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.

Keywords: Bacillus subtilis; Core promoter region; Nattokinase; Recombinant enzyme; Tandem promoter.

Fig. 1

Schematic representation of the expression cassettes. a Map of the pSG(x)-NK vectors. All of the expression cassettes were cloned into the pMA0911-wapA-pro-NK, and the sites of the relevant restriction enzymes were shown. b The schematic diagram of the expression cassettes with tandem promoters. The signal peptide (SP) and the NK gene are represented by gray and black, respectively. The promoters, P_X, are represented by arrows. c The expression cassettes with repetitive core regions of promoters. The sequences of core regions (− 35 and − 10) are shown

Fig. 2

Effects of different single promoters on the overexpression of NK. (a) Fibrinolytic activities of NK in the supernatant. The recombinant strains having different single promotes were cultured in TB medium for 72 h with periodical sampling. b SDS-PAGE analysis. Recombinant strains having different single promoters were cultured in the TB medium for 36 h, and then the cells and the supernatant culture were separated by centrifugation. Supernatant (15 μL) was loaded into each lane. Lane M: standard marker proteins; Lane 1–6: P_HpaII; P_P43; P_BcaprE; P_luxS; P_gsiB and P_yxiE. The arrow indicates that the NK bands correspond to 36-h supernatant. c Fibrin plate analysis. Transparent zones produced by the enzyme activity of NK and its variants in the supernatant, which was induced for 36 h, were examined by the fibrin plate method, which was conducted at 37 °C for 4 h. 1–6: P_HpaII; P_P43; P_BcaprE; P_luxS; P_gsiB and P_yxiE

Fig. 3

Overproduction of NK under the control of the dual-promoter systems. a Fibrinolytic activities of NK in the supernatant. b SDS-PAGE analysis. Lane M: standard marker proteins. The position of the NK protein bands is indicated by an arrow. Recombinant strains having different dual-promotes were cultured in the TB medium for 36 h, and then the cells and the supernatant culture were separated by centrifugation

Fig. 4

Analysis of the NK production mediated by different triple-promoter systems. a Fibrinolytic activities of NK in the supernatant. b SDS-PAGE analysis of the culture supernatant. Recombinant strains promoted by different triple-promoters were cultured in the TB medium for 36 h, and then cells and the supernatant culture were separated by centrifugation. Lane 1–8: P_P43-P_P43-P_HpaII, P_P43-P_HpaII-P_HpaII, P_HpaII-P_P43-P_P43, P_P43-P_HpaII-P_P43, P_P43-P_P43-P_P43, P_HpaII-P_HpaII-P_P43, P_HpaII-P_P43-P_HpaII, and P_HpaII-P_HpaII-P_HpaII; Lane M: standard marker proteins. The arrow indicates NK bands

Fig. 5

Effects of the multi core regions of P_HpaII and P_P43 in tandem on NK production. a The fibrinolytic activities of NK in the supernatant. Recombinant strains harboring promoters of repetitive core regions were cultured in TB medium for 36 h, and then cells and the supernatant culture were separated by centrifugation. The SDS-PAGE analysis of the NK expression mediated by the repetitive core regions of P_HpaII (b) and P_P43 (c). The arrow indicates the NK bands corresponding to the 36-h supernatant, and 15 μL supernatant was loaded into each lane

Fig. 6

Analysis of fermentation of NK in the recombinant strain harboring pSG-P_HpaII-P_HpaII-P_P43. The fermentation was carried out in a 5-L fermenter, and the cell growth and NK activity were measured by taking a sample every 2 h