Accepted Manuscript Title: A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis Author: Ruangurai Promchai Boonhiang Promdonkoy Sutipa Tanapongpipat Wonnop Visessanguan Lily Eurwilaichitr Plearnpis Luxananil PII: DOI: Reference:

S0168-1656(16)30061-X http://dx.doi.org/doi:10.1016/j.jbiotec.2016.02.019 BIOTEC 7413

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

10-10-2015 18-1-2016 8-2-2016

Please cite this article as: Promchai, Ruangurai, Promdonkoy, Boonhiang, Tanapongpipat, Sutipa, Visessanguan, Wonnop, Eurwilaichitr, Lily, Luxananil, Plearnpis, A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.02.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis Ruangurai Promchai1, Boonhiang Promdonkoy2, Sutipa Tanapongpipat1, Wonnop Visessanguan3, Lily Eurwilaichitr4, Plearnpis Luxananil1* [email protected] 1

Microbial Cell Factory Laboratory, National Center for Genetic Engineering and Biotechnology,

National Science and Technology Development Agency, 113, Thailand Science Park, Phahonyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand. 2

Biocontrol Research Laboratory, National Center for Genetic Engineering and Biotechnology,

National Science and Technology Development Agency 3

Food Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology,

National Science and Technology Development Agency 4

Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology, National

Science and Technology Development Agency *

Corresponding author.

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Highlights New salt-inducible expression and secretion vector for Bacillus subtilis Inducible expression system with safe, simple, and cost-effective inducer Development of a safe process for heterologous protein production in B. subtilis

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Abstract Bacillus subtilis is commonly used as a host for heterologous protein production via plasmid-based expression system. In order to improve product safety, avoid carbon catabolite repression and lower production cost, a novel salt-inducible vector, pSaltExSePR5, was developed based on a natural plasmid of Lactobacillus plantarum BCC9546. Salt-inducible promoter opuAA and a DNA fragment encoding a signal peptide of subtilisin E (SubE) were sequentially added to the core shuttle vector to facilitate expression and secretion of a target protein in B. subtilis. To evaluate the effectiveness of this system under salt induction, a protease gene from Halobacillus sp. without its native signal sequence was inserted in the pSaltExSePR5 plasmid downstream of SubE signal sequence and transformed into B. subtilis WB800. Protease activities from cell-free supernatants of the recombinant bacteria cultures induced with 0.5-6% NaCl were analyzed. The highest protease activity of 9.1 U/ml was obtained after induction with 4% NaCl, while the non-induced culture exhibited activity of 0.128 U/ml. The results demonstrated that pSaltExSePR5 provides an alternative vector for efficient and simple production of heterologous proteins in B. subtilis with a safer and more economic inducer.

Keywords: Salt-inducible promoter; Expression and secretion vector; Heterologous protein production; Bacillus subtilis

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1. Introduction The use of recombinant DNA technology in protein production has raised several safety issues of consumer concern. Host-vector system for heterologous gene expression using safe hosts with chemical- or antibiotic-free expression system is of great interest. B. subtilis is an attractive host due to its GRAS (Generally Recognized As Safe) status approved by the U.S. Food and Drug Administration (FDA). The bacterium possesses good physiological and genetic properties for genetic manipulation, such as naturally competent cell, high level of protein secretion and available genomic data. In addition, large-scale production is well established (Kunst et al., 1997; Westers et al., 2004). Recently, expression vectors used for heterologous gene expression in B. subtilis have been developed by using various types of promoters. The inducible promoters, including grac, spac, lial, spaS, amy, xylA, glv and sacB, have been commonly used for the expression systems (Bhavsar et al., 2001; Bongers et al., 2005; Phan et al., 2015; Toymentseva et al., 2012; Vavrova et al., 2010; Yansura and Henner, 1984). However, many chemicals, antibiotics, polysaccharides or sugars used as inducers for these systems such as isopropyl β-D-1-thiogalactopyranoside (IPTG), bacitracin, subtilin, amylose, xylose, maltose or sucrose, respectively, have some drawbacks. Using of chemical or antibiotic as an inducer has raised concerns about safety for product utilization, especially in food industries. On the other hand, the use of sugar-inducible system may face a problem with carbon catabolite repression (CCR) when glucose is present in the system (Marciniak et al., 2012). The utilization of those inducers in large-scale production also leads to high costs. Hence, a novel inducible expression system, using inducer which is safe, CCR-independent and cost-effective, is required. As B. subtilis is naturally found in soil which usually encounters diverse climates and environmental conditions, its stress response systems to these stimuli have been readily developed to cope with the changes (Kust et al., 1997; Price et al., 2001). Indeed, utilizing natural stress-response system of B. subtilis for construction of the new expression and secretion vector is of great advantage. In this work, the function of seven stress-inducible promoters, including adhA, gsiB, gspA, htrA, yvyD, opuAA and opuE, of the genes that respond to physical stresses of B. subtilis was studied via green fluorescent protein (GFP) expression under various stress conditions. These promoters were previously found to be important for production of proteins involved in general or specific stress response system. They were upregulated when the cells encounter stress. The results showed that the opuAA promoter gave the highest green fluorescence intensity under saltinduced condition. Hence, the salt-inducible opuAA promoter is an attractive promoter to construct the new safe vector for B. subtilis. Previously, the salt-inducible proU promoter of E. coli was used for construction of pOSEX vectors and engineered the E. coli GJ1158 which carries a single chromosomally integrated copy of the gene for phage T7 RNA polymerase under transcriptional control of the osmoresponsive proU operon for heterologous gene expression in E. coli (Herbst et al., 1994; Bhandari and Gowrishankar, 1997). NaCl was used as the inducer for these systems. However, the desired product was produced as intracellular protein that required extensive downstream extraction and purification processes. The contamination of E. coli endotoxin (Lipopolysaccharide) may be concerned for safe use of products. Hence, the salt-inducible heterologous gene expression system in B. subtilis is an attractive alternative system. Thus the opuAA promoter was selected to construct the new

