Abstract
BACKGROUND: Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800. RESULTS: Six recombinant strains harboring different plasmids with a single promoter (P(P43), P(HpaII), P(BcaprE), P(gsiB), P(yxiE) or P(luxS)) were constructed, and the analysis of the fibrinolytic activity showed that P(P43) and P(HpaII) exhibited a higher expression activity than that of the others. The NK yield that was mediated by P(P43) and P(HpaII) reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive P(P43) or P(HpaII) increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of P(P43) or P(HpaII) could effectively enhance NK production. Eight triple-promoters with P(P43) and P(HpaII) in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter P(HpaII)-P(HpaII)-P(P43). The scale-up production of NK that was promoted by P(HpaII)-P(HpaII)-P(P43) was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL. CONCLUSIONS: Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by P(HpaII)-P(HpaII)-P(P43), which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.