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Oyster mushroom spherical virus (OMSV) is a mycovirus that inhibits mycelial growth, induces malformation symptoms, and decreases the yield of fruiting bodies in Pleurotus ostreatus. However, the pathogenic mechanism of OMSV infection in P. ostreatus is poorly understood. In this study, RNA sequencing (RNA-seq) was conducted, identifying 354 differentially expressed genes (DEGs) in the mycelium of P. ostreatus during OMSV infection. Verifying the RNA-seq data through quantitative real-time polymerase chain reaction on 15 DEGs confirmed the consistency of gene expression trends. Both Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses highlighted the pivotal role of primary metabolic pathways in OMSV infection. Additionally, significant changes were noted in the gene expression levels of carbohydrate-active enzymes (CAZymes), which are crucial for providing the carbohydrates needed for fungal growth, development, and reproduction by degrading renewable lignocellulose. The activities of carboxymethyl cellulase, laccase, and amylase decreased, whereas chitinase activity increased, suggesting a potential mechanism by which OMSV influenced mycelial growth through modulating CAZyme activities. Therefore, this study provided insights into the pathogenic mechanisms triggered by OMSV in P. ostreatus.

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Perfluorooctanoic acid (PFOA), an emerging pollutant, has been frequently detected in organic solid waste. It becomes a major concern for compost application, but studies on its toxic effects during composting are rare. This study evaluated the impact of PFOA presence at the environmentally relevant level on the humification process and microbiology during composting. The results showed that the PFOA presence (15.5 µg/kg dry) caused 45.5 % and 40.5 % decreases in the total organic carbon and humic acid-like substances, respectively. PFOA negatively affected microbial activity during the thermophilic period, as evidenced by the increases in reactive oxygen species and lactate dehydrogenase concentration. It altered the microbial community with an enrichment of Bacteroidota, conducive to resisting press. Unexpectedly, the PFOA presence induced hormesis at the maturity period, consistent with stimulated carbon metabolism (i.e., glycolysis and oxidative phosphorylation). The modulated microbial metabolism stimulated the catabolic metabolism of small-molecule humus precursors and reduced intracellular quinone availability. Furthermore, the secretion of auxiliary activities for crude fiber degradation was suppressed, which decreased the generation of extracellular quinone, and thereby impeded the humification process. These findings deciphered the metabolic response of composting to PFOA presence and highlighted the potential carbon loss of PFOA-containing composting.

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The human gastrointestinal microbiota, densely populated with a diverse array of microorganisms primarily from the bacterial phyla Bacteroidota, Bacillota, and Actinomycetota, is crucial for maintaining health and physiological functions. Dietary fibers, particularly pectin, significantly influence the composition and metabolic activity of the gut microbiome. Pectin is fermented by gut bacteria using carbohydrate-active enzymes (CAZymes), resulting in the production of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, which provide various health benefits. The gastrointestinal microbiota has evolved to produce CAZymes that target different pectin components, facilitating cross-feeding within the microbial community. This review explores the fermentation of pectin by various gut bacteria, focusing on the involved transport systems, CAZyme families, SCFA synthesis capacity, and effects on microbial ecology in the gut. It addresses the complexities of the gut microbiome's response to pectin and highlights the importance of microbial cross-feeding in maintaining a balanced and diverse gut ecosystem. Through a systematic analysis of pectinolytic CAZyme production, this review provides insights into the enzymatic mechanisms underlying pectin degradation and their broader implications for human health, paving the way for more targeted and personalized dietary strategies.

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Six bacterial strains, Mut1T, Mut2, Alt1, Alt2, Alt3T, and Alt4, were isolated from soil samples collected in parks in Gothenburg, Sweden, based on their ability to utilize the insoluble polysaccharides α-1,3-glucan (mutan; Mut strains) or the mixed-linkage α-1,3/α-1,6-glucan (alternan; Alt strains). Analysis of 16S rRNA gene sequences identified all strains as members of the genus Streptomyces. The genomes of the strains were sequenced and subsequent phylogenetic analyses identified Mut2 as a strain of Streptomyces laculatispora and Alt1, Alt2 and Alt4 as strains of Streptomyces poriferorum, while Mut1T and Alt3T were most closely related to the type strains Streptomyces drozdowiczii NBRC 101007T and Streptomyces atroolivaceus NRRL ISP-5137T, respectively. Comprehensive genomic and biochemical characterizations were conducted, highlighting typical features of Streptomyces, such as large genomes (8.0-9.6 Mb) with high G+C content (70.5-72.0%). All six strains also encode a wide repertoire of putative carbohydrate-active enzymes, indicating a capability to utilize various complex polysaccharides as carbon sources such as starch, mutan, and cellulose, which was confirmed experimentally. Based on phylogenetic and phenotypic characterization, our study suggests that strains Mut1T and Alt3T represent novel species in the genus Streptomyces for which the names Streptomyces castrisilvae sp. nov. and Streptomyces glycanivorans sp. nov. are proposed, with strains Mut1T (=DSM 117248T=CCUG 77596T) and Alt3T (=DSM 117252T=CCUG 77600T) representing the respective type strains.

