Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system
- Suttipong Sak-Ubol†1, 2,
- Peenida Namvijitr†1,
- Phornsiri Pechsrichuang1,
- Dietmar Haltrich2,
- Thu-Ha Nguyen2,
- Geir Mathiesen3,
- Vincent G. H. Eijsink3 and
- Montarop Yamabhai1Email authorView ORCID ID profile
© The Author(s). 2016
Received: 29 January 2016
Accepted: 3 May 2016
Published: 12 May 2016
Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially food-grade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications.
We studied overexpression of a chitosanase (CsnA) and a β-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84 % for ManB, and 79 kU/L and 56 % for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host.
ManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker.
Heterologous production of hydrolytic enzymes is important for green and white biotechnology since such enzymes serve as green industrial biocatalysts for the conversion of biomass into value-added products . Lactic acid bacteria (LAB) are interesting hosts for the production of such enzymes because many of these bacteria are generally recognized as safe (GRAS), carry the qualified presumption of safety (QPS) status, and are easy to cultivate . While LAB may not be the most efficient cell factories, their safety and food-grade status make them particularly attractive for producing enzymes that are to be used in e.g. food processing. One attractive host is Lactobacillus plantarum, as it had been widely used for foods, and hence is food-grade and even considered a probiotic  with potential benefits to human health . To facilitate downstream processing in large-scale biotechnological applications, secretion of the over-expressed enzymes into the culture medium is desirable . Therefore, lactobacillal expression systems based on the so-called pSIP vectors [6, 7] have been developed recently for the efficient secretion of heterologous proteins in L. plantarum .
In the present study we selected two extracellular, hydrolytic enzymes from Bacillus, a β-mannanase from Bacillus licheniformis (BlManB) and a chitosanase from Bacillus subtilis (BsCsnA), to study secretory production in L. plantarum. Both enzymes are of interest for biotechnological applications, namely the conversion of hemicelluloses (mannans) and chitosan into manno-oligosaccharides (MOS)  and chito-oligosaccharides (CHOS) , respectively. For comparative purposes, these enzymes were overproduced in three forms: with no signal peptide, with their native (Bacillus) signal peptide, and with a signal peptide derived from the Escherichia coli OmpA protein. In addition, we compared two different selection markers, one based on antibiotic (erythromycin) resistance, and the other based on complementation selection using alanine racemase (alr). The engineered production strains were evaluated in terms of enzyme yields and secretion efficiencies.
Construction of expression vectors
Expression and secretion of BlManB and BsCsnA in L. plantarum
Yields and secretion efficiency for production of BlManB and BsCsnA in L. plantarum WCFS1 harboring various expression constructs based on the pSIP409 vector
Type of SP
Volumetric activity (per liter)
% Secretion efficiency
Specific activity (U/mg)
42 ± 1.3 kU
8 ± 0.2 kU
50 ± 1.5 kU
16 ± 0.6 kU
7 ± 0.3 kU
23 ± 0.9 kU
5 ± 0.2 kU
20 ± 0.9 kU
25 ± 1.1 kU
63 ± 0.5 kU
36 ± 1.4 kU
99 ± 1.9 kU
9 ± 0.03 kU
13 ± 0.1 kU
22 ± 0.13 kU
1 ± 0.05 kU
15 ± 0.7 kU
16 ± 0.8 kU
The genome of L. plantarum WCFS1 does not encode any known β-mannanase or chitosanase (www.cazy.org; ). Since mannanase activity in certain L. plantarum strains has however been reported , we checked the intrinsic β-mannanase and chitosanase activities in L. plantarum WCFS1 and its alr derivative TGL02, which were used as host strains in this study. Using the same fermentation and analytical procedures as above, we could not detect any chitosanase activity in the cell lysate or the culture supernatant of either strain. As for β-mannanase activity, we did not detect any activity in the lysates, while a trace amount of β-mannanase activity was detected in culture supernatants (about 150 units/L; i.e. less than 1 % of typical values shown in Fig. 2). Thus, the enzyme activities reported and discussed in this study are essentially devoid of background activity from the host bacterium.
