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The economically important edible basidiomycete mushroom A garicus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A . bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A . bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush. [ABSTRACT FROM AUTHOR]

The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant‐based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose‐degrading ascomycetes for their capacity to react with G‐type and S‐type phenolic compounds. All six PPOs catalyze the ortho‐hydroxylation of G‐type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy‐ortho‐diphenols. Remarkably, a subset of these PPOs is also active towards S‐compounds (syringol, syringic acid, and sinapic acid) resulting in identical methoxy‐ortho‐diphenols. Assays with 18O2 demonstrate that these PPOs in particular catalyze ortho‐hydroxylation and ortho‐demethoxylation of S‐compounds and generate methanol as a co‐product. Oxidative (ortho−) demethoxylation of S‐compounds is a novel reaction for PPOs, which we propose occurs by a distinct reaction mechanism as compared to aryl‐O‐demethylases. We further show that addition of a reducing agent can steer the PPO reaction to form methoxy‐ortho‐diphenols from both G‐ and S‐type substrates rather than reactive quinones that lead to unfavorable polymerization. Application of PPOs opens for new routes to reduce the heterogeneity and methoxylation degree of mixtures of G and S lignin‐derived compounds. [ABSTRACT FROM AUTHOR]

Penicillium subrubescens has an expanded set of genes encoding putative endoxylanases (Ps XLNs) compared to most other Penicillia and other fungi. In this study, all GH10 and GH11 Ps XLNs were produced heterologously in Pichia pastoris and characterized. They were active towards beech wood xylan (BWX) and wheat flour arabinoxylan (WAX), and showed stability over a wide pH range. Additionally, Ps XLNs released distinct oligosaccharides from WAX, and showed significant cooperative action with P. subrubescens α- L -arabinofuranosidases (Ps ABFs) from GH51 or GH54 for WAX degradation, giving insight into a more diverse XLN and ABF system for the efficient degradation of complex hemicelluloses. Homology modeling analysis pointed out differences in the catalytic center of Ps XLNs, which are discussed in view of the different modes of action observed. These findings facilitate understanding of structural requirements for substrate recognition to contribute to recombinant XLN engineering for biotechnological applications. [Display omitted] • The P. subrubescens genome harbors an expanded set of genes encoding endoxylanases. • P. subrubescens endoxylanases (Ps XLNs) are xylan-specific and base-tolerant. • Ps XLNs display a diverse hydrolysis pattern and 3D-structure. • The diversity amongst Ps XLNs is attractive for various biotechnology applications. [ABSTRACT FROM AUTHOR]

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for l-tryptophan and l-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37–55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On l-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. Key points: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fungal enzymes of GH30 are only present in four subfamilies based on phylogenetic analysis. • Products from fungal GH30 enzymes are mainly short non-digestible di- and oligosaccharides. • A novel xylobiohydrolase from GH30_SF7 shows potential for xylobiose production. Efficient bioconversion of agro-industrial side streams requires a wide range of enzyme activities. Glycoside Hydrolase family 30 (GH30) is a diverse family that contains various catalytic functions and has so far been divided into ten subfamilies (GH30_1-10). In this study, a GH30 phylogenetic tree using over 150 amino acid sequences was contructed. The members of GH30 cluster into four subfamilies and eleven candidates from these subfamilies were selected for biochemical characterization. Novel enzyme activities were identified in GH30. GH30_3 enzymes possess β-(1→6)-glucanase activity. GH30_5 targets β-(1→6)-galactan with mainly β-(1→6)-galactobiohydrolase catalytic behavior. β-(1→4)-Xylanolytic enzymes belong to GH30_7 targeting β-(1→4)-xylan with several activities (e.g. xylobiohydrolase, endoxylanase). Additionally, a new fungal subfamily in GH30 was proposed, i.e. GH30_11, which displays β-(1→6)-galactobiohydrolase. This study confirmed that GH30 fungal subfamilies harbor distinct polysaccharide specificity and have high potential for the production of short (non-digestible) di- and oligosaccharides. [ABSTRACT FROM AUTHOR]

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and puri fied water-soluble lignin fractions, but no remarkable changes in the carbohydrate pro file were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive. [ABSTRACT FROM AUTHOR]