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Analysis of bacterial carbohydrate-active enzymes (CAZymes) contributes significantly to comprehending the response exhibited by coral symbionts to the external environment. This study explored the impact of bleaching on the bacteria and their CAZymes in coral Favites sp. through metagenomic sequencing. Notably, principal coordinates analysis (PCoA) unveiles substantial difference in bacterial communities between bleached and unbleached corals. Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidota, and Chloroflexi, exhibit noteworthy alterations during coral bleaching. CAZymes profiles in bleached coral disclosed a significant increase in Glycosyltransferases (GTs) abundance, suggesting an intensified biosynthesis of polysaccharides. Conversely, there is a marked reduction in other CAZymes abundance in bleached coral. Proteobacteria, Bacteroidota, Chlorobi, and Planctomycetota exhibit greater contributions to CAZymes in bleached corals, with Rhodobacterales, Cytophagales, Burkholderiales, Caulobacterales, and Hyphomicrobiales being the main contributors. While Acidobacteria, Actinobacteria, and Chloroflexi demonstrate higher contributions to CAZymes in unbleached corals. The changes in bacteria and their CAZymes reflect the ecological adaptability of coral holobionts when facing environmental stress. The alterations in CAZymes composition caused by bleaching events may have profound impacts on coral nutrient absorption and ecosystem stability. Therefore, understanding the dynamic changes in CAZymes is crucial for assessing the health and recovery potential of coral ecosystems.

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Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis.IMPORTANCEIn the present study, we developed a strategy to identify the key fungal enzymatic activities involved in the improvement of soybean meal (SBM) digestibility. Our data unravel the importance of pectin degradation for the release of nutrients from SBM and provide some insights regarding the degradation of rhamnogalacturonan-I (RG-I) by ascomycetes. Indeed, the hydrolysis of pectins and RG-I by human microbiota is well documented in the literature, but our knowledge of the fungal CAZymes at play for the degradation of soybean pectins remains hitherto underexplored. Due to its wide use in animal feed, improving the digestibility of SBM by enzymatic treatments is a current challenge for feed additive suppliers. Since non-starch polysaccharides and pectins have often been reported for their anti-nutritional role in SBM, we believe this study will provide new avenues toward the improvement of enzymatic cocktails for animal nutrition and health.

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Xyloglucan oligosaccharides (XyGOs) are highly branched, complex carbohydrates with a variety of chemical and biotechnological applications. Due to the regular repeating pattern of sidechain substitution of the xyloglucan backbone, well-defined XyGOs are readily accessed for analytical and preparative purposes by specific hydrolysis of the polysaccharide with endo-glucanases. To broaden the application potential of XyGOs, we present here an optimized, scalable method to access large quantities of galactosylated XyGOs by treatment of the bulk agricultural by-product, tamarind kernel powder (TKP), with a highly specific endo-xyloglucanase at high-solids content. Subsequent ß-galactosidase treatment reduced XyGO complexity to produce exclusively the branched heptasaccharide XXXG (Xyl3Glc4: [α-D-Xylp-(1 → 6)]-ß-D-Glcp-(1 → 4)-[α-D-Xylp-(1 → 6)]-ß-D-Glcp-(1 → 4)-[α-D-Xylp-(1 → 6)]-ß-D-Glcp-(1 → 4)-D-Glcp). The challenge of removing the co-product galactose was overcome by fermentation with baker's yeast, thereby avoiding chromatography and other fractionation steps to yield highly pure XXXG. This simplified approach employs many of the core concepts of green chemistry and engineering, enables facile production of 100 g quantities of XyGOs and XXXG for laboratory use, and serves as a guide to further production scale-up for applications, including as prebiotics, plant growth effectors and elicitors, and building blocks for glycoconjugate synthesis.

