Traditionally, acetate formation in *E. coli* K12 strains is tackled by blocking the acetate pathways or avoiding overflow metabolism through limiting the glucose uptake rate or redirecting the fluxes around the bottleneck, the phosphoenolpyruvate-pyruvateoxaloacetate node. Alternatively, we propose to copy similar physiological and metabolic properties of *E. coli* BL21 in *E. coli* K12. This was achieved by combined deletion of the global transcriptional regulator ArcA and the local regulator IclR. Albeit this metabolically engineered *E. coli* K12 derivate displayed higher biomass yield and lower acetate yield resulting in a substantially increase in recombinant protein yield, the protein yield was still considerably lower than the yield observed in *E. coli* BL21. This difference in recombinant

acetate in aerobic cultures which retards growth and impedes protein production.

protease Lon (Gottesman, 1989) and the periplasmic OmpT (Gottesman, 1996).

in bioreactor experiments approximates the GFP yield of *E. coli* BL21 (DE3).

suggesting proteases activity (Waegeman et al., 2011b).

Δ*iclR* strain.

**4. Conclusion**

bioprocesses in both industries.

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**7**

**Improvement of Heterologous**

Hiroshi Kakeshita1,2, Yasushi Kageyama1,

*2Graduate School of Life and Environmental Sciences,*

*1Biological Science Laboratories,*

*Kao Corporation,*

*Japan*

*University of Tsukuba,*

**Protein Secretion by** *Bacillus subtilis*

Katsuya Ozaki1, Kouji Nakamura2 and Katsutoshi Ara1

The Gram-positive bacterium, *Bacillus subtilis* and related species are widely used as hosts for the extracellular production of industrially worthy enzymes, such as amylases, proteases, xylanase, and lipases (Braun et al., 1999; Tjalsma et al., 2000; Westers et al., 2004). These species possess a very high capacity for secreting a variety of exoenzymes into the growth medium, thereby reducing downstream purification processes. In addition, many of these are generally regarded as safe (GRAS) microorganisms, and do not produce endotoxins. Therefore, the secretion system of these species presents many advantages in terms of production capacity, structural authenticity, product purification, and safety. Nevertheless, the secretion of heterologous proteins from eukaryotes by these species is frequently inefficient (Table1). Hence, these species are never selected as the best cell factory

In pharmaceutical industry, the production of recombinant proteins in *Escherichia coli* is well established. In many cases, proteins are produced in cytoplasm of *E. coli*, and therefore, the production of recombinant proteins involves refolding and purification from inclusion bodies. However, the production of soluble recombinant proteins is relatively more costeffective and less time-consuming. In fact, many studies have been performed regarding methods to overcome the problem of inclusion bodies and to improve protein solubility for the expression of heterologous proteins (Kapust & Waugh, 1999; Baneyx & Mujacic, 2004; Sørensen & Mortensen, 2005; Rabhi-Essafi et al., 2007). Therefore, developing human protein producing hosts is a major challenge in the field of biotechnology and protein production in

In *B. subtilis*, one long-standing major problem is the presence of high levels of extracellular protease for the production of heterologous proteins. In recent years, many proteases have been identified via the completed genome sequence of *B. subtilis* (Kunst et al., 1997; Westers et al., 2004), thereby allowing the construction of many protease-depleted strains for the

**1. Introduction**

*Bacilli*.

for pharmaceutical proteins (Westers et al., 2004).

production of heterologous proteins.

### **Improvement of Heterologous Protein Secretion by** *Bacillus subtilis*

Hiroshi Kakeshita1,2, Yasushi Kageyama1, Katsuya Ozaki1, Kouji Nakamura2 and Katsutoshi Ara1 *1Biological Science Laboratories, Kao Corporation, 2Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan*

#### **1. Introduction**

144 Advances in Applied Biotechnology

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*Biotechnology Progress*, in press

*Microbiology and Biotechnology*, accepted

production. *Metab Eng*, Vol. 10, pp. 97-108

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*Biotechnol*, Vol. 24, pp. 179-185

The Gram-positive bacterium, *Bacillus subtilis* and related species are widely used as hosts for the extracellular production of industrially worthy enzymes, such as amylases, proteases, xylanase, and lipases (Braun et al., 1999; Tjalsma et al., 2000; Westers et al., 2004). These species possess a very high capacity for secreting a variety of exoenzymes into the growth medium, thereby reducing downstream purification processes. In addition, many of these are generally regarded as safe (GRAS) microorganisms, and do not produce endotoxins. Therefore, the secretion system of these species presents many advantages in terms of production capacity, structural authenticity, product purification, and safety. Nevertheless, the secretion of heterologous proteins from eukaryotes by these species is frequently inefficient (Table1). Hence, these species are never selected as the best cell factory for pharmaceutical proteins (Westers et al., 2004).

In pharmaceutical industry, the production of recombinant proteins in *Escherichia coli* is well established. In many cases, proteins are produced in cytoplasm of *E. coli*, and therefore, the production of recombinant proteins involves refolding and purification from inclusion bodies. However, the production of soluble recombinant proteins is relatively more costeffective and less time-consuming. In fact, many studies have been performed regarding methods to overcome the problem of inclusion bodies and to improve protein solubility for the expression of heterologous proteins (Kapust & Waugh, 1999; Baneyx & Mujacic, 2004; Sørensen & Mortensen, 2005; Rabhi-Essafi et al., 2007). Therefore, developing human protein producing hosts is a major challenge in the field of biotechnology and protein production in *Bacilli*.

In *B. subtilis*, one long-standing major problem is the presence of high levels of extracellular protease for the production of heterologous proteins. In recent years, many proteases have been identified via the completed genome sequence of *B. subtilis* (Kunst et al., 1997; Westers et al., 2004), thereby allowing the construction of many protease-depleted strains for the production of heterologous proteins.

Improvement of Heterologous Protein Secretion by *Bacillus subtilis* 147

yields. The relationship between signal peptides and target proteins remains unknown. Accordingly, previous studies have indicated the need for individually optimal signal

Recently, Brockmeier et al. (2006) established a new strategy for the optimization of heterologous protein secretion in *B. subtilis*, by screening a library of all natural signal peptides of the strain. Accordingly, the best signal peptide for the secretion of one target protein is not automatically the best, or even sufficient, for the secretion of a different target

In our study, human interferon- (hIFN-) was used as a heterologous model protein, to investigate the secretion of the *B. subtilis* several major signal peptides. (Fig. 2). We found that for the secretion production of hIFN-α, the AmyE signal peptide is one of the best

Fig. 1. The amino acid sequence of N-terminus-pre-pro AmyE. The putative SPase cleavage site is indicated by a closed arrowhead, and the post-secretory processing site is indicated by an open arrowhead, as described in the references (Takase et al., 1988; Sasamoto et al., 1989). Numbers above the AmyE amino acid sequence refer to the locations of the encoded amino acid residues of AmyE (adapted from Kakeshita et al.,

Some secreted bacterial proteins have cleavage propeptides located between their signal peptide and the mature protein. The propeptide is processed after translocation. Long propeptides (60 to 200 residues) are present for most bacterial extracellular proteases, which are auto-catalytically cleaved and possess intramolecular chaperon activities, for example, *B. subtilis* AprE (Braun et al., 1996; Ikemura & Inouye, 1998; Wang et al., 1998; Yabuta et al., 2001; Yabuta et al., 2002; Zhu et al., 1989). On the other hand, short propeptides (with fewer than 60 residues) are present for a few secreted proteins, including *B. subtilis* α-amylase (AmyE) (Davis et al., 1977; Mezes et al., 1983; Takase et al., 1998) (Fig. 1). In *B. subtilis*, the AmyE propeptide is cleaved by unknown proteins, and is dispensable for secretion, folding,

However, the secretion efficiency of the *Staphylococcus aureus* nuclease (Nuc) was found to be enhanced by a propeptide in *E. coli* (Suciu & Inouye 1996) and *Lactococcus lactis* (Le Loir et al., 1998). In addition, in *L. lactis*, the nine-residue synthetic propeptide, LEISSTCDA, which is fused immediately after the signal peptide cleavage site, is known to enhance heterologous protein secretion (Le Loir et al., 1998; Le Loir et al., 2005; Zhuang et al., 2008; Zhang et al., 2010). Therefore, we evaluated whether the fusion of the AmyE signal peptide and the propeptide could improve the secretion of hIFNα-2b, compared to that with only

peptides for every heterologous secretion target.

and stability (Takase et al., 1998; Sasamoto et al., 1989).

protein (Brockmeier et al., 2006).

signal peptides (unpublished data).

