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

Nattokinase (NK, E.C. 3.4.21.62) was first identified by Sumi et al. from “Natto”, which is a popular traditional Japanese soybean food [1]. 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]. Compared with other thrombolytic reagents, including urokinase, tissue type plasminogen activator (t-PA) and streptokinase, NK has advantages in preventative and prolonged effects, with few side effects and stability in the gastrointestinal tract [5]. The NK gene was cloned and characterized, and protein engineering techniques and site-directed mutagenesis were carried out to improve NK stability [6–10]. The NK enzyme is usually industrially produced by the wild-type Bacillus subtilis natto (B. subtilis natto) [11].

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. B. subtilis is a gram-positive bacterium and is a well-studied host for the expression of heterologous proteins because of its many attractive features [12]. As a model organism, B. subtilis is widely used in laboratory studies because it is easy to culture and has a high-level secretory system. In addition, B. subtilis is a food-grade safety strain and presents no safety concerns, as reviewed by the U.S. FDA Center. Some efficient expression systems have been constructed to promote the production of homologous and heterologous proteins in B. subtilis, because of its well-characterized physiological and biochemical properties and nonpathogenicity [13–15]. B. subtilis strains has been engineered as extracellular-protease deficient strains for the overexpression of subtilisin and β-lactamase in B. subtilis WB600 [16, 17], the overexpression of staphylokinase and xylanase in B. subtilis WB700 [18, 19], and the overexpression of phospholipase C in B. subtilis WB800 [20]. In addition, several studies have reported the secretory overexpression of NK in recombinant B. subtilis strains [21, 22].

As is well known, the promoter-regulated gene transcription is usually located upstream of the gene. There are two kinds of promoters: the constitutive promoter that is active in all circumstances and the regulated promoter that become active only in response to specific stimulation in the cell. Because the promoter is a crucial aspect of the expression system, many strong promoters have been screened and characterized in B. subtilis [23–26]. Recent studies have increasingly focused on the strategy to improve the expression level of recombinant proteins or peptides by the construction of tandem promoters and promoter engineering. Using engineered promoters by altering the − 10 or − 35 region led to a much higher production of recombinant proteins [27, 28]. Widner et al. had studied the gene expression in B. subtilis and found that the expression level of the gene could increase by using expression systems that contain two or three tandem promoters in contrast to a single promoter. The study demonstrated that the expression of aprL achieved a high level by combining the mutant amyQ promoter with the promoter of the cry3A gene [29]. The thermostable 4-α-glucanotransferase from Thermus scotoductus was overexpressed in B. subtilis, and its productivity was elevated by more than ten-fold when promoted by a dual-promoter system, compared to that of the single HpaII promoter system [30]. Researchers have investigated the strength of single and dual promoters for overexpression of aminopeptidase in B. subtilis. In addition, the dual-promoter P_gsiB–P_HpaII gave the best performance, which was much higher than P_HpaII and P_gsiB [31]. The system containing a dual-promoter P_HpaII-P_amyQ′ was found to sustain superior expression of β-cyclodextrin glycosyltransferase in a B. subtilis strain (CCTCC M 2016536) [32]. Okegawa and Motohashi successfully expressed the functional ferredoxin-thioredoxin reductase by using a system containing tandem T7 promoters in Escherichia coli [33].

In this study, we aimed to increase the secretory expression of NK in B. subtilis WB800 by mediating the gene expression promotion by tandem promoters. Six constitutive promoters, P_HpaII, P_P43, P_BcaprE, P_luxS, P_gsiB and P_yxiE, were selected, and a series of expression cassettes containing single promoters, dual-promoters and triple-promoters was achieved by arranging promoters in different orders. The efficacies of these multiple tandem promoters for controlling the expression of NK are presented.