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO2. Recent research, however, demonstrated fungal conversion of 13C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13C-labeled lignin served as substrate for Agaricus bisporus, the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi. [ABSTRACT FROM AUTHOR]

Background: The white-rot fungi Ceriporiopsis subvermispora (Cs), Pleurotus eryngii (Pe), and Lentinula edodes (Le) have been shown to be high-potential species for selective delignification of plant biomass. This delignification improves polysaccharide degradability, which currently limits the efficient lignocellulose conversion into biochemicals, biofuels, and animal feed. Since selectivity and time efficiency of fungal delignification still need optimization, detailed understanding of the underlying mechanisms at molecular level is required. The recently developed methodologies for lignin quantification and characterization now allow for the in-depth mapping of fungal modification and degradation of lignin and, thereby, enable resolving underlying mechanisms. Results: Wheat straw treated by two strains of Cs (Cs1 and Cs12), Pe (Pe3 and Pe6) and Le (Le8 and Le10) was characterized using semi-quantitative py-GC–MS during fungal growth (1, 3, and 7 weeks). The remaining lignin after 7 weeks was quantified and characterized using 13C lignin internal standard based py-GC–MS and whole cell wall HSQC NMR. Strains of the same species showed similar patterns of lignin removal and degradation. Cs and Le outperformed Pe in terms of extent and selectivity of delignification (Cs ≥ Le >> Pe). The highest lignin removal [66% (w/w); Cs1] was obtained after 7 weeks, without extensive carbohydrate degradation (factor 3 increased carbohydrate-to-lignin ratio). Furthermore, though after treatment with Cs and Le comparable amounts of lignin remained, the structure of the residual lignin vastly differed. For example, Cα-oxidized substructures accumulated in Cs treated lignin up to 24% of the total aromatic lignin, a factor two higher than in Le-treated lignin. Contrarily, ferulic acid substructures were preferentially targeted by Le (and Pe). Interestingly, Pe-spent lignin was specifically depleted of tricin (40% reduction). The overall subunit composition (H:G:S) was not affected by fungal treatment. Conclusions: Cs and Le are both able to effectively and selectively delignify wheat straw, though the underlying mechanisms are fundamentally different. We are the first to identify that Cs degrades the major β-O-4 ether linkage in grass lignin mainly via Cβ–O–aryl cleavage, while Cα–Cβ cleavage of inter-unit linkages predominated for Le. Our research provides a new insight on how fungi degrade lignin, which contributes to further optimizing the biological upgrading of lignocellulose. [ABSTRACT FROM AUTHOR]

Background: Acetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive. Results: An unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase. Conclusion: This study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion. [ABSTRACT FROM AUTHOR]

BACKGROUND: Arabinoxylan is the main fiber component in corn and corn co‐products that are commonly included in pig diets. However, this fiber fraction is resistant to enzymatic degradation in the gastrointestinal tract of pigs. Ferulic acid and p‐coumaric acid are covalently linked to arabinoxylan, so it is likely that the majority of these hydroxycinnamic acids are excreted in feces. However, data to confirm this have not been reported. The objective of this research was therefore to quantify the ferulic and p‐coumaric acids in a diet based on corn and soybean meal (SBM) and in a diet based on corn, SBM, and distillers' dried grains with solubles, as well as in feces from pigs fed these diets. RESULTS: The concentration of bound ferulic and coumaric acids in diets was greater in the corn‐SBM‐DDGS diet and in feces from pigs fed this diet than in the corn‐SBM diet and feces from pigs fed that diet. The disappearance of free coumaric acids was greater (>85%) than that of bound phenolic acids (<50%) in both diets. The disappearance of free coumaric acid and bound ferulic acid in the intestinal tract of pigs was not different between the two diets. In contrast, disappearance of bound coumaric acid was greater (P < 0.05) in the corn‐SBM diet than in the corn‐SBM‐DDGS diet. CONCLUSION: A diet based on corn and SBM contains less hydroxycinnamic acid than a corn‐SBM‐DDGS diet but bound phenolic acids are more resistant to digestion by pigs than free phenolic acids. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Insoluble fiber degradation by supplemented enzymes was previously shown to improve fermentation in poultry, and has been further postulated to disrupt the cereal cell wall matrix, thus improving nutrient digestion. Here, we characterized insoluble feed-derived polysaccharides and lignin in digesta from broilers fed wheat-soybean and maize-soybean diets without or with xylanase/glucanase supplementation. Enzyme supplementation in wheat-soybean diet increased the yield of water-extractable arabinoxylan (AX) in the ileum. Still, most AX (> 73 %) remained insoluble across wheat-soybean and maize-soybean diets. Analysis of so-far largely ignored lignin demonstrated that a lignin-rich fiber fraction accumulated in the gizzard, while both insoluble AX and lignin reaching the ileum appeared to be excreted unfermented. More than 20 % of water-insoluble AX was extracted by 1 M NaOH and 11–20 % was sequentially extracted by 4 M NaOH, alongside other hemicelluloses, from ileal digesta and excreta across all diets. These findings showed that enzyme-supplementation did not impact AX extractability by alkali, under the current experimental conditions. It is, therefore, suggested that the degradation of insoluble AX by dietary xylanase in vivo mainly results in arabinoxylo-oligosaccharide release, which is not accompanied by a more loose cell wall architecture. [ABSTRACT FROM AUTHOR]

