Development of a Bacillus subtilis expression system using the improved Pglv promoter

BACKGROUND: B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter P(glv). The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the P(glv )promoter system and enhance its expression strength. RESULTS: Here, site-directed mutagenesis was facilitated to enhance the expression strength of P(glv). The transcription level from four mutants was increased. Production of β-Gal from the mutants reached the maximum 1.8 times as high as that of wildtype promoter. When induced by 5% maltose, the production of β-Gal from two mutants reached 14.3 U/ml and 13.8 U/ml, 63.5% and 57.5% higher than wildtype promoter (8.8 U/ml) respectively. Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity. To further improve the promoter system, the B. subtilis expression host was reconstructed, in which B. subtilis well-characterized constitutive promoter P43 replaced the promoter of the glv operon in B. subtilis chromosome through a double crossover event. The β-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system. Meanwhile, the repression caused by glucose was further alleviated. CONCLUSIONS: In this study, we obtained a mutated promoter Pglv-M1 through site-directed mutagenesis, which demonstrated high expression strength and alleviated the repression caused by glucose. Moreover, we alleviated the repression and enhanced the expression activity of the Pglv-M1 promoter system via reconstruction of the B. subtilis host. Thus, we provided a valuable expression system in B. subtilis.