Purification and analysis of secreted BlManB and BsCsnA
Expression of BlManB and BsCsnA using a food-grade vector system
Yields and secretion efficiency for production of BlManB_nt and BsCsnA_nt by L. plantarum TLG02 (d-alanine auxotroph), measured 20 h after induction
Volumetric activity (per liter)
% Secretion efficiency
Cell extract (kU)
31 ± 0.8
9 ± 0.2
40 ± 1
31 ± 0.5
38 ± 0.7
69 ± 1.2
33 ± 0.1
50 ± 2.2
83 ± 2.3
In this study we show that a β-mannanase (BlManB) from B. licheniformis as well as a chitosanase (BsCsnA) from B. subtilis can be expressed and secreted efficiently in L. plantarum using the pSIP expression system. Both enzymes were produced and secreted at high levels compared to the levels previously obtained using E. coli expression systems [11, 12], also when using a potentially food-grade vector system that does not depend on the use of an antibiotic resistance selection marker. When estimated from the specific activities of the purified enzymes (1800 U/mg for ManB and 800 U/mg for CsnA), total yields of recombinant proteins were ~28 mg/L medium for ManB produced with its native signal and a pSIP409-based vector, and ~127 mg/L of recombinant CsnA, again when using the native signal and a pSIP409-derived vector, 24 h after induction (Table 1). The amount of secreted, extracellular recombinant protein for these constructs was ~23 mg/L medium for ManB and ~79 mg/L for CsnA, showing again the efficient secretion of these recombinant enzymes. Replacement of the selection marker from erm R to alr led to slightly lower expression levels (~22 mg of total ManB and ~17 mg of secreted ManB per L medium; ~86 mg of total CsnA and ~39 mg of secreted CsnA per L medium; calculated from data given in Table 2 and the specific activities of the purified enzymes). The lower overall expression levels with the alr marker could reflect different plasmid copy numbers due to different selective pressures. Indeed, a previous comparative study on intracellular expression of β-galactosidases showed that alr selection led to lower plasmid copy numbers and slightly lower protein production levels . Since the difference in expression level primarily concerned the secreted fraction of the produced protein, one could also speculate about connections between cell wall metabolism (which is effected by the alr mutation) and protein secretion.
It should be noted that further optimization can be done to obtain higher production levels with the various expression set-ups developed here. Possible variables include the glucose concentration and the glucose-feeding regime, the amount of inducing peptide added as well as the time point of induction, the cultivation temperature, and the harvesting time .
The potential of using pSIP vectors for both intracellular protein production and protein secretion has been previously reported for model enzymes such as nuclease (NucA) and amylase (AmyA)  as well as other enzymes including β-glycosidases , oxalate decarboxylase [22, 23], cellulases and xylanases , and L-arabinose and D-xylose isomerases . Most of these studies used pSIP vectors with the erm antibiotic selection marker, with the exception of studies on the expression of intracellular β-galactosidase , and L-arabinose and D-xylose isomerase , in which the food-grade alr selection marker was used. The present results underpin the usefulness of the alr selection marker for food-grade applications.
Notably, since the pSIP vector is a modular plasmid, existing constructs could easily be modified to suit desired purposes . For example, the p256 replicon, which only functions in a limited range of lactobacilli  could be exchanged to allow broader host range. This could be useful because lactobacilli have different properties when it comes to e.g. probiotic activity, acid production, production of antimicrobial compounds such as bacteriocins, and the ability to interact with the human mucosa . Notably, if the alr selection marker is to be used, application of pSIP vectors in other lactobacilli would require the engineering of d-alanine auxotrophs for each host strain .
