Summary: The Bacteroidetes phylum is renowned for its ability to degrade a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use polysaccharide utilization loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum‐exclusive type IX secretion system (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL‐encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non‐PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that the literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions that can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems.

The production of cellulose nanofibres (CNFs) typically requires harsh chemistry and strong mechanical fibrillation, both of which have negative environmental impacts. A possible solution is offered by lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes that boost cellulose fibrillation. Although the role of LPMOs in oxidative modification of cellulosic substrates is rather well established, their use in the production of cellulose nanomaterials is not fully explored, and the effect of the carbohydrate-binding module (CBM) on nanofibrillation has not yet been reported. Herein, we studied the activity of two LPMOs, one of which was appended to a CBM, on delignified softwood fibres for green and energy-efficient production of CNFs. The CNFs were used to prepare cellulose nanopapers, and the structure and properties of both nanofibres and nanopapers were determined. Both enzymes were able to facilitate nanocellulose fibrillation and increase the colloidal stability of the produced CNFs. However, the CBM-lacking LPMO was more efficient in introducing carboxyl groups (0.53 mmol g⁻¹) on the cellulose fibre surfaces and releasing CNFs with a thinner width (4.3 ± 1.5 nm) from delignified spruce fibres than the modular LPMO (carboxylate content of 0.38 mmol g⁻¹ and nanofibre width of 6.7 ± 2.5 nm) through LPMO-pretreatment followed by mild homogenisation. The prepared nanopapers showed improved mechanical properties (tensile strength of 262 MPa and modulus of 16.2 GPa) compared to those obtained by conventional CNF preparation methods, demonstrating the potential of LPMOs as green alternatives for cellulose nanomaterial preparation.

Commensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment.

Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. We report the characterisation of a recombinant α-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me)GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an α-l-arabinofuranosidase (AbfA), and a β-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Our GH115 α-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.

The genome and natural habitat of Chitinophaga pinensis suggest the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of β-glucanase activities. We therefore conducted a screen of growth on and hydrolysis of β-glucans isolated from different sources. Most non-crystalline β-glucans supported good growth, with variable efficiency of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, β-glucan was the only type of polysaccharide that could be effectively hydrolysed by secreted enzymes. This contrasts with the secretion of a broad range of enzyme activities we observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass, and suggest that the species may have two metabolic modes: a ‘scavenging mode’ where multiple different types of glycan may be degraded, and a more ‘focussed mode’ of β-glucan metabolism. The significant accumulation of some types of β-gluco-oligosaccharide in growth medium may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed Polysaccharide Utilisation Loci. We present a hypothetical model for β-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community. IMPORTANCE It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis, a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source, but that it is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota.