Constitutive expression and cell-surface display of a bacterial β-mannanase in Lactobacillus plantarum

Abstract

Background: Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production.

Results: Two strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS).

Conclusion: This work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.

Keywords: Cell-surface display; Constitutive promoter; Lactobacillus plantarum; Lipoprotein anchor; Mannanase; Pgm; SlpA; Whole-cell biocatalyst.

Fig. 1

The construction of the constitutive expression vectors for N-terminal lipoprotein anchoring of mannanase (ManB) in L. plantarum. a Schematic overview of the pSIP409 expression vector, the starting point for construction. The sppK and sppR genes encode the proteins in the two-component regulatory system. The gene of interest is placed under control of a strong inducible bacteriocin promoter P_sppQ-sakacin Q. The multiple cloning site (MCS), the replicon region consists of two determinants pUC(pGEM)-ori for E. coli and 256_rep for L. sakei and L. plantarum. The selection marker is the erythromycin resistance gene (ery), the lollipops indicate transcription terminators, L indicates the SalI-linker sequence. b Schematic overview of the constitutive expression cassette for N-terminal lipoprotein anchoring of mannanase (ManB) with BglII and EcoRI cloning sites. The inserts containing the manB sequence were fused with a myc tag for protein detection. P_pgm and P_slpA: the promoters of a phosphoglycerate mutase (pgm) from L. acidophilus NCFM and a S-layer protein SlpA of L. acidophilus ATCC 4356, respectively; SP1261 and Lp_1261: a signal peptide and a lipoprotein anchor; SPase: lipobox withSignal Peptidase II cleavage site (SPase); L: linker (SalI site)

Fig. 2

Western blot analysis of cell-free extracts from L. plantarum cells harboring constitutive expression vectors for lipoprotein anchoring of mannanase (ManB) (1) pSIP_1261ManB: L. plantarum harboring inducible expression vector for lipoprotein anchoring of ManB as positive control (protein size 51 kDa); (2) pEV: L. plantarum harboring an empty vector as negative control; (3) pSlpA_1261ManB (expected protein size 51 kDa); (4) pPgm_1261ManB (expected protein size 51 kDa). Lane M indicates molecular mass markers

Fig. 3

Surface localization of ManB in L. plantarum cells analyzed by flow cytometry (a) and immunofluorescent microscopy (b). L. plantarum strains harboring ManB-encoding plasmids are depicted by different colors in the flow cytometry histograms (a) and different numbers in the micrographs (b): pPgm_1261Man (blue, 1, an arrow indicates the ‘shoulder’ of surface mannanase detected by slight shift in the fluorescent signal); pSlpA_1261Man, (green, 2); L. plantarum harboring an empty vector pEV was used as negative control (black, 3)

Fig. 4

Enzymatic activity of ManB-displaying cells. a, b Time course of cultivations of ManB-displaying L. plantarum recombinant strains harboring the plasmids pPgm_1261ManB (closed symbols) and pSlpA_1261ManB (open symbols) in MRS medium at 37 °C with activities of surface displayed mannanase expressed as U/g dry cell weight and U/l fermentation (a) and OD₆₀₀ and dry cell weights (mg/l fermentation) (b). c Maximum activities of ManB-displaying L. plantarum carrying various plasmids: pEV (negative control); pSIP_1261ManB for inducible expression of ManB; pSlpA_1261ManB and pPgm_1261ManB for constitutive expression of ManB

Fig. 5

Enzyme activity of surface displayed ManB in viable L. plantarum. a Results of repeated activity measurements of L. plantarum cells harboring pSlpA_1261ManB in comparison with pSIP_1261ManB, with 0 indicating freshly harvested cells, while 1, 2, 3, 4 indicated the number of repetitions; b Enzyme activity of ManB displayed on L. plantarum harboring pSlpA_1261ManB at various period of incubation in PBS and at 37 °C. The activity is relative to time point 0 (freshly harvested cells)

Fig. 6

Formation of manno-oligosaccharides (MOS) from LBG (5%) by mannanase-displaying L. plantarum cells harboring pSlpA_1261ManB at 37 °C. a Thin layer chromatography (TLC) analysis; Standards: mannose, M1, mannobiose, M2, mannotriose, M3, mannnotetraose (M4), mannopentose (M5), mannohexaose, M6. b HPAEC chromatogram of the reaction mixture after 4 h of conversion (black line); Standards: O-GGM5 (pink line), M1–M6 as used in TLC analysis (blue line)