Construction And Application Of Multi-gene Expression Regulation System In Corynebacterium Glutamicum

Corynebacterium glutamicum is an important strain for industrial production of diversiform bioproducts.Multiplex control of metabolic pathway genes is crucial for maximizing biosynthesis of desired products.However,few tools for simultaneously regulating multiple genes in C.glutamicum have been reported.Therefore,it is important to develop a new CRISPR-FndCpf1(Francisella novicida DNase deactivated Cpf1)based multi-gene expression regulation tool in C.glutamicum.The results are mainly divided into two parts:construction and method application.Firstly,after optimizing the expression of FndCpf1,the toxicity of FndCpf1 system was solved.Therefore,a system based on CRISPR-FndCpf1 was successfully constructed in C.glutamicum.Second,after optimizing different point mutations of the FndCpf1 protein,the system’s weakening efficiency was successfully improved.The system’s weakening efficiency was increased from 63%to 90%.For the first time,the weakening efficiency of the double-point mutation FndCpf1 system was significantly higher in C.glutamicum than single-point mutations.Third,in the dual-fluorescence reporter strain,the weakening effect of the FndCpf1 system on both genes was more than 90%,confirming that the FndCpf1 system can be used for the regulation of multiple gene expression.Fourth,the CRISPR-FndCpf1 system was applied to metabolic engineering.Weakening the four genes separately and in combination could increase the titer of lysine and screen out the optimal combination strain.The weakening efficiency of each gene was higher than 90%.It was determined that the FndCpf1 system can be used to regulate the expression of endogenous genes,and can easily and quickly achieve the regulation of multi-gene expression.