Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2.

BACKGROUND: Utilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins. RESULTS: Whole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35-37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences. CONCLUSION: All experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles.

The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1.

Enterocin K1 (EntK1), a bacteriocin that is highly potent against vancomycin-resistant enterococci, depends on binding to an intramembrane protease of the site-2 protease family, RseP, for its antimicrobial activity. RseP is highly conserved in both EntK1-sensitive and EntK1-insensitive bacteria, and the molecular mechanisms underlying the interaction between RseP and EntK1 and bacteriocin sensitivity are unknown. Here, we describe a mutational study of RseP from EntK1-sensitive Enterococcus faecium to identify regions of RseP involved in bacteriocin binding and activity. Mutational effects were assessed by studying EntK1 sensitivity and binding with strains of naturally EntK1-insensitive Lactiplantibacillus plantarum-expressing various RseP variants. We determined that site-directed mutations in conserved sequence motifs related to catalysis and substrate binding, and even deletion of two such motifs known to be involved in substrate binding, did not abolish bacteriocin sensitivity, with one exception. A mutation of a highly conserved asparagine, Asn359, in the extended so-called LDG motif abolished both binding of and killing by EntK1. By constructing various hybrids of the RseP proteins from sensitive E. faecium and insensitive L. plantarum, we showed that the extracellular PDZ domain is the key determinant of EntK1 sensitivity. Taken together, these data may provide valuable insight for guided construction of novel bacteriocins and may contribute to establishing RseP as an antibacterial target.

The Fish Pathogen Aliivibrio salmonicida LFI1238 Can Degrade and Metabolize Chitin despite Gene Disruption in the Chitinolytic Pathway.

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source, since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that A. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose [(GlcNAc)2], the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate than on the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and ß-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity than other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on ß-chitin, whereas the LPMO gene deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of A. salmonicida LFI1238 growth on ß-chitin showed expression of all three chitinolytic enzymes and, intriguingly, also three of the disrupted chitinases. In conclusion, our results show that A. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study, we demonstrate that A. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation.

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants.

Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP TG Anchor.

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.

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

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.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE: This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice.

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose ß-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu(2+) center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation.

Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.

BACKGROUND: 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. RESULTS: We studied overexpression of a chitosanase (CsnA) and a ß-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. CONCLUSIONS: 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.

Display of a ß-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using ß-galactosidase from Streptococcus thermophilus.

ß-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000 U of ß-galactosidase activity per liter of culture corresponding to approximately 170 mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205 g L-1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

EF0176 and EF0177 from Enterococcus faecalis V583 are substrate-binding lipoproteins involved in ABC transporter mediated ribonucleoside uptake.

One of the ABC transporter systems in Enterococcus faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knockout mutants, we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI 1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad and overlapping, but not identical, substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.

Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells.

BACKGROUND: Chemokines are attractive candidates for vaccine adjuvants due to their ability to recruit the immune cells. Lactic acid bacteria (LAB)-based delivery vehicles have potential to be used as a cheap and safe option for vaccination. Chemokine produced on the surface of LAB may potentially enhance the immune response to an antigen and this approach can be considered in development of future mucosal vaccines. RESULTS: We have constructed strains of Lactobacillus plantarum displaying a chemokine on their surface. L. plantarum was genetically engineered to express and anchor to the surface a protein called CCL3Gag. CCL3Gag is a fusion protein comprising of truncated HIV-1 Gag antigen and the murine chemokine CCL3, also known as MIP-1α. Various surface anchoring strategies were explored: (1) a lipobox-based covalent membrane anchor, (2) sortase-mediated covalent cell wall anchoring, (3) LysM-based non-covalent cell wall anchoring, and (4) an N-terminal signal peptide-based transmembrane anchor. Protein production and correct localization were confirmed using Western blotting, flow cytometry and immunofluorescence microscopy. Using a chemotaxis assay, we demonstrated that CCL3Gag-producing L. plantarum strains are able to recruit immune cells in vitro. CONCLUSIONS: The results show the ability of engineered L. plantarum to produce a functional chemotactic protein immobilized on the bacterial surface. We observed that the activity of surface-displayed CCL3Gag differed depending on the type of anchor used. The chemokine which is a part of the bacteria-based vaccine may increase the recruitment of immune cells and, thereby, enhance the reaction of the immune system to the vaccine.

Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo.

BACKGROUND: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. RESULTS: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. CONCLUSION: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.

