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Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo- β-galactosidase activity with regiopreference for hydrolyzing β- d -(1,6) glycosidic linkages. Notably, it exhibited a preference for d -glucopyranosyl (d -Glc p) over d -galactopyranosyl (d -Gal p) and d -fructofuranosyl (d -Fru f) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-β-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (α/α) 6 topology, which has not been observed before for β-galactosidases. BD-β-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-β-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from Bacteroides thetaiotaomicron , described as β-glucuronidase, displayed exo -β-galactosidase activity like BD-β-Gal but lacked a tetrameric structure. [Display omitted] • New β-galactosidase activity in Glycoside Hydrolase family 154 (GH154). • BD-β-Gal shows β-galactosidase activity with preference for β-(1,6)-linkages. • First crystal structure of GH154 with novel (α/α) 6 topology for β-galactosidase. • Novel active site topology for BD-β-Gal, different from canonical β-galactosidases. • BD-β-Gal shows transglycosylation activity on lactose. [ABSTRACT FROM AUTHOR]

[Display omitted] • Firmicutes shared common substrates like starch, xylan, chitin, and arabinan. • Species coexisted through utilizing less common substrates in anaerobic digesters. • Limnochordia and Clostridia were the most versatile, utilizing 6 to 8 substrates. • Unique CAZymes and PULs like GH177, GH179, and CBM91 facilitated niches of microbes. • Larger genomes conferred higher CAZyme number and greater metabolic versatility. Fermentative Firmicutes species are key players in anaerobic digestion; however, their niche differentiation based on carbohydrate utilization in full-scale systems remains unclear. In this study, we investigated niche differentiation among four major Firmicutes classes using a genome-centric approach, reconstructing 39 high-quality metagenome-assembled genomes. Limnochordia and Clostridia exhibited the broadest substrate versatility, utilizing 24% and 18% of the predicted substrates, respectively. Although common substrates were shared, each class demonstrated unique substrate preferences driven by distinct functional and metabolic differences. Limnochordia and Clostridia possess unique carbohydrate-active enzyme families, such as GH177 and CBM91, which enable xylan and arabinan degradation. Bacilli were abundant with the GH1 and GH3 families, which are critical for cellulose degradation. Overall, the Firmicutes classes showed low overlap in substrate use and functional profiles, confirming significant niche differentiation. Our results demonstrate that Firmicutes occupy distinct dietary niches supporting insights into bacterial coexistence in anaerobic digestion systems. [ABSTRACT FROM AUTHOR]

Symbiotic bacteria, including members of the Bacteroides genus, are known to digest dietary fibers in the gastrointestinal tract. The metabolism of complex carbohydrates is restricted to a specified subset of species and is likely orchestrated by polysaccharide utilization loci (PULs) in these microorganisms. β-Mannans are plant cell wall polysaccharides that are commonly found in human nutrients. Here, we report the structural basis of a PUL cluster, BdPUL12, which controls β-mannan-like glycan catabolism in Bacteroides dorei. Detailed biochemical characterization and targeted gene disruption studies demonstrated that a key glycoside hydrolase, BdP12GH26, performs the initial attack on galactomannan or glucomannan likely via an endo -acting mode, generating mannooligosaccharides and mannose. Importantly, coculture assays showed that the B. dorei promoted the proliferation of Lactobacillus helveticus and Bifidobacterium adolescentis , likely by sharing mannooligosaccharides and mannose with these gut probiotics. Our findings provide new insights into carbohydrate metabolism in gut-inhabiting bacteria and lay a foundation for novel probiotic development. • The gene cluster, BdPUL12, exclusively contributes to β-mannan-like substrates utilization in Bacteroides dorei. • The mannanase BdP12GH26 involved in BdPUL12 hydrolyzes galactomannan or glucomannan likely via an endo -acting mode, generating mannooligosaccharides and mannose. • B. dorei is capable of promoting proliferation of gut probiotics. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs), as a class of biopolymers, play pivotal roles in various biological metabolisms such as cell signaling, tissue development, cell apoptosis, immune modulation, and growth factor activity. They are mainly present in the colon in free forms, which are essential for maintaining the host's health by regulating the colonization and proliferation of gut microbiota. Therefore, it is important to explain the specific members of the gut microbiota for GAGs' degradation and their enzymatic machinery in vivo. This review provides an outline of GAGs-utilizing entities in the Bacteroides , highlighting their polysaccharide utilization loci (PULs) and the enzymatic machinery involved in chondroitin sulfate (CS) and heparin (Hep)/heparan sulfate (HS). While there are some variations in GAGs' degradation among different genera, we analyze the reputed GAGs' utilization clusters in lactic acid bacteria (LAB), based on recent studies on GAGs' degradation. The enzymatic machinery involved in Hep/HS and CS metabolism within LAB is also discussed. Thus, to elucidate the precise mechanisms utilizing GAGs by diverse gut microbiota will augment our understanding of their effects on human health and contribute to potential therapeutic strategies for diseases. [ABSTRACT FROM AUTHOR]