Thermo-assisted NaOH pretreatment to deconstruct xylan and lignin in sugar cane bagasse (SCB) is poorly understood. Hence, in this research it is was aimed to study the effect of NaOH pretreatment on the insoluble remaining lignin structures. Hereto, SCB milled fibres were pretreated using different dosages of NaOH at different temperatures and residence times. Of untreated SCB about 63% of the lignin compounds were assigned as p -coumarates and ferulates, analysed by pyrolysis-GC/MS as 4-vinyl phenol and 4-vinyl guaiacol, and designated as non-core lignin (NCL) compounds. More severe NaOH pretreatments resulted in lower xylan and lower lignin recoveries in the insoluble residues. Especially, the relative abundance of NCL decreased and this decrease followed a linear trend with the decrease in xylan. Core lignin compounds, analysed as phenol, guaiacol and syringol, accumulated in the residues. The decrease in residual xylan and NCL correlated positively with the enzymatic hydrolysis of the residual glucan. [ABSTRACT FROM AUTHOR]

Recently, a pyrolysis-GC-MS methodology for specific lignin quantification and structural characterisation was developed, relying on the use of uniformly 13C-labeled polymeric lignin isolate as internal standard (IS). The 13C-IS py-GC-MS method has been validated for grasses, woods, and applied in various showcases. To study the fate of lignin in animals, this method still requires careful validation in animal feeds and, especially complex faecal samples, hence the aim of this work. Hereto, faecal material was collected from pigs fed with wheat straw as lignin source and subjected to the py-GC-MS analytical platform for thorough examination of IS pyrolysis behaviour in terms of response and structural features. Next, 13C-ISpy-lignin contents and corrected Klason lignin contents were compared. Most importantly, we revealed that pyrolysis behaviour of 13C-IS lignin and 12C-sample lignin was differently affected in the faecal matrix, resulting in the ultimate underestimation of 13C-ISpy-lignin contents. In-depth examination and evaluation of matrix constituents showed that predominantly matrix ash was responsible for the effects observed. We further demonstrated that said matrix effects can be overcome by water extraction of the samples prior to analysis. Our validation and approach extend the use of the specific 13C-IS py-GC-MS methodology for accurate quantitative lignin analysis to biomass samples with complex matrices like pig faeces, and now call for application in future digestibility studies. • 13C-IS py-GC-MS underestimated lignin content in pig faeces and ash-rich wheat straw. • Matrix effects were not similarly exerted on 13C-IS lignin and sample lignin. • Ash was identified as main driver of the matrix effects observed. • Matrix effects can be overcome by water extraction prior to 13C-IS py-GC-MS. [ABSTRACT FROM AUTHOR]

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues. [ABSTRACT FROM AUTHOR]

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate‐Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation. [ABSTRACT FROM AUTHOR]