Six promoters having high expression strength were selected to overexpress the NK enzyme in B. subtilis WB800, and the overexpression of NK mediated by those single promoter systems exhibited significantly different levels. In our study, the highest expression level of NK driven by a single promoter was 140.5 ± 3.9 FU/ml as induced by P_P43. The order of the strength of the six single promoters mediating NK expression in B. subtilis was P_P43 > P_HpaII > P_BcaprE > P_luxS > P_gsiB > P_yxiE. However, Guan et al. reported that the activity of the single promoter P_P43 was lower than that of P_luxS and P_yxiE for aminopeptidase expression in B. subtilis [31]. In addition, Zhang et al. reported that P_yxiE exhibited higher expression strengths than P_P43, both in B. subtilis and E. coli [23]. The expression level of the target gene is naturally determined by the promoter, signal peptide and host, and many studies have suggested that the effect of the promoter strength on the heterologous expression varies. Our results are consistent with the effect of a promoter varying with the change in the target gene [34]. The growth curves of strains containing a single promoter were approximately same (Additional file 1: Figure S1), and this result confirmed that the expression cassettes with different promoters, not the cell amount, caused the different expression levels of NK.

The NK expression level driven by a dual-promoter P_P43-P_P43 reached 157.2 ± 3.0 FU/ml, which was 10% higher than that induced by the single promoter P_P43. Similar results were observed between the dual-promoter P_BcaprE-P_BcaprE and the single promoter P_BcaprE and the dual-promoter P_gsiB-P_gsiB and the single promoter P_gsiB. However, the strength of the dual-promoter containing two P_HpaII was 1.8-fold higher than that of the single promoter. The two promoters P_P43 and P_HpaII exhibited higher promoter activity for NK expression than that of the other promoters. We further carried out the experiments of arranging P_HpaII and P_P43 by combining three or four promoters in tandem. As shown in Table 2, our results indicated that the NK expression level was not associated with the numbers of tandem repeats of the promoters. The NK production under the control of P_P43-P_P43 was increased by 11.9% compared with that promoted by P_P43, and the NK production mediated by P_P43-P_P43-P_P43 increased by 39.4% compared with that promoted by P_P43-P_P43. However, the NK expression level increased by only 1.7% when promoted by P_P43-P_P43-P_P43-P_P43. Furthermore, NK production decreased under the control of four P_HpaII in tandem compared with the expression controlled by three tandem promoters. The results were in agreement with studies that suggested that the length of the promoter affects its expression activity [35]. Although the cooperation mechanism of the tandem promoters was not clear, the increased production of NK suggested that this strategy of gene expression based on tandem promoter is an effective way to improve promoter activity.

The core region of a promoter plays an important role in regulating transcription initiation and is the minimal portion of a promoter that is required to properly initiate transcription [36]. To understand the effect of the length of repetitive whole-sequence promoters containing P_P43 or P_HpaII in tandem on the expression level of NK, a series of promoters with core-region repeats (nCP_P43 and nCP_HpaII) were constructed. The whole sequence of P_HpaII is 284 bps; however, the core-region sequence of P_HpaII is only 31 bps. The NK production mediated by 2P_HpaII and 2CP_HpaII almost reached the same level, which suggested that the core region of P_HpaII could efficiently initiate the NK overexpression. However, it was unexpected that the strength of 3CP_HpaII for NK expression was 35.2% lower than that of 3P_HpaII. Further studies will be needed to explore the difference between the whole sequence and the core regions of P_HpaII for the level of gene expression. In addition, the whole sequence of P_P43 is 300 bps, and the core region of P_P43 is 29 bps. The NK expression mediated by the whole sequence of P_P43 in tandem increased to that mediated by 4P_P43. The NK production that was initiated by core promoters of P_P43 in tandem gradually increased as the number of core regions increased. It was found that both strong promoters, P_P43 and P_HpaII, have distinct characterization and differential expressions of NK. The analysis of the expression level of NK induced by more core regions of P_P43 in tandem will be carried out.