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salt-inducible expression and secretion vector, pSaltExSePR5, based on the natural plasmid of Lactobacillus plantarum BCC9546. The effectiveness of this new vector was then evaluated in B. subtilis.

2. Materials and methods 2.1. Bacterial strains and growth conditions L. plantarum BCC9546 (LpBCC9546) was previously isolated from Nham, a Thai-fermented sausage (Valyasevi et al., 1999). The bacterium contains indigenous plasmids and the smallest plasmid named pLpB9 was isolated, sequenced, and used as plasmid backbone in this study (Table 1). The indigenous plasmids of LpBCC9546 were subsequently removed by Novobiocin treatment to obtain plasmid-free L. plantarum strain named L. plantarum BMGC152. L. plantarum was grown in de Man, Rogosa and Sharpe (MRS) medium (BD Difco, MD, USA) while recombinant L. plantarum was grown in MRS containing 5 g/ml erythromycin and incubated at 30 °C without shaking. Escherichia coli JM109, B. subtilis ANA-1 (Lee et al., 1994) and B. subtilis WB800 (Wu et al., 2002) were grown in LB and incubated at 37 °C with shaking. Recombinant E. coli was grown in LB supplemented with 100 g/ml ampicillin or 300 g/ml erythromycin whereas recombinant B. subtilis was grown in LB supplemented with 20 g/ml erythromycin. Growth was monitored by optical density (OD600 nm) measurement of the cultures using a spectrophotometer (UV-VIS RS, Labomed, Inc., California, USA) and plate count on LB agar plate. 2.2. Plasmid constructions 2.2.1. pLPPR vectors A series of broad-host-range vectors were constructed based on the pLpB9 and pUC18 plasmids. First, the plasmid pLpB9 was cut with SphI and ligated to pUC18 (Yanisch-Perron et al., 1985) at SphI site. The recombinant plasmid, pLpB9-pUC18, was transformed into E. coli JM109 for plasmid amplification (Dagert and Ehrlich, 1979; Sambrook et al., 1989). The plasmid was purified and then sequenced by Macrogen (Macrogen, Seoul, Korea) to obtain the entire sequence of pLpB9. Subsequently, the pLpB9 was cut with MluI and the ends of the DNA fragment were blunted by Klenow fragment (New England BioLabs, MA, USA). The blunted fragment was joined with pUC18Ery plasmid that was previously digested with XbaI and blunted. The pUC18-Ery was constructed by insertion of erythromycin resistant gene (Gory et al., 2001) into pUC18 at BamHI site. The newly constructed plasmid containing pLpB9-pUC18-Ery was designated as pLPPR1. Subsequently, the gfp gene linked to L-ldh promoter of L. plantarum BCC9546 was introduced into the pLPPR1 vector at EcoRI site, resulting in a new plasmid named pLPPR3. The plasmid was amplified in E. coli and the purified plasmid was transformed into L. plantarum BMGC152 or B. subtilis ANA-1 (Serror et al., 2002) in order to examine the function of the new vector. Afterward, the pLPPR1 was modified by

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deleting the ampicillin resistant gene. The deletion was performed via PCR using the primers, p18Del-1 and 2 (Table S1) with KOD DNA polymerase (Toyobo, Osaka, Japan). The purified PCR fragment was then self-ligated, resulting in pLPPR2 vector. The NcoI recognition site of the pLPPR2 was disrupted by cutting with NcoI and blunted. Then, the DNA fragment was self-ligated, yielding pLPPR5 plasmid. The pLPPR5 was used as the plasmid backbone of pSaltExSePR5. 2.2.2. pSaltExSePR5 The opuAA promoter that regulates the gene encoding glycine betaine ABC transporter ATP binding subunit and a DNA fragment encoding the signal peptide sequence of subtilisin E gene (SubE) of B. subtilis ANA-1 were amplified by PCR using primers opuAA-F/opuAA-R and SubES-F/SubESR, respectively (Table S1). The opuAA promoter fragment was purified and cut with SphI and XbaI, while the SubE signal sequence was digested with XbaI and NcoI. The opuAA promoter was inserted into pET15b plasmid that was also digested with SphI and XbaI. Then, the digested SubE signal sequence was placed downstream of the opuAA promoter, resulting in pET15b-opuAA-SubE plasmid. The DNA fragment comprising the opuAA promoter and SubE signal sequence was amplified by PCR using the pET15b-opuAA-SubE plasmid as a template and opuAA-F and SubES-RKpn primers. The PCR fragment was purified and cut with KpnI prior to ligation with the linearized pLPPR5, resulting in pSaltExSePR5* plasmid. The linearized pLPPR5 was previously prepared by digesting with BamHI and the ends of the fragment were blunted before being subjected to KpnI cutting. Subsequently, the pSaltExSePR5* was cut with EcoRI and the ends were blunted prior to additional digestion with KpnI. In addition a DNA fragment containing 13 restriction enzyme recognition sequences of KpnI, EcoRI, BglI, EcoRV, MluI, NcoI, BsrGI, NotI, StuI, SpeI, XhoI, BamHI, and SmaI, which was synthesized by GenScript via pUC57-Simple (GenScript, NJ, USA), was digested with SmaI and KpnI. The DNA fragment was subsequently ligated to the digested pSaltExSePR5*, yielding pSaltExSePR5 vector. 2.2.3. pSaltExSePR5-Pro The protease gene of Halobacillus sp. SR5-3 (Namwong et al., 2006) was amplified by PCR using genomic DNA of Halobacillus sp. SR5-3 as a template and SR53-Kpn and SR53-Eco primers. The PCR product was purified and digested with KpnI and EcoRI before ligation to the pSaltExSePR5 vector which was also cut with both enzymes. The recombinant plasmid, named pSaltExSePR5-Pro, was amplified in E. coli prior to transformation into B. subtilis WB800. 2.2.4. pGFP373 The promoter probe vector, pGFP373, containing gfp gene was constructed by insertion of the gfp gene into plasmid pRB373 (Bruckner, 1992). The gfp gene was amplified by PCR using the plasmid pRV85 (Gory et al., 2001) as the DNA template and GFP-Kpn and GFP-2 primers. The gfp