Dietary fiber-degrading enzyme supplementation in broilers aims at off-setting the anti-nutritive effect of non-starch polysaccharides and at promoting broiler health. Recently, we demonstrated that xylanase/glucanase addition in wheat-based diet improved nutrient digestibility, arabinoxylan fermentability and broiler growth. Conversely, maize arabinoxylan was found to be recalcitrant to xylanase action. These findings suggested that enzyme-mediated improvement of nutrient digestion and carbohydrate fermentation depended on the cereal type present in the diet, and may have contributed to broiler growth. Hence, we aimed at further investigating the link between dietary enzymes and carbohydrate fermentation in broilers, by studying the impact of enzyme supplementation in cereal-based diets, to the microbial communities in the ileum and ceca of broilers. For that purpose, 96 one-day-old male broilers were randomly reared in two pens and received either wheat-based or maize-based starter and grower diets. At d 20, the broilers were randomly assigned to one out of four dietary treatments. The broilers received for 8 d the wheat-based or maize-based finisher diet as such (Control treatments; WC, MC) or supplemented with a xylanase/glucanase combination (Enzyme treatments; WE, ME). At d 28, samples from the digestive tract were collected, and the ileal and cecal microbiota composition was determined by 16S ribosomal RNA gene amplicon sequencing. A similar phylogenetic (alpha) diversity was observed among the four treatments, both in the ileal and the cecal samples. Furthermore, a similar microbial composition in the ileum (beta diversity) was observed, with lactobacilli being the predominant community for all treatments. In contrast, both cereal type and enzyme supplementation were found to influence cecal communities. The type of cereal (i.e., wheat or maize) explained 47% of the total variation in microbial composition in the ceca. Further stratifying the analysis per cereal type revealed differences in microbiota composition between WC and WE, but not between MC and ME. Furthermore, the prevalence of beneficial genera, such as Faecalibacterium and Blautia, in the ceca of broilers fed wheat-based diets coincided with arabinoxylan accumulation. These findings indicated that fermentable arabinoxylan and arabinoxylo-oligosaccharides released by dietary xylanase may play an important role in bacterial metabolism. [ABSTRACT FROM AUTHOR]

• Fungal genome sequencing aids functionality prediction of carbohydrate-active enzymes. • Genome comparison revealed significant differences in the enzyme sets of fungi. • An expansion of CAZyme-encoding genes was observed in specific fungi. • Xylanolytic enzymes have high potential in sustainable bioprocesses. As important polysaccharide degraders in nature, fungi can diversify their extensive set of carbohydrate-active enzymes to survive in ecological habitats of various composition. Among these enzymes, xylanolytic ones can efficiently and sustainably degrade xylans into (fermentable) monosaccharides to produce valuable chemicals or fuels from, for example relevant for upgrading agro-food industrial side streams. Moreover, xylanolytic enzymes are being used in various industrial applications beyond biomass saccharification, e.g. food, animal feed, biofuel, pulp and paper. As a reference for researchers working in related areas, this review summarized the current knowledge on substrate specificity of xylanolytic enzymes from different families of the Carbohydrate-Active enZyme database. Additionally, the diversity of enzyme sets in fungi were discussed by comparing the number of genes encoding xylanolytic enzymes in selected fungal genomes. Finally, to support bio-economy, the current applications of fungal xylanolytic enzymes in industry were reviewed. [ABSTRACT FROM AUTHOR]

Background: The polyphyletic group of seagrasses shows an evolutionary history from early monocotyledonous land plants to the marine environment. Seagrasses form important coastal ecosystems worldwide and large amounts of seagrass detritus washed on beaches might also be valuable bioeconomical resources. Despite this importance and potential, little is known about adaptation of these angiosperms to the marine environment and their cell walls. Results: We investigated polysaccharide composition of nine seagrass species from the Mediterranean, Red Sea and eastern Indian Ocean. Sequential extraction revealed a similar seagrass cell wall polysaccharide composition to terrestrial angiosperms: arabinogalactans, pectins and different hemicelluloses, especially xylans and/or xyloglucans. However, the pectic fractions were characterized by the monosaccharide apiose, suggesting unusual apiogalacturonans are a common feature of seagrass cell walls. Detailed analyses of four representative species identified differences between organs and species in their constituent monosaccharide composition and lignin content and structure. Rhizomes were richer in glucosyl units compared to leaves and roots. Enhalus had high apiosyl and arabinosyl abundance, while two Australian species of Amphibolis and Posidonia, were characterized by high amounts of xylosyl residues. Interestingly, the latter two species contained appreciable amounts of lignin, especially in roots and rhizomes whereas Zostera and Enhalus were lignin-free. Lignin structure in Amphibolis was characterized by a higher syringyl content compared to that of Posidonia. Conclusions: Our investigations give a first comprehensive overview on cell wall composition across seagrass families, which will help understanding adaptation to a marine environment in the evolutionary context and evaluating the potential of seagrass in biorefinery incentives. [ABSTRACT FROM AUTHOR]

