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The cell walls of red and green algae contain β-1,3-xylan, which is hydrolysed by the endo-type enzyme β-1,3-xylanase. Notably, only marine-bacteria-derived β-1,3-xylanases have been functionally characterised to date. In this study, we characterised the enzymatic properties of a potential β-1,3-xylanase ( Bc Xyn26B) derived from the human gut bacterium, Bacteroides cellulosilyticus WH2. The codon optimized Bc Xyn26B gene was synthesised and expressed in Escherichia coli BL21(DE3). The recombinant protein was purified by a two-step purification process using Ni-affinity chromatography followed by anion exchange chromatography, and its enzymatic properties were characterised. The recombinant Bc Xyn26B exhibited specific hydrolytic activity against β-1,3-xylan and released various β-1,3-xylooligosaccharides, with β-1,3-xylobiose as the primary product. The optimum reaction temperature was 50 °C, higher than that for other enzymes derived from marine bacteria. This study represents the first report on the identification, heterologous expression, and characterisation of β-1,3-xylanase from human gut microbes. Notably, the substrate specificity of Bc Xyn26B indicates that human gut Bacteroides species possess an unknown β-1,3-xylan utilisation system.

Xylanolytic enzyme systems in ascomycetous yeasts remain underexplored, despite the presence of yeasts in various xylan-rich ecological niches. In this study, we investigated the secreted xylanolytic machineries of three Blastobotrys species— B. mokoenaii , B. illinoisensis , and B. malaysiensis —by integrating genome annotation, bioinformatics, and secretome analyses of cultures grown on beechwood glucuronoxylan. Our findings demonstrate that these yeasts effectively hydrolyze xylan through the secretion of xylanases from the glycoside hydrolase (GH) family 11, which play a central role in cleaving the xylan backbone. Additionally, the yeasts produce a diverse array of other CAZymes, including members of GH families 3, 5, and 67, with putative roles in xylan degradation. We also report on the heterologous expression and functional characterization of the GH30_7 xylanase Bm Xyn30A from B. mokoenaii , which exhibits both glucuronoxylanase and xylobiohydrolase activities. We demonstrate additive effects between GH family 30 Bm Xyn30A and GH family 11 Bm Xyn11A during the hydrolysis of beechwood glucuronoxylan, where the enzymes exhibit complementary roles that enhance the deconstruction of this complex hemicellulose substrate. These findings broaden our understanding of the xylanolytic systems in yeasts and underscore the potential of Blastobotrys species as cell factories and natural xylanase producers. The enzymes they produce hold promise for biorefining applications, enabling efficient utilization of renewable xylan-rich plant biomass resources. Key points • Extracellular GH11 xylanases dominate glucuronoxylan degradation in Blastobotrys yeasts. • Yeast GH30_7 enzyme shows multifaceted activity, supporting complex xylan breakdown. • Blastobotrys yeasts show promise as cell factories for industrial biotechnology applications.

Secondarywalls are synthesizedin specializedcells, suchas tracheary elements andfibers, and their remarkable strength andrigidityprovide strongmechanical support tothe cells andthe plant body. The main components of secondary walls are cellulose, xylan, glucomannan and lignin. Biochemical, molecular and genetic studies have led to the discovery of most of the genes involved in the biosynthesis of secondary wall components. Cellulose is synthesized by cellulose synthase complexes in the plasma membrane and the recent success of in vitro synthesis of cellulose microfibrils by a single recombinant cellulose synthase isoform reconstituted into proteoliposomes opens new doors to further investigate the structure and functions of cellulose synthase complexes. Most genes involved in the glycosyl backbone synthesis, glycosyl substitutions and acetylation of xylan and glucomannan have been genetically characterized and the biochemical properties of some of their encoded enzymes have been investigated. The genes and their encoded enzymes participating in monolignol biosynthesis andmodification have been extensively studied both genetically and biochemically. A full understanding of how secondary wall components are synthesized will ultimately enable us to produce plants with custom-designed secondary wall composition tailored to diverse applications.

