(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 CRIB^T. 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.

The gut microbiota of mammals possess distinctive metabolic pathways with untapped therapeutic potential. Using molecular insights into dietary fiber metabolism by the human gut microbiota, we designed a targeted drug delivery system, called GlycoCaging, that is based on bespoke glycoconjugates of a complex plant oligosaccharide. GlycoCaging of exemplar anti-inflammatory drugs enabled release of active molecules triggered by specific glycosidases of autochthonous gut bacteria. GlycoCaging ensured that drug efficacy was potentiated, and off-target effects were eliminated in murine models of inflammatory bowel disease. Biochemical and metagenomic analyses of gut microbiota of individual humans confirmed the broad applicability of this strategy.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique’s potential to differentiate various red seaweed species.

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

This study introduces a novel approach to analyze glycosidic linkages in unfractionated polysaccharides from alcohol-insoluble residues (AIRs) of five brown seaweed species. GC-MS analysis of partially methylated alditol acetates (PMAAs) enables monitoring and comparison of structural variations across different species, harvest years, and tissues with and without blanching treatments. The method detects a wide array of fucose linkages, highlighting the structural diversity in glycosidic linkages and sulfation position in fucose-containing sulfated polysaccharides. Additionally, this technique enhances cellulose quantitation, overcoming the limitations of traditional monosaccharide composition analysis that typically underestimates cellulose abundance due to incomplete hydrolysis of crystalline cellulose. The introduction of a weak methanolysis-sodium borodeuteride reduction pretreatment allows for the detection and quantitation of uronic acid linkages in alginates.

β-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of β-chitin-derived high-value enzymes and products.