Search

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]

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]

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]

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. [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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

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. [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]

Laccase-mediator systems (LMS) are potential green tools to improve the valorization of lignocellulosic biomass by selective degradation of lignin. Despite extensive attention devoted to lignin degradation by LMS in literature, knowledge on the underlying mechanisms is largely limited to model compound studies. Here, we report a mechanistic study on the delignification of wheat straw (WS) and corn stover (CS) by a laccase/HBT system. Quantitative 13C-IS py-GC-MS analysis revealed that WS and CS were delignified in the range of 28–51% (w/w). Based on a combination of py-GC-MS, 2D NMR, SEC and RP-UHPLC-MS, extensive structural characterization of both residual and solubilized lignin structures was performed, from which we reconstructed the degradation pathway of native lignin by laccase/HBT. For the first time, we show that degradation of native lignin in the plant cell wall matrix by LMS occurs via both Cα–Cβ cleavage and ether cleavage of β-O-4′ aryl ethers, and that the latter primarily occurs via cleavage of the β-O bond. Cγ-Coumaroylated substructures were found to be more recalcitrant towards degradation than non-acylated substructures. In addition to lignin degradation, our results provide evidence for grafting of HBT onto lignin. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two‐photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti‐inhibitory agents. [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]

BACKGROUND White rot fungi have been used to improve the nutritive value of lignocellulose for ruminants. In feed analysis, the Van Soest method is widely used to determine the cell wall contents. To assess the reliability of this method (Method A) for determination of cell wall contents in fungal‐treated wheat straw, we compared a combined monosaccharide analysis and pyrolysis coupled to gas chromatography with mass spectrometry (Py‐GC/MS) (Method B). Ruminal digestibility, measured as in vitro gas production (IVGP), was subsequently used to examine which method explains best the effect of fungal pretreatment on the digestibility of wheat straw. RESULTS: Both methods differed considerably in the mass recoveries of the individual cell wall components, which changed on how we assess their degradation characteristics. For example, Method B gave a higher degradation of lignin (61.9%), as compared to Method A (33.2%). Method A, however, showed a better correlation of IVGP with the ratio of lignin to total structural carbohydrates, as compared to Method B (Pearson's r of −0.84 versus −0.69). Nevertheless, Method B provides a more accurate quantification of lignin, reflecting its actual modification and degradation. With the information on the lignin structural features, Method B presents a substantial advantage in understanding the underlying mechanisms of lignin breakdown. Both methods, however, could not accurately quantify the cellulose contents – among others, due to interference of fungal biomass. CONCLUSION: Method A only accounts for the recalcitrant residue and therefore is more suitable for evaluating ruminal digestibility. Method B allows a more accurate quantification of cell wall, required to understand and better explains the actual modification of the cell wall. The suitability of both methods, therefore, depends on their intended purposes. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: This study investigated the ruminal degradability of various wheat straw types by the white‐rot fungi Ceriporiopsis subvermispora (CS) and Lentinula edodes (LE). Different cultivars (CV) of wheat straw at different maturity stages (MS) were treated with the fungi for 7 weeks and assessed for chemical composition and in vitro gas production (IVGP). RESULTS: Both fungi showed a more pronounced degradation of lignin on a more mature straw (MS3; 89.0%) in comparison with the straw harvested at an earlier stage (MS1; 70.7%). Quantitative pyrolysis coupled to gas chromatography and mass spectrometry, using 13C lignin as an internal standard 13C‐IS Py‐GC/MS revealed that lignin in more mature straw was degraded and modified to a greater extent. In contrast, cellulose was less degraded in MS3, as compared to MS1 (8.3% versus 14.6%). There was no effect of different MS on the IVGP of the fungus‐treated straws. Among the different straw cultivars, the extent of lignin degradation varied greatly (47% to 93.5%). This may explain the significant (P < 0.001) effect of cultivar on the IVGP of the fungal‐treated straws. Regardless of the factors tested, both fungi were very capable of improving the IVGP of all straw types by 15.3% to 47.6%, (as compared to untreated straw), with CS performing better than LE – on different MS (33.6% versus 20.4%) and CVs (43.2% versus 29.1%). CONCLUSION: The extent of lignin degradation caused by fungal treatment was more pronounced on the more mature and lignified straw, while variable results were obtained with different cultivars. Both fungi were capable of improving the IVGP of various straw types. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Agaricus bisporus consumes carbohydrates contained in wheat straw based compost used for commercial mushroom production. Double substituted arabinoxylan is part of the ~40% of the compost polysaccharides that are not degraded by A. bisporus during its growth and development. Genes encoding α-1,3--arabinofuranosidase (AXHd3) enzymes that act on xylosyl residues doubly substituted with arabinosyl residues are absent in this mushroom forming fungus. Here, the AXHd3 encoding hgh43 gene of Humicola insolens was expressed in A. bisporus with the aim to improve its substrate utilization and mushroom yield. Transformants secreted active AXHd3 in compost as shown by the degradation of double substituted arabinoxylan oligomers in an in vitro assay. However, carbohydrate composition and degree of arabinosyl substitution of arabinoxylans were not affected in compost possibly due to inaccessibility of the doubly substituted xylosyl residues. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave glycosidic bonds in polysaccharides. The ability of these copper enzymes to boost the degradation of lignocellulose has greatly stimulated research efforts and biocatalytic applications within the biorefinery field. Initially found as oxidizing recalcitrant substrates, such as chitin and cellulose, it is now clear that LPMOs cleave a broad range of oligo- and poly-saccharides and make use of various electron-donating systems. Herein, substrate specificities and electron-donating systems of fungal LPMOs are summarized. A closer look at LPMOs as part of the fungal enzyme machinery might provide insights into their role in fungal growth and plant-pathogen interactions to further stimulate the search for novel LPMO applications. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a β-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature. [ABSTRACT FROM AUTHOR]

