Establishment Of Tightly Regulated Promoter System In Corynebacterium Glutamicum

Corynebacterium glutamicum,a utilitarian workhorse,has been used for the industrial production of amino acids,heterologous proteins and compounds to the food,medicine and chemical industry.Metabolic engineering and genetic engineering take an approach at the development of strains with improved properties,in which tightly regulated inducible system is important for the expression of endogenous genes and heterologous genes.However,there are few suitable inducible promoters for C.glutamicum and all of them have leaky expression to a certain extent,which cannot achieve strict regulation of target gene expression.In this study,two induction systems,tetracycline-inducible system and Cumate-inducible system,were constructed and tested in C.glutamicum.In the tetracycline-inducible system,the bidirectional promoter P_{tet R/tet A}was selected which could simplify the system.Compared with the commonly used IPTG-inducible system,the leaky expression level of the tetracycline-inducible system was lower.We developed a novel Cumate-inducible system in C.glutamicum.The Cumate-inducible system was constructed in which the fluorescent protein sf GFP was the reporter protein.The expression system worked well,of which the background fluorescence intensity was extremely low,and could tightly control the expression of the reporter protein in C.glutamicum.And the Cumate-inducible system was dose reponsed to the concentration of cumate and relyed on the induction time.Based on the above two inducible systems,an AND Gate,Cumate-ATc inducible system,was constructed.The switch was open only when the two inducer cumate and ATc exist simultaneously.The level of background expression of Cumate-ATc inducible system was 4-5 times lower than that of Cumate-inducible system and tetracycline-inducible system.And the Cumate-ATc inducible system can regulate the expression level of target gene with different concentration of inducer and induction time.We successfully applied the Cumate-ATc inducible system for the expression of Cre recombinase in C.glutamicum.The DNA fragments between two lox Psym sites did not be deleted or reversed without the inducer,which indicated that the expression of Cre recombinase was strictly inhibited.In this work,we developed a new tightly regulated Cumate-inducible system and an AND Gate struction named Cumate-ATc inducible system,which expands the inducible expression system for C.glutamicum.And these systems can be used as a tightly regulated genetic tool in gene engineering and metabolic engineering.