Obviously different effects on NK production are caused by different arrangements in the dual-promoter system. The promoter is recognized by the σ factor of RNA polymerase to initiate gene transcription. Several σ factors have been defined in B. subtilis. It has been reported that σ^A- and σ^B-promoters can function cooperatively. The promoter synergism resulting from the double promoters was found only when the σ^B-promoter was located upstream of the σ^A-promoter, and the expression level of reporter gene was severely reduced by switching the locations of the σ^A- and σ^B-promoters [37]. Since P_gsiB is σ^B-dependent (Additional file 1: Table S1), NK production promoted by P_gsiB-P_HpaII (164.9 ± 3.0 FU/ml) compared with that by P_HpaII (110.8 ± 3.6 FU/ml) and that by P_HpaII-P_gsiB (0 FU/ml), suggested that P_HpaII might be a σ^B-dependent promoter. Similar phenomena were observed in the results of NK expression mediated by P_BcaprE-P_HpaII (175.5 ± 5.0 FU/ml) and P_HpaII-P_BcaprE (0 FU/ml), by P_luxS-P_HpaII (77.5 ± 4.0 FU/ml) and P_HpaII-P_luxS (0 FU/ml), predicting that P_luxS and P_BcaprE are σ^B-dependent promoters. Whereas P_yxiE is σ^A-dependent (Additional file 1: Table S1), results of NK production promoted by P_HpaII-P_yxiE (166.7 ± 2.5 FU/ml), compared with that by P_yxiE-P_HpaII (0 FU/ml) and that by P_yxiE (20.2 ± 2.0 FU/ml), suggested that P_HpaII might also be recognized by σ^A RNA polymerase. Therefore, promoters P_HpaII and P_P43 might be recognized by both σ^A and σ^B RNA polymerases. Our results showed that the NK expression that was promoted by the dual-promoter system makes a large difference, which could be due to the synergistic effect of the double promoters.

Studies have shown that triple-promoters could markedly increase the expression level of heterogeneous genes [29, 38]. We operated by combining both strong promoters in the form of three promoters in tandem. Eight strains harboring a triple-promoter system containing P_HpaII and P_P43 were generated, from which the NK production showed different levels. Among these 8 strains, one strain harboring the plasmid pSG-P_HpaII-P_P43-P_P43 lost the ability to express NK, and one strain harboring the plasmid pSG-P_P43-P_HpaII-P_HpaII exhibited low activity of NK expression (47.5 ± 3.1 FU/ml). The other six strains harboring the plasmid containing a triple-promoter exhibited relatively high production of the secreted NK, and the NK expression of four strains were higher than 200 FU/ml. The growth curves of strains containing triple-promoters were approximately the same (Additional file 1: Figure S2), and these results confirmed that the expression cassettes, but not the cell numbers, caused the different levels of NK production with different promoters. On account of the RNA polymerase gene transcription mechanism under the promoter action being very complex, the problem of how to produce this synergy has yet to be further studied. In this study, the highest NK production was mediated by a triple-promoter P_HpaII-P_HpaII-P_P43 and achieved 264.2 ± 7.0 FU/ml, which is much higher than that reported in previous studies [39, 40]. This strain is a potential strain for the industrial production of NK. In addition, the high yield of NK could promote its application in medicine and in supplementary nutrition. A series of plasmids for NK expression in B. subtilis were constructed in this study, and they have great potential to be used for NK expression or the expression of other genes in industrial applications. The results of the various initial activities of multiple tandem promoters for NK expression also provide additional information on the synergistic interaction of promoters.

In this study, we generated and characterized the secretory expression of NK under the control of different promoters, including six single promoters and a series of promoters with the whole sequence or core regions in tandem. The expression level of NK mediated by one of these different promoters led to a remarkable difference in B. subtilis WB800. Among the six single promoters, NK production mediated by P_HpaII and P_P43 exhibited a higher level than the others. The arrangement of these promoters in tandem produced various effects on NK expression. We successively used the triple-promoter P_HpaII-P_HpaII-P_P43 to increase the production of NK to 264.2 ± 7.0 FU/ml in B. subtilis WB800, which was the highest expression level ever reported. Our study provided an efficient way to increase NK production in Bacillus subtilis based on tandem promoters.