Endo -xylanase and endo -glucanase are supplemented to poultry diets in order to improve nutrient digestion and non-starch polysaccharide (NSP) fermentation. Here, the action of these enzymes on alcohol insoluble solids (AIS) from wheat and maize grains as well as its implications for starch digestion in milled grains were evaluated in vitro , under conditions mimicking the poultry digestive tract. For wheat AIS, GH11 endo -xylanase depolymerized soluble arabinoxylan (AX) during the gizzard phase, and proceeded to release insoluble AX under small intestine conditions. At the end of the in vitro digestion (480 min), the endo -xylanase, combined with a GH7 endo -β-1,4-glucanase, released 30.5 % of total AX and 18.1 % of total glucan in the form of arabinoxylo- and gluco-oligosaccharides, as detected by HPAEC-PAD and MALDI-TOF-MS. For maize AIS, the combined enzyme action released 2.2 % and 7.0 % of total AX and glucan, respectively. Analogous in vitro digestion experiments of whole grains demonstrated that the enzymatic release of oligomers coincided with altered grain microstructure, as examined by SEM. In the present study, cell wall hydrolysis did not affect in vitro starch digestion kinetics for cereal grains. This study contributes to understanding the action of feed enzymes on cereal NSP under conditions mimicking the poultry digestive tract. [Display omitted] [ABSTRACT FROM AUTHOR]

Simple β- O -4' linked dimeric model compounds are often targeted as substrate to mimic the reactivity of lignin in enzymatic or chemical treatments. These models mimic the structure and reactivity of regular β- O -4′ linkages in lignin, but are less suitable to predict the reactivity of acylated β- O -4′ substructures, which are abundant in various types of lignin. Here, we present a one-step lipase-catalyzed acylation of a commercially available lignin model compound with p -coumaric acid, p -hydroxybenzoic acid, cinnamic acid and acetic acid as acyl donors. This facile procedure allows to obtain new and relevant lignin model compounds at milligram scale, with simple purification of products and unreacted substrate. Unlabelled Image • New acylated lignin model compounds relevant for grass and wood lignin research were produced. • A facile one-step approach was used with Candida antarctica Lipase B as catalyst. • Coumaroylation, hydroxybenzoylation and cinnamoylation occur selectivity on the primary alcohol of the lignin model dimer. [ABSTRACT FROM AUTHOR]

Background: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs. Results: Two Neurospora crassa (Nc) LPMOs were found to represent different mode-of-action towards XG. Interestingly, the configuration of active site segments of these LPMOs differed as well, with a shorter Segment 1 (−Seg1) and a longer Segment 2 (+Seg2) present in NcLPMO9C and the opposite for NcLPMO9M (+Seg1−Seg2). We confirmed that NcLPMO9C cleaved the non-reducing end of unbranched glucosyl residues within XG via the oxidation of the C4-carbon. In contrast, we found that the oxidative cleavage of the XG backbone by NcLPMO9M occurred next to both unbranched and substituted glucosyl residues. The latter are decorated with xylosyl, xylosyl–galactosyl and xylosyl–galactosyl–fucosyl units. The relationship between active site segments and the mode-of-action of these NcLPMOs was rationalized by a structure-based phylogenetic analysis of fungal AA9 LPMOs. LPMOs with a −Seg1+Seg2 configuration clustered together and appear to have a similar XG substitution-intolerant cleavage pattern. LPMOs with the +Seg1−Seg2 configuration also clustered together and are reported to display a XG substitution-tolerant cleavage pattern. A third cluster contained LPMOs with a −Seg1−Seg2 configuration and no oxidative XG activity. Conclusions: The detailed characterization of XG degradation products released by LPMOs reveal a correlation between the configuration of active site segments and mode-of-action of LPMOs. In particular, oxidative XG-active LPMOs, which are tolerant and intolerant to XG substitutions are structurally and phylogenetically distinguished from XG-inactive LPMOs. This study contributes to a better understanding of the structure–function relationship of AA9 LPMOs. [ABSTRACT FROM AUTHOR]

