Expression of transcriptional fusions between the htrA and htrB promoters and lacZ reporter gene in various genetic backgrounds.
Bacillus subtilis is a Gram-positive, nonpathogenic organism which is widely used as a host for enzyme production, due to its ability to secrete large amounts of proteins into the growth medium (Simonen et al., 1993; Westers et al., 2004). The secretion of a target protein leads to the natural separation of the product from cell components, which simplifies downstream processing of the protein. Accordingly, there has been a great deal of research performed regarding protein production in B. subtilis (Simonen et al., 1993; Westers et al., 2004). Nevertheless, the yields of heterologous protein obtained from this organism are often insufficient (Harwood, 1992). Several bottlenecks in the B. subtilis secretion pathway have been reported, including poor targeting to the translocase, degradation of the secretory protein, and incorrect folding (Westers et al., 2004). One of the major bottlenecks involves the degradation of the produced protein by extracellular proteases; therefore, inactivation of extracellular proteases is essential for improvement of protein production with B. subtilis as the host.
2. Inhibition of proteolysis of heterologous and nature proteins after the translocation process by inactivation of multiple proteases
Eight extracellular proteases have been identified in B. subtilis to date, which are encoded by the following genes: aprE (Stahl et al., 1984; Wong et al., 1984), bpr (Sloma et al., 1990b; Wu et al., 1990), epr (Bruckner et al., 1990; Sloma et al., 1988), mpr (Rufo et al., 1990; Sloma et al., 1990a), nprB (Tran et al., 1991), nprE (Yang et al., 1984), vpr (Sloma et al., 1991), and wprA (Margot et al., 1996). Deletions in the aprE (encoding subtilisin, alkaline protease) and nprE (encoding neutral protease) genes were the first such mutations, whose mutants show lower activities of extracellular proteases (Sloma et al., 1991). In addition, a deletion mutation in the epr gene resulted in low protease activity in the culture supernatant. wprA encodes a 96-kDa protein that is processed to the CWBP23 propeptide and CWBP52 mature protease, forming a complex associated with the cell wall (Margot et al., 1996).
This complex was also found in the culture supernatant of B. subtilis WB600 (Babe et al., 1998; Wu et al., 1991). Whether it is present in the cell wall or in the culture medium is therefore a critical factor in the degradation of heterologous proteins (Lee et al., 2000). Strains with deletion mutations in multiple extracellular proteases have since been constructed with extracellular protease activities of less than 0.5%, compared to the parental strain (Wu et al., 1991). It was recently reported that an eight protease-deficient strain, WB800, was a useful host for the production of various heterologous proteins (Murashima et al., 2002; L. Westers et al., 2006 ). However, the use of B. subtilis as a host has remained limited to bulk industrial enzyme production. Further optimization is necessary to develop production systems for heterologous proteins. This chapter focuses on the inhibition of proteolysis of secreted proteins after the translocation process by inactivation of multiple proteases which are extracellular (AprE, Bpr, Epr, Mpr, NprB, NprE, Vpr, and WprA), leaked outside from intracellular (AprX), and membrane-bound (HtrA and HtrB).
2.1. The intracellular protease, AprX is involved in degradation of a heterologous protein
In B. subtilis, extracellular protease-deficient mutants have been used in attempts to increase the productivity of heterologous proteins. We detected the protease activity of AprX using protease zymography in the culture medium at the late stationary growth phase. Construction of multiple-protease-deficient mutant without antibiotic-resistance markers and the effect of AprX on the heterologous protein production are descrived in detail in the following sections.
2.1.1. Construction of an eight-extracellular-protease-deficient mutant by marker-free deletion in B. subtilis
Antibiotic-resistance marker genes were used to create new bacterial strains. However, the number of markers available for use in B. subtilis and other bacteria is limited. We used the “ampicillin concentration” method for the creation of eight-extracellular-protease-deficient mutant with marker-free deletion (Fig. 1, Kodama et al., 2007a). Recently, several useful methods were developed to produce unmarked mutations in B. subtilis (Liu et al., 2008; Morimoto et al., 2008; Morimoto et al., 2009). These systems are more convenient for the introduction of multiple mutations.