2011a).

**2.2 Propeptide**

AmyE signal peptide.

In addition, considerable efforts have been targeted at developing *B. subtilis* as a host for the production of heterologous proteins (Wu & Wong, 2002; Li et al., 2004; Westers et al., 2004; Kodama et al., 2007a, 2007b). However, many problems still remain for the secretion of human proteins, and these should be analyzed from the complementary perspectives of both the target protein and the secretion pathway, in order to improve human protein secretion.

We have used human interferon-α and interferon-β as heterologous model proteins to investigate the effects of *B. subtilis* secretion.

In this report, the knowledge which has become available in recent years aimed at improving heterologous protein secretion is discussed, and co-production of a Tat system is shown to provide a useful tool to enhance the secretion of heterologous proteins.

Table 1. Protein products from *B. subtilis*

#### **2. Signal peptide and propeptide**

The major of Bacterial secreted proteins are translocated across the cytoplasmic membrane via the Sec pathway (Antelmann et. al. 2004). Secretory proteins are identified by a signal peptide at the protein's N-terminus. A signal peptide consists of a positively charged Ndomain, a hydrophobic H domain, and a C domain containing a specific cleavage site. Most signal peptides are Sec dependent signal peptides, which are cleaved by a type I signal peptidase at the AXA cleavage site (Tjalsma et al., 2000), as an example, *B. subtilis* α-amlyase (AmyE) (Fig. 1).

#### **2.1 Signal peptide**

For the production of a heterologous protein in the culture medium of *B. subtilis*, it is necessary to use a signal peptide that directs the protein very efficiently to the translocase. However, heterologous protein secretion often results in inefficient and unsatisfyingly low yields. The relationship between signal peptides and target proteins remains unknown. Accordingly, previous studies have indicated the need for individually optimal signal peptides for every heterologous secretion target.

Recently, Brockmeier et al. (2006) established a new strategy for the optimization of heterologous protein secretion in *B. subtilis*, by screening a library of all natural signal peptides of the strain. Accordingly, the best signal peptide for the secretion of one target protein is not automatically the best, or even sufficient, for the secretion of a different target protein (Brockmeier et al., 2006).

In our study, human interferon- (hIFN-) was used as a heterologous model protein, to investigate the secretion of the *B. subtilis* several major signal peptides. (Fig. 2). We found that for the secretion production of hIFN-α, the AmyE signal peptide is one of the best signal peptides (unpublished data).

$$\begin{array}{l} \mathsf{BDS \mathsf{A}m\mathsf{y}\mathsf{E}} \quad \mathsf{\hbox{\footnotesize{\footnotesize{\footnotesize{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\cdots}\big{\cdots}}}}}}}}}}}}}}}}}}}}}}} \,\mathsf{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\texttt{\cdots}\big{\cdots}}}}}}}}}}}}}}}}}}\,\mathsf{\texttt{\texttt{\texttt{\tilde{\texttt{\texttt{\tilde{\texttt{\texttt{\texttt{\texttt{\big{\beta}}}}}}}}}}}}}\mathsf{\texttt{\tilde{\texttt{\tilde{\texttt{\tilde{\beta}}}}}}}} $$

Fig. 1. The amino acid sequence of N-terminus-pre-pro AmyE. The putative SPase cleavage site is indicated by a closed arrowhead, and the post-secretory processing site is indicated by an open arrowhead, as described in the references (Takase et al., 1988; Sasamoto et al., 1989). Numbers above the AmyE amino acid sequence refer to the locations of the encoded amino acid residues of AmyE (adapted from Kakeshita et al., 2011a).

#### **2.2 Propeptide**

146 Advances in Applied Biotechnology

In addition, considerable efforts have been targeted at developing *B. subtilis* as a host for the production of heterologous proteins (Wu & Wong, 2002; Li et al., 2004; Westers et al., 2004; Kodama et al., 2007a, 2007b). However, many problems still remain for the secretion of human proteins, and these should be analyzed from the complementary perspectives of both the target protein and the secretion pathway, in order to improve human protein

We have used human interferon-α and interferon-β as heterologous model proteins to

In this report, the knowledge which has become available in recent years aimed at improving heterologous protein secretion is discussed, and co-production of a Tat system is

The major of Bacterial secreted proteins are translocated across the cytoplasmic membrane via the Sec pathway (Antelmann et. al. 2004). Secretory proteins are identified by a signal peptide at the protein's N-terminus. A signal peptide consists of a positively charged Ndomain, a hydrophobic H domain, and a C domain containing a specific cleavage site. Most signal peptides are Sec dependent signal peptides, which are cleaved by a type I signal peptidase at the AXA cleavage site (Tjalsma et al., 2000), as an example, *B. subtilis* α-amlyase

For the production of a heterologous protein in the culture medium of *B. subtilis*, it is necessary to use a signal peptide that directs the protein very efficiently to the translocase. However, heterologous protein secretion often results in inefficient and unsatisfyingly low

shown to provide a useful tool to enhance the secretion of heterologous proteins.

secretion.

investigate the effects of *B. subtilis* secretion.

Table 1. Protein products from *B. subtilis*

**2. Signal peptide and propeptide**

(AmyE) (Fig. 1).

**2.1 Signal peptide**

Some secreted bacterial proteins have cleavage propeptides located between their signal peptide and the mature protein. The propeptide is processed after translocation. Long propeptides (60 to 200 residues) are present for most bacterial extracellular proteases, which are auto-catalytically cleaved and possess intramolecular chaperon activities, for example, *B. subtilis* AprE (Braun et al., 1996; Ikemura & Inouye, 1998; Wang et al., 1998; Yabuta et al., 2001; Yabuta et al., 2002; Zhu et al., 1989). On the other hand, short propeptides (with fewer than 60 residues) are present for a few secreted proteins, including *B. subtilis* α-amylase (AmyE) (Davis et al., 1977; Mezes et al., 1983; Takase et al., 1998) (Fig. 1). In *B. subtilis*, the AmyE propeptide is cleaved by unknown proteins, and is dispensable for secretion, folding, and stability (Takase et al., 1998; Sasamoto et al., 1989).

However, the secretion efficiency of the *Staphylococcus aureus* nuclease (Nuc) was found to be enhanced by a propeptide in *E. coli* (Suciu & Inouye 1996) and *Lactococcus lactis* (Le Loir et al., 1998). In addition, in *L. lactis*, the nine-residue synthetic propeptide, LEISSTCDA, which is fused immediately after the signal peptide cleavage site, is known to enhance heterologous protein secretion (Le Loir et al., 1998; Le Loir et al., 2005; Zhuang et al., 2008; Zhang et al., 2010). Therefore, we evaluated whether the fusion of the AmyE signal peptide and the propeptide could improve the secretion of hIFNα-2b, compared to that with only AmyE signal peptide.

Improvement of Heterologous Protein Secretion by *Bacillus subtilis* 149

We showed that the secretion production and activity of hIFN-α2b with propeptide increased by more than 3-fold, compared to that without propeptide. The amount of secreted hIFN-α2b with propeptide was 15mg /L. This result indicated that the propeptide

of AmyE enhanced the secretion of hIFNα-2b (Fig. 3, Kakeshita et al., 2011a).

