Construction of a Shuttle Vector for Protein Secretory Expression in Bacillus subtilis and the Application of the Mannanase Functional Heterologous Expression

Abstract

We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.

Introduction

Gram-positive bacterial strains are well known for their contributions to agricultural, medical, and commercial enzyme production. Among them, Bacillus subtilis has been widely used in recombinant protein production such as enzymes, biochemicals, antibiotics, and insecticides. B. subtilis has been developed as an attractive host because of several reasons: nonpathogenic and considered as a safe organism, no significant bias in codon usage, and capable of secreting functional extracellular proteins directly into the culture medium [10, 15]. At present, about 60% of the commercially available enzymes are produced by Bacillus species [11]. A wide variety of promoters used in B. subtilis have been reported [2, 11, 18]. Among them, the constitutive promoter P43 is active during the exponential and lag phases of growth and shows the highest expression capability compared with other promoters [18]. Furthermore, P43 promoter has been successfully used in recombinant proteins production, such as thermostable β-galactosidase, staphylokinase, and dehydrogenase [18].

Endo-1,4-β-mannanases (β-mannanases, E.C. 3.2.1.78) catalyze the random hydrolysis of manno-glycosidic bonds in mannans and heteromannans. Based upon the amino acid sequence alignment and hydrophobic cluster analysis, most β-mannanases belong to glycoside hydrolase (GH) families 5, 26, and 113. Endo-1,4-β-mannanases play important roles in basic research, the bioconversion of biomass materials, and various potential industrial applications [3, 8, 13]. β-Mannanases from bacteria (Bacillus sp., Aeromonas sp., Streptomyces sp., Pseudomonas sp., or Vibrio sp.), fungi (Penicillium sp., Tyromices sp., Trichosporum sp., Sclerotium sp., and Aspergillus sp.), plants (Amorphophallus konjac), animals, and in the colonic region of humans were reported in previous research. During recent years, the production of mannanases in recombinant Escherichia coli has been well studied [3, 17]. However, there have been few reports of the mannanases expression in Bacillus subtilis.

In this study, we report the construction of two B. subtilis expression vectors, pBNS1 and pBNS2, both with the strong constitutive promoter P43. The two newly constructed vectors can be efficiently transformed into B. subtilis by electroporation and be used for intra- and extracellular production of recombinant proteins in B. subtilis. In our study, the GFP gene and mannanase gene were expressed successfully. Both vectors can be used to express exogenous genes in B. subtilis and are useful for the large-scale gene expression industry.

 

Materials and Methods

Bacterial Strains, Plasmids, and Culture Conditions

The following strains were used: Escherichia coli DH5α, Escherichia coli BL21(DE3)/pLysS (Invitrogen), B. subtilis 1A751 (Lab collection), and Bacillus pumilus Nsic-2 (wild strain, accession number: CCTCC AB 2013050. China Center for Type Culture Collection). The bacterial strains and plasmids used in this study are listed in Table 1. Both E. coli cells and B. subtilis cells were grown in Luria-Bertani (LB) medium at 37℃. Antibiotics were used in this study at the following concentrations: ampicillin at 100 μg/ml for E. coli and kanamycin at 30 μg/ml for B. subtilis.

Table 1.AmpR, ampicillin resistance; KanR, kanamycin resistance.

Chemicals and Manipulation of DNA

Taq DNA polymerase, restriction endonucleases, T4 DNA ligase, and pMDTM19-T vector were purchased from Takara (Takara Biotechnology, Shanghai, China). Isolated chromosomal DNA from B. subtilis, plasmids, and gel extraction of DNA were prepared using the AXYGEN kit (BIOSCIENCE, Shanghai, China). PCR products and DNA bands were separated by electrophoresis on a 1.0% agarose gel. Transformation of E. coli was carried out using CaCl2-treated 50 μl aliquots of competent cells, and Bacillus subtilis cells were transformed by electroporation. Primers used in this study are listed in Table 2.

Table 2.Primers P3/P4, A1/A2, pBNS1-gfp-U/pBNS1-gfp-D, pBNS2-gfp-U/pBNS2-gfp-D, and manA-F/manA-D were used to amplify the P43 promoter, the amyQ signal peptide, the GFP reporter gene, and the β-mannanase gene, respectively. Primers F1/R1, R2, and R3 were used for sequential PCR to amplify the 188 bp fusion fragment. The underlined sequences are the restriction sites. The bold underlined sequence are the modified Shine-Dalgarno sequences.