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Carbohydrates, which are a vital dietary component, undergo digestion and gut fermentation through microbial enzymes to produce beneficial short-chain fatty acids. Certain carbohydrates selectively modulate the gut microbiota, impacting host health. Carbohydrate-active enzymes within the gut microbiota significantly contribute to carbohydrate utilization and microbial diversity. Despite their importance, the structural complexity of carbohydrates poses analytical challenges. However, recent advancements, notably, mass spectrometry, have allowed for their characterization and functional analysis. This review examines the intricate relationship between dietary carbohydrates and the gut microbiota, highlighting the crucial role of advanced analytical techniques in understanding their diversity and implications. These advancements provide valuable insights into carbohydrate bioactivity. Integrating high-throughput analysis with next-generation sequencing provides deeper insights into gut microbial interactions, potentially revealing which carbohydrate structures are beneficial for gut health.

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Polysaccharides are one of the most important components of traditional Chinese medicine (TCM) and have been extensively studied for their immunomodulatory properties. The functions and effects of TCM polysaccharides are closely related to the gut microbiota, making the study of their interaction a hot topic in the field of TCM metabolism. This review follows two main inquiries: first, how the gut microbiota breaks down TCM polysaccharides to produce bioactive metabolites; and second, how TCM polysaccharides reshape the gut microbiota as a carbon source. Understanding the interaction mechanism involves a challenging equation of the structural association of TCM polysaccharides with the metabolic activities of the microbiota. This review has meticulously searched, partially organized literature spanning the past decade, that delves into the interaction mechanism between TCM polysaccharides and gut microbiota. It also gives an overview of the complex factors of the elusive "polysaccharides-bond-bacteria-enzyme" equation: the complexity of polysaccharide structures, the diversity of glycosidic bond types, the communal nature of metabolizing microbiota, the enzymes involved in functional degradation by microbiota, and the hierarchical roles of polysaccharide utilization locus and gram-positive PULs. Finally, this review aims to facilitate discussion among peers in the field of TCM microbiota and offers prospects for research in related fields, paving the way for pharmacological studies on TCM polysaccharides and gut microbiota therapeutics, and providing a reference point for further clinical research.

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Carbohydrates are chemically and structurally diverse, composed of a wide array of monosaccharides, stereochemical linkages, substituent groups, and intermolecular associations with other biological molecules. A large repertoire of carbohydrate-active enzymes (CAZymes) and enzymatic activities are required to form, dismantle, and metabolize these complex molecules. The software SACCHARIS (Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity) provides a rapid, easy-to-use pipeline for the prediction of potential CAZyme function in new datasets. We have updated SACCHARIS to (i) simplify its installation by re-writing in Python and packaging for Conda; (ii) enhance its usability through a new (optional) interactive GUI; and (iii) enable semi-automated annotation of phylogenetic tree output via a new R package or the commonly-used webserver iTOL. Significantly, SACCHARIS v2 has been developed with high-throughput omics in mind, with pipeline automation geared toward complex (meta)genome and (meta)transcriptome datasets to reveal the total CAZyme content ("CAZome") of an organism or community. Here, we outline the development and use of SACCHARIS v2 to discover and annotate CAZymes and provide insight into complex carbohydrate metabolisms in individual organisms and communities.

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Filamentous plant pathogens, including fungi and oomycetes, pose significant threats to cultivated crops, impacting agricultural productivity, quality and sustainability. Traditionally, disease control heavily relied on fungicides, but concerns about their negative impacts motivated stakeholders and government agencies to seek alternative solutions. Biocontrol agents (BCAs) have been developed as promising alternatives to minimize fungicide use. However, BCAs often exhibit inconsistent performances, undermining their efficacy as plant protection alternatives. The eukaryotic cell wall of plants and filamentous pathogens contributes significantly to their interaction with the environment and competitors. This highly adaptable and modular carbohydrate armor serves as the primary interface for communication, and the intricate interplay within this compartment is often mediated by carbohydrate-active enzymes (CAZymes) responsible for cell wall degradation and remodeling. These processes play a crucial role in the pathogenesis of plant diseases and contribute significantly to establishing both beneficial and detrimental microbiota. This review explores the interplay between cell wall dynamics and glycan interactions in the phytobiome scenario, providing holistic insights for efficiently exploiting microbial traits potentially involved in plant disease mitigation. Within this framework, the incorporation of glycobiology-related functional traits into the resident phytobiome can significantly enhance the plant's resilience to biotic stresses. Therefore, in the rational engineering of future beneficial consortia, it is imperative to recognize and leverage the understanding of cell wall interactions and the role of the glycome as an essential tool for the effective management of plant diseases.

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We report the complete genome of Priestia filamentosa H146 isolated from tobacco leaves. H146 contained a circular chromosome and five circular plasmids. A total of 4,669 genes were predicted, of which 4,372 genes were in the chromosome and other genes were located on plasmids. The genome sequence data provide an important basis for studying Priestia filamentosa.