2.1.2. Detection of AprX activity in the culture supernatant with protease zymography
Zymography has been used to detect proteolytic enzymes after electrophoretic separation in gels. Recently, the activities of some proteases, including Vpr have been detected by fibrin zymography of the extracellular proteins of B. subtilis (Murashima et al., 2002; L. Westers et al., 2006 ). The supernatant proteins of B. subtilis culture in modified 2xL broth (Kodama et al., 2007b) at 8 h (exponential growth phase), 25 h (early stationary phase), 50 h (mid-stationary phase), and 75 h (late stationary phase) (Fig. 2A) were analyzed by gelatin zymography (Fig. 2B). The resulting zymogram shows the protease activities as clear bands (or zones). In the exponential growth phase (8 h), no protease activity was detected (Fig. 2B, lane 1). However, protease activity increased during the stationary phase, and was highest at 75 h of the late stationary phase (Fig. 2B, lane 4). We examined the zymogram profile of the supernatant from the eight-extracellular-protease-deficient mutant (Dpr8) at 75 h, and found one clear band in the zymogram (Fig. 2C, lane 2). Protease activity disappeared in the aprX mutant at 75 h (Fig. 2C, lane 3). All of the protease activities completely disappeared in the KA8AX strain, in which nine genes (eight extracellular protease genes and aprX) were disrupted (Fig. 2C, lane 4). These results support the idea that the protease is serine protease AprX. To determine the serine and metal protease activities of this protease by zymography, PMSF and EDTA (2 mM each) were added to the supernatant of the 75 h culture of Dpr8 (Fig. 2D). EDTA decreased the activity of the protease slightly, whereas 2 mM PMSF completely inhibited the protease activity (Fig. 2D). These results suggest that the gelatin-degrading protease from the supernatant of Dpr8 culture is AprX. To determine whether AprX is the gelatin-degrading protease in the supernatant of the Dpr8 culture at 75 h, the AprX-FLAG fusion protein was constructed. The fusion gene was expressed with the original promoter and ribosomal binding site. On a zymographic gel, the activity bands corresponding to AprX-FLAG from both 168/AprX-FLAG and Dpr8/AprX-FLAG strains were located at slightly larger positions in size than those of AprX (Fig. 3). The size of the band corresponded to the size of the FLAG peptide. These results indicate that the activity of AprX is detectable as a single band by gelatin zymography of the supernatant of a 75 h culture of B. subtilis strains.
2.1.3. Intracellular AprX leaked to the culture medium during the late stationary phase
It has been supposed that AprX is a serine protease belonging to the subtilase superfamily, and that it is an intracellular protease, because a canonical signal sequence for secretion has not been found in this protease (Valbuzzi et al.; 1999). However, AprX was detected in the culture medium by gelatin zymography (Fig. 3). aprX is transcribed during the stationary phase, and the regulator of SinR exerts negative effect on its transcription directly or indirectly (Valbuzzi et al.; 1999). However, aprX is not essential for either growth or sporulation (Valbuzzi et al.; 1999). As a result, the function of AprX has remained poorly understood. The Western blotting of AprX-FLAG from the intracellular fraction showed that the expression of AprX-FLAG began at 25 h, and that the expression level markedly increased after 50 h (Fig. 4). Our results agreed with a previous report that aprX is transcribed during the stationary phase. In contrast, a weak AprX-FLAG expression was detected in the supernatant only in the late stationary phase at 75 h (Fig. 4). This result agreed with the zymogram pattern of wild-type AprX (Fig. 2B). A slight decrease in cell density was observed after 50 h for the wild-type (Fig. 2A) and 168/AprX-FLAG strains (data not shown). The bands corresponding to AprX-FLAG from both the intra- and extracellular fractions were located at the same position. This result suggests that there is insufficient secretion of AprX, due to the absence of an obvious signal sequence. These observations also suggest that AprX is localized intracellularly by nature, and is leaked to the culture medium during the late stationary phase due to cell lysis.