Construction of Shuttle Expression Vector pBNS1 and pBNS2

First, the original plasmid pBEn (7.4 kb) was constructed using plasmids pUB110 (4.5 kb) from B. subtilis and pGEM3 (2.9 kb) from E. coli. Then, plasmid pBE2a (6.3 kb) was constructed by removing a 1.1 kb PvuII cleavage fragment in pBEn. Primers P43-U/P43-D and A1/A2 were used to clone the P43 promoter sequence (from B. subtilis 168) and signal peptide (from B. amyloliquefaciens), respectively. The PCR products were gel extracted and cloned into the vector pMD19-T to generate pMT-P43 and pMT-amyQ, respectively. Next, the 0.3 kb P43 promoter gene obtained from pMT-P43 was subcloned into the EcoRI/XbaI site of pBE2a to generate expression plasmid pBNS1 (B. subtilis intracellular expression plasmid).

To construct the B. subtilis secreted expression vector pBNS2, the method of sequential PCR with specific primers F1/R1/R2/R3 was used. In this way, a fragment of designed SD sequence was added to the upstream of the amyQ signal peptide, while a multiple cloning site sequence including BamHI, SalI, SacI, XhoI, PstI, and SphI and the optional C-terminal His-Tag sequence were inserted into the downstream of the amyQ signal peptide. Finally, the 188 bp target fragment was obtained and inserted into pMDTM19-T vector (named pMD19-SP). Next, the 188 bp fragment was subcloned into the XbaI/Hind site of pBNS1. The constructed plasmid, named pBNS2 (6,781 bp), contains the amyQ signal peptide and C-terminal His-Tag.

Construction of GFP Reporter Vector and Transformation

Using the pGFP1 (Clontech) plasmid as the template, the GFP gene (717 bp) was amplified with the primers pBNS1-gfp-U/pBNS1-gfp-D and pBNS2-gfp-U/pBNS2-gfp-D. Next, the 0.7 kb GFP gene was subcloned into the XbaI/HindIII site of pBNS1 and the BamHI/XhoI sites of pBNS2 to generate pBNS1-GFP (7,359 bp) and pBNS2-GFP (7,483 bp), respectively (Fig. 1). Sequence verification and sequence analysis of the GFP gene in pBNS1-GFP and pBNS2-GFP was performed (HuaDa Gene Company, Shanghai, china). The constructed plasmids were transformed into B. subtilis by electroporation. The positive strains were selected based on 30 μg/ml kanamycin and shaken at 37℃ overnight in Luria-Bertani (LB) broth. The obtained transformants were examined for the expression of GFP under a fluorescence microscope.

Fig. 1.Schematic diagram of the constructed plasmids in B. subtilis. SD, amyQ, MCS, gfp, Ori, Amp, and Kan represent the B. subtilis Shine-Dalgarno sequence, signal peptide of alpha-amylase from Bacillus amyloliquefaciens, multiple cloning site sequence, green fluorescent protein gene, replication origin, ampicillin resistance marker, and kanamycin resistance marker, respectively.

Measurement of Green Fluorescent Protein

Recombinant B. subtilis strains were cultured for 24 h at 37℃. After centrifuged at 10,000 rpm for 10 min (at 4℃), the supernatant and the precipitated cells were collected, respectively. The precipitated cells were diluted in 100 mM sodium phosphate buffer (pH 7.0). Both supernatant and precipitated cells were analyzed for GFP expression on a microtiter plate reader (GENios Pro), using an excitation wavelength of 485 nm and an emission wavelength of 535 nm [7].

Construction of pBNS2-Man and Enzyme Assays

To further verify the constructed plasmids, the β-mannanase gene (GeneBank Accession No. KC436314) was inserted into the SD sequence of pBNS2, and the β-mannanase activity was measured. Using primers manA-F/manA-D, the β-mannanase (named manB) gene was amplified from Bacillus pumilus Nsic-2. Next, the manB gene was subcloned into the BamHI/XhoI site of pBNS2 to generate pBNS2-man. Sequence verification of the manB gene in pBNS2-man was performed. The recombinant plasmid was then transformed into the B. subtilis 1A751 strain.