Background: The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome. Results: Quantitative 13C lignin internal standard py-GC–MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC–MS and HSQC NMR analyses demonstrated that Cα-oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions: For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms. [ABSTRACT FROM AUTHOR]

Summary: Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network of branching and fusing hyphae that are often considered to be relatively uniform entities in which nutrients can freely move through the hyphae. In nature, different parts of a colony are often exposed to different nutrients. We have investigated, using a multi‐omics approach, adaptation of A. niger colonies to spatially separated and compositionally different plant biomass substrates. This demonstrated a high level of intra‐colony differentiation, which closely matched the locally available substrate. The part of the colony exposed to pectin‐rich sugar beet pulp and to xylan‐rich wheat bran showed high pectinolytic and high xylanolytic transcript and protein levels respectively. This study therefore exemplifies the high ability of fungal colonies to differentiate and adapt to local conditions, ensuring efficient use of the available nutrients, rather than maintaining a uniform physiology throughout the colony. [ABSTRACT FROM AUTHOR]

Fungi are main lignin degraders and the edible white button mushroom, Agaricus bisporus , inhabits lignocellulose-rich environments. Previous research hinted at delignification when A. bisporus colonized pre-composted wheat straw-based substrate in an industrial setting, assumed to aid subsequent release of monosaccharides from (hemi-)cellulose to form fruiting bodies. Yet, structural changes and specific quantification of lignin throughout A. bisporus mycelial growth remain largely unresolved. To elucidate A. bisporus routes of delignification, at six timepoints throughout mycelial growth (15 days), substrate was collected, fractionated, and analyzed by quantitative pyrolysis-GC–MS, 2D-HSQC NMR, and SEC. Lignin decrease was highest between day 6 and day 10 and reached in total 42 % (w/w). The substantial delignification was accompanied by extensive structural changes of residual lignin, including increased syringyl to guaiacyl (S/G) ratios, accumulated oxidized moieties, and depleted intact interunit linkages. Hydroxypropiovanillone and hydroxypropiosyringone (HPV/S) subunits accumulated, which are indicative for β-| O -4′ ether cleavage and imply a laccase-driven ligninolysis. We provide compelling evidence that A. bisporus is capable of extensive lignin removal, have obtained insights into mechanisms at play and susceptibilities of various substructures, thus we were contributing to understanding fungal lignin conversion. [Display omitted] • Agaricus bisporus removes lignin from pre-composted biomass during mycelial growth. • Structural changes indicate laccase-driven delignification. • Agaricus bisporus preferentially cleaves the lignin β- O -4 ether linkage via β- O. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides. [ABSTRACT FROM AUTHOR]

• Techno-economic parameters of this pretreatment are presented. • Plant cell wall and composition of biomass are shown. • Environmental aspects of sustainability for hydrothermal are discuses. • Process design integration for hydrothermal pretreatment is presented. The development and sustainability of second-generation biorefineries are essential for the production of high added value compounds and biofuels and their application at the industrial level. Pretreatment is one of the most critical stages in biomass processing. In this specific case, hydrothermal pretreatments (liquid hot water [LHW] and steam explosion [SE]) are considered the most promising process for the fractionation, hydrolysis and structural modifications of biomass. This review focuses on architecture of the plant cell wall and composition, fundamentals of hydrothermal pretreatment, process design integration, the techno-economic parameters of the solubilization of lignocellulosic biomass (LCB) focused on the operational costs for large-scale process implementation and the global manufacturing cost. In addition, profitability indicators are evaluated between the value-added products generated during hydrothermal pretreatment, advocating a biorefinery implementation in a circular economy framework. In addition, this review includes an analysis of environmental aspects of sustainability involved in hydrothermal pretreatments. [ABSTRACT FROM AUTHOR]

Previously, arabinoxylan (AX) depolymerization by dietary endo-xylanase was observed in the broiler ileum, but released arabinoxylo-oligosaccharides (AXOS) were not characterized in detail. This study aimed at extracting and identifying AXOS released in vivo in broilers, in order to delineate the influence of endo-xylanase on AX utilization. Hereto, digesta from the gizzard, ileum, ceca and excreta of broilers fed a wheat-soybean diet without (Con) or with endo-xylanase supplementation (Enz) were assessed. Soluble AX content in the ileum was higher for Enz diet (26.9%) than for Con diet (18.8%), indicating a different type and amount of AX entering the ceca. Removal of maltodextrins and fructans enabled monitoring of AX depolymerization to AXOS (Enz diet) using HPSEC-RI and HPAEC-PAD. A recently developed HILIC-MSn methodology allowed AXOS (DP 4–10) identification in ileal digesta and excreta. Xylanase-induced AXOS formation coincided with decreased total tract AX recovery, which indicated improved AX hindgut utilization. [Display omitted] [ABSTRACT FROM AUTHOR]