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PCR product was cut with KpnI and EcoRI before ligation to pRB373 which was also digested with KpnI and EcoRI, respectively. 2.3. Functional analysis of stress-inducible promoters Seven promoters, adhA, gsiB, gspA, htrA, opuAA, opuE and yvyD from the genes encode dehydrogenase, general stress protein, glycosyl transferase, membrane bound serine protease, glycine betaine ABC transporter ATP-binding protein, proline transporter and ribosome-associated sigma 54 modulation protein, respectively, were amplified by PCR. The B. subtilis ANA-1 genomic DNA was used as the template with specific primers, adhA-F/R, gsiB-F/R, gspA-F/R, htrA-F/R, opuAA-F/R, opuE-F/R and yvyD-F/R, respectively (Table S1). The PCR fragments were digested with SphI and XbaI and ligated separately to the pGFP373 plasmid that was also cut with SphI and XbaI. Each recombinant plasmid was transformed into E. coli to amplify the plasmid prior to transformation into B. subtilis WB800. The recombinant B. subtilis was grown in LB containing 20 g/ml erythromycin and incubated at 37 C for 18 h with 200 rpm shaking. Then, three hundred microliters of the culture was inoculated independently into 30 ml of fresh LB and grown for 6 h before the cell pellets were divided into 4 equal parts for induction process. The first part was suspended in 5 ml LB and grown at 37 C for 2 h with shaking. This culture was used as a non-induced control. The second part was suspended in 5 ml LB and grown at 48 C for 2 h with shaking. The third and fourth parts were distributed separately into 5 ml of LB pH 5 and LB plus 5% (w/v) NaCl, respectively, and then grown at 37 C for 2 h with shaking. After two-hour induction, cells from each culture was collected and approximately 108 cells (equivalent of 1 OD at 600 nm) were individually resuspended in 1 ml of sterile distilled water prior to measurement of green fluorescence emission with a spectrofluorometer (FP-6500, Jasco, UK) by setting the excitation and emission wavelengths at 395 and 509 nm, respectively. 2.4. Protease activity assay The protease activity was assayed using sodium caseinate as a substrate and activity was determined by the TCA-Lowry assay (Lowry et al., 1951). The cell-free supernatant with an appropriate dilution (100 μl) was mixed with 900 μl of standard assay reaction mixture consisting of 25 mM Tris-HCl, pH 7.5 and 4 mg sodium caseinate. The reaction was incubated at 37 °C for exactly 30 minutes and terminated by adding 400 l of 440 mM Trichloroacetic acid (TCA). The reaction mixture was incubated at 4 °C for 15 min to precipitate unhydrolyzed proteins and the mixture was then centrifuged at 15,000 ×g for 10 minutes to collect the supernatant. Activity was expressed as tyrosine equivalents in TCA supernatant as measured by the Lowry assay. A blank was run in the same manner, except the sample was added after addition of 400 l of 440 mM TCA. One unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 nanomole of tyrosine per minute under the specified conditions.