BACKGROUND The relationship between the chemical and molecular properties - in particular the (acid detergent) lignin (ADL) content and composition expressed as the ratio between syringyl and guaiacyl compounds (S:G ratio) - of maize stems and in vitro gas production was studied in order to determine which is more important in the degradability of maize stem cell walls in the rumen of ruminants. Different internodes from two contrasting maize cultivars (Ambrosini and Aastar) were harvested during the growing season. RESULTS The ADL content decreased with greater internode number within the stem, whereas the ADL content fluctuated during the season for both cultivars. The S:G ratio was lower in younger tissue (greater internode number or earlier harvest date) in both cultivars. For the gas produced between 3 and 20 h, representing the fermentation of cell walls in rumen fluid, a stronger correlation ( R2 = 0.80) was found with the S:G ratio than with the ADL content ( R2 = 0.68). The relationship between ADL content or S:G ratio and 72-h gas production, representing total organic matter degradation, was weaker than that with gas produced between 3 and 20 h. CONCLUSION The S:G ratio plays a more dominant role than ADL content in maize stem cell wall degradation. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Wheat straw based composting generates a selective substrate for mushroom production. The first phase of this process requires 5 days, and a reduction in time is wished. Here, we aim at understanding the effect of gypsum on the duration of the first phase and the mechanism behind it. Hereto, the regular process with gypsum addition and the same process without gypsum were studied during a 5-day period. The compost quality was evaluated based on compost lignin composition analysed by py-GC/MS and its degradability by a commercial (hemi-)cellulolytic enzyme cocktail. The composting phase lead to the decrease of the pyrolysis products 4-vinylphenol and 4-vinylguaiacol that can be associated with p-coumarates and ferulates linking xylan and lignin. In the regular compost, the enzymatic conversion reached 32 and 39% for cellulose, and 23 and 32% for xylan after 3 and 5 days, respectively. In absence of gypsum similar values were reached after 2 and 4 days, respectively. Thus, our data show that in absence of gypsum the desired compost quality was reached 20% earlier compared to the control process. [ABSTRACT FROM AUTHOR]