Identification of arabinoxylo-oligosaccharides (AXOS) within complex mixtures is an ongoing analytical challenge. Here, we established a strategy based on hydrophilic interaction chromatography coupled to collision induced dissociation-mass spectrometry (HILIC-MSn) to identify a variety of enzyme-derived AXOS structures. Oligosaccharide reduction with sodium borohydride remarkably improved chromatographic separation of isomers, and improved the recognition of oligosaccharide ends in MS-fragmentation patterns. Localization of arabinosyl substituents was facilitated by decreased intensity of Z ions relative to corresponding Y ions, when fragmentation occurred in the vicinity of substituents. Interestingly, the same B fragment ions (MS2) from HILIC-separated AXOS isomers showed distinct MS3 spectral fingerprints, being diagnostic for the linkage type of arabinosyl substituents. HILIC-MSn identification of AXOS was strengthened by using specific and well-characterized arabinofuranosidases. The detailed characterization of AXOS isomers currently achieved can be applied for studying AXOS functionality in complex (biological) matrices. Overall, the present strategy contributes to the comprehensive carbohydrate sequencing. [Display omitted] [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic conversion of plant cell wall polysaccharides. Continuous discovery and functional characterization of LPMOs highly contribute to the tailor-made design and improvement of hydrolytic-activity based enzyme cocktails. In this context, a new Mt LPMO9F was characterized for its substrate (xyloglucan) specificity, and Mt LPMO9H was further delineated. Aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis, we found that both Mt LPMOs released predominately C4-oxidized, and C4/C6-double oxidized xylogluco-oligosaccharides. Further characterization showed that Mt LPMO9F, having a short active site segment 1 and a long active site segment 2 (−Seg1+Seg2), followed a "substitution-intolerant" xyloglucan cleavage profile, while for Mt LPMO9H (+Seg1−Seg2) a "substitution-tolerant" profile was found. The here characterized xyloglucan specificity and substitution (in)tolerance of Mt LPMO9F and Mt LPMO9H were as predicted according to our previously published phylogenetic grouping of AA9 LPMOs based on structural active site segment configurations. [Display omitted] [ABSTRACT FROM AUTHOR]

The enzymatic conversion of cellulosic-rich waste streams of white asparagus into cellobiose and cello-oligosaccharides (COS) is proposed for producing a natural carrier agent. The enzyme cocktail 'Celluclast' was used to steer towards maximum conversion and minimum formation of monosaccharides to obtain an enzymatic hydrolysate with a high glass transition temperature (T g). Different enzyme loadings and hydrolysis times were tested in combination with a sodium hydroxide pre-treatment of the asparagus fibre. A loading of 700 nkat/g substrate and 7 h of hydrolysis time resulted in the best yield/purity combination, namely a conversion of 36 g/100 g cellulose with 81% celllobiose/COS. The same hydrolysis conditions were tested in a larger bench-scale experiment (conversion of 45 g/100 g cellulose) and the soluble hydrolysates were concentrated and spray-dried. The high T g (108 °C) of the spray-dried hydrolysates of asparagus fibre proves its potential usage as a carrier agent for spray drying. [Display omitted] • The abundant asparagus waste stream is rich in cellulose. • Steering formation of cellobiose and cello-oligosaccharides (COS) was feasible. • Alkaline pre-treatment of asparagus fibre increased the hydrolysis yield. • Cellobiose and COS have potential as carrier agent for spray drying. [ABSTRACT FROM AUTHOR]