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2.5. Optimization of induction conditions 2.5.1. Optimal salt concentration The recombinant B. subtilis containing the popuAAGFP373 plasmid was grown in LB supplemented with 10 g/ml kanamycin for 18 h at 37 C. The cells were centrifuged and washed twice with LB before resuspension in LB to obtain OD600 of 1. Cells were then cultivated by inoculating 10% (v/v) in fresh LB broth and incubated at 37 C for 4 h prior to being divided into 7 equal portions. Cells from each portion were suspended separately in LB containing 0.5, 1, 2, 3, 4, 5, or 6% (w/v) NaCl and incubated at 37 C for 2 h. After induction, treated cells (OD600 1) were collected by centrifugation. The cells were suspended with 25 mM Tris-HCl, pH 7.5 and sonicated at 80% power by using a Bandelin sonopuls sonicator (Bandelin, Berlin, Germany). The cell suspension was sonicated for 15 s, followed by a 10 sec rest interval with a total of 1 min sonication. The soluble proteins were collected by centrifugation at 15,000×g for 5 min. Ten micrograms of protein were loaded to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (Laemmli and Favre, 1973). Native PAGE was performed in the same way as SDS-PAGE without SDS in the system. GFP expression was determined by observing green fluorescence emission under UV excitation at 302 nm using an UV transilluminator (UVP LM-20E, CA, USA). The intensity of GFP of protein solution (10 µg/ml) was measured using a spectrofluorometer (FP-6500, Jasco, UK) at the excitation and emission wavelengths of 395 and 509 nm, respectively. For protease expression, the recombinant B. subtilis containing the pSaltExSePR5-Pro plasmid was grown in LB supplemented with 20 g/ml erythromycin for 18 h at 37 C. The cells were centrifuged and washed twice with LB before resuspension in LB to obtain OD600 of 1. Cells were then cultivated by inoculating 10% (v/v) in fresh LB broth and incubated at 37 C for 4 h prior to being divided into 7 equal portions. Cells from each portion were suspended separately in LB containing 0.5, 1, 2, 3, 4, 5, or 6% (w/v) NaCl and incubated at 37 C for 2 h. After induction, proteolytic activity of the cell-free supernatants of each culture was assayed and number of cells was monitored by plate count. 2.5.2. Optimal induction time in the presence of 4% NaCl Approximately 107 activated cells of the recombinant B. subtilis containing pSaltExSePR5-Pro were added to fresh LB broth and incubated at 37 C for 4 h. After the incubation, cells were divided into 5 equal parts. Cells from each part were suspended separately in LB containing 4% (w/v) NaCl and each culture was incubated individually at 37 C for 0.5, 1, 2, 3 or 4 h. After the incubation, the cellfree supernatant of each culture was analyzed for levels of protease activity. 2.6. Effect of exogenous glycine betaine on protease expression from the pSaltExSePR5-Pro plasmid

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The recombinant B. subtilis containing pSaltExSePR5 or pSaltExSePR5-Pro plasmid were grown under different conditions. Cells containing pSaltExSePR5 was grown in LB at 37 C for 4 h before addition of 4% NaCl and further growth for 3 h. The culture supernatant was then collected and used as a baseline control of protease production. Cells harboring the pSaltExSePR5-Pro were grown in LB broth at 37 C for 4 h prior to being separated into five parts. Each part was individually suspended in LB, LB plus 4% (w/v) NaCl, or LB plus 4% (w/v) NaCl with 0.001, 0.01 or 1 mM glycine betaine, respectively. The cultures were incubated at 37 C for 3 h before analysis of protease activity.

3. Results and discussion 3.1. Broad-host-range cloning vector, pLPPRs A new, salt-inducible expression system based on the natural plasmid namely pLpB9 of L. plantarum BCC9546 originally isolated from Thai fermented sausage, Nham, an opuAA promoter and signal peptide of subtilisin E gene was developed for use in Bacillus subtilis. pLpB9 contained 2,269 bp with 37% GC content and 4 open reading frames of 221, 153, 118 and 108 amino acids which were similar to Rep protein, acetyltransferase and two hypothetical proteins of L. plantarum, respectively. DNA sequence of pLpB9 was deposited in GenBank under the accession number EU391630. The pLpB9 was linked to the modified pUC-Ery, resulting in a new shuttle vector designated as pLPPR1 (Fig. 1). To evaluate the function of the vector, L-ldh promoter of L. plantarum BCC9546 was linked to the gfp gene prior to insertion into pLPPR1 at EcoRI site, yielding pLPPR3 plasmid. The plasmid was transformed into E. coli, L. plantarum and B. subtilis. All three recombinant strains were able to emit green fluorescence after excitation with ultraviolet light (data not shown). Hence, the pLPPR1 could be used as a new broad-host-range cloning vector in both Gram-positive and Gram-negative bacteria. However, the large size of pLPPR1 (6083 bp) may cause inefficient use of vector. Therefore, the ampicillin resistant gene was eliminated by PCR-generated deletion resulting in a smaller vector namely pLPPR2 (4916 bp). The vector was further developed to be pLPPR5 via disruption of NcoI site. The pLPPR5 (4920 bp) was then used as a core vector for construction of pSaltExSePR5. 3.2. Expression and secretion vector, pSaltExSePR5 In order to develop a new expression-secretion vector, several promoters were tested for their ability to induce an expression of the gfp gene. Seven stress-inducible promoters of the genes encoding of dehydrogenase (adhA), general stress protein (gsiB), glycosyl transferase (gspA), membrane-bound serine protease Do (htrA), ribosome-associated sigma 54 modulation protein (yvyD), glycine betaine ABC transporter ATP-binding subunit (opuAA) and proline transporter (opuE) were amplified by PCR using genomic DNA of B. subtilis ANA-1 as a template. All promoters were introduced individually into the pGFP373 promoter probe vector containing gfp reporter gene and transformed into B. subtilis WB800, resulting in seven recombinant strains. The green fluorescence emission from each strain was evaluated under four different growth conditions including 1) LB at 37 °C, 2) heat shock treatment in

