Abstract
BackgroundNattokinase (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.ResultsSix recombinant strains harboring different plasmids with a single promoter (PP43, PHpaII, PBcaprE, PgsiB, PyxiE or PluxS) were constructed, and the analysis of the fibrinolytic activity showed that PP43 and PHpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by PP43 and PHpaII 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 PP43 or PHpaII 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 PP43 or PHpaII could effectively enhance NK production. Eight triple-promoters with PP43 and PHpaII 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 PHpaII-PHpaII-PP43. The scale-up production of NK that was promoted by PHpaII-PHpaII-PP43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL.ConclusionsOur studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by PHpaII-PHpaII-PP43, 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.
Highlights
Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy
Construction of expression cassettes for overexpression of nattokinase Six strong and widely used promoters, HpaII promoter (PHpaII), P43 promoter (PP43), aprE promoter (PBcaprE), luxS promoter (PluxS), gsiB promoter (PgsiB) and yxiE promoter (PyxiE), were selected as targets for enhancing the production of NK, and their origins and characteristics are listed in Additional file 1: Table S1
The plasmid pSG-pro-NK with no promoter was constructed first, and five promoters were employed to construct the plasmids pSG-PP43, pSG-PBcaprE, pSG -PluxS, pSG-PgsiB and pSG-PyxiE following the MEGAWHOP method (Fig. 1a)
Summary
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. NK, as a potent fibrinolytic enzyme, can directly cleave cross-linked fibrin in vitro and inactivate the fibrinolysis inhibitor or catalyze the conversion of plasminogen to plasmin [2, 3]. Studies in rats showed that NK exhibited 5-fold more fibrinolytic activity than that of plasmin [4]. The species B. subtilis is a good host strain for the industrial production of the NK enzyme, as NK was isolated from B. subtilis natto.
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