Evaluation of Microbial-Fructo-Oligosaccharides Metabolism by Human Gut Microbiota Fermentation as Compared to Commercial Inulin-Derived Oligosaccharides.

The prebiotic potential of fructo-oligosaccharides (microbial-FOS) produced by a newly isolated Aspergillus ibericus, and purified by Saccharomyces cerevisiae YIL162 W, was evaluated. Their chemical structure and functionality were compared to a non-microbial commercial FOS sample. Prebiotics were fermented in vitro by fecal microbiota of five healthy volunteers. Microbial-FOS significantly stimulated the growth of Bifidobacterium probiotic strains, triggering a beneficial effect on gut microbiota composition. A higher amount of total short-chain fatty acids (SCFA) was produced by microbial-FOS fermentation as compared to commercial-FOS, particularly propionate and butyrate. Inulin neoseries oligosaccharides, with a degree of polymerization (DP) up to 5 (e.g., neokestose and neonystose), were identified only in the microbial-FOS mixture. More than 10% of the microbial-oligosaccharides showed a DP higher than 5. Differences identified in the structures of the FOS samples may explain their different functionalities. Results indicate that microbial-FOS exhibit promising potential as nutraceutical ingredients for positive gut microbiota modulation.

PcCesA1 is involved in the polar growth, cellulose synthesis, and glycosidic linkage crosslinking in the cell wall of Phytophthora capsici.

Phytophthora capsici is a destructive plant pathogen that infects a wide range of hosts worldwide. The P. capsici cell wall, rich in cellulose, is vital for hyphal growth and host interactions. However, the enzymes involved in its synthesis remain largely unelucidated. In the current study, we functionally characterized the cellulose synthase gene PcCesA1, which is highly conserved in Phytophthora. By using CRISPR/Cas9-mediated gene replacement and in situ complementation system, it was found PcCesA1 is essential for the mycelial growth, cystospore germination, and pathogenicity of P. capsici. The normal deposition of newly synthesized cell wall components and the polar growth point formation were disrupted in PcCesA1 knockout mutants, suggesting that PcCesA1 plays an important role in the polar growth of P. capsici. Compared with the wild-type strains, PcCesA1 knockout mutants displayed a thicker inner layer cell wall and were more sensitive to carboxylic acid amide fungicides (CAAs). The contents of the cell wall polysaccharides 1,4-Glc, 1,4,6-Glc, and 1,3,4-Glc were reduced in PcCesA1 knockout mutants, suggesting that PcCesA1 affected cellulose content and glycosidic linkage crosslinking in the cell wall. Our findings demonstrate that PcCesA1 is required for cell wall biogenesis. Therefore, PcCesA1 may be a potential target for Phytophthora disease control.

Combined whole cell wall analysis and streamlined in silico carbohydrate-active enzyme discovery to improve biocatalytic conversion of agricultural crop residues.

The production of biofuels as an efficient source of renewable energy has received considerable attention due to increasing energy demands and regulatory incentives to reduce greenhouse gas emissions. Second-generation biofuel feedstocks, including agricultural crop residues generated on-farm during annual harvests, are abundant, inexpensive, and sustainable. Unlike first-generation feedstocks, which are enriched in easily fermentable carbohydrates, crop residue cell walls are highly resistant to saccharification, fermentation, and valorization. Crop residues contain recalcitrant polysaccharides, including cellulose, hemicelluloses, pectins, and lignin and lignin-carbohydrate complexes. In addition, their cell walls can vary in linkage structure and monosaccharide composition between plant sources. Characterization of total cell wall structure, including high-resolution analyses of saccharide composition, linkage, and complex structures using chromatography-based methods, nuclear magnetic resonance, -omics, and antibody glycome profiling, provides critical insight into the fine chemistry of feedstock cell walls. Furthermore, improving both the catalytic potential of microbial communities that populate biodigester reactors and the efficiency of pre-treatments used in bioethanol production may improve bioconversion rates and yields. Toward this end, knowledge and characterization of carbohydrate-active enzymes (CAZymes) involved in dynamic biomass deconstruction is pivotal. Here we overview the use of common "-omics"-based methods for the study of lignocellulose-metabolizing communities and microorganisms, as well as methods for annotation and discovery of CAZymes, and accurate prediction of CAZyme function. Emerging approaches for analysis of large datasets, including metagenome-assembled genomes, are also discussed. Using complementary glycomic and meta-omic methods to characterize agricultural residues and the microbial communities that digest them provides promising streams of research to maximize value and energy extraction from crop waste streams.