Fig. 4. Western blot analysis of hIFN-β production by *B. subtilis* Dpr8 with pHKK3111 (AmyE SP-hIFN-β) or pHKK3211 (AmyE SP-Pro hIFN-β). Samples were collected at 20 h after xylose induction, separated by 15% SDS-PAGE, and stained with Western blotting using anti hIFN-β polyclonal antibodies. Dpr8 with pHKK3111 (lanes 1 and 2); Dpr8 with pHKK3211 (lanes 3 and 4); 0.6% xylose induced (lanes 1 and 3), none induced (lanes 2 and 4), and commercially purified hIFN-β 50 ng (lane 5). Arrowheads indicate the positions of

In *L. lactis*, directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety (Le Loir et al., 2001). In hIFN-α2b with AmyE propeptide, the first 10 amino acid residues of this mature protein have a net charge of -1. On the other hand, hIFN-α2b without propeptide has a net charge of 0. In addition, we demonstrated that propeptide mutants of neutral or positive charge resulted in a reduction in the amount of secreted hIFN-α2b, compared with propeptides of negative charge. This result suggested that negative charges in the mature protein can enhance the secretion of

We then indicated that the AmyE propeptide enhanced the secretion of the hIFN-β protein from *B. subtilis*, as well. The secretion production and activity of hIFN-β with propeptide increased by more than 4-fold (Fig. 4, Kakeshita et al., 2011b). The amount of secreted hIFN-

the Pro-hIFN-β and hIFN-β. (adapted from Kakeshita et al., 2011b)

hIFN-α2b (Kakeshita et al., 2011a).

Fig. 2. Construction of plasmids for production and secretion of heterologous proteins. The restriction sites used for each construction are indicated. P*xylA*, promoter of *xylA*; RBS, ribosome binding site; SP, signal peptide; Pro, propeptide.

Fig. 3. Western blot analysis of hIFN-α production by *B. subtilis* Dpr8 with pHKK3101 (AmyE SP-hIFN-α) or pHKK3201 (AmyE SP-Pro-hIFN-α). Samples were collected at 20 h after xylose induction, separated by 15% SDS-PAGE, and stained with Western blotting using anti hIFN-α2b polyclonal antibodies. Dpr8 with pHKK3101 (lanes 1 and 2); Dpr8 with pHKK3201 (lanes 3 and 4); 0.6% xylose induced (lanes 1 and 3), none induced (lanes 2 and 4), and commercially purified hIFN-α 10 ng (lane 5). Arrowheads indicate the positions of the Pro-hIFN-α2b and hIFN-α2b. (adapted from Kakeshita et al., 2011a)

Fig. 2. Construction of plasmids for production and secretion of heterologous proteins. The restriction sites used for each construction are indicated. P*xylA*, promoter of *xylA*; RBS,

Fig. 3. Western blot analysis of hIFN-α production by *B. subtilis* Dpr8 with pHKK3101 (AmyE SP-hIFN-α) or pHKK3201 (AmyE SP-Pro-hIFN-α). Samples were collected at 20 h after xylose induction, separated by 15% SDS-PAGE, and stained with Western blotting using anti hIFN-α2b polyclonal antibodies. Dpr8 with pHKK3101 (lanes 1 and 2); Dpr8 with pHKK3201 (lanes 3 and 4); 0.6% xylose induced (lanes 1 and 3), none induced (lanes 2 and 4), and commercially purified hIFN-α 10 ng (lane 5). Arrowheads indicate the positions of

the Pro-hIFN-α2b and hIFN-α2b. (adapted from Kakeshita et al., 2011a)

ribosome binding site; SP, signal peptide; Pro, propeptide.

We showed that the secretion production and activity of hIFN-α2b with propeptide increased by more than 3-fold, compared to that without propeptide. The amount of secreted hIFN-α2b with propeptide was 15mg /L. This result indicated that the propeptide of AmyE enhanced the secretion of hIFNα-2b (Fig. 3, Kakeshita et al., 2011a).

Fig. 4. Western blot analysis of hIFN-β production by *B. subtilis* Dpr8 with pHKK3111 (AmyE SP-hIFN-β) or pHKK3211 (AmyE SP-Pro hIFN-β). Samples were collected at 20 h after xylose induction, separated by 15% SDS-PAGE, and stained with Western blotting using anti hIFN-β polyclonal antibodies. Dpr8 with pHKK3111 (lanes 1 and 2); Dpr8 with pHKK3211 (lanes 3 and 4); 0.6% xylose induced (lanes 1 and 3), none induced (lanes 2 and 4), and commercially purified hIFN-β 50 ng (lane 5). Arrowheads indicate the positions of the Pro-hIFN-β and hIFN-β. (adapted from Kakeshita et al., 2011b)

In *L. lactis*, directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety (Le Loir et al., 2001). In hIFN-α2b with AmyE propeptide, the first 10 amino acid residues of this mature protein have a net charge of -1. On the other hand, hIFN-α2b without propeptide has a net charge of 0. In addition, we demonstrated that propeptide mutants of neutral or positive charge resulted in a reduction in the amount of secreted hIFN-α2b, compared with propeptides of negative charge. This result suggested that negative charges in the mature protein can enhance the secretion of hIFN-α2b (Kakeshita et al., 2011a).

We then indicated that the AmyE propeptide enhanced the secretion of the hIFN-β protein from *B. subtilis*, as well. The secretion production and activity of hIFN-β with propeptide increased by more than 4-fold (Fig. 4, Kakeshita et al., 2011b). The amount of secreted hIFN-

Improvement of Heterologous Protein Secretion by *Bacillus subtilis* 151

Fig. 5. Comparison of the amounts of secreted hIFN-β from *B. subtilis* D8C and D8PA, PrsA co-expressing strains. (a) Schematic representation of the gene structure around the amyE locus in the *B. subtilis* mutant strains D8PA and D8C. *PspoVG* and *prsA* represent the *B. subtilis*

chloramphenicol-resistant gene promoter and coding region, respectively. (b) Western blot analysis of PrsA protein from *B. subtilis* D8C, D8PA, and Dpr8. (c) Western blot analysis of hIFN-β production by *B. subtilis* D8C, D8PA, and Dpr8. D8C with pHKK3211 (lane 1); D8PA with pHKK3211 (lane 2); Dpr8 with pHKK3211 (lane 3). Arrowheads indicate the positions

The majority of bacterial secreted proteins are translocated across the cytoplasmic membrane via the Sec pathway, which acts on unfolded proteins using the energy provided by ATP hydrolysis (Tajalsma et al., 2000; Antelman et al., 2000). Recently, a novel and different secretion protein translocation pathway, the twin-arginine translocation (Tat) pathway was discovered (Santini et al., 1998; Berks et al., 2000; van Dijl et al., 2002). The bacterial twin-arginine translocation (Tat) machinery is able to transport folded proteins across the cytoplasmic membrane (Robinson et al., 2001). The Tat pathway might have advantages over the Sec pathway for the production of heterologous proteins, because many proteins fold tightly before they reach the Sec machinery, and thus cannot engage with it for

*B. subtilis* contains two substrate specific Tat systems, TatAyCy and TatAdCd. The TatAyCy translocase is required for translocation of YwbN. On the other hand, a TatAdCd translocase

translocates the phosphodiesterase PhoD (Jongbloed JD et al., 2002; Pop et al., 2002).

*spoVG* promoter and *B. subtilis PrsA*, respectively. *Pcat* and Cmr represent the

of Pro-IFN-β. (Adapted from Kakeshita et al., 2011b).

translocation across the cytoplasmic membrane.

**3. Tat pathway**

β with propeptide was 3.7mg /L. These results indicated that the propeptide of AmyE enhanced the secretion and extracellular production of a heterologous protein in *B. subtilis*.