2.1.4. AprX involved in degradation of the α-amylase-A522-PreS2 hybrid protein
AprX in the supernatant was able to degrade gelatin. Therefore, we considered that AprX may affect the production of secreted proteins. pTUBE522-preS2 has already been developed for the extracellular production of small peptides of the human hepatitis B virus preS2 antigen (42 amino acids) fused with B. subtilis α-amylase (deleting the C-terminal region to construct a peptide carrier) (Honda et al., 1993). To confirm the effect of AprX on the degradation of heterologous proteins, we examined the production of α-amylase-A522-PreS2 as a model of heterologous proteins, in multiple-protease-deficient B. subtilis strains. Cells carrying pTUBE522-preS2 were cultured in modified 2xL broth for 25, 50, and 75 h. α-amylase-A522-PreS2 in the supernatants from the cultures of Dpr7, Dpr8, and KA8AX strains was analyzed by Western blotting with the anti-PreS2 antibody (Fig. 5A). The Dpr7 strain lacked seven extracellular proteases (AprE deficiency excluded). No positive band corresponding to α-amylase-A522-PreS2 was detected at any phase for Dpr7 (pTUBE522-preS2) (Fig. 5A, lanes 1-3). Dpr8 (pTUBE522-preS2) produced α-amylase-A522-PreS2 at detectable levels, and production of the hybrid protein attained high levels after 50 h (Fig. 5A, lane 5). However, when AprX was produced in the supernatant of Dpr8 (pTUBE522-preS2) at 75 h, the amount of α-amylase-A522-PreS2 decreased markedly (Fig. 6A, lane 6). As expected, the degradation of α-amylase-A522-PreS2 was markedly inhibited in KA8AX (pTUBE522-preS2) at 75 h, with the relative amount of the hybrid protein produced by this strain being 1.8-times higher than that of Dpr8 at 50 h (Fig. 5A, lanes 5 and 9; Fig. 5B). KA8AX produced α-amylase-A522-PreS2 up to 80 mg/L, which is at least eightfold higher than the amount produced by the improved strain in a previous study (Lee et al., 2000; Fig. 5, lane 9). We also examined the degradation of α-amylase-A522-PreS2 by AprX protease. First, we prepared AprX protease from KA8AX (pDG-AprX) that was grown in a medium containing 1 mM IPTG for 4 h. The overexpression of AprX was confirmed by gelatin zymography (Fig. 6A, lane 2). Afterwards, the α-amylase-A522-PreS2 protein prepared from the supernatant of KA8AX (pTUBE522-preS2) at 75 h was mixed with AprX protease, and the mixture was incubated at 37ºC for 60 min. The degradation of α-amylase-A522-PreS2 was analyzed by Western blotting using the anti-PreS2 antibody. H2O and intracellular proteins extracted from KA8AX (pDG-AprX) cells cultured without IPTG did not decrease the amount of the α-amylase-A522-PreS2 protein (Figs. 6B, C, lanes 2 and 4), but intracellular proteins extracted from KA8AX (pDG-AprX) cells cultured with 1 mM IPTG decreased the amount to 70% (Figs. 6B, C, lanes 5 and 6).
These results indicate that the AprX protease directly degraded the α-amylase-A522-PreS2 protein. One bottleneck of the production of α-amylase-A522-PreS2 was partially solved by the disruption of eight extracellular proteases and AprX, as shown in this chapter. However, the supernatant from the KA8AX culture at 75 h contained not only a small amount of α-amylase-A522-PreS2, but also a large amount of α-amylase protein (determined by Western blotting with the anti-α-amylase antibody; data not shown). On other hand, no PreS2 peptide was detected by Western blotting with the anti-PreS2 antibody (Fig. 5A). These results indicate that the degradation of α-amylase-A522-PreS2 was not inhibited completely in the KA8AX strain, and that there were as yet unidentified protease(s) involved in the proteolysis of the PreS2 region. Therefore, there is still room for improving the inhibition of hybrid protein degradation. It has been reported that IspA (Isp) was identified as a major intracellular serine protease (Koide et al., 1986). We evaluated the inhibition of the degradation of α-amylase-A522-PreS2 by the inactivation of IspA in the KA8AX strain. However, the productivity of α-amylase-A522-PreS2 in the ten-protease deficient mutant was almost same as that in the KA8AX strain. A search in the GenoList database for B. subtilis 168 genome (http://genodb.pasteur.fr/cgi-bin/WebObjects/GenoList) of proteases and peptidases revealed the presence of 31 known and 11 putative proteases, and 38 known and 12 putative peptidases, respectively. Section 2.2 describes the investigation of membrane-bound proteases involved in protein degradation.