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Wood-rotting fungi play an important role in the global carbon cycle because they are the only known organisms that digest wood, the largest carbon stock in nature. In the present study, we used linear discriminant analysis and random forest (RF) machine learning algorithms to predict white- or brown-rot decay modes from the numbers of genes encoding Carbohydrate-Active enZymes with over 98% accuracy. Unlike other algorithms, RF identified specific genes involved in cellulose and lignin degradation, including auxiliary activities (AAs) family 9 lytic polysaccharide monooxygenases, glycoside hydrolase family 7 cellobiohydrolases, and AA family 2 peroxidases, as critical factors. This study sheds light on the complex interplay between genetic information and decay modes and underscores the potential of RF for comparative genomics studies of wood-rotting fungi. IMPORTANCE: Wood-rotting fungi are categorized as either white- or brown-rot modes based on the coloration of decomposed wood. The process of classification can be influenced by human biases. The random forest machine learning algorithm effectively distinguishes between white- and brown-rot fungi based on the presence of Carbohydrate-Active enZyme genes. These findings not only aid in the classification of wood-rotting fungi but also facilitate the identification of the enzymes responsible for degrading woody biomass.

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Lignocellulose biomass raw materials have a high value in energy conversion. Recently, there has been growing interest in using microorganisms to secret a series of enzymes for converting low-cost biomass into high-value products such as biofuels. We previously isolated a strain of Penicillium oxalicun 5-18 with promising lignocellulose-degrading capability. However, the mechanisms of lignocellulosic degradation of this fungus on various substrates are still unclear. In this study, we performed transcriptome-wide profiling and comparative analysis of strain 5-18 cultivated in liquid media with glucose (Glu), xylan (Xyl) or wheat bran (WB) as sole carbon source. In comparison to Glu culture, the number of differentially expressed genes (DEGs) induced by WB and Xyl was 4134 and 1484, respectively, with 1176 and 868 genes upregulated. Identified DEGs were enriched in many of the same pathways in both comparison groups (WB vs. Glu and Xly vs. Glu). Specially, 118 and 82 CAZyme coding genes were highly upregulated in WB and Xyl cultures, respectively. Some specific pathways including (Hemi)cellulose metabolic processes were enriched in both comparison groups. The high upregulation of these genes also confirmed the ability of strain 5-18 to degrade lignocellulose. Co-expression and co-upregulated of genes encoding CE and AA CAZy families, as well as other (hemi)cellulase revealed a complex degradation strategy in this strain. Our findings provide new insights into critical genes, key pathways and enzyme arsenal involved in the biomass degradation of P. oxalicum 5-18.

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Interactions between proteins and glycans are critical to various biological processes. With databases of carbohydrate-interacting proteins and increasing amounts of structural data, the three-sided right-handed ß-helix (RHBH) has emerged as a significant structural fold for glycan interactions. In this review, we provide an overview of the sequence, mechanistic, and structural features that enable the RHBH to interact with glycans. The RHBH is a prevalent fold that exists in eukaryotes, prokaryotes, and viruses associated with adhesin and carbohydrate-active enzyme (CAZyme) functions. An evolutionary trajectory analysis on structurally characterized RHBH-containing proteins shows that they likely evolved from carbohydrate-binding proteins with their carbohydrate-degrading activities evolving later. By examining three polysaccharide lyase and three glycoside hydrolase structures, we provide a detailed view of the modes of glycan binding in RHBH proteins. The 3-dimensional shape of the RHBH creates an electrostatically and spatially favorable glycan binding surface that allows for extensive hydrogen bonding interactions, leading to favorable and stable glycan binding. The RHBH is observed to be an adaptable domain capable of being modified with loop insertions and charge inversions to accommodate heterogeneous and flexible glycans and diverse reaction mechanisms. Understanding this prevalent protein fold can advance our knowledge of glycan binding in biological systems and help guide the efficient design and utilization of RHBH-containing proteins in glycobiology research.