Degradation of lignin by fungi enhances availability of cellulose and hemicellulose in plant waste and thereby increases the amount of carbon source available to these microorganisms. The button mushroom Agaricus bisporus degrades only about half of the lignin in compost and about 40% of the carbohydrates remain unutilized during mushroom cultivation. Here it was assessed whether over-expression of the manganese peroxidase gene mnp1 improves lignin degradation and, as a consequence, carbohydrate breakdown by A. bisporus. Transformants expressing mnp1 under the control of actin regulatory sequences produced MnP activity in malt extract medium, while the parental strain A15 did not. MnP activity was increased 0.3- and 3-fold at casing and after the 2nd flush of a semi-commercial cultivation, respectively, when compared to strain A15. Pyrolysis-GC-MS showed that overexpression of MnP decreased phenylmethane and phenylethane type lignin relative to the phenylpropane type after the 2nd flush. However, it neither affected the syringyl/guaiacyl derived residue ratio nor the ratio of oxidized to non-oxidized lignin residues. Moreover, the carbohydrate content and accessibility was not affected in compost. Notably, the capacity of compost extract to consume the MnP co-factor HO was 4- to 8-fold higher than its production. This may well explain why over-expression of mnp1 did not improve carbohydrate degradation in compost. In fact, availability of HO may limit lignin degradation by wild-type A. bisporus. [ABSTRACT FROM AUTHOR]

The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed. [ABSTRACT FROM AUTHOR]

Background: Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential. Results: A HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. Conclusions: The G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 °C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates. [ABSTRACT FROM AUTHOR]

Background: Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry. Results: Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase- activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes. Conclusions: The activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy. [ABSTRACT FROM AUTHOR]

Interactions among dietary ingredients are often assumed non-existent when evaluating the nutritive value and health effects of dietary fiber. Specific fibers can distinctly affect digestive processes; therefore, digestibility and fermentability of the complete diet may depend on fiber types present. This study aimed to evaluate the effects of readily fermentable fibers (β-glucans and resistant starch) on the degradation of feed ingredients containing more persistent, recalcitrant, fibers. Six semi-synthetic diets with recalcitrant fibers from rapeseed meal (pectic polysaccharides, xyloglucans, and cellulose) or corn distillers dried grain with solubles (DDGS; (glucurono)arabinoxylans and cellulose) with or without inclusion of β-glucans (6%) or retrograded tapioca (40%) substituted for corn starch were formulated. Six ileal-cannulated pigs (BW 28±1.4 kg) were assigned to the diets according to a 6×6 Latin square. β-glucan-extract increased apparent total tract digestibility (ATTD) of non-glucosyl polysaccharides (accounting for ~40% of the fiber-fraction) from rapeseed meal (6%-units, P<0.001), but did not affect non-glucosyl polysaccharides from DDGS. Retrograded tapioca reduced ATTD of non-glucosyl polysaccharides from rapeseed meal and DDGS (>10%-units, P<0.001), indicating that the large amount of resistant starch entering the hindgut was preferentially degraded over recalcitrant fibers from rapeseed meal and DDGS, possibly related to reduced hindgut-retention time following the increased intestinal bulk. Fermentation of fiber sources was not only dependent on fiber characteristics, but also on the presence of other fibers in the diet. Hence, interactions in the gastrointestinal tract among fibrous feed ingredients should be considered when evaluating their nutritive value. [ABSTRACT FROM AUTHOR]

Background: Lytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity. Results: Here, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds. Conclusions: Several of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ. [ABSTRACT FROM AUTHOR]