The rumen of ruminants is a natural anaerobic fermentation system that efficiently degrades lignocellulosic biomass and mainly depends on synergistic interactions between multiple microbes and their secreted enzymes. Ruminal microbes have been employed as biomass waste converters and are receiving increasing attention because of their degradation performance. To explore the application of ruminal microbes and their secreted enzymes in biomass waste, a comprehensive understanding of these processes is required. Based on the degradation capacity and mechanism of ruminal microbes and their secreted lignocellulose enzymes, this review concentrates on elucidating the main enzymatic strategies that ruminal microbes use for lignocellulose degradation, focusing mainly on polysaccharide metabolism-related gene loci and cellulosomes. Hydrolysis, acidification, methanogenesis, interspecific H 2 transfer, and urea cycling in ruminal metabolism are also discussed. Finally, we review the research progress on the conversion of biomass waste into biofuels (bioethanol, biohydrogen, and biomethane) and value-added chemicals (organic acids) by ruminal microbes. This review aims to provide new ideas and methods for ruminal microbe and enzyme applications, biomass waste conversion, and global energy shortage alleviation. • Rumen microbes have genetic potential in degradation and exhibit diversity in enzymes. • Cellulosome and polysaccharide utilization loci explain enzyme degradation mechanism. • Rumen microbes together contribute to metabolism exchange of ecosystem functions. • Synergy of rumen microbes and enzymes effectively converts biomass to bioenergy. • A future biorefinery based on rumen microbes and digestive strategies is proposed. [ABSTRACT FROM AUTHOR]

• Homogenization releases agar molecules from Gelidium amansii. • Agarases are discovered using predicted agar polysaccharide utilization loci. • Process causes no damage to other nutrition. • Enzyme supported one step process is time saving, cost saving and energy saving. Neoagarooligosaccharides (NAOs) are drawing more and more attention because of their numerous bioactivities, yet limited number of agarases impedes NAOs production from red algae. In this study, predicted agar polysaccharide utilization loci (agar-PUL) were firstly used as inventory for agarase. 6 agarases were identified from agar-PULs and two of them were successfully expressed and analyzed. Then enzyme cocktail (GH16-1:GH16-2:Aga50D = 2:1:1) was proved to have highest synergistic effect. Finally homogenization was applied to G. amansii and proved to be an efficient way to release agar from tissues. When liquid-to-solid ratio was 9 g/150 mL, homogenization time was 20 min, and enzyme cocktail loading was 150 U/150 mL, maximum NAOs production (90.2 mg per 9 g wet G. amansii) could be achieved. Enzyme supported one-step process (ESOP) proposed in study is environment-friendly, time saving, cost saving and none-destructive, therefore has a potential industrial application in red algae utilization. [ABSTRACT FROM AUTHOR]