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BACKGROUND: Members of the Planctomycetota phylum harbour an outstanding potential for carbohydrate degradation given the abundance and diversity of carbohydrate-active enzymes (CAZymes) encoded in their genomes. However, mainly members of the Planctomycetia class have been characterised up to now, and little is known about the degrading capacities of the other Planctomycetota. Here, we present a comprehensive comparative analysis of all available planctomycetotal genome representatives and detail encoded carbohydrolytic potential across phylogenetic groups and different habitats. RESULTS: Our in-depth characterisation of the available planctomycetotal genomic resources increases our knowledge of the carbohydrolytic capacities of Planctomycetota. We show that this single phylum encompasses a wide variety of the currently known CAZyme diversity assigned to glycoside hydrolase families and that many members encode a versatile enzymatic machinery towards complex carbohydrate degradation, including lignocellulose. We highlight members of the Isosphaerales, Pirellulales, Sedimentisphaerales and Tepidisphaerales orders as having the highest encoded hydrolytic potential of the Planctomycetota. Furthermore, members of a yet uncultivated group affiliated to the Phycisphaerales order could represent an interesting source of novel lytic polysaccharide monooxygenases to boost lignocellulose degradation. Surprisingly, many Planctomycetota from anaerobic digestion reactors encode CAZymes targeting algal polysaccharides - this opens new perspectives for algal biomass valorisation in biogas processes. CONCLUSIONS: Our study provides a new perspective on planctomycetotal carbohydrolytic potential, highlighting distinct phylogenetic groups which could provide a wealth of diverse, potentially novel CAZymes of industrial interest.

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BACKGROUND: Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp. RESULTS: Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20-25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. CONCLUSION: This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

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Teredinibacter turnerae is a cultivable cellulolytic Gammaproeteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood-eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose, and pectin and contribute to lignocellulose digestion in the shipworm gut. However, the mechanism by which symbiont-made enzymes are secreted by T. turnerae and subsequently transported to the site of lignocellulose digestion in the shipworm gut is incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce outer membrane vesicles (OMVs) that contain a variety of proteins identified by LC-MS/MS as carbohydrate-active enzymes with predicted activities against cellulose, hemicellulose, and pectin. Reducing sugar assays and zymography confirm that these OMVs retain cellulolytic activity, as evidenced by hydrolysis of CMC. Additionally, these OMVs were enriched with TonB-dependent receptors, which are essential to carbohydrate and iron acquisition by free-living bacteria. These observations suggest potential roles for OMVs in lignocellulose utilization by T. turnerae in the free-living state, in enzyme transport and host interaction during symbiotic association, and in commercial applications such as lignocellulosic biomass conversion.

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BACKGROUND: Ruminal microbial communities enriched on lignocellulosic biomass have shown considerable promise for the discovery of microorganisms and enzymes involved in digesting cell wall compounds, a key bottleneck in the development of second-generation biofuels and bioproducts, enabling a circular bioeconomy. Cardoon (Cynara cardunculus) is a promising inedible energy crop for current and future cellulosic biorefineries and the emerging bioenergy and bioproducts industries. The rumen microbiome can be considered an anaerobic "bioreactor", where the resident microbiota carry out the depolymerization and hydrolysis of plant cell wall polysaccharides (PCWPs) through the catalytic action of fibrolytic enzymes. In this context, the rumen microbiota represents a potential source of microbes and fibrolytic enzymes suitable for biofuel production from feedstocks. In this study, metatranscriptomic and 16S rRNA sequencing were used to profile the microbiome and to investigate the genetic features within the microbial community adherent to the fiber fractions of the rumen content and to the residue of cardoon biomass incubated in the rumen of cannulated cows. RESULTS: The metatranscriptome of the cardoon and rumen fibre-adherent microbial communities were dissected in their functional and taxonomic components. From a functional point of view, transcripts involved in the methanogenesis from CO2 and H2, and from methanol were over-represented in the cardoon-adherent microbial community and were affiliated with the Methanobrevibacter and Methanosphaera of the Euryarchaeota phylum. Transcripts encoding glycoside hydrolases (GHs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and glycoside transferases (GTs) accounted for 1.5% (6,957) of the total RNA coding transcripts and were taxonomically affiliated to major rumen fibrolytic microbes, such as Oscillospiraceae, Fibrobacteraceae, Neocallimastigaceae, Prevotellaceae, Lachnospiraceae, and Treponemataceae. The comparison of the expression profile between cardoon and rumen fiber-adherent microbial communities highlighted that specific fibrolytic enzymes were potentially responsible for the breakdown of cardoon PCWPs, which was driven by specific taxa, mainly Ruminococcus, Treponema, and Neocallimastigaceae. CONCLUSIONS: Analysis of 16S rRNA and metatranscriptomic sequencing data revealed that the cow rumen microbiome harbors a repertoire of new enzymes capable of degrading PCWPs. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential approach for accelerating the discovery of novel cellulolytic enzymes that could be harnessed for biotechnology. This research contributes a relevant perspective towards degrading cellulosic biomass and providing an economical route to the production of advanced biofuels and high-value bioproducts.

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The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and ß-xylosidase activities (5 ×103 U/L and 1.7 ×105 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach.

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