Background: The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are performed to enable access for the subsequent added carbohydrate-degrading enzymes. Nevertheless, partly substituted xylan structures remain after pretreatment, in particular the ones substituted with (4-O-methyl-)glucuronic acids (UAme). Hence, α-glucuronidases play an important role in the degradation of UAmexylan structures facilitating the complete utilisation of plant biomass. The characterisation of α-glucuronidases is a necessity to find the right enzymes to improve degradation of recalcitrant UAmexylan structures. Results: The mode-of-action of two α-glucuronidases was demonstrated, both obtained from the fungus Rasamsonia emersonii; one belonging to the glycoside hydrolase (GH) family 67 (ReGH67) and the other to GH115 (ReGH115). Both enzymes functioned optimal at around pH 4 and 70 °C. ReGH67 was able to release UAme from UAme-substituted xylo-oligosaccharides (UAmeXOS), but only the UAme linked to the non-reducing end xylosyl residue was cleaved. In particular, in a mixture of oligosaccharides, UAmeXOS having a degree of polymerisation (DP) of two were hydrolysed to a further extent than longer UAmeXOS (DP 3-4). On the contrary, ReGH115 was able to release UAme from both polymeric UAmexylan and UAmeXOS. ReGH115 cleaved UAme from both internal and non-reducing end xylosyl residues, with the exception of UAme attached to the non-reducing end of a xylotriose oligosaccharide. Conclusion: In this research, and for the first time, we define the mode-of-action of two α-glucuronidases from two different GH families both from the ascomycete R. emersonii. To date, only four α-glucuronidases classified in GH115 are characterised. ReGH67 showed limited substrate specificity towards only UAmeXOS, cleaving UAme only when attached to the non-reducing end xylosyl residue. ReGH115 was much less substrate specific compared to ReGH67, because UAme was released from both polymeric UAmexylan and UAmeXOS, from both internal and non-reducing end xylosyl residues. The characterisation of the mode-of-action of these two α-glucuronidases helps understand how R. emersonii attacks UAmexylan in plant biomass and the knowledge presented is valuable to improve enzyme cocktails for biorefinery applications. [ABSTRACT FROM AUTHOR]

In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained. [ABSTRACT FROM AUTHOR]

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. [ABSTRACT FROM AUTHOR]

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]

Background: Many agricultural and industrial food by-products are rich in cellulose and xylan. Their enzymatic degradation into monosaccharides is seen as a basis for the production of biofuels and bio-based chemicals. Lytic polysaccharide monooxygenases (LPMOs) constitute a group of recently discovered enzymes, classified as the auxiliary activity subgroups AA9, AA10, AA11 and AA13 in the CAZy database. LPMOs cleave cellulose, chitin, starch and ß-(1 ? 4)-linked substituted and non-substituted glucosyl units of hemicellulose under formation of oxidized gluco-oligosaccharides. Results: Here, we demonstrate a new LPMO, obtained from Myceliophthora thermophila C1 (MtLPMO9A). This enzyme cleaves ß-(1 ? 4)-xylosyl bonds in xylan under formation of oxidized xylo-oligosaccharides, while it simultaneously cleaves ß-(1 ? 4)-glucosyl bonds in cellulose under formation of oxidized gluco-oligosaccharides. In particular, MtLPMO9A benefits from the strong interaction between low substituted linear xylan and cellulose. MtLPMO9A shows a strong synergistic effect with endoglucanase I (EGI) with a 16-fold higher release of detected oligosaccharides, compared to the oligosaccharides release of MtLPMO9A and EGI alone. Conclusion: Now, for the first time, we demonstrate the activity of a lytic polysaccharide monooxygenase (MtLPMO9A) that shows oxidative cleavage of xylan in addition to cellulose. The ability of MtLPMO9A to cleave these rigid regions provides a new paradigm in the understanding of the degradation of xylan-coated cellulose. In addition, MtLPMO9A acts in strong synergism with endoglucanase I. The mode of action of MtLPMO9A is considered to be important for loosening the rigid xylan-cellulose polysaccharide matrix in plant biomass, enabling increased accessibility to the matrix for hydrolytic enzymes. This discovery provides new insights into how to boost plant biomass degradation by enzyme cocktails for biorefinery applications. [ABSTRACT FROM AUTHOR]

Background: Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. Results: In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. Conclusions: The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples. [ABSTRACT FROM AUTHOR]

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes. [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]