#### **2.3 Deletion of the C-terminus of SecA**

In *B. subtilis*, most secreted proteins are translocated across the cytoplasmic membrane via the Sec system (Tjalsma et al., 2000; Tjalsma et al., 2004; Yamane et al., 2004). Nearly all of the components of the Sec system identified in *E. coli* have also been identified in *B. subtilis* and are biochemically well-characterized (van Wely et al., 2001; Harwood et al., 2008). Among these components, the peripheral membrane protein, SecA is considered to play a pivotal role in secretion. The SecYEG complex acts as a receptor for SecA, and functions as a preprotein conducting channel (Hartl et al., 1990; Fekkes et al., 1997). In *E. coli*, SecB is a molecular chaperone that functions in the post-translational protein translocation pathway, and binds to the C-terminal SecB binding site of *E. coli* SecA. In *B. subtilis*, this region of SecA is highly conserved. However, genome sequencing revealed that SecB is absent in *B. subtilis* (Kunst et al., 1997). *B. subtilis* Ffh interacts directly with SecA, and promotes the formation of soluble SecA-preprotein complexes (Bunai et al., 1999). These results suggest that the signal recognition particle (SRP) of *B. subtilis* not only acts as a targeting factor in co-translational translocation, but also stimulates the process of post-translocation across the membrane (Harwood & Cranenburgh, 2008; Ling et al., 2007; Tjalsma et al., 2000; Yamane et al., 2004). In additon, it has been shown that SecB binding site of *B. subtilis* SecA is not essential for viability and protein secretion (van Wely et al., 2000). The SecB binding site is connected by a C-terminal Linker (CTL) with the α-helical scaffold domain (HSD) in SecA. A cross-species comparison of the amino acid sequence of SecA revealed that the CTL is not well-conserved between *B. subtilis* and other species, including *E. coli*. We examined the effects of modifying the C-terminal region of SecA on growth and the extracellular production of heterologous proteins in *B. subtilis*, and demonstrated that the C-terminal domain (CTD) of SecA is not essential for viability or protein secretion. Furthermore, we showed that the productivity of hINF-α2b increased by 2.2-fold, compared to wild type SecA (Kakeshita et al., 2010). The crystal structure of *B. subtils* SecA indicated that CTL binds to the surface of NBF-I. The CTL-binding grove is a highly conserved and hydrophobic surface, and this grove is predicted to be one of the mature preprotein binding sites in SecA (Hunt et al., 2002). Therefore, deletion of the CTL of SecA is likely to affect SecA - preprotein interaction, and likely caused an increase in the secretion of heterologous proteins.

#### **2.4 Co-expression of PrsA**

PrsA is essential for viability and protein secretion. In protein secretion, PrsA is suggested to mediate protein folding at the late stage of secretion (Konitinen et al., 1991; Kontinen & Sarvas, 1993; Vitikainen et al., 2001). We examined the effect of co-expression of an extracytoplasmic molecular chaperone, PrsA. It is known that co-expression of an extracytoplasmic molecular chaperone, PrsA enhances the secretion of several model proteins: α amylase, Single-chain antibody (SCA), and recombinant Protective antigen (rPA) (Kontinen & Sarvas, 1993; Vitikainen et al., 2001; Wu et al., 1998; Williams et al., 2003).

We demonstrated that co-expression of PrsA can act in concert with the AmyE propeptide to enhance the secretion production of hIFN-β. The amount of secreted hIFN-β with propeptide was 5.5mg /L. (Fig. 5, Kakeshita et al., 2011b).

Fig. 5. Comparison of the amounts of secreted hIFN-β from *B. subtilis* D8C and D8PA, PrsA co-expressing strains. (a) Schematic representation of the gene structure around the amyE locus in the *B. subtilis* mutant strains D8PA and D8C. *PspoVG* and *prsA* represent the *B. subtilis spoVG* promoter and *B. subtilis PrsA*, respectively. *Pcat* and Cmr represent the chloramphenicol-resistant gene promoter and coding region, respectively. (b) Western blot analysis of PrsA protein from *B. subtilis* D8C, D8PA, and Dpr8. (c) Western blot analysis of hIFN-β production by *B. subtilis* D8C, D8PA, and Dpr8. D8C with pHKK3211 (lane 1); D8PA with pHKK3211 (lane 2); Dpr8 with pHKK3211 (lane 3). Arrowheads indicate the positions of Pro-IFN-β. (Adapted from Kakeshita et al., 2011b).

#### **3. Tat pathway**

150 Advances in Applied Biotechnology

β with propeptide was 3.7mg /L. These results indicated that the propeptide of AmyE enhanced the secretion and extracellular production of a heterologous protein in *B. subtilis*.

In *B. subtilis*, most secreted proteins are translocated across the cytoplasmic membrane via the Sec system (Tjalsma et al., 2000; Tjalsma et al., 2004; Yamane et al., 2004). Nearly all of the components of the Sec system identified in *E. coli* have also been identified in *B. subtilis* and are biochemically well-characterized (van Wely et al., 2001; Harwood et al., 2008). Among these components, the peripheral membrane protein, SecA is considered to play a pivotal role in secretion. The SecYEG complex acts as a receptor for SecA, and functions as a preprotein conducting channel (Hartl et al., 1990; Fekkes et al., 1997). In *E. coli*, SecB is a molecular chaperone that functions in the post-translational protein translocation pathway, and binds to the C-terminal SecB binding site of *E. coli* SecA. In *B. subtilis*, this region of SecA is highly conserved. However, genome sequencing revealed that SecB is absent in *B. subtilis* (Kunst et al., 1997). *B. subtilis* Ffh interacts directly with SecA, and promotes the formation of soluble SecA-preprotein complexes (Bunai et al., 1999). These results suggest that the signal recognition particle (SRP) of *B. subtilis* not only acts as a targeting factor in co-translational translocation, but also stimulates the process of post-translocation across the membrane (Harwood & Cranenburgh, 2008; Ling et al., 2007; Tjalsma et al., 2000; Yamane et al., 2004). In additon, it has been shown that SecB binding site of *B. subtilis* SecA is not essential for viability and protein secretion (van Wely et al., 2000). The SecB binding site is connected by a C-terminal Linker (CTL) with the α-helical scaffold domain (HSD) in SecA. A cross-species comparison of the amino acid sequence of SecA revealed that the CTL is not well-conserved between *B. subtilis* and other species, including *E. coli*. We examined the effects of modifying the C-terminal region of SecA on growth and the extracellular production of heterologous proteins in *B. subtilis*, and demonstrated that the C-terminal domain (CTD) of SecA is not essential for viability or protein secretion. Furthermore, we showed that the productivity of hINF-α2b increased by 2.2-fold, compared to wild type SecA (Kakeshita et al., 2010). The crystal structure of *B. subtils* SecA indicated that CTL binds to the surface of NBF-I. The CTL-binding grove is a highly conserved and hydrophobic surface, and this grove is predicted to be one of the mature preprotein binding sites in SecA (Hunt et al., 2002). Therefore, deletion of the CTL of SecA is likely to affect SecA - preprotein interaction, and

likely caused an increase in the secretion of heterologous proteins.

& Sarvas, 1993; Vitikainen et al., 2001; Wu et al., 1998; Williams et al., 2003).

propeptide was 5.5mg /L. (Fig. 5, Kakeshita et al., 2011b).

PrsA is essential for viability and protein secretion. In protein secretion, PrsA is suggested to mediate protein folding at the late stage of secretion (Konitinen et al., 1991; Kontinen & Sarvas, 1993; Vitikainen et al., 2001). We examined the effect of co-expression of an extracytoplasmic molecular chaperone, PrsA. It is known that co-expression of an extracytoplasmic molecular chaperone, PrsA enhances the secretion of several model proteins: α amylase, Single-chain antibody (SCA), and recombinant Protective antigen (rPA) (Kontinen

We demonstrated that co-expression of PrsA can act in concert with the AmyE propeptide to enhance the secretion production of hIFN-β. The amount of secreted hIFN-β with

**2.4 Co-expression of PrsA**

**2.3 Deletion of the C-terminus of SecA**

The majority of bacterial secreted proteins are translocated across the cytoplasmic membrane via the Sec pathway, which acts on unfolded proteins using the energy provided by ATP hydrolysis (Tajalsma et al., 2000; Antelman et al., 2000). Recently, a novel and different secretion protein translocation pathway, the twin-arginine translocation (Tat) pathway was discovered (Santini et al., 1998; Berks et al., 2000; van Dijl et al., 2002). The bacterial twin-arginine translocation (Tat) machinery is able to transport folded proteins across the cytoplasmic membrane (Robinson et al., 2001). The Tat pathway might have advantages over the Sec pathway for the production of heterologous proteins, because many proteins fold tightly before they reach the Sec machinery, and thus cannot engage with it for translocation across the cytoplasmic membrane.

*B. subtilis* contains two substrate specific Tat systems, TatAyCy and TatAdCd. The TatAyCy translocase is required for translocation of YwbN. On the other hand, a TatAdCd translocase translocates the phosphodiesterase PhoD (Jongbloed JD et al., 2002; Pop et al., 2002).

Improvement of Heterologous Protein Secretion by *Bacillus subtilis* 153

pHKK3101 (AmyE signal), pHKK4004 (WprA), pHKK4005 (LipA), and pHKK4006 (WapA) by Western blot and immunoblot. This result suggested that the obtained three

signal peptides (WprA, LipA, WapA) directed efficient secretion expression.

