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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.

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 purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive.

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. Graphical abstract: (Figure presented.)

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 convert 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. We show that the priming reaction occurs with catalytic amounts of MtPPO7 products and that those compounds do not generate substantial amounts of H2O2in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2O2 can provide the means for controlling the LPMO catalysis through exogenous H2O2 and thereby minimize any enzyme inactivation.

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 H2O2 in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2O2 can provide the means for controlling the LPMO catalysis through exogenous H2O2 and thereby minimize any enzyme inactivation.

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 MtAA16A from Myceliophthora thermophila and AnAA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the MtAA16A 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 H2O2. The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila (MtLPMO9s) but not by three AA9 LPMOs from Neurospora crassa (NcLPMO9s). The interplay with MtLPMO9s is explained by the H2O2-producing capability of the AA16s, which, in the presence of cellulose, allows the MtLPMO9s to optimally drive their peroxygenase activity. Replacement of MtAA16A by glucose oxidase (AnGOX) with the same H2O2-producing activity could only achieve less than 50% of the boosting effect achieved by MtAA16A, and earlier MtLPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H2O2to the MtLPMO9s 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.

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.

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.

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.

Xyloglucan is a prominent matrix heteropolysaccharide binding to cellulose microfibrils in primary plant cell walls. Hence, the hydrolysis of xyloglucan facilitates the overall lignocellulosic biomass degradation. Xyloglucanases (XEGs) are key enzymes classified in several glycoside hydrolase (GH) families. So far, family GH44 has been shown to contain bacterial XEGs only. Detailed genome analysis revealed GH44 members in fungal species from the phylum Basidiomycota, but not in other fungi, which we hypothesized to also be XEGs. Two GH44 enzymes from Dichomitus squalens and Pleurotus ostreatus were heterologously produced and characterized. They exhibited XEG activity and displayed a hydrolytic cleavage pattern different from that observed in fungal XEGs from other GH families. Specifically, the fungal GH44 XEGs were not hindered by substitution of neighboring glucosyl units and generated various "XXXG-type," "GXXX(G)-type," and "XXX-type" oligosaccharides. Overall, these fungal GH44 XEGs represent a novel class of enzymes for plant biomass conversion and valorization.

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.

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 (Tg). 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 Tg (108 °C) of the spray-dried hydrolysates of asparagus fibre proves its potential usage as a carrier agent for spray drying.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard-to-convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello-oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H218O, it was confirmed that the C6 gem-diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem-diol, could not be excluded. These findings can be extended to how the reactive LPMO-cosubstrate complex is positioned towards the substrate.

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 MtLPMO9F was characterized for its substrate (xyloglucan) specificity, and MtLPMO9H was further delineated. Aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis, we found that both MtLPMOs released predominately C4-oxidized, and C4/C6-double oxidized xylogluco-oligosaccharides. Further characterization showed that MtLPMO9F, 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 MtLPMO9H (+Seg1−Seg2) a “substitution-tolerant” profile was found. The here characterized xyloglucan specificity and substitution (in)tolerance of MtLPMO9F and MtLPMO9H were as predicted according to our previously published phylogenetic grouping of AA9 LPMOs based on structural active site segment configurations.

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.

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.

Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC). We observed that attachment of a carbohydrate-binding module 1 (CBM1) did not change the substrate specificity of LPMO9B from Myceliophthora thermophila C1 (MtLPMO9B) but stimulated the degradation of all three types of cellulose. A detailed quantification of oxidized ends in both soluble and insoluble fractions, as well as characterization of oxidized cello-oligosaccharide patterns, suggested that MtLPMO9B generates mainly oxidized cellobiose from BC, while producing oxidized cello-oligosaccharides from AVI and RAC ranged more randomly from DP2-8. Comparable product profiles, resulting from BC, AVI, and RAC oxidation, were found for three other AA9 LPMOs. These distinct cleavage profiles highlight cellulose specificity rather than an LPMO-dependent mechanism and may further reflect that the product profiles of AA9 LPMOs are modulated by different cellulose types.

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers. Graphic abstract: [Figure not available: see fulltext.].