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LB at 48 °C, 3) acid treatment in LB adjusted to pH 5, and 4) salt-induction in LB containing 5% NaCl. Results showed that upon salt induction, GFP expression was found to be induced by all tested promoters. However, induction rate were considerably lower than that of the opuAA promoter (Fig. 2). The opuE promoter, which is a A and B-dependent promoter, regulates the expression of proline uptake involving in the osmotic stress response mechanism whereas the yvyD promoter, a B and Hdependent promoter, controls the transcription of ribosome-associated sigma 54 modulation protein and functions in starvation and general stress response mechanism (Drzewiecki et al., 1998; Hoper et al., 2005; Spiegelhalter and Bremer, 1998). The activities of the single promoters either binding to A (htrA) or B (gspA and gsiB) were also found to be weaker than the opuAA activity when cells encountered high salt condition. The htrA promoter regulates the transcription of htrA gene which encodes serine protease functioning in protein quality control in response to heat or severe secretion stress (Hyyrylainen et al., 2001; Noone et al., 2000). The gspA and gsiB promoters control expression of general stress proteins which play important roles in coping with starvation and physical stresses (Maul et al., 1995; Petersohn et al., 2001). As A-dependent promoters (P1 and P2), opuAA promoter regulates an opuA operon controlling the transcription of opuAA, opuAB and opuAC which encode the structural proteins of glycine betaine ABC transporter: ATP-binding protein, permease and substratebinding protein, respectively. These proteins play a role in binding and uptake of glycine betaine when B. subtilis faces osmotic stress conditions. However, only P1 promoter which is in closer proximity to the gene than the P2 promoter is reported to be upregulated when the cell encounters high osmotic condition (Hoffmann et al., 2013; Horn et al., 2005; Kempf et al., 1995). Unexpectedly, the heat or acid stress condition displayed lower GFP intensity than non-induced condition in all recombinant strains (Fig. 2). It is hypothesized that heat or acid stress condition may stimulate various stress response proteins especially ATP-dependent proteases (Hecker et al., 1996; Hecker and Volker, 2001; Hecker et al., 2007; Petersohn et al., 2001; Wilks et al, 2009) that could degrade or affect GFP expression. Therefore, the opuAA promoter was further used as the salt-inducible promoter for construction of the new expression and secretion vector. In order to facilitate the recovery of target protein from cultured broth, the signal peptide sequence of aprE-encoded subtilisin E (SubE) was selected for construction of the new plasmid vector. Since the subtilisin E (alkaline serine protease) is naturally secreted by B. subtilis, its signal peptide should be efficient in this system (Kunst et al., 1997). Hence, a new expression and secretion vector, designated pSaltExSePR5, was constructed by inserting the opuAA promoter and the signal peptide sequence SubE into pLPPR5 (Fig. 1). In addition, the multiple cloning site of pSaltExSePR5 was modified to possess twelve recognition sites of common restriction enzymes. The sequence of pSaltExSePR5 was deposited in GenBank under the accession number KT822451. 3.3. Functional analysis of pSaltExSePR5 3.3.1. Optimal salt concentration for induction system The soluble proteins extracted from the recombinant B. subtilis containing both pGFP373 and popuAAGFP373 were compared (Fig 3.1). Electrophoretic analyses by SDS-PAGE indicated two

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distinctive patterns of proteins expressed in low salt (0.5- 2%) and high salt (3-6%) conditions based on the increased band intensity of several protein bands and the GFP band with the estimated apparent molecular weight of 27 kDa (Fig 3.1A). The GFP expression was also reconfirmed by the increased fluorescence intensity of protein band resolved at the almost the same position in native PAGE as observed upon visualization with an UV transilluminator (Fig 3.1 B). Quantitatively, the GFP intensities of protein solutions obtained from all conditions were compared at the same protein concentration of 10 µg/ml by using a spectrofluorometer (Fig 3.1C). The results evidently indicated the highest expression of GFP in the recombinant B. subtilis containing popuAAGFP373 at 4% salt induction, while no fluorescent intensity was detected in protein extracted from the recombinant B. subtilis containing pGFP373. To examine function of the pSaltExSePR5 plasmid in B. subtilis, the protease gene without its native signal peptide sequence from Halobacillus sp. SR5-3 was introduced into pSaltExSePR5 downstream of the opuAA promoter and signal peptide SubE, yielding the recombinant plasmid named as pSaltExSePR5-Pro. The pSaltExSePR5-Pro was transformed into B. subtilis WB800 which is deficient in eight extracellular proteases (Wu et al., 2002). The recombinant B. subtilis WB800 containing the pSaltExSePR5-Pro plasmid was induced in LB broth with different concentrations of salts (0.5-6% NaCl) for 2 h. After the induction, the cell-free supernatant of each culture was analyzed for protease activity. The results showed that protease activity increased to the highest value of 9.1 U/ml with the increased concentration of NaCl up to 4% (Fig. 3.2). Upon induction with 2 to 5% NaCl, higher levels of protease activity indicated opuAA promoter response to osmotic pressure. Unexpectedly, the 6% NaCl condition showed strikingly lower level of protease activity, although the number of cells after treatment with 6% NaCl condition was lower than those challenged with lower NaCl concentrations. These results indicated that the opuAA promoter is activated at 2% NaCl and that the optimum concentration for the strongest induction is 4% resulting in an approximately 9-fold increase in proteolytic activity when compared with non-induced condition. Previously, there was a report of molecular cloning of an extracellular protease from four B. subtilis strains in E. coli using pGEX vector with IPTG induction system (Han et al., 2013). However, the highest proteolytic activity of the cell extract of the recombinant E. coli expressing the alkaline extracellular protease of B. subtilis CN2 revealed only at approximately 4-fold higher proteolytic activity than B. subtilis 168. 3.3.2. Optimal induction time To maximize expression and secretion, optimal induction time of this new salt-inducible system was investigated under 4% NaCl condition. The highest proteolytic activity of 9.656 U/ml was observed at 3 h after salt treatment (Fig. S1). 3.4. The effect of glycine betaine Since the opuA system plays a functional role in glycine betaine transport in B. subtilis, the induction effect of glycine betaine toward the expression system of pSaltExSePR5-Pro was

