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Summary 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., Graphical abstract, Highlights • The fungal members of GH44 only belong to the phylum Basidiomycota • The characterized fungal GH44 xyloglucanases (XEGs) are xyloglucan-specific • These XEGs cleave xyloglucan at both sides of unsubstituted glucosyl units, Enzymology; Mycology; Biomass

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.

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate specificity is an indispensability for selection of most suitable candidates. Hence, linking distinct substrate specificities to a FAE classification, such as the recently classified FAE subfamilies (SF), is a promising approach to improve the application of these enzymes for a variety of industrial applications. In total, 14 FAEs that are classified members of SF1, 5, 6, 7, 9, and 13 were tested in this research. All FAEs were investigated for their activity toward a variety of substrates: synthetic model substrates, plant cell wall-derived substrates, including lignin, and natural substrates. Released HCAs were determined using reverse phase-ultra high performance liquid chromatography coupled to UV detection and mass spectrometry. Based on this study, FAEs of SF5 and SF7 showed the highest release of FA, pCA, and diFAs over the range of substrates, while FAEs of SF6 were comparable but less pronounced for diFAs release. These results suggest that SF5 and SF7 FAEs are promising enzymes for biorefinery applications, like the production of biofuels, where a complete degradation of the plant cell wall is desired. In contrast, SF6 FAEs might be of interest for industrial applications that require a high release of only FA and pCA, which are needed as precursors for the production of biochemicals. In contrast, FAEs of SF1, 9 and 13 showed an overall low release