The native signal peptides of the two Bacillus hydrolytic enzymes functioned well in that they gave good secretion efficiencies, comparable to those obtained with the better performing signal peptides of L. plantarum itself, as assessed in previous genome-wide signal peptide-screening studies . Since Bacillus also is a Gram-positive bacterium, it is not surprising that the native Bacillus signal peptides were efficient in directing secretion of heterologous proteins in Gram-positive L. plantarum. The Bacillus ManB signal peptide seemed particularly efficient reaching secretion efficiencies in the order of 80 %, and should perhaps be considered for use in secretion of other heterologous proteins in L. plantarum.
The data presented above show that the choice of the signal peptide not only affects the secretion efficiency but also the total expression level. This has been observed before [8, 20] and is likely due to effects of the 5′ part of the gene sequence and/or the amino acid sequence of the N-terminal part of the translated protein on overall translation efficiency . The level of protein production apparently determines the secretion efficiency as well. It is likely that when the protein is expressed above a certain critical amount, saturation of the secretion machinery will occur [28, 29]. The latter could be the case for BsCsnA, which is expressed at more than threefold higher levels and even secreted in higher amounts, but with a lower overall secretion efficiency compared to BlManB (Table 1). Possibly, enzyme size also plays a role; the better produced BsCsnA (~30 kDa) is smaller than BlManB (~41 kDa).
An efficient expression and secretion system for food-grade production of a β-mannanase (ManB) and a chitosanase (CsnA) in L. plantarum has been established. Our results indicate that native Bacillus signal peptides can be used for efficient expression and secretion of heterologous proteins in L. plantarum, providing an alternative for homologous, and, in a sense, more “food-grade” lactobacillal signal peptides. The modular pSIP vectors and the alr selection marker provide useful tools for the expression of heterologous proteins in L. plantarum.
Plasmids CsnNative-pMY202 and CsnOmpA-pMY202 containing the chitosanase (csnA) gene from B. subtilis with its native or the OmpA signal peptide, respectively, and a C-terminal His-tag  were used as templates for amplification of chitosanase constructs in this study. The plasmid manBOmpA-pMY202  was used as template for amplification of the B. licheniformis manB gene containing the OmpA signal peptide. B. licheniformis DSM13 (NCBI accession number NC006322.1) genomic DNA was used as a template for amplifying a manB variant containing the native signal peptide. E. coli Top10 and MB1259 cells were used for molecular cloning with the pSIP409 and pSIP609 vectors, respectively. L. plantarum strains WCFS1  and TGL02  were used for expression studies with pSIP409 and pSIP609, respectively.
Constructs based on pSIP409 (antibiotic selection)
Oligonucleotide primers used in this study
BsCsn_OmpA & BlManB_OmpA
Plasmid pSIP409  was digested with NcoI and XhoI, and gel-purified before being used in ligation reactions. In these reactions the molar ratio of linearized vector to re-annealed insert was approximately 1:15. The amount of linearized vector used for each ligation reaction was 50–100 ng. Ligations were performed for 16 h at 16 °C in the presence of T4 DNA ligase in a final volume of 25 µL. T4 DNA ligase was heat-inactivated (65 °C for 15 min) before transformation of the ligation mixtures into competent E. coli TOP10 cell. Transformants were selected on LB agar containing 800 µg/mL erythromycin; plates were incubated at 37 °C for 16 h.