Mannanase activity was assayed using the DNS method with some modifications [17]. The reaction mixture containing 100 μl of appropriately diluted enzyme sample and 900 μl of 0.5% (w/v) locust bean gum in 0.05 M sodium phosphate buffer (pH 7.0) was incubated at 50℃ for 10 min, and terminated by the addition of 0.5 ml of DNS reagent. After boiling in a water bath for 10 min, the absorbance was measured at 540 nm. The reaction system, with the same enzyme sample added after DNS reagent, was treated as the control. Activities were expressed as mean values in U (mg protein). One unit of enzyme activity is defined as the amount of enzyme releasing 1 μmol of reducing sugar per minute under the assay experimental conditions.

 

Results

Construction of Expression Vectors pBNS1 and pBNS2

In this study, we constructed two shuttle expression vectors that are based on the strong promoter P43. The pBNS2 vector was constructed by modification of pBNS1. Using the method of sequential PCR, the XbaI-Hindrestriction fragment of pBNS1 was replaced by a 188 bp DNA cassette (see Fig. 2). The constructed plasmid pBNS2 contains the SD sequence, B. amyloliquefaciens amyQ signal peptide-encoding sequence, multiple cloning site sequence (BamHI, SalI, SacI, XhoI, PstI, SphI), and optional C-terminal His-Tag sequence (see Fig. 2). Promoter P43 was amplified from B. subtilis 168 chromosomal DNA. The homology analysis showed that the length of P43 promoter is 305 bp and 100% identical to the reported B. subilis genome DNA (GenBank Accession No. AL009126.3).

Fig. 2.Construction of plasmid pBNS2. (A) Part of the pBNS2 sequence. The sequence between the EcoRI site and XbaI site is the P43 promoter (contains transcription factors σA and σB). The SD sequence is marked by blue underline. The sequences in the black and red boxes are the B. amyloliquefaciens amyQ signal peptide and MCS, respectively. The C-terminal His-Tag sequence is indicated by the bold underline. (B) Schematic diagram of sequential PCR. SD, amyQ, and MCS represent the B. subilis SD sequence, the signal peptide of alpha-amylase from Bacillus amyloliquefaciens, and the multiple cloning site sequence, respectively.

Construction of the GFP Reporter Recombinant Strains and Growth Curve Analysis

We used the GFP gene as a reporter to test the applicability of the constructed vectors pBNS1 and pBNS2 in Bacillus subtilis. Expression vectors pBNS1-GFP and pBNS2-GFP were first transformed into E. coli and then transformed into B. subtilis 1A751 by electroporation. The transformants that carried the desired vectors were directly screened on ampicillin-containing solid plates for E. coli and kanamycin-containing solid plates for B. subtilis 1A751, respectively. The resultant plasmid and mutant strain were verified by restriction enzyme digestion and nucleotide sequencing (Fig. 3).

The growth curve was determined by measuring the turbidity of wild strain and recombinant strains pBNS1- GFP-1A751/pBNS2-GFP-1A751. The results show that the stationary phase and the death phase of recombinant strains were consistent with wild strain 1A751 (Fig. 3). The cell concentration reached the maximum after 24-27 h cultured and then decreased as the cells ran out of nutrients and died. There was a noticeable lag phase for recombinant strains as the additional kanamycin. After culture for 2 h, wild strain 1A751 reached the exponential phase, whereas recombinant strains pBNS1-GFP-1A751/pBNS2-GFP-1A751 reached the exponential phase after 6 h of culture.

Fig. 3.The growth curve and restriction enzyme analysis of recombinant strains. (A) The growth curve of wild strain and recombinant strains pBNS1-GFP-1A751/pBNS2-GFP-1A751. The wild strain 1A751 is indicated by filled diamonds, and filled squares are for recombinant strain pBNS1-GFP-1A751, and filled triangles for pBNS2-GFP-1A751. (B) Lanes 1 and 2 represent plasmid pBNS2 digested by EcoRI/XbaI and EcoRI/Hind, respectively; M, DNA ladder. (C) Lanes 1 and 2 represent plasmid pBNS2-GFP digested by XbaI/Hind and BamHI/XhoI, respectively; lane 3, plasmid pBNS1-GFP digested by BamHI/XhoI.

