Dataset Information

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

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.

SUBMITTER: Liu Z

PROVIDER: S-EPMC6505213 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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