Fig. 7. Comparison of the amounts of secreted hIFN-α using the Twin arginine signal peptides from *B. subtilis* Dpr8. (a) Western blot analysis of hIFN-α production in *B. subtilis* Dpr8 harboring seven recombinants. Cells were grown at 30 °C in 2xL medium. Samples were collected at 20 h after xylose induction, separated by 15% SDS-PAGE, and subjected to Western blotting using anti hIFN-β polyclonal antibodies. Protein samples present in the supernatant (lanes 1, 2, 3, 4, 5, and 6) and cell fractions (lanes 7, 8, 9, 10, 11, and 12) of stationary-phase cultures were prepared by centrifugation, analyzed by SDS-PAGE, and

immunodetected with anti-hIFN-α antibodies. Dpr8/pHKK3101 (lanes 1 and 8);

4 and 11); Dpr8/pHKK4004 (lanes 5 and 12); Dpr8/pHKK4005 (lanes 6 and 13);

three experiments, and error bars represent standard error.

Dpr8/pHKK4001 (lanes 2 and 9); Dpr8/pHKK4002 (lanes 3 and 10); Dpr8/pHKK4003 (lanes

Dpr8/pHKK4006 (lanes 7 and 14); precursor, pre hIFN-α; mature, hIFN-α. S, supernatant; C, cell fractions. (b) Quantification of secreted hIFN-α mature form in the culture medium and cell fraction. The hIFN-α production corresponding to the supernatant of *B. subtilis* Dpr8 carrying pHKK3101 (AmyE signal peptide) was set as 100%. Data represent the mean of

#### **3.1 Twin-arginine signal peptide**

Proteins are targeted to the Tat pathway by tripartite N-terminal signal peptides, the aminoterminal portion (n region) of which contain a conserved twin-arginine (RR) motif (R-R-X-#- #, where # is a hydrophobic residue).

In a previous study by Jongbloed et al., a database search for the presence of this motif in amino-terminal protein sequences identified a total number of 27 putative RR-signal peptides.

Fig. 6. Schematic representation of the signal sequences used for secretion of human Interferon-α in *B. subtilis*. Schematic structure of the proteins encoded by each indicated plasmid. The twin-arginine motif is boxed, and the residues at positions -3 to -1 relative to the predicted SPase I cleavage site are underlined. The six base pairs of the KpnI site add the amino acids Gly–Thr to the end of each signal peptide coding sequence; therefore, in the table, each sequence ends with GT. Numbers under the signal peptides refer to the respective locations of the encoded amino acid residues.

We therefore selected six candidate Tat signal peptides, shown in Fig. 6, from the list generated by Jongbloed et al. for testing in the hIFN-α secreted assay. To determine the secretion ability for hINF-α2b, the six signal peptide genes considered to belong to the Tat pathway of *B. subtilis* were PCR-amplified. The PCR-amplified signal peptide genes were inserted upstream of the hIFN-α mature peptide gene in pHKK3101, yielding six secretion expression vectors. pHKK3101 expressing hIFN-α with the AmyE signal peptide, as the Sec-type signal peptide, was used as the control plasmid. The resultant recombinants were transformed into *B. subtilis* Dpr8, respectively, and the secretion expression of hIFNα mediated by these signal peptides was detected by immunoblotting analysis. The hIFNα was expressed in these strains and hIFN-α production was induced with the addition of 0.6% of xylose to the exponentially growing cultures (OD660 = 0.3), and both culture supernatants and intracellular lysates were analyzed as described in Kakeshita et al. (2010). As shown in Fig. 7a, in the extracellular fraction, only one band corresponding to mature protein (16 kDa) was detected for the samples of *B. subtilis* Dpr8 cells harboring

Proteins are targeted to the Tat pathway by tripartite N-terminal signal peptides, the aminoterminal portion (n region) of which contain a conserved twin-arginine (RR) motif (R-R-X-#-

In a previous study by Jongbloed et al., a database search for the presence of this motif in amino-terminal protein sequences identified a total number of 27 putative RR-signal

Fig. 6. Schematic representation of the signal sequences used for secretion of human Interferon-α in *B. subtilis*. Schematic structure of the proteins encoded by each indicated plasmid. The twin-arginine motif is boxed, and the residues at positions -3 to -1 relative to the predicted SPase I cleavage site are underlined. The six base pairs of the KpnI site add the amino acids Gly–Thr to the end of each signal peptide coding sequence; therefore, in the table, each sequence ends with GT. Numbers under the signal peptides refer to the

We therefore selected six candidate Tat signal peptides, shown in Fig. 6, from the list generated by Jongbloed et al. for testing in the hIFN-α secreted assay. To determine the secretion ability for hINF-α2b, the six signal peptide genes considered to belong to the Tat pathway of *B. subtilis* were PCR-amplified. The PCR-amplified signal peptide genes were inserted upstream of the hIFN-α mature peptide gene in pHKK3101, yielding six secretion expression vectors. pHKK3101 expressing hIFN-α with the AmyE signal peptide, as the Sec-type signal peptide, was used as the control plasmid. The resultant recombinants were transformed into *B. subtilis* Dpr8, respectively, and the secretion expression of hIFNα mediated by these signal peptides was detected by immunoblotting analysis. The hIFNα was expressed in these strains and hIFN-α production was induced with the addition of 0.6% of xylose to the exponentially growing cultures (OD660 = 0.3), and both culture supernatants and intracellular lysates were analyzed as described in Kakeshita et al. (2010). As shown in Fig. 7a, in the extracellular fraction, only one band corresponding to mature protein (16 kDa) was detected for the samples of *B. subtilis* Dpr8 cells harboring

respective locations of the encoded amino acid residues.

**3.1 Twin-arginine signal peptide**

#, where # is a hydrophobic residue).

peptides.

pHKK3101 (AmyE signal), pHKK4004 (WprA), pHKK4005 (LipA), and pHKK4006 (WapA) by Western blot and immunoblot. This result suggested that the obtained three signal peptides (WprA, LipA, WapA) directed efficient secretion expression.

Fig. 7. Comparison of the amounts of secreted hIFN-α using the Twin arginine signal peptides from *B. subtilis* Dpr8. (a) Western blot analysis of hIFN-α production in *B. subtilis* Dpr8 harboring seven recombinants. Cells were grown at 30 °C in 2xL medium. Samples were collected at 20 h after xylose induction, separated by 15% SDS-PAGE, and subjected to Western blotting using anti hIFN-β polyclonal antibodies. Protein samples present in the supernatant (lanes 1, 2, 3, 4, 5, and 6) and cell fractions (lanes 7, 8, 9, 10, 11, and 12) of stationary-phase cultures were prepared by centrifugation, analyzed by SDS-PAGE, and immunodetected with anti-hIFN-α antibodies. Dpr8/pHKK3101 (lanes 1 and 8); Dpr8/pHKK4001 (lanes 2 and 9); Dpr8/pHKK4002 (lanes 3 and 10); Dpr8/pHKK4003 (lanes 4 and 11); Dpr8/pHKK4004 (lanes 5 and 12); Dpr8/pHKK4005 (lanes 6 and 13); Dpr8/pHKK4006 (lanes 7 and 14); precursor, pre hIFN-α; mature, hIFN-α. S, supernatant; C, cell fractions. (b) Quantification of secreted hIFN-α mature form in the culture medium and cell fraction. The hIFN-α production corresponding to the supernatant of *B. subtilis* Dpr8 carrying pHKK3101 (AmyE signal peptide) was set as 100%. Data represent the mean of three experiments, and error bars represent standard error.