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investigated. A slight increase in protease production was observed in the presence of 0.001 mM glycine betaine. However, addition of glycine betaine at concentrations higher than 0.001 mM resulted in a significant decrease in protease activity (Fig. S2). The negative effect of high concentrations of exogenous glycine betaine on the function of opuAA promoter was reported by Hoffmann et al. (2013) who studied the osmotic control of opuA expression in B. subtilis and its modulation in response to intracellular glycine betaine and proline pools.

4. Conclusions A new expression and secretion vector, designated pSaltExSePR5, contains two origins of replication for Gram-negative and Gram-positive bacteria to support plasmid amplification in both E. coli and B. subtilis. This shuttle vector contains erythromycin resistant gene, twelve recognition sites of common restriction enzymes and active part of salt-inducible promoter with signal peptide sequence to facilitate effective expression and utilization of protein product. Production of protein of interest by this system is achieved by a simple induction protocol with sodium chloride. This vector would provide a basis for further development of a safe process for heterologous protein production in B. subtilis.

Acknowledgments This research was granted by National center for genetic engineering and biotechnology, National Science and Technology Development Agency, Thailand (P1100813). We thank Dr. Piyanun Harnpicharnchai for proof-reading of the manuscript.

Conflict of interest The authors declare that they have no conflict of interest.

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References Bhandari, P., Gowrishankar, J., 1997. An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer. J. Bacteriol. 179, 4403-4406. Bhavsar, A.P., Zhao, X., Brown, E.D., 2001. Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis: conditional complementation of a teichoic acid mutant. Appl. Environ. Microbiol. 67, 403-410. Bongers, R.S., Veening, J.W., Van Wieringen, M., Kuipers, O.P., Kleerebezem, M., 2005. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl. Environ. Microbiol. 71, 8818-8824. Bruckner, R., 1992. A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene. 122, 187-192. Dagert, M., Ehrlich, S.D., 1979. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 6, 23-28. Drzewiecki, K., Eymann, C., Mittenhuber, G., Hecker, M., 1998. The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH. J. Bacteriol. 180, 6674-6680. Gory, L., Montel, M.C., Zagorec, M., 2001. Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products. FEMS. Microbiol. Lett. 194, 127-133. Han, X., Shiwa, Y., Itoh, M., Suzuki, T., Yoshikawa, H., Nakagawa, T., Nagano, H., 2013. Molecular cloning and sequence analysis of an extracellular protease from four Bacillus subtilis strains. Biosci. Biotechnol. Biochem. 77, 870-873. Hecker, M., Schumann, W., Volker, U., 1996. Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19, 417-428. Hecker, M., Volker, U., 2001. General stress response of Bacillus subtilis and other bacteria. Adv. Microb. Physiol. 44, 35-91. Hecker, M., Pane-Farre, J., Volker, U., 2007. SigB-dependent general stress response in Bacillus subtilis and relates gram-positive bacteria. Annu. Rev. Microbiol. 61, 215-236. Herbst, B., Kneip, S., Bremer, E., 1994. pOSEX: vectors for osmotically controlled and finely tuned gene expression in Escherichia coli. Gene. 151, 137-142. Hoffmann, T., Wensing, A., Brosius, M., Steil, L., Völker, U., Bremer, E., 2013. Osmotic control of opuA expression in Bacillus subtilis and its modulation in response to intracellular glycine betaine and proline pools. J. Bacteriol. 195, 510-522. Hoper, D., Volker, U., Hecker, M., 2005. Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis. J. Bacteriol. 187, 2810-2826. Horn, C., Jenewein, S., Sohn-Bosser, L., Bremer, E., Schmitt, L., 2005. Biochemical and structural analysis of the Bacillus subtilis ABC transporter OpuA and its isolated subunits. J. Mol. Microbiol. Biotechnol. 10, 76-91. Hyyrylainen, H.L., Bolhuis, A., Darmon, E., Muukkonen, L., Koski, P., Vitikainen,

