Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially ‘prebiotic’ oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.

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. 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). 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.

This study aimed to investigate the specific growth stimulation of certain desired intestinal bacteria by a novel galacto-oligosaccharide mixture, which was produced with a β-galactosidase from a potential probiotic Lactobacillus isolate that contained mainly oligosaccharides of β-1,3 and β-1,6 glycosidic linkages (termed Lb-GOS) using single-strain fermentations. The composition of this Lb-GOS mixture was 33.5% disaccharides, 60.5% trisaccharides, 4.8% tetrasaccharides, and 1.0% pentasaccharides with a negligible amount of monosaccharides, lactose, and lactobionic acid (0.3%). Eight Lactobacillus spp. strains and three Bifidobacterium spp. strains were used in single-strain fermentations to determine the fermentation activity scores of this Lb-GOS preparation compared to two commercially available prebiotic mixtures, 4′GOS-P and Vivinal GOS (V-GOS). The highest scores were obtained when L. reuteri Lb46 and the two Bifidobacterium strains, B. animalis subsp. lactis Bif1 and Bif3, were grown on these galacto-oligosaccharide mixtures. In addition, the Lb-GOS mixture was found to have higher fermentation activity scores; hence, it stimulated the growth of these probiotic strains more than 4′GOS-P and V-GOS, which may be attributed to the different glycosidic linkage types that are found in the Lb-GOS mixture compared to the other two commercial preparations. These findings suggested that the Lb-GOS mixture that is described in this work should be of interest for the formulations of new carbohydrate-based functional food ingredients.

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.

Previously, β-mannanase (ManB) was successfully immobilized on the surface of the Lactobacillus plantarum using two constitutive promoters, Pgm and SlpA. The highest activity of immobilized ManB (3010 U/g dry cell weight) was obtained after 4 hours of induction culture, extending cultivation to 8 hours led to a 4.2-fold decrease. This decline might be due to lactic acid production by L. plantarum, which lowered the pH of the culture medium. This study evaluated the impact of pH-controlled cultivation on the activity of ManB displayed on L. plantarum WCFS1. Under these conditions, the highest ManB activity decreased by only ~1.5-fold after 8 hours. This demonstrates that maintaining a constant pH level is a critical factor for improving bacterial cell growth and enhancing the overall yield of enzymes displayed L. plantarum surface. This is the first study to investivate the effect of pH on constitutive immobilization of ManB on the Lactobacillus cell surface.