Improvement of Heterologous Protein Secretion by *Bacillus subtilis* 155

Fig. 8. Comparison of the amounts of secreted hIFN-α from *B. subtilis* Dpr8 and Tat overexpressing strains. (a) Schematic representation of the gene structure around the *amyE* locus in the *B. subtilis* D8tatD and D8tatY mutant strain genomes. Construction of strains D8tatD and D8tatY was by double crossover integration of plasmids pHKK2001 (*tatAd-Cd*) and pHKK2002 (*tatAy-Cy*) into the *amyE* locus of *B. subtilis* Dpr8. The resulting strain contains the native *phoD-tatAd-tatCd* locus, as well as one copy of *tatAd-Cd* and *tatAy-Cy* under the control of the P*spoVG* promoter. The stem-loop structures and the bent arrows indicate the putative Rho-independent terminators and promoters, respectively. (b) Western

blot analysis of hIFN-α production by *B. subtilis* Dpr8, D8tatD, and D8tatY (carrying pHKK3101, pHKK4004, pHKK4005, or pHKK4006) was performed in the same manner as for hIFN-α. (c) Quantification of secreted hIFN-α in mature form in the culture medium. The hIFN-α production corresponding to the *B. subtilis* Dpr8 strain was set as 100%. Data represent the mean of three experiments, and error bars represent standard error.

Especially, WapA demonstrated the highest efficiency of hIFN-α secretion expression, which was 1.5-fold as high as the Sec dependent signal peptide, AmyE (Fig. 7b).

However, No hIFN-α was detected in the supernatants of Dpr8/pHKK4001 (YvhJ), Dpr8/pHKK4002 (YwbN), or Dpr8/pHKK4003 (PhoD). In the intracellular lysates of Dpr8/pHKK3101, Dpr8/pHKK4004, Dpr8/pHKK4005, and Dpr8/pHKK4006, two bands were detected. As deduced from the molecular mass of each band, these bands ware assigned to the unprocessed precursor (17 kDa) and the mature protein (16 kDa), respectively. On the other hand, only one band corresponding to the unprocessed protein was detected for the samples of Dpr8/pHKK4001 (YvhJ), Dpr8/pHKK4002 (YwbN), and Dpr8/pHKK4003 (PhoD).

These results suggested that the three obtained signal peptides, YvhJ, YwbN, and PhoD cannot be secreted hIFN-α2b into the supernatant.

#### **3.2 Co-expression of the tat system**

We examined the effect of co-expression of the Tat-machinary, TatAd/Cd or TatAy/Cy. To examine the effects of the co-expression of *B. subtilis* tat genes on hIFN-α secretion, we constructed TatAd/TatCd and TatAy/TatCy under the control of the *spoVG* promoter in plasmids. It is known that the *spoVG* promoter is a powerful promoter (Zuber & Losick 1983). The resulting constructs were subsequently integrated into the chromosome of *B. subtilis* strain Dpr8 via a double crossover event at the *amyE* locus, leaving the native tat genes intact (Fig. 8a).

The resultant strains, D8tatD and D8tatY were transformed with pHKK3101, pHKK4001, pHKK4002, pHKK4003, pHKK4004, pHKK4005, and pHKK4006 for expression of hIFN-α.

As shown in Fig. 8b and c, when the LipA signal peptide was fused to hIFN-α, a densitometric analysis of the western blotting demonstrated that the amounts of hIFN-α secreted by D8tatD and D8tatY were increased by roughly 2-fold, compared with that in strain Dpr8 (Fig. 8c). When the WprA signal peptide was fused to hIFN-α, in D8tatD, the amount of secreted hIFNα was increased by 71% compared with that in the parental strain, Dpr8, whereas the enhanced production of hIFN-α increased by 29%. On the other hand, When the WapA signal peptide was fused to hIFN-α, the amounts of hIFN-α secreted by D8tatD and D8tatY were increased by only 10-20%, compared with that in strain Dpr8 (Fig. 8c). Then, when the AmyE signal peptide was fused to hIFN-α, the amounts of hIFN-α secreted by D8tatD and D8tatY were increased by 37% and 25%, respectively compared with that in strain Dpr8 (Fig. 8c). Therefore, WapA signal peptide and AmyE signal peptide are not able to enhance of secretion by co–expression of Tat system. In addition, when the YvhJ, YwbN, and PhoD signal peptides, respectively were fused to hIFN-α, the bands of hIFN-α secreted by D8tatD and D8tatY could not be detected in the resulting supernatants (data not shown).

We demonstrated that co-expression of TatAd/Cd or TatAy/Cy with LipA signal peptide can act in concert to enhance the secretion production of hIFN-α. In addition, WprA signal peptide was enhanced the secretion production of hIFNα by co-expression of TatAd/Cd, not TatAy/Cy. On the other hands, AmyE signal peptide and WapA peptide are Tat pathway independent.

Especially, WapA demonstrated the highest efficiency of hIFN-α secretion expression,

However, No hIFN-α was detected in the supernatants of Dpr8/pHKK4001 (YvhJ), Dpr8/pHKK4002 (YwbN), or Dpr8/pHKK4003 (PhoD). In the intracellular lysates of Dpr8/pHKK3101, Dpr8/pHKK4004, Dpr8/pHKK4005, and Dpr8/pHKK4006, two bands were detected. As deduced from the molecular mass of each band, these bands ware assigned to the unprocessed precursor (17 kDa) and the mature protein (16 kDa), respectively. On the other hand, only one band corresponding to the unprocessed protein was detected for the samples of Dpr8/pHKK4001 (YvhJ), Dpr8/pHKK4002 (YwbN), and

These results suggested that the three obtained signal peptides, YvhJ, YwbN, and PhoD

We examined the effect of co-expression of the Tat-machinary, TatAd/Cd or TatAy/Cy. To examine the effects of the co-expression of *B. subtilis* tat genes on hIFN-α secretion, we constructed TatAd/TatCd and TatAy/TatCy under the control of the *spoVG* promoter in plasmids. It is known that the *spoVG* promoter is a powerful promoter (Zuber & Losick 1983). The resulting constructs were subsequently integrated into the chromosome of *B. subtilis* strain Dpr8 via a double crossover event at the *amyE* locus, leaving the native tat

The resultant strains, D8tatD and D8tatY were transformed with pHKK3101, pHKK4001, pHKK4002, pHKK4003, pHKK4004, pHKK4005, and pHKK4006 for expression of hIFN-α.

As shown in Fig. 8b and c, when the LipA signal peptide was fused to hIFN-α, a densitometric analysis of the western blotting demonstrated that the amounts of hIFN-α secreted by D8tatD and D8tatY were increased by roughly 2-fold, compared with that in strain Dpr8 (Fig. 8c). When the WprA signal peptide was fused to hIFN-α, in D8tatD, the amount of secreted hIFNα was increased by 71% compared with that in the parental strain, Dpr8, whereas the enhanced production of hIFN-α increased by 29%. On the other hand, When the WapA signal peptide was fused to hIFN-α, the amounts of hIFN-α secreted by D8tatD and D8tatY were increased by only 10-20%, compared with that in strain Dpr8 (Fig. 8c). Then, when the AmyE signal peptide was fused to hIFN-α, the amounts of hIFN-α secreted by D8tatD and D8tatY were increased by 37% and 25%, respectively compared with that in strain Dpr8 (Fig. 8c). Therefore, WapA signal peptide and AmyE signal peptide are not able to enhance of secretion by co–expression of Tat system. In addition, when the YvhJ, YwbN, and PhoD signal peptides, respectively were fused to hIFN-α, the bands of hIFN-α secreted by D8tatD and D8tatY could

We demonstrated that co-expression of TatAd/Cd or TatAy/Cy with LipA signal peptide can act in concert to enhance the secretion production of hIFN-α. In addition, WprA signal peptide was enhanced the secretion production of hIFNα by co-expression of TatAd/Cd, not TatAy/Cy. On the other hands, AmyE signal peptide and WapA peptide are Tat

which was 1.5-fold as high as the Sec dependent signal peptide, AmyE (Fig. 7b).

Dpr8/pHKK4003 (PhoD).

genes intact (Fig. 8a).

pathway independent.

cannot be secreted hIFN-α2b into the supernatant.

not be detected in the resulting supernatants (data not shown).