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M., Sarvas, M., Prágai, Z., Bron, S., van Dijl, J.M., Kontinen, V.P., 2001. A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress. Mol. Microbiol. 41, 1159-1172. Kempf, B., Bremer, E., 1995. OpuA, an osmotically regulated binding proteindependent transport system for the osmoprotectant glycine betaine in Bacillus subtilis. J. Biol. Chem. 270, 16701-16713. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessières, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S.C., Bron, S., Brouillet, S., Bruschi, C.V., Caldwell, B., Capuano, V., Carter, N.M., Choi, S.K., Codani, J.J., Connerton, I.F., Danchin, A, et al., 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 390, 249-256. Laemmli, U.K., Favre, M. 1973. Maturation of the head of bacteriophage T4:DNA packaging events, J. Mol. Biol. 80, 575-599. Lee, S.P., Morikawa, M., Takagi, M., Imanaka, T., 1994. Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601. Appl. Environ. Microbiol. 60, 3764-3773. Liu, S.L., Du, K., 2012. Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme. Protein. Expr. Purif. 83,164-168. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Marciniak, B.C., Pabijaniak, M., de Jong, A., Dűhring, R., Seidel, G., Hillen, W., Kuipers, O.P., 2012. High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis. BMC. Genomics. 13, 401. Maul, B., Volker, U., Riethdorf, S., Engelmann, S., Hecker, M., 1995. sigma Bdependent regulation of gsiB in response to multiple stimuli in Bacillus subtilis. Mol. Gen. Genet. 248, 114-120. Ming, Y.M., Wei, Z.W., Lin, C.Y., Sheng, G.Y., 2010. Development of a Bacillus subtilis expression system using the improved Pglv promoter. Microb. Cell. Fact. 10,9:55. Namwong, S., Hiraga, K., Takada, K., Tsunemi, M., Tanasupawat, S., Oda, K., 2006. A halophilic serine proteinase from Halobacillus sp. SR5-3 isolated from fish sauce: purification and characterization. Biosci. Biotechnol. Biochem. 70, 13951401. Noone, D., Howell, A., Devine, K.M., 2000. Expression of ykdA, encoding a Bacillus subtilis homologue of HtrA, is heat shock inducible and negatively autoregulated. J. Bacteriol. 182, 1592-1599. Petersohn, A., Brigulla, M., Haas, S., Hoheisel, J.D., Volker, U., Hecker, M., 2001. Global analysis of the general stress response of Bacillus subtilis. J. Bacteriol. 183, 5617-5631. Phan, T.T., Tran, L.T., Schumann, W., Nguyen, H.D., 2015. Development of 14

Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb. Cell. Fact. 14, 72. Price, C.W., Fawcett, P., Ceremonie, H., Su, N., Murphy, C.K., Youngman, P., 2001. Genome-wide analysis of the general stress response in Bacillus subtilis. Mol. Microbiol. 41, 757-774. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Serror, P., Sasaki, T., Ehrlich, S.D., Maguin, E., 2002. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids. Appl. Environ. Microbiol. 68, 46-52. Spiegelhalter, F., Bremer, E., 1998. Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A- and sigma B-dependent stress-responsive promoters. Mol. Microbiol. 29, 285-296. Toymentseva, A.A., Schrecke, K., Sharipova, M.R., Mascher, T., 2012. The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter. Microb. Cell. Fact. 11, 143. Valyasevi, R., Smitinond, T., Praphailog, W., Chowalitnitithum, C., Kunawasen, S., Chavasith, V., 1999. The microbiology and development of starter culture for Nham, the traditional Thai pork sausage. Proceedings of the Seventeenth International Conference of the International Committee on Food Microbiology and Hygience (ICFMH), Veldhoven, The Netherlands. p. 709-711. Vavrova, L., Muchova, K., Barak, I., 2010. Comparison of different Bacillus subtilis expression systems. Res. Microbiol. 161, 791-797. Westers, L., Westers, H., Quax, W.J., 2004. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim. Biophys. Acta. 1694, 299-310. Wilks, J.C., Kitko, R.D., Cleeton, S.H., Lee, G.E., Ugwu, C.S., Jones, B.D., BonDurant, S.S., Slonczewski, J.L., 2009. Acid and base stress and transcriptomic responses in Bacillus subtilis. Appl. Environ. Microbiol. 75; 981-990. Wu, S.C., Yeung, J.C., Duan, Y., Ye, R., Szarka, S.J., Habibi, H.R., Wong, S.L., 2002. Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl. Environ. Microbiol. 68, 3261– 3269. Yanisch-Perron, C., Vieira, J., Messing, J., 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequencing of the M13mp18 and pUC9 vectors. Gene. 33, 103-119. Yansura, D.G., Henner, D.J., 1984. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. U S

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A. 81, 439-443. Yin, S., Hao, Y., Zhai, Z., Li, R., Huang, Y., Tian, H., Luo, Y., 2008. Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4. FEMS. Micobiol. Lett. 285, 183-187.

