Effects Of Gene Pepc And Pck On Accumulation Of L-valine In Corynebacterium Glutamicum V1

This paper was focused on a strain named V1, which can product L-valine from sugarsubstances. By using morphology, physiology and biochemisry and analysis of16S rDNAgene sequence of bacterial V1, determined the V1is a Corynebacterium glutamicum（C.glutmicum） in the status of taxonomy.Pyruvic acid is precursor of L-Valine, phosphoenolpyruvic acid carboxylase(encoded bygene pepc) and phosphoenolpyruvic acid carboxykinase(encoded by gene pck) affect themetablic of pyruvic acid to some extent, so in this work overexpression of pepc and pck andgene knockout of pepc were executed, and the results are as follows:(1) The homologous fragment Δpepc was amplified through crossover PCR, and wasligased to pK18mobsacB, then the mutant V1-Δpepc was constructed through homologousrecombination.(2) The recombinant strain V1-Δpepc can grow in the medium with2%peptone. Butafter the fermentation, there was no accumulation of L-valine while L-agrine was7.48g/L.(3) The physiological characteristics of the mutant V1-Δpepc were investigated byenzymes activity measurement of pyruvate carboxylase (PC)、pyruvate dehydrogenase (PDH)and pyruvate kinase (PK). The pepc knock out mutant resulted in increase of PDH activityand PC activity in C. glutamicum V1. The results shows that PDH of V1is36.5%of C.glutmicum ATCC13032, at the same time39.6%of V1Δpepc, and PC of V1is29.2%of C.glutmicum ATCC13032and50%of V1Δpepc. And PK and PEPC of V1are no apparentdifference with C. glutmicum ATCC13032.(4) The recombinant strain V1(pJC1-tac-pepc) with overexpression of pepc gene wasconstructed, and PEPC crude enzyme activity was raised by27.50%compared to V1. Andfermentationtation experiments with shake flask were consducted, the results showed that therecombinant strain V1(pJC1-tac-pepc) grow slower, at the end of the fermentation, the twostrains biomass was no apparent difference; accumulation of L-valine was decreased by18.48%compared to V1.(5) The recombinant strain V1(pJC1-tac-pck) with overexpression of pck gene wasconstructed, and PCK crude enzyme activity was raised by22.86%compared to V1. Andfermentationtation experiments with shake flask were consducted, the results showed that therecombinant strain V1(pJC1-tac-pck) grow slower, at the end of the fermentation, the twostrains biomass was no apparent difference; accumulation of L-valine was raised by17.41%compared to V1.