**3.2 Co-expression of the tat system**

Fig. 8. Comparison of the amounts of secreted hIFN-α from *B. subtilis* Dpr8 and Tat overexpressing strains. (a) Schematic representation of the gene structure around the *amyE* locus in the *B. subtilis* D8tatD and D8tatY mutant strain genomes. Construction of strains D8tatD and D8tatY was by double crossover integration of plasmids pHKK2001 (*tatAd-Cd*) and pHKK2002 (*tatAy-Cy*) into the *amyE* locus of *B. subtilis* Dpr8. The resulting strain contains the native *phoD-tatAd-tatCd* locus, as well as one copy of *tatAd-Cd* and *tatAy-Cy* under the control of the P*spoVG* promoter. The stem-loop structures and the bent arrows indicate the putative Rho-independent terminators and promoters, respectively. (b) Western blot analysis of hIFN-α production by *B. subtilis* Dpr8, D8tatD, and D8tatY (carrying pHKK3101, pHKK4004, pHKK4005, or pHKK4006) was performed in the same manner as for hIFN-α. (c) Quantification of secreted hIFN-α in mature form in the culture medium. The hIFN-α production corresponding to the *B. subtilis* Dpr8 strain was set as 100%. Data represent the mean of three experiments, and error bars represent standard error.

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### **4. Conclusions**

In recent years, considerable efforts have been targeted at developing *B. subtilis* as a host for the production of heterologous proteins. However, the secretion of heterologous proteins from eukaryotes by these species produces small yields and is frequently inefficient. Initially, we considered the major problem to be the presence of high levels of extracellular protease in *B. subtilis*. Nevertheless, even after obtaining many depleted protease strains, the problem of inefficient secretion was not resolved. Currently, it is considered that the largest problem is the detection of the pre-mature form of human protein in cell lysate, when human proteins with signal peptide are over expressed in *B. subtilis* (Fig. 7a). Normally, the pre-mature forms of target secretion proteins are not detected in cell lysates. If the premature form of target a secretion protein is detected, it indicates a problem in the secretion pathway, for example, non-functional or depleted SecA, SecY, Ffh, or FtsY (Sadaie et al. 1991; Takamatsu et al., 1992; Honda et al., 1993; Oguro et al., 1995; Tjalsma et al., 2000; Tjalsma et al., 2004; Yamane et al., 2004). Therefore, we must solve this primary problem, which is the accumulation of the precursor of human proteins in *B. subtilis* cells.

We indicated that the propeptide of AmyE enhanced the secretion of the extracellular production of a heterologous protein in *B. subtilis*. In *L. lactis*, the nine-residue synthetic propeptide, LEISSTCDA, which is fused immediately after the signal peptide cleavage site, is known to enhance heterologous protein secretion (Le Loir et al., 1998). In addition, LEISSTCDA enhances secretion efficiency (Le Loir et al., 2001). Therefore, it is considered that a short type propeptide may be one answer to improve the accumulation of precursor.

On the other hand, we indicated that the deletion of the C-terminal domain of SecA enhanced the secretion of heterologous proteins. *secA* is an essential gene, and SecA is considered to play a pivotal role in secretion (Sadaie et al. 1991; Takamatsu et al., 1992; Tjalsma et al., 2000; Tjalsma et al., 2004; Yamane et al., 2004). In addition, we exhibited that the co-expression of PrsA or the Tat system can be able to enhance the secretion production. In the future, it may be necessary to modify the components of the secretion machinery for higher secretion efficiency.

#### **5. Acknowledgments**

We are grateful to Naotake Ogasawara, Junichi Sekiguchi, Fujio Kawamura, Kunio Yamane and members of MGP group in Kao Corporation for valuable discussions.

This work is the subproject, 'Development of a Technology for Creation of a Host Cell' included within the industrial technology project, 'Development of a Generic Technology for Production Process Starting Productive Function' of the Ministry of Economy, Trade and Industry, entrusted by the New Energy and Industrial Technology Development Organization (NEDO), Japan.

#### **6. References**

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In recent years, considerable efforts have been targeted at developing *B. subtilis* as a host for the production of heterologous proteins. However, the secretion of heterologous proteins from eukaryotes by these species produces small yields and is frequently inefficient. Initially, we considered the major problem to be the presence of high levels of extracellular protease in *B. subtilis*. Nevertheless, even after obtaining many depleted protease strains, the problem of inefficient secretion was not resolved. Currently, it is considered that the largest problem is the detection of the pre-mature form of human protein in cell lysate, when human proteins with signal peptide are over expressed in *B. subtilis* (Fig. 7a). Normally, the pre-mature forms of target secretion proteins are not detected in cell lysates. If the premature form of target a secretion protein is detected, it indicates a problem in the secretion pathway, for example, non-functional or depleted SecA, SecY, Ffh, or FtsY (Sadaie et al. 1991; Takamatsu et al., 1992; Honda et al., 1993; Oguro et al., 1995; Tjalsma et al., 2000; Tjalsma et al., 2004; Yamane et al., 2004). Therefore, we must solve this primary problem,

which is the accumulation of the precursor of human proteins in *B. subtilis* cells.

We indicated that the propeptide of AmyE enhanced the secretion of the extracellular production of a heterologous protein in *B. subtilis*. In *L. lactis*, the nine-residue synthetic propeptide, LEISSTCDA, which is fused immediately after the signal peptide cleavage site, is known to enhance heterologous protein secretion (Le Loir et al., 1998). In addition, LEISSTCDA enhances secretion efficiency (Le Loir et al., 2001). Therefore, it is considered that a short type propeptide may be one answer to improve the accumulation of precursor. On the other hand, we indicated that the deletion of the C-terminal domain of SecA enhanced the secretion of heterologous proteins. *secA* is an essential gene, and SecA is considered to play a pivotal role in secretion (Sadaie et al. 1991; Takamatsu et al., 1992; Tjalsma et al., 2000; Tjalsma et al., 2004; Yamane et al., 2004). In addition, we exhibited that the co-expression of PrsA or the Tat system can be able to enhance the secretion production. In the future, it may be necessary to modify the components of the secretion machinery for

We are grateful to Naotake Ogasawara, Junichi Sekiguchi, Fujio Kawamura, Kunio Yamane

This work is the subproject, 'Development of a Technology for Creation of a Host Cell' included within the industrial technology project, 'Development of a Generic Technology for Production Process Starting Productive Function' of the Ministry of Economy, Trade and Industry, entrusted by the New Energy and Industrial Technology Development

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**4. Conclusions**

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**5. Acknowledgments**

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**8**

*Japan*

**Approaches for Improving Protein**

Katsutoshi Ara1, Katsuya Ozaki1 and Junichi Sekiguchi2

*Bacillus subtilis* **Host Strains**

*1Biological Science Laboratories, Kao Corporation, 2Department of Bioscience and Textile Technology,*

**Production in Multiple Protease-Deficient**

Takeko Kodama1\*, Kenji Manabe1, Yasushi Kageyama1, Shenghao Liu1,

*Interdisciplinary Graduate School of Science and Technology, Shinshu University,*

*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

**2. Inhibition of proteolysis of heterologous and nature proteins after the**

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

**translocation process by inactivation of multiple proteases**

**1. Introduction**

the host.

### **Approaches for Improving Protein Production in Multiple Protease-Deficient**  *Bacillus subtilis* **Host Strains**

Takeko Kodama1\*, Kenji Manabe1, Yasushi Kageyama1, Shenghao Liu1, Katsutoshi Ara1, Katsuya Ozaki1 and Junichi Sekiguchi2 *1Biological Science Laboratories, Kao Corporation, 2Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, Japan*

#### **1. Introduction**

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*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

Approaches for Improving Protein Production in

membrane-bound (HtrA and HtrB).

**protein**

following sections.

**deletion in** *B***.** *subtilis*

introduction of multiple mutations.

Multiple Protease-Deficient *Bacillus Subtilis* Host Strains 165

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

**2.1 The intracellular protease, AprX is involved in degradation of a heterologous**

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

**2.1.1 Construction of an eight-extracellular-protease-deficient mutant by marker-free**

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

**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 (midstationary 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

Fig. 1. Strategy for construction of a ∆epr mutant. (A) Construction of a chloramphenicolresistance (Cmr) plasmid, pUC118-Cmr∆epr. Fragment 3 was amplified with fragment a (containing the repU promoter of pUB110) and fragment b (containing the chloramphenicol resistance gene of pC194), and primers 7 and 8. Fragment 4 was amplified with fragments 1, 2, 3, and primers 5 and 6. The amplified fragment 4 was prepared by blunting and kination, and then cloned into the *Sma*I site of pUC118 to generate pUC118-Cmr∆epr. (B) Construction of the ∆epr mutant. *B*. *subtilis* 168 cells were transformed with pUC118-Cmr∆epr, followed by selection for chloramphenicol resistance, obtaining Cmr∆epr. To obtain a ∆epr mutant (deleted chloramphenicol-resistance cassette), the ampicillin concentration method was used (Kodama et al., 2007a). The chloramphenicol-sensitive (Cms) ∆epr mutant was confirmed by PCR using primers 5 and 4.