Heterologous expression of a recombinant lactobacillal ß-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

BACKGROUND: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric ß-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri ß-galactosidase. RESULTS: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum ß-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), ß-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant ß-galactosidase. CONCLUSIONS: The over-expression of recombinant L. reuteri ß-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

BACKGROUND: Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. RESULTS: In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. CONCLUSIONS: We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

Effects of Lactobacillus rhamnosus LA68 on the immune system of C57BL/6 mice upon oral administration.

Probiotic bacteria have been used in human nutrition for centuries and are now attracting more attention. In order to examine the immunological aspects of probiotic consumption, Lactobacillus rhamnosus LA68 was orally administrated using gavage to healthy C57BL/6 mice. After one month splenocytes were isolated, and analysed by flow cytometry. The magnitude of splenocyte proliferation upon stimulation with lipopolysaccharide and peptidoglycan and cytokine levels (IFN-γ, IL-6, IL-10 and IL-17) was assessed. Cytokine levels in the serum were also analysed. Oral application of strain LA68 leads to a significant decrease of CD3+, CD25+ and CD19+ cells, and an increase of CD11b+ and CD16/CD32+ positive cell populations in the mouse spleen. Increased sensitivity to stimulation through proliferation and IL-6 secretion was detected. Increased serum IFN-γ and decreased IL-10 levels were found. Our results show increased responsiveness of splenocytes, activation of the Th1 type of immune response, and a shift of leucocyte populations towards monocyte/granulocyte populations.

Lactobacillus plantarum WCFS1 O-linked protein glycosylation: an extended spectrum of target proteins and modification sites detected by mass spectrometry.

It has recently been shown that the major autolysin Acm2 from Lactobacillus plantarum WCFS1 undergoes intracellular O-GlcNAcylation [Fredriksen L, Mathiesen G, Moen A, Bron PA, Kleerebezem M, Eijsink VG, Egge-Jacobsen W. 2012. The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation. J Bacteriol. 194(2):325-333]. To gain more insight into the occurrence of this protein modification, methods based on the higher energy collisional fragmentation of the Orbitrap XL mass spectrometer to generate both diagnostic oxonium (glycan) ions and significant peptide sequencing information were used to detect and identify novel glycoproteins. This led to the identification of 10 novel glycoproteins, including four proteins with well-known functions in the cytoplasm, a compartment not previously recognized to contain glycosylated proteins in bacteria: the molecular chaperone DnaK, the E2 subunit of the pyruvate dehydrogenase complex PdhC, the signal recognition particle receptor FtsY and the DNA translocase FtsK1. Among the other, glycosylated proteins were two extracellular peptidoglycan hydrolases and a mucus-binding protein. In total, 49 glycosylation sites for N-acetylhexosamine (HexNAc) were detected in the 11 Lactobacillus glycoproteins found so far. Most of the attached glycans consisted of a single HexNAc per site, whereas hexose moieties were also found in a few cases (in both of the peptidoglycan hydrolases and in DnaK).

Establishment of a simple Lactobacillus plantarum cell consortium for cellulase-xylanase synergistic interactions.

Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD600) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.

The role of site-2-proteases in bacteria: a review on physiology, virulence, and therapeutic potential.

Site-2-proteases are a class of intramembrane proteases involved in regulated intramembrane proteolysis. Regulated intramembrane proteolysis is a highly conserved signaling mechanism that commonly involves sequential digestion of an anti-sigma factor by a site-1- and site-2-protease in response to external stimuli, resulting in an adaptive transcriptional response. Variation of this signaling cascade continues to emerge as the role of site-2-proteases in bacteria continues to be explored. Site-2-proteases are highly conserved among bacteria and play a key role in multiple processes, including iron uptake, stress response, and pheromone production. Additionally, an increasing number of site-2-proteases have been found to play a pivotal role in the virulence properties of multiple human pathogens, such as alginate production in Pseudomonas aeruginosa, toxin production in Vibrio cholerae, resistance to lysozyme in enterococci and antimicrobials in several Bacillus spp, and cell-envelope lipid composition in Mycobacterium tuberculosis. The prominent role of site-2-proteases in bacterial pathogenicity highlights the potential of site-2-proteases as novel targets for therapeutic intervention. In this review, we summarize the role of site-2-proteases in bacterial physiology and virulence, as well as evaluate the therapeutic potential of site-2-proteases.