Screening Of Endogenous Promoters And Modification Of L-arginine Synthesis Pathway Of Corynebacterium Glutamicum ATCC 14067

Corynebacterium glutamicum(C.glutamicum)is an important microbial host in the microbial fermentation industry and is widely used in the production of various amino acids.The promoter is an important regulatory element in the strain,but there are still few constitutive endogenous promoters reported in C.glutamicum,so firstly,we performed transcriptome sequencing of samples in logarithmic growth phase during fermentation of wild-type C.glutamicum ATCC 14067,and then used the red fluorescent protein(m Cherry)reporter system to characterize the strength of the promoter in C.glutamicum ATCC 14067.29 promoters of relatively high intensity with different expression intensities were screened.In previous work,the feedback inhibition of L-arginine on Cg NAGK was successfully abolished,and the genome editing system of Rec ET-Cre/lox and CRISPR/Cpf1 which was suitable for C.glutamicum ATCC 14067 was successfully established.In this work,we carried out metabolic engineering on C.glutamicum ATCC 14067,which has relieved the feedback inhibition of L-arginine to Cg NAGK,to enhance the ability of the strain to synthesize L-arginine.The main results can be summarized as follows:(i)29 endogenous promoters of C.glutamicum ATCC 14067 with different strengths were screened.To analyze the transcriptome data of C.glutamicum ATCC 14067,construct a m Cherry-based promoter reporter system,and screen out 29 endogenous constitutive promoters of different strengths in C.glutamicum ATCC 14067.(ii)It was proved that the key enzyme in the synthesis pathway of overexpression of L-arginine can promote the metabolic flow of the strain into the synthesis pathway of L-arginine.In C.glutamicum ATCC 14067,overexpression of key enzymes of L-arginine synthesis approach,can promote the metabolism of strain flow into Larginine synthetic ways and enhance the capacity of strains of synthesis of Larginine,and L-arginine synthesis operon gene's expression of strain had the greatest influence the synthesis of L-arginine;(iii)Metabolic engineering of L-arginine synthesis pathway in C.glutamicum.The gene cluster and synthetic precursors that enhance the L-arginine synthesis operon directly enhance the synthesis of L-arginine;enhance isocitrate dehydrogenase and down-regulate ?-ketoglutarate dehydrogenase to optimize TCA Circulation;weakening the synthesis pathway of by-products L-valine and L-lysine to enhance the metabolic flux of L-arginine synthesis in the strain,and finally directly overexpressing the transporter protein to enhance the intracellular L-arginine outside.Under the condition of flask fermentation for 72 h,the accumulation of L-arginine in the constructed strain PMQBD-LP was up to 5.65 g/L,12 times that of the original strain.