Validation of the Vector Applicability by the GFP Fluorescence Detection and SDS-PAGE

The two newly constructed plasmids were Escherichia coli-Bacillus subtilis shuttle expression vectors and can replicate in both E. coli and B. subtilis. As there is no signal peptide (http://www.cbs.dtu.dk/services/SignalP/) in the green fluorescent protein gene, the GFP gene was expressed intra- and extracellular directly. Positive transformants, pBNS1-GFP-1A751 and pBNS2-GFP-1A751, were selected and then screened for further observation of GFP expression under a fluorescence microscope. The recombinant strain glowed with a bright green color and was visible by the naked eye. The results (Fig. 4) indicated that both vectors were capable of expressing the exogenous gene in B. subtilis.

After culture for 24 h, the fluorescence values in the fermentation supernatant and precipitated cells were analyzed. Based on data obtained from the fluorescence microplate reader (Table 3), we concluded that the GFP gene was well expressed and secreted from the constructed expression cassette. Almost all the GFP was expressed intracellularly with vector pBNS1, whereas most of the GFP was secreted extracellularly with vector pBNS2 (64% from culture medium). The result indicates that vector pBNS2 has the capacity of secreting functional extracellular proteins directly into the culture medium.

Fig. 4.Detection of recombinant strains by fluorescence microscopy and SDS-PAGE analysis. (A) The fluorescence detection of the recombinant strain pBNS1-GFP-1A751. (B) The fluorescence detection of the recombinant strain pBNS2-GFP-1A751. (C) The result of precipitated cells (pBNS1-GFP-1A751). Lane 1, control strains (empty vector); Lanes 2-6, SDS-PAGE results of target protein grow at different times (12 h, 15 h, 18 h, 21 h, 24 h); (D) The result of fermentation supernatant (pBNS2-GFP-1A751). Lane 1, control strains (empty vector); Lanes 2-7, SDS-PAGE results of target protein grow at different times (3 h, 6 h, 9 h, 12 h, 15 h, 18 h). (E) Protein purification of GFP by Ni-NTA superflow column (pBNS2-GFP-1A751).

Table 3.The fermentation supernatant and precipitated cells of three strains collected at 24 h; the results are the average from three flasks. The standard errors are shown.

We collected the precipitated cells of pBNS1-GFP-1A751 at different times and the proteins were separated by SDS– PAGE. The result (Fig. 4C) showed a protein band with an apparent molecular mass of about 30 kDa, which was identical to the calculated value mass of the GFP. Meanwhile, culture samples of pBNS2-GFP-1A751 (fermentation supernatant) were collected at different times. The results of SDS-PAGE (Fig. 4D) showed that the GFP band was present in the fermentation supernatant, and the expression level of GFP was decreased compared with the intracellular expression vector pBNS1.

Usage of Secretion Expression Vector pBNS2 for Mannanase Expression

The applicability of pBNS2 vector was tested by first generating the expression vector pBNS2-man and then detecting the mannanase activity of the positive transformants. The resultant plasmid and mutant strain were verified by restriction enzyme digestion and nucleotide sequencing (Fig. 5A). We collected the fermentation supernatant and the precipitated cells of pBNS2-man-1A751 after culture for 24 h and samples were separated by SDS–PAGE. The result (Fig. 5C) showed a protein band with an apparent molecular mass of about 40 kDa, which was identical to the calculated value mass of the mannanase. Furthermore, most of the recombinant protein was secreted extracellularly with vector pBNS2.

Fig. 5.Mannanase expression and SDS-PAGE analysis. (A) Restriction enzyme analysis of recombinant strains. Lanes 1 and 2 represent plasmid pBNS2-man digested by BamHI/XhoI and BamHI, respectively. (B) The restriction map of plasmid pBNS2-man. (C) The SDS-PAGE analysis of pBNS2-man-1A751. Lane 1, fermentation supernatant; Lane 2, precipitated cells.