2.2. The effect of HtrA and HtrB on the degradation of secreted proteins
In this section we describe the effects of membrane-bound proteases and a two-component system on degradation of secreted proteins, and transcriptional regulation of the membrane-bound protease genes.
2.2.1. Cell envelope-associated quality control proteases
In B. subtilis, the accumulation of misfolded proteins at the membrane-cell wall interface is sensed by the CssR–CssS two-component system, which consists of the membrane-embedded sensor kinase, CssS and the response regulator, CssR (Hyyryläinen et al., 2001). This system responds to general protein secretion stresses, which can be triggered by either homologous (e.g., overproduction of LipA) or heterologous (e.g., overproduction of AmyQ and hIL-3) proteins, and consequently activates the transcription of the monocistronic htrA and htrB genes (Darmon et al., 2002; H. Westers et al., 2006 ; Hyyryläinen et al., 2007). HtrA and HtrB are membrane-bound serine proteases that are responsible for the degradation of misfolded proteins, and can thereby rescue the cell from a lethal accumulation of misfolded proteins in the cell envelope. In addition, HtrA has a dual localization, because it can be detected in the membrane-associated cellular fraction as well as the growth medium. Therefore, HtrA has a chaperone-like activity that might assist misfolded proteins in recovering their conformation, while also targeting unsuccessful protein for degradation (Antelmann et al., 2003). Induction of htrA and htrB expressions is responsive to secretion stress in a manner dependent on the CssRS two-component system. In addition, htrA and htrB expressions are negatively autoregulated and reciprocally cross-regulated (Noone et al., 2000, Noone et al., 2001). Therefore, the absence of HtrA leads to the increased synthesis of HtrB, and vice versa (Noone et al., 2001).
2.2.2. High-level lipase A (LipA) production in eleven proteases mutant
We examined the production of lipase A (LipA) of B. subtilis (van Pouderoyen et al., 2001), as a valuable model for industrial enzyme production, in a nine-protease-deficient B. subtilis strain. Therefore, we constructed the pHLApm plasmid, in which LipA with the promoter and ribosomal binding site of an alkaline cellulase gene, egl-237 (Hakamada et al., 2000) was cloned into pHY300PLK (Takara, Japan). LipA was overproduced in B. subtilis. Cells carrying pHLApm were cultured in modified 2xL broth for 12, 24, 36, 48, 60, and 75 h. The productivity of LipA in the supernatants from cultures of the 168 and Dpr9 (in which nine genes encoding eight extracellular proteases and AprX were precisely and completely deleted from the chromosome) strains was calculated based on the activity of LipA (Fig. 7). In 24 h cultivation, the production level of the LipA in 168 and Dpr9 could be obtained at 860 mg/L, an excellent yield which is 1.4-times higher than that of previously reported (Lesuisse et al., 1993). After 24 h, the amount of LipA markedly decreased in the 168 strain (Fig. 7). In contrast, degradation of LipA in the Dpr9 was effectively inhibited, compared with the 168 strain. However, after 36 h, the production of LipA in Dpr9 was reduced by approximately 10% (Fig. 7). These results showed that LipA was also degraded in the Dpr9 strain. Overproduction of both homologous (LipA) and heterologous (AmyQ and hIL-3) proteins induces the expression of htrA and htrB by the CssRS system (Darmon et al., 2002; H. Westers et al., 2006 ). From the currently available data, it seems most likely that limitation of both proteases of HtrA and HtrB improved the yield of heterologous proteins (Vitikainen, M., H. L. et al., 2005). To confirm the effect of HtrA and HtrB on the degradation of secreted proteins, we examined the production of LipA of B. subtilis in the htrA and/or htrB deficient B. subtilis strains.
We constructed Dpr9∆htrA, Dpr9∆htrB, and Dpr9∆htrA/B (with eleven inactivated proteases), and evaluated each strain for the production of LipA. No effect on LipA production was observed in Dpr9∆htrA and Dpr9∆htrB. However, the production of LipA by the Dpr9∆htrA/B strain was at 1100 mg/L, which is 1.2-times higher than that of the Dpr9 strain (Fig. 8). These results suggest that inactivation of both htrA and htrB, as well as the nine proteases, has improved the productivity of B. subtilis for the production of LipA.