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Figure Captions Fig. 1. Plasmid maps of pLPPR1 and pSaltExSePR5. The smallest indigenous plasmid of L. plantarum BCC9546 named pLpB9 was isolated and linked to pUC18-Ery, resulting in new broad-host-range shuttle vector named pLPPR1. The ampicillin resistant gene of pLPPR1 was deleted, resulting smaller vector named pLPPR2. Disruption of NcoI site in pLPPR2 yielded pLPPR5. The opuAA promoter and a DNA fragment encoding signal peptide of subtilisin E (SubE) of B. subtilis were introduced into pLPPR5, resulting in pSaltExSePR5. The multiple cloning sites (MCS) of pSaltExSePR5 were modified to achieve twelve recognition sites of common restriction enzymes. The nucleotide sequences of opuAA promoter (Single underline), RBS (Bold with single underline), start codon (Large letter with single underline), DNA encoding Subtilisin E signal peptide (Double underline) and restriction enzyme recognition sequences (Bold) are depicted. M denotes 1 kb DNA ladder and Lp stands for plasmid isolated from L. plantarum BCC9546. The pLpB9 and pMB1 are the origins of replication of pLpB9 and pUC18, respectively. Rep2 is a gene encoding replication protein. AmpR and EryR represent ampicillin and erythromycin resistant genes, respectively. Fig. 2. Functional analysis of stress-inducible promoters. Functions of selected promoters under different stress conditions were investigated. Seven promoters, adhA, gsiB, gspA, htrA, opuAA, opuE and yvyD of B. subtilis were inserted separately upstream of gfp gene in pGFP373 and transformed into B. subtilis. Each recombinant strain was grown in LB at 37 C for 6 h and then incubated in LB at 37 C (control) or subjected to stress conditions for 2 h. The stress conditions tested were high temperature (48 C), acidic pH (pH 5), and presence of salt (5% NaCl). After incubation, approximately 108 cells (equivalent to 1 OD600) were suspended in 1 ml of sterile distilled water. The green fluorescence was measured at emission and excitation wavelengths at 509 and 395 nm, respectively. Fig. 3.1. Functional analysis of the opuAA promoter by GFP expression. The soluble proteins extracted from the recombinant B. subtilis containing pGFP373 or popuAAGFP373 plasmid strains which were cultured in medium containing different salt concentrations were analyzed by SDS-PAGE (A) and native PAGE followed by visualization upon UV light at 302 nm (B) to monitor the expression of GFP (27 kDa). The GFP intensities were measured using a spectrofluorometer at the excitation and emission wavelengths at 395 and 509 nm, respectively (C). M denotes standard protein marker (Thermo Fisher Scientific, MA, USA). V0.5 represents proteins of B. subtilis containing pGFP373 that was cultured in LB containing 0.5% NaCl. S0.5-S6 represent the proteins of B. subtilis containing popuAAGFP373 which was cultured in LB obtaining 0.5-6% NaCl, respectively. Fig. 3.2. Optimal salt concentration for induction condition. The activated recombinant B. subtilis containing pSaltExSePR5-Pro was grown in LB broth and incubated at 37 C for 4 h prior divided into 7 equal portions. The cells from each portion were resuspended separately in LB containing 0.5, 1, 2, 3, 4, 5, or 6% NaCl and the cultures were incubated at 37 C for 2 h. After incubation, the cell-free supernatant of each culture was analyzed for level of protease activity. Bars show the protease activities

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of each salt concentration. Values are mean ± SD from three independent experiments. Line indicates the number of cells after salt induction at different salt concentrations.

18

Fig. 1.

19

GFP intensity (AU)

1200

LB

pH 5

48 °C

gspA

htrA

opuAA

5% NaCl

1000 800 600 400 200 0 adhA

gsiB

opuE

yvyD

Promoters

Fig. 2.

20

1400

GFP intensity (AU)

C

1148

1200 1000

837

830

800 556

600 401

548

450

400 200 0 0 V0.5

S0.5

S1

S2

S3

S4

S5

S6

Fig. 3.1.

21

10

1.00E+09

9.1 8.584

9

7.725

1.00E+08

8

1.00E+07

7

6

1.00E+05 5 1.00E+04 4

Cell count (CFU/ml)

Protease activity (U/ml)

1.00E+06 5.579

1.00E+03 3 1.00E+02

2 0.729

1.00E+01

0.257

1 0.128 0

1.00E+00 0.5

1

2

3

4

5

6

NaCl concentration (%)

Fig. 3. 2.

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Tables Table 1 Bacterial strains and plasmids used in this study. Bacterial strains and plasmids L. plantarum BCC9546

Information

Reference

The bacterium was isolated from

Valyasevi et al. (1999)

traditional Thai fermented pork, Nham L. plantarum BMGC152

Plasmid-free strain of L. plantarum BCC9546 -

-

B. subtilis ANA-1

arg-15 hsdR hsdM ΔaprA3 Amy Npr

B. subtilis WB800

Eight-extracellular protease-deficient strain

E. coli JM109

+ +

q

F´ traD36 proA B lacI Δ(lacZ)M15/ Δ(lac-

This study Lee et al. (1994) Wu et al. (2002) Yanisch-Perron et al. (1985)

-

proAB) glnV44 e14 gyrA96 recA1 relA1 endA1 thi hsdR17 pUC18

pMB1 origin of replication from pBR322, lacI

Yanisch-Perron et al. (1985)

coding sequence, bla coding sequence pET15b

pBR322 origin, T7 promoter, lacI coding

Novagen, Wisconsin, USA

sequence, T7 terminator, His-Tag coding sequence, bla coding sequence pLpB9

Indigenous plasmid of L. plantarum BCC9546

This study

pLPPR1

pLpB9-pUC18-Ery containing ampicillin and

This study

erythromycin resistant genes pLPPR2

pLPPR1-ampicillin resistant gene deletion

This study

pLPPR3

pLPPR1-pldhgfp

This study

pLPPR5

pLPPR2-NcoI deletion

This study

pGFP373

pRB373-gfp

This study

pSaltExSePR5

pLPPR5-opuAA promoter-SubE

This study

pSaltExSePR5-Pro

pLPPR5-opuAA promoter-SubE-Protease gene

This study

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A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis.

Bacillus subtilis is commonly used as a host for heterologous protein production via plasmid-based expression system. In order to improve product safe...
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