• LPMO-generated C1- and C4-oxidised cello-oligomers have distinct MS/MS patterns. • C4-oxidised cello-oligomers showed B-/Y- and X-type fragments at the oxidised end. • Diagnostic 2,4X n fragments were obtained for C4-oxidised cello-oligomers. • Extensive A-type cleavage was typical for C1-oxidised cello-oligomers. • Reduced and non-reduced cello-oligomers yield distinct fragmentation behaviours. Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that degrade recalcitrant polysaccharides, such as cellulose. However, the identification of LPMO-generated C1- and/or C4-oxidised oligosaccharides is far from straightforward. In particular, their fragmentation patterns have not been well established when using mass spectrometry. Hence, we studied the fragmentation behaviours of non-, C1- and C4-oxidised cello-oligosaccharides, including their sodium borodeuteride-reduced forms, by using hydrophilic interaction chromatography and negative ion mode collision induced dissociation - mass spectrometry. Non-oxidised cello-oligosaccharides showed predominantly C- and A-type cleavages. In comparison, C4-oxidised ones underwent B-/Y- and X-cleavage close to the oxidised non-reducing end, while closer to the reducing end C-/Z- and A-fragmentation predominated. C1-oxidised cello-oligosaccharides showed extensively A-cleavage. Reduced oligosaccharides showed predominant glycosidic bond cleavage, both B-/Y- and C-/Z-, close to the non-reducing end. Our findings provide signature mass spectrometric fragmentation patterns to unambiguously elucidate the catalytic behaviour and classification of LPMOs. [ABSTRACT FROM AUTHOR]

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO 2 . Recent research, however, demonstrated fungal conversion of 13 C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13 C-labeled lignin served as substrate for Agaricus bisporus , the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13 C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13 C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13 C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Arabinoxylo-oligosaccharides (AXOS) are non-digestible dietary fibers that potentially confer a health benefit by stimulating beneficial bacteria in the gut. Still, a detailed overview of the diversity of gut bacteria and their specificity to utilize structurally different AXOS has not been provided to date and was aimed for in this study. Moreover, we assessed the genetic information of summarized bacteria, and we extracted genes expected to encode for enzymes that are involved in AXOS hydrolysis (based on the CAZy database). The taxa involved in AXOS fermentation in the gut display a large variety of AXOS-active enzymes in their genome and consequently utilize AXOS to a highly different extent. Clostridia and Bacteroidales are generalists that consume many structurally diverse AXOS, whereas Bifidobacterium are specialists that specifically consume AXOS with a low degree of polymerization. Further complexity is evident from the fact that the exact bacterial species, and in some cases even the bacterial strains (e.g. in Bifidobacterium longum ) that are stimulated, highly depend on the specific AXOS molecular structure. Furthermore, certain species in Bifidobacterium and Lactobacillaceae are active as cross-feeders and consume monosaccharides and unbranched short xylo-oligosaccharides released from AXOS. Our review highlights the possibility that (enzymatic) fine-tuning of specific AXOS structures leads to improved precision in targeting growth of specific beneficial bacterial species and strains in the gut.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila converts lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. The priming reaction is shown to occur with catalytic amounts of MtPPO7 products and those compounds do not generate substantial amounts of H 2 O 2 in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H 2 O 2 can provide the means for controlling the LPMO catalysis through exogenous H 2 O 2 and thereby minimize any enzyme inactivation., (© 2023 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro . To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-β-guaiacyl ether (SBG) cleavage, guaiacylglycerol-β-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-β-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that Mt AA16A from Myceliophthora thermophila and An AA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the Mt AA16A crystal structure showed a fairly LPMO-typical histidine brace active site, but the cellulose-acting LPMO-typical flat aromatic surface parallel to the histidine brace region was lacking. Further, we showed that both AA16 proteins are able to oxidize low-molecular-weight reductants to produce H 2 O 2 . The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila ( Mt LPMO9s) but not by three AA9 LPMOs from Neurospora crassa ( Nc LPMO9s). The interplay with Mt LPMO9s is explained by the H 2 O 2 -producing capability of the AA16s, which, in the presence of cellulose, allows the Mt LPMO9s to optimally drive their peroxygenase activity. Replacement of Mt AA16A by glucose oxidase ( An GOX) with the same H 2 O 2 -producing activity could only achieve less than 50% of the boosting effect achieved by Mt AA16A, and earlier Mt LPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H 2 O 2 to the Mt LPMO9s is facilitated by protein-protein interaction. Our findings provide new insights into the functions of copper-dependent enzymes and contribute to a further understanding of the interplay of oxidative enzymes within fungal systems to degrade lignocellulose., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)