain GDK-19 decreased 25.84%,which compared with GDK-18.It demonstrates that the deletion of pknG and aceA can’t contribute to the a-KG synethsis.We odtained GDK-20(△opqo△aceA)and GDK-21(△pqo△pknG)from the starting strain C.glutamicumGDK-15.The fermentation experiments showed the a-KG concentration of strain GDK-20 decreased 33.03%,which compared with GDK-15.It demonstrates double gene deletion resulted in decrease of α-KG accumulation.But despite the a-KG concentration of strain GDK-21 decreased by 6.87%,conversion rate of glucose can increase obviously. |