Fig. 1. Strategy for construction of a ∆epr mutant. (A) Construction of a chloramphenicolresistance (Cmr) plasmid, pUC118-Cmr∆epr. Fragment 3 was amplified with fragment a (containing the repU promoter of pUB110) and fragment b (containing the chloramphenicol resistance gene of pC194), and primers 7 and 8. Fragment 4 was amplified with fragments 1, 2, 3, and primers 5 and 6. The amplified fragment 4 was prepared by blunting and kination, and then cloned into the *Sma*I site of pUC118 to generate pUC118-Cmr∆epr. (B) Construction of the ∆epr mutant. *B*. *subtilis* 168 cells were transformed with pUC118-Cmr∆epr, followed by selection for chloramphenicol resistance, obtaining Cmr∆epr. To obtain a ∆epr mutant (deleted chloramphenicol-resistance cassette), the ampicillin concentration method was used (Kodama et al., 2007a). The chloramphenicol-sensitive (Cms) ∆epr mutant was confirmed by

PCR using primers 5 and 4.

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 (midstationary 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

Approaches for Improving Protein Production in

Multiple Protease-Deficient *Bacillus Subtilis* Host Strains 167

Fig. 3. Zymography of AprX-FLAG proteases. (A) Lane 1, 168; lane 2, 168/AprX-FLAG. (B) Lane 1, Dpr8; lane 2, Dpr8/AprX-FLAG. Proteins from the supernatants of the 75-h cultures (equivalent to 0.3 µl) were applied to the lanes for panels A and B. Arrowheads indicate the

**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

Fig. 4. Western blot analysis of AprX-FLAG. Western blot analysis was carried out to detect AprX-FLAG in the 168/AprX-FLAG strain with the anti-FLAG antibody. Proteins of cells (lanes 1-3) and supernatants (lanes 4-6) from 168/AprX-FLAG (0.02 OD600 units) were prepared as described in Materials and Methods. The arrowhead indicates the position of

AprX-FLAG. The times of harvest of cells and supernatants are shown at the top.

positions of AprX (closed symbol) and AprX-FLAG (open symbol).

the culture medium during the late stationary phase due to cell lysis.

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 gelatindegrading 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.

Fig. 2. (A) Cells from the wild-type were cultured in modified 2xL broth at 30ºC. (B) Protein samples were prepared from the supernatants of the wild-type, cultured for various incubation times. Lane 1, 8 h culture; lane 2, 25 h; lane 3, 50 h; lane 4, 75 h. (C). Protein samples were prepared from the supernatants of the protease-deficient mutants after a 75 h culture. Lane 1, 168; lane 2, Dpr8; lane 3, AprXdd; lane 4, KA8AX. (D) PMSF or EDTA (2 mM each) was added to the supernatants of Dpr8 after a 75 h culture. Lane 1, Control (no addition); lane 2, addition of 2 mM PMSF; lane 3, addition of 2 mM EDTA. The samples were analyzed on SDS-12% polyacrylamide gels with 0.1% (w/v) gelatin. Proteins from the culture supernatants (equivalent to 0.3 µl) were applied to each lane for panels B, C, and D.

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 gelatindegrading 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

Fig. 2. (A) Cells from the wild-type were cultured in modified 2xL broth at 30ºC. (B) Protein samples were prepared from the supernatants of the wild-type, cultured for various incubation times. Lane 1, 8 h culture; lane 2, 25 h; lane 3, 50 h; lane 4, 75 h. (C). Protein samples were prepared from the supernatants of the protease-deficient mutants after a 75 h culture. Lane 1, 168; lane 2, Dpr8; lane 3, AprXdd; lane 4, KA8AX. (D) PMSF or EDTA (2 mM each) was added to the supernatants of Dpr8 after a 75 h culture. Lane 1, Control (no addition); lane 2, addition of 2 mM PMSF; lane 3, addition of 2 mM EDTA. The samples were analyzed on SDS-12% polyacrylamide gels with 0.1% (w/v) gelatin. Proteins from the culture supernatants (equivalent to 0.3 µl) were applied to each lane for

culture of *B*. *subtilis* strains.

panels B, C, and D.

Fig. 3. Zymography of AprX-FLAG proteases. (A) Lane 1, 168; lane 2, 168/AprX-FLAG. (B) Lane 1, Dpr8; lane 2, Dpr8/AprX-FLAG. Proteins from the supernatants of the 75-h cultures (equivalent to 0.3 µl) were applied to the lanes for panels A and B. Arrowheads indicate the positions of AprX (closed symbol) and AprX-FLAG (open symbol).

#### **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.

Fig. 4. Western blot analysis of AprX-FLAG. Western blot analysis was carried out to detect AprX-FLAG in the 168/AprX-FLAG strain with the anti-FLAG antibody. Proteins of cells (lanes 1-3) and supernatants (lanes 4-6) from 168/AprX-FLAG (0.02 OD600 units) were prepared as described in Materials and Methods. The arrowhead indicates the position of AprX-FLAG. The times of harvest of cells and supernatants are shown at the top.

Approaches for Improving Protein Production in

membrane-bound proteases involved in protein degradation.

Multiple Protease-Deficient *Bacillus Subtilis* Host Strains 169

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

Fig. 5. Western blot analysis of the α-amylase-A522-PreS2 hybrid protein in the extracellular fractions of Dpr7, Dpr8, and KA8AX. (A) Western blot analysis was carried out to detect αamylase-A522-PreS2 with the anti-PreS2 antibody. Culture supernatants from Dpr7 (lanes 1-3), Dpr8 (lanes 4-6), and KA8AX (lanes 7-9) were collected after 25, 50 h, and 75 h of cultivation, and subjected to Tricine-SDS-PAGE and Western blotting, as described in the Materials and Methods. Proteins from the culture supernatants (equivalent to 1 µl) were applied to each lane. The arrowhead indicates the position of α-amylase-A522-PreS2. The times of harvest of supernatants are shown at the top. (B) The relative α-amylase-A522-PreS2 protein amounts were compared on the basis of band intensities on Western blots (the amount of α-amylase-A522-PreS2 at 50 h in the Dpr8 strain was set to 100%). The presented results are the average of three individual experiments. Error bars correspond to the standard errors of the means

(SEM). Lane numbers in panel A correspond to those in panel B.

#### **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. αamylase-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 αamylase-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

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. αamylase-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

**2.1.4 AprX involved in degradation of the** α**-amylase-A522-PreS2 hybrid protein**

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 αamylase-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.

Fig. 5. Western blot analysis of the α-amylase-A522-PreS2 hybrid protein in the extracellular fractions of Dpr7, Dpr8, and KA8AX. (A) Western blot analysis was carried out to detect αamylase-A522-PreS2 with the anti-PreS2 antibody. Culture supernatants from Dpr7 (lanes 1-3), Dpr8 (lanes 4-6), and KA8AX (lanes 7-9) were collected after 25, 50 h, and 75 h of cultivation, and subjected to Tricine-SDS-PAGE and Western blotting, as described in the Materials and Methods. Proteins from the culture supernatants (equivalent to 1 µl) were applied to each lane. The arrowhead indicates the position of α-amylase-A522-PreS2. The times of harvest of supernatants are shown at the top. (B) The relative α-amylase-A522-PreS2 protein amounts were compared on the basis of band intensities on Western blots (the amount of α-amylase-A522-PreS2 at 50 h in the Dpr8 strain was set to 100%). The presented results are the average of three individual experiments. Error bars correspond to the standard errors of the means (SEM). Lane numbers in panel A correspond to those in panel B.

Approaches for Improving Protein Production in

HtrB, and vice versa (Noone et al., 2001).

Relative activity (%)

Multiple Protease-Deficient *Bacillus Subtilis* Host Strains 171