Abstract:

【Objective】 Corynebacterium glutamicum is an important industrial microorganism, and its metabolic engineering modification by gene editing may effectively broaden the diversity of its fermentation products. The lack of high-intensity, low-leakage, and low-cost inducible promoters limits the metabolic engineering modification, thereby the design and application of novel inducible promoters are necessary. 【Method】 An cumate-induced promoter P_H10-CuO was constructed by embedding the operator sequence CuO on the constitutive promoter P_H10. 【Result】 Using green fluorescent protein as a quantitative reporter, the relative fluorescence intensity was low due to the basal leakage of P_H10-CuO when without the inducer 4-isopropylbenzoic acid; and gfp expression significantly increased when the fluorescence intensity was up to 62 000 at presence of 25 μg/mL 4-isopropylbenzoic acid for 12 h. This proved that PH 10-CuO has very good rigor and induced expression intensity. Meanwhile, in Constructing the gene editing plasmid for C. glutamicum with P_H10-CuO regulating the expression of recET and cas12a, The accurate editing of target gene and insertion of foreign genes in C. glutamicum chromosome. 【Conclusion】 The inducible promoter PH_10-CuO presented the advantages with high intensity and low leakage, which enables it as an useful element to regulate temporal expression of specific genes in C. glutamicum.

Key words: Corynebacterium glutamicum, inducible promoter, repressor protein CymR, 4-isopropylbenzoic acid