2.2.3. Transcriptional regulation of htrB and htrA by reciprocal cross regulation
We predicted that there was no difference between the productivities of LipA in the Dpr9∆htrA and Dpr9∆htrB strains, because the inactivation of either htrA or htrB results in a compensating overexpression of the other gene (Noone et al., 2001). To confirm that the overexpressions of htrA and htrB are caused by the inactivation of the other gene, we examined the level of expression of the htrB-lacZ fusion for the Dpr9∆htrA mutant, as well as the similar expression of the htrA-lacZ fusion for the Dpr9∆htrB mutant. Cells carrying pHY300PLK (control) and pHLApm (LipA overexpression) were cultured in modified 2xL broth for 48 h. As shown in Table 1, Dpr9∆htrA cells harboring pHLApm transcribed htrB-lacZ at a 4-fold increased level, compared with Dpr9 harbouring pHLApm (from 0.51 to 2.30 U). Similarly, a 10-fold increase in the htrA-lacZ expression level was observed in the Dpr9∆htrB mutant (from 0.41 to 4.26 U). The expressions of htrB-lacZ and htrA-lacZ also demonstrated reciprocal cross regulation in cells carrying pHY300PLK. These observations suggest that the overexpression of htrB in Dpr9∆htrA and of htrA in Dpr9∆htrB might affect LipA production. The expression level of htrB-lacZ in LipA-producing Dpr9 was 2.4-times higher than that of non-LipA-producing Dpr9 (Table 1). There was almost no change in the expression level of htrA-lacZ, between Dpr9 cells harboring pHLApm and Dpr9 cells harbouring pHY300PLK. The expression of the htrB-lacZ reporter gene fusion has previously been shown to be more sensitive to secretion stress than the htrA-lacZ reporter gene fusion (Hyyryläinen et al., 2001). These results suggest that Dpr9 produced LipA in weak response to secretion stress.
|Expressed gene||Strain||Plasmid||Expression a|
|a One activity unit is defined as 1 nmol of o-nitrophenyl-ß-d-galactopyranoside hydrolysed per min per µg of OD600. The results presented are the average of three individual experiments. Plus/minus values represent standard deviations.|
This chapter focused on biotechnological approaches to optimization of heterologous protein and enzyme production by multiple protease-deficient mutations in B. subtilis. Section 2.2 described the identification of AprX protease using gelatin zymography and the effects of AprX on heterologous protein production. The nine-protease-deficient KA8AX strain (lacking nine genes encoding eight extracellular proteases and AprX) effectively prevented proteolysis of α-amylase-A522-PreS2 [PreS2 antigen of human hepatitis B virus (HBV) fused with the N-terminal 522 amino acids of B. subtilis α-amylase] in the late stationary growth phase and improved the yield of the fusion protein. In addition, AprX was detected in the culture medium due to leakage on cell lysis during the late stationary growth phase. Section 2.3 described that the inactivation of nine-proteases and both htrA and htrB (resulting the Dpr9∆htrA/B mutant) improved the productivity of LipA in B. subtilis. In particular, the productivity of the LipA in the Dpr9∆htrA/B strain was 1100 mg/L, an optimal yield which is 1.8-times higher than that of previously reported. There was no difference in the productivities of LipA in the Dpr9∆htrA and Dpr9∆htrB strains, compared with that of Dpr9. Because the transcriptions of htrA and htrB are controlled by reciprocal cross regulation, overexpression of htrB in the Dpr9∆htrA strain and of htrA in the Dpr9∆htrB strain might affect LipA production. The previous approach for effective protein production was to generate a strain which has the inactivation of eight extracellular proteases in B. subtilis as the host. We reported that AprX leaked outside of cells, and HtrA/HtrB membrane-bond proteases of B. subtilis were also key proteases involved in the degradation of natural and heterologous proteins. In addition, nine- or eleven-protease-deficient strains of B. subtilis were helpful in improving protein productivity. Our findings, described in this chapter should contribute to the generation of hosts to be further optimized for protein production.
We would like to thank Mr. Keiji Endo, Mr. Kazuhisa Sawada, Dr. Koji Nakamura, Dr. Yasutaro Fujita, Dr. Fujio Kawamura, and Dr. Naotake Ogasawara for useful advice and discussions, and Dr. Hiroshi Kakeshita and Dr. Kunio Yamane for their generous gift of plasmid of pTUBE522-PreS2, and for useful advice and discussions. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO).