The development of bacterial vaccines is a complex challenge due to the substantial serological diversity of protective antigens. One promising antigenic target is the conserved surface polysaccharide poly-β-(1,6)- N -acetyl-D-glucosamine (PNAG). Despite its widespread distribution, antibodies raised against PNAG have shown restricted efficacy in promoting microbial elimination in vitro and safeguarding against infections in vivo. Systematic studies and vaccine development have been hindered by limited knowledge of optimal antigenic features, such as chain length and degree of N -acetylation. Here, we describe an effective n + 2 glycosylation strategy enabling controlled synthesis of partially (dPNAG) and fully deacetylated PNAG glycans. Glycan microarray analysis shows that dPNAG glycans with DP8 and DP12 are optimal, with corresponding protein conjugates eliciting the highest IgG titers. Sera containing antibodies against the dPNAG DP8 conjugate with 40% acetylation exhibit the best opsonic activity against three prevalent nosocomial pathogens and confer the highest protection in female BALB/c mice against Staphylococcus aureus , supporting its potential as a vaccine candidate.

Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.

Plant-based expression systems have emerged as promising avenues for the production of recombinant N-linked glycoproteins. This review offers insights into the evolution and progress of plant glycoengineering. It delves into the distinctive features of plant-derived N-glycans, the diverse range of plant hosts employed for glycoprotein synthesis, and the advancements in glycoengineering strategies aimed at generating glycoproteins with N-glycan structures akin to those produced in mammalian cell lines. Furthermore, alternative strategies for augmenting glycoengineering efforts and the current spectrum of applications for plant-produced N-glycan recombinant proteins are examined, underscoring their potential significance in biopharmaceutical manufacturing.

Barley (1,3;1,4)-β-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-β-d-glucanase, enabling the hydrolysis of (1,3;1,4)-β-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-β-d-glucan endohydrolase [(1,3;1,4)-β-d-glucanase] isoenzyme EII (HvEII) and (1,3)-β-d-glucan endohydrolase [(1,3)-β-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-β-d-glucanase and one variant that hydrolyzed both (1,3)-β-d-glucans and (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-β-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-β-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-β-d-glucans or (1,3)-β-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-β-d-glucans and (1,3)-β-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

The exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis have recently been shown to include (1,3;1,4)-β-D-glucans. In the present study, we examined another Clostridia bacterium Clostridium ventriculi that has long been considered to contain abundant amounts of cellulose in its exopolysaccharides. We treated alcohol insoluble residues of C. ventriculi that include the exopolysaccharides with the enzyme lichenase that specifically hydrolyses (1,3;1,4)-β-D-glucans, and examined the oligosaccharides released. This showed the presence of (1,3;1,4)-β-D-glucans, which may have previously been mistaken for cellulose. Through genomic analysis, we identified the two family 2 glycosyltransferase genes CvGT2–1 and CvGT2–2 as possible genes encoding (1,3;1,4)-β-D-glucan synthases. Gain-of-function experiments in the yeast Saccharomyces cerevisiae demonstrated that both of these genes do indeed encode (1,3;1,4)-β-D-glucan synthases.

The aquatic Araceae species Lemna minor was earlier shown to have xyloglucans with a different structure from the fucogalactoxyloglucans of other non-commelinid monocotyledons. We investigated 26 Araceae species (including L. minor), from five of the seven subfamilies. All seven aquatic species examined had xyloglucans that were unusual in having one or two of three features: < 77% XXXG core motif [L. minor (Lemnoideae) and Orontium aquaticum (Orontioideae)]; no fucosylation [L. minor (Lemnoideae), Cryptocoryne aponogetonifolia, and Lagenandra ovata (Aroideae, Rheophytes clade)]; and > 14% oligosaccharide units with S or D side chains [Spirodela polyrhiza and Landoltia punctata (Lemnoideae) and Pistia stratiotes (Aroideae, Dracunculus clade)]. Orontioideae and Lemnoideae are the two most basal subfamilies, with all species being aquatic, and Aroideae is the most derived. Two terrestrial species [Dieffenbachia seguine and Spathicarpa hastifolia (Aroideae, Zantedeschia clade)] also had xyloglucans without fucose indicating this feature was not unique to aquatic species.

(1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.