After culture for 24 h, the mannanase activity was measured by the DNS method as previously described. The mannanase total enzyme activity was 8.65 U/ml, and in the supernatant was 6.49 U/ml (about 75% of the total activity), but no enzyme activity was detected in the transformant harboring the empty pBNS2 plasmid (negative control). The expression level of the mannanase gene in the constructed B. subtilis was 6 times higher than that of parent strain Bacillus pumilus Nsic-2. The results indicate that the plasmid pBNS2 was capable of secreting heterologous proteins directly into the culture medium in B. subtilis.

 

Discussion

Bacillus subtilis has received widespread attention because of several advantages, the most important being the ability of secreting proteins directly into the culture medium. These abilities have been largely successful in producing correctly folded and soluble heterologous proteins [15], and make Bacillus subtilis with the potential application as a gene expression host [12]. However, compared with E. coli, the expression level of Bacillus subtilis is still low. Research scholars have used various methods to improve the expression system of B. subtilis. The common way is using a strong promoter, including inducible promoters PsacB, Pspac[4], Pxyl [18], and constitutive promoters, such as PlepA, PamyE, P43 [18]. Other ways, like incorporating a fused signal sequence to improving protein secretion or deleting various host proteases, were also reported as efficient methods [5]. A strong promoter is the essential element to achieve highlevel expression in B. subtilis genetic engineering. The endogenous B. subtilis promoter P43 is a cytidine/deoxycytidine deaminase promoter, and was originally found using a promoter-probing vector. The promoter P43 contains transcription factors σA (a house-keeping sigma factor that is active during the exponential growth phase) and σB (transcribes stress-related genes in B. subtilis and is active at the end of the exponential cell growth phase). Besides the strong promoter, an efficient Shine-Dalgarno sequence was also depended in the expression system [11]. In these two constructed plasmids, not only the P43 promoter but also the SD sequence worked efficiently in Bacillus subtilis.

The green fluorescent protein has been used as a reporter for many applications [1]. GFP detection only requires irradiation with blue light, and the results can be clearly visualized and rapidly analyzed by fluorescence microscopy [14]. Nowadays, green fluorescent protein as a novel marker gene has been widely used in prokaryotic and eukaryotic cells. We use the GFP gene as a reporter to test the applicability of the two newly constructed vectors in Bacillus subtilis 1A751, and the result indicated that the GFP gene was successfully expressed in B. subtilis. The applicability of the vectors was tested by the expression of the mannanase gene from B. pumilus Nsic-2. β-Mannanase is the key enzyme that catalyzes the random hydrolysis of the β-1,4-D-mannopyranosyl linkages in mannans/heteromannans to release manno-oligosaccharides and plays important roles in industrial applications. Many microorganisms can utilize mannans as the source of carbon, and among these strains Bacillus spp. occupy an important position and have been well studied in previous research [6, 9, 16]. However, there have been few reports of the mannanases expression in Bacillus subtilis. In this work, we focus our attention on the construction and application of the constructed plasmids. Although the expression level in B. subtilis was lower than E. coli, the results indicated that the exogenous gene was successfully expressed in B. subtilis and pBNS2 has the capacity of secreting functional extracellular proteins directly into the culture medium. As the cell concentration reached the maximum after 24-27 h culture and then decreased as cells ran out of nutrients and died, we collected all the samples within 24 h. As is known, the amount of recombinant proteins is dependent on four major factors: efficiency of transcription, mRNA stability, efficiency of translation, and stability of the protein. That may be due to incorrect folding of the target protein, or extracellular protease secreted by B. subtilis host cell, which recognizes and degrades heterologous proteins. In subsequent research, we will further improve the plasmids by increasing the plasmid stability. Moreover, the biochemical characterization of the recombinant mannanase will be studied.

In this research, pBE2a was used as the basic plasmid to constructe two expression vectors, pBNS1 and pBNS2. First, the vector pBNS1 was used to express exogenous genes in high level under the control of the P43 promoter and did not contain signal peptide. Meanwhile, as it contains the B. amyloliquefaciens amyQ signal peptide and unique endonuclease restriction sites, the vector pBNS2 can be directly used for the secretory expression and is convenient to further construct the desired vector in future research. In summary, we have constructed two pBE2a-based expression vectors to achieve intracellular or secretory expression under the control of the P43 promoter. The two expression vectors pBNS1 and pBNS2 showed potential value in industrial applications.