Construction of food-grade expression vectors (pSIP609 series)
Plasmids used in this study
erm, pSIP401 derivative, gusA controlled by PsppQ
pSIP409 derivative, erm replaced by alr
pFLAG-CTS derivative, csn_nt controlled by tac
pFLAG-CTS derivative, csn_OmpA controlled by tac
pFLAG-CTS derivative, manB_ompA controlled by tac
erm, pSIP409 derivative, csn_nt controlled by PsppQ
erm, pSIP409 derivative, csn_OmpA controlled by PsppQ
erm, pSIP409 derivative, csn controlled by PsppQ
alr, pSIP409 derivative, csn_nt controlled by PsppQ, erm replaced by alr
erm, pSIP409 derivative, manB_nt controlled by PsppQ
erm, pSIP409 derivative, manB_OmpA controlled by PsppQ
erm, pSIP409 derivative, manB controlled by PsppQ
alr, pSIP409 derivative, manB_nt controlled by PsppQ, erm replaced by alr
Transformation of L. plantarum
Lactobacillus plantarum competent cells were prepared and transformed by electroporation as previously described . To transform the competent cells, 2–5 µg of plasmid DNA was added to 40 µL of electrocompetent cells. The mixture was then transferred to chilled cuvettes. After drying and cleaning the outside of the cuvette it was placed into the electroporator, after which the cells were electroporated at 1.5 kV, followed by incubation on ice for 2 min. After adding 500 µL of MRS medium containing 0.5 M glucose and 0.1 M MgCl2 the cells were transferred to a clean 1.5 mL tube and incubated at 30 °C without agitation for 1–2 h. Finally, the cells were plated out on MRS agar plates containing 200 µg/mL of erythromycin (for pSIP409-type vectors transformed to L. plantarum WCFS1) or containing no antibiotics (for pSIP609-type vectors transformed to L. plantarum TGL02). Colonies appeared after incubation at 37 °C for 16 h.
Expression of BlManB and BsCsnA in L. plantarum
Batch fermentations with pH control were carried out in 3-L MRS medium using a BIOSTAT B plus bioreactor (Sartorius, Germany). Recombinant L. plantarum strains were taken from a glycerol stock stored at −80 °C, re-streaked on appropriate MRS plates (with or without antibiotic, depending on the L. plantarum strain; see above) and grown overnight at 37 °C. Five to ten colonies were picked and grown in 5 mL MRS broth overnight, then sub-cultured into two flasks of 100 mL of MRS, and cultivated at 37 °C without shaking for 18–24 h. The two overnight cultures were pooled together, mixed well and after measuring the cell density at 600 nm (Ultrospec 2000, Pharmacia biotech, UK) they were used to inoculate 3 L of MRS medium to an OD600 of ~0.1. After incubation at 30 °C with 100 rpm agitation under anaerobic condition to an OD600 of ~0.3, the cultures were induced with 12.5 ng/mL of IP-673 (amino acid sequence of IP-673 is Met-Ala-Gly-Asn-Ser–Ser-Asn-Phe-Ile-His-Lys-Ile-Lys-Gln-Ile-Phe-Thr-His-Arg; ). During further cultivation (30 °C with 100 rpm), the pH was controlled at pH 6.5 using 3.0 M sodium hydroxide. To monitor enzyme production, 40–50 mL of culture broth were harvested at 0, 3, 6, 9, 12, 18, 20, and 24 h after induction. The cells and culture supernatant were separated by centrifugation at 4000 rpm for 15 min at 4 °C (swing angle rotor, Centrifuge 5804, Eppendorf, Belgium), after which the cells were washed twice with lysis buffer (20 mM Tris–HCl, 150 mM NaCl, pH 8.0), and re-suspended in 3–4 mL of the same buffer. The cells were broken using a sonicator (Vibra-Cell Sonicator, Sonics & Materials, Inc, USA) at 25 % amplitude, pulse 5 s, 3 min for 2 rounds on ice. The cell lysate fraction was collected by centrifugation at 13,000 rpm, 4 °C for 45 min (Thermo Scientific, USA).
To measure the enzyme activity in culture supernatants, 3–5 mL of culture supernatant containing secreted enzymes were dialyzed with 10 mM Tris–HCl buffer, pH 8.0 with stirring at 250 rpm, at 4 °C for 8–12 h, using the snake skin dialysis tubing, 10 kDa kit (Thermo scientific, USA). The dialyzed fraction of approx. 4–7 mL was collected and kept on ice for no longer than 6 h before the enzyme activity was determined.