BACKGROUND: Variability in the health effects of dietary fiber might arise from inter-individual differences in the gut microbiota's ability to ferment these substrates into beneficial metabolites. Our understanding of what drives this individuality is vastly incomplete and will require an ecological perspective as microbiomes function as complex inter-connected communities. Here, we performed a parallel two-arm, exploratory randomized controlled trial in 31 adults with overweight and class-I obesity to characterize the effects of long-chain, complex arabinoxylan (n = 15) at high supplementation doses (female: 25 g/day; male: 35 g/day) on gut microbiota composition and short-chain fatty acid production as compared to microcrystalline cellulose (n = 16, non-fermentable control), and integrated the findings using an ecological framework. RESULTS: Arabinoxylan resulted in a global shift in fecal bacterial community composition, reduced α-diversity, and the promotion of specific taxa, including operational taxonomic units related to Bifidobacterium longum, Blautia obeum, and Prevotella copri. Arabinoxylan further increased fecal propionate concentrations (p = 0.012, Friedman's test), an effect that showed two distinct groupings of temporal responses in participants. The two groups showed differences in compositional shifts of the microbiota (p ≤ 0.025, PERMANOVA), and multiple linear regression (MLR) analyses revealed that the propionate response was predictable through shifts and, to a lesser degree, baseline composition of the microbiota. Principal components (PCs) derived from community data were better predictors in MLR models as compared to single taxa, indicating that arabinoxylan fermentation is the result of multi-species interactions within microbiomes. CONCLUSION: This study showed that long-chain arabinoxylan modulates both microbiota composition and the output of health-relevant SCFAs, providing information for a more targeted application of this fiber. Variation in propionate production was linked to both compositional shifts and baseline composition, with PCs derived from shifts of the global microbial community showing the strongest associations. These findings constitute a proof-of-concept for the merit of an ecological framework that considers features of the wider gut microbial community for the prediction of metabolic outcomes of dietary fiber fermentation. This provides a basis to personalize the use of dietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features to account for the inconsistent health effects in human intervention studies. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

This review examines the recent models describing the mode of action of various xylanolytic enzymes and how these enzymes can be applied (sequentially or simultaneously) with their distinctive roles in mind to achieve efficient xylan degradation. With respect to homeosynergy, synergism appears to be as a result of β-xylanase and/or oligosaccharide reducing-end β-xylanase liberating xylo-oligomers (XOS) that are preferred substrates of the processive β-xylosidase. With regards to hetero-synergism, two cross relationships appear to exist and seem to be the reason for synergism between the enzymes during xylan degradation. These cross relations are the debranching enzymes such as α-glucuronidase or side-chain cleaving enzymes such as carbohydrate esterases (CE) removing decorations that would have hindered back-bone-cleaving enzymes, while backbone-cleaving-enzymes liberate XOS that are preferred substrates of the debranching and side-chain-cleaving enzymes. This interaction is demonstrated by high yields in co-production of xylan substituents such as arabinose, glucuronic acid and ferulic acid, and XOS. Finally, lytic polysaccharide monooxygenases (LPMO) have also been implicated in boosting whole lignocellulosic biomass or insoluble xylan degradation by glycoside hydrolases (GH) by possibly disrupting entangled xylan residues. Since it has been observed that the same enzyme (same Enzyme Commission, EC, classification) from different GH or CE and/or AA families can display different synergistic interactions with other enzymes due to different substrate specificities and properties, in this review, we propose an approach of enzyme selection (and mode of application thereof) during xylan degradation, as this can improve the economic viability of the degradation of xylan for producing precursors of value added products.

Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.

Summary Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end‐use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE‐AX), then xylanase action (XE‐AX) leaving an unextractable (XU‐AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild‐type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3‐linked to mono‐substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5–7 and 13–19 mg bound ferulate (g−1 AX), and their content of diferulates (diFA), <0.3, 1–1.7 and 4–5 mg (g−1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE‐AX fraction than in the WE‐AX fraction and largely unaffected in the XU‐AX fraction. As the majority of diFA is associated with the XU‐AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.