Combinatorial Glycomic Analyses to Direct CAZyme Discovery for the Tailored Degradation of Canola Meal Non-Starch Dietary Polysaccharides.

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.

Analysis of Active Site Architecture and Reaction Product Linkage Chemistry Reveals a Conserved Cleavage Substrate for an Endo-alpha-mannanase within Diverse Yeast Mannans.

Yeast α-mannan (YM) is a densely branched N-linked glycan that decorates the surface of yeast cell walls. Owing to the high degree of branching, cleavage of the backbone of YM appears to rely on the coupled action of side-chain-cleaving enzymes. Upon examining the genome sequences of bovine-adapted Bacteroides thetaiotaomicron strains, isolated for their ability to degrade YM, we have identified a tandem pair of genes inserted into an orphan pathway predicted to be involved in YM metabolism. Here, we investigated the activity of one of these enzymes, a predicted endo-mannanase from glycoside hydrolase (GH) family 76 (BtGH76-MD40). Purified recombinant BtGH76-MD40 displayed activity on structurally distinct YMs from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Linkage analysis of released oligosaccharide products from S. cerevisiae and S. pombe mannan determined BtGH76-MD40 targets a specific linkage that is conserved in structurally diverse YM substrates. In addition, using two differential derivatization methods, we have shown that there is an absolute requirement for undecorated d-mannopyranose in the -1 subsite. Determination of the BtGH76-MD40 X-ray crystal structure and structural superimposition and molecular docking of a branched alpha-mannopentatose substrate supported these findings. In contrast, BtGH76-MD40 can accommodate extended side chains in the +1 and -2 subsites, highlighting that a single alpha-1,6-mannosyl residue is a prerequisite for activity, and cleavage occurs at the reducing end of the undecorated monosaccharide. Collectively these results demonstrate how acquisition of new enzymes within extant pathways contributes to the functional abilities of saccharolytic bacteria persisting in complex digestive ecosystems.

Process development for the production of prebiotic fructo-oligosaccharides by penicillium citreonigrum.

A new isolated P. citreonigrum URM 4459 was selected to produce fructooligosaccharides (FOS) in an efficient, economical and fast one-step fermentation. Optimal culture conditions were stablished by experimental design. Experiments run in bioreactor resulted in a high yield, content, productivity and purity of FOS (0.65 ±â€¯0.06 gFOS/ginitial Sucrose, 126.3 ±â€¯0.1 g/L, 2.28 ±â€¯0.08 g/L.h and 61 ±â€¯0%). The FOS mixture was purified up to 92% (w/w) with an activated charcoal column. FOS produced were able to promote lactobacilli and bifidobacteria growth. Higher bacteria cell density was obtained for microbial-FOS mixtures than commercial Raftilose® P95. Some strains grew even faster in the FOS mixture produced than in all other carbon sources. FOS were resistant to the simulated gastrointestinal conditions. A high amount of a reducing trisaccharide was identified in the FOS produced mixture, possibly neokestose, which may explain the great prebiotic potential of the FOS.

Chemical studies on the polysaccharides of Salicornia brachiata.

A group of 12 polysaccharide extracts were prepared from the tips, stem and roots of an Indian halophyte Salicornia brachiata Roxb. obtained by sequential extractions with cold water (CW), hot water (HW), aqueous ammonium oxalate (OX) and aqueous sodium hydroxide (ALK) solutions. Monosaccharide composition analysis revealed that all the polysaccharide extract samples consisted primarily of rhamnose, arabinose, mannose, galactose, glucose, whereas ribose and xylose were present only in some of the extracts. All the extracts exhibited low apparent viscosity (1.47-2.02 cP) and sulphate and contained no prominent toxic metal ions. Fucose was detected only in OX extract of the roots. These polysaccharides were found to be heterogeneous and highly branched (glycoside linkage analysis, size-exclusion chromatography, (13)C-NMR, FT-IR, circular dichroism and optical rotation data). Physico-chemical analyses of these polysaccharides including uronic acid, sulphate and protein contents were also carried out. This constitutes the first report on the profiling of Salicornia polysaccharides.