Enzyme activity assay
ManB and CsnA activities in both lysates and supernatants were determined using the DNS method as previously described [11, 12]. For BlManB, an appropriately diluted enzyme solution (0.1 mL) was incubated with 0.9 mL of pre-heated 0.5 % (w/v) locust bean gum (dissolved in 50 mM sodium citrate buffer, pH 6.0) at 50 °C for exactly 5 min, with mixing at 800 rpm. The amount of reducing sugars liberated in the enzyme reaction was assayed by mixing 100 μL of the reaction mixture with 100 μL DNS solution, followed by heating at 100 °C for 20 min, cooling on ice, and dilution with 300 μL of de-ionized water, before measuring the absorbance at 540 nm, using 1–5 µmol/mL of d-mannose as standards. The reactions were done in triplicate and we report mean values together with their standard deviation. The substrate solution was prepared by suspending 0.5 % (w/v) locust bean in 50 mM sodium citrate buffer, pH 6.0. The suspension was then dissolved at 80 °C, using hot plate stirrer at 200 rpm. (RCT CL, IKA Laboratory, Germany), followed by heating to the boiling point, cooled and stored overnight with continuous stirring. After that insoluble material was removed by centrifugation.
For BsCsnA, the reaction mixture consisted of 40 µL of appropriately diluted sample and 160 µL of 0.5 % chitosan (w/v) (in 200 mM sodium acetate buffer, pH 5.5, and pre-incubated at 50 °C for 30 min). The reaction was incubated in a Thermomixer Comfort (Eppendorf AG, Hamburg, Germany) at 50 °C for 5 min, with mixing at 900 rpm. The reaction was stopped by adding 200 µL of DNS solution, and the mixture was centrifuged at 12,000g for 5 min to remove the remaining chitosan that was precipitated. The colour in the supernatant was developed by heating at 100 °C for 20 min and cooling on ice. The reducing sugar in the supernatant was determined by measuring OD at 540 nm, using 1–5 µmol/mL of d-glucosamine as standards. The reactions were done in triplicate and we report mean values with standard deviations.
The final volume of culture supernatant after dialysis was taken into account when the volumetric enzyme activity was determined. Units of enzyme activity were defined as the amount of enzyme that liberates 1 μmol of reducing sugar (using d-mannose or d-glucosamine as a standard) per minute under the standard assay conditions.
Denaturing sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) was performed using the method of Laemmli  with 12 % (w/v) polyacryamide gels. The protein samples were briefly heated (3 min) in the loading buffer at 100 °C using a heat block (Eppendorf), and then cooled on ice before loading. Protein bands were visualized by staining with Coomassie brilliant blue R-250.
Protein concentrations were determined using the method of Bradford  with bovine serum albumin as standard.
N-terminal protein sequencing
Proteins in culture supernatants were separated by SDS-PAGE and electroblotted onto a PVDF membrane (Bio-Rad) in 50 mM borate buffer containing 10 % (v/v) methanol, pH 9. After blotting, the membrane was stained with Coomassie blue for 3 min, followed by destaining with 40 % (v/v) methanol, 10 % (v/v) acetic acid. Bands were cut out of the membrane and analyzed by a commercial provider using Edman degradation on an Applied Biosystems Procise 492 protein sequencer (Protein Micro-Analysis Facility, Medical University of Innsbruck, Austria).
SS and PN performed most of the ManB and CsnA experiments, respectively. SS and PN helped drafting the manuscript. PP helped with the CsnA assay and drafted the manuscript. DH and THN helped in designing the experiments, analyzed the data and edited the manuscript. GM and VGH participated in the generation of the pSIP vectors, analyzed data, and wrote parts of the manuscript. MY conceived of the study, supervised the cloning, expression and analysis of the enzymes, and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by Suranaree University of Technology (SUT) and by the Office of the Higher Education Commission under NRU project of Thailand as well as the National Research Council (NRCT) of Thailand. It is the outcome of the MoU between SUT and BOKU. SS was supported by Royal Golden Jubilee (RGJ) scholarship from Thailand Research Fund (TRF), PN was supported by TRF-MAG scholarship, and PS was supported by SUT-Ph.D. scholarship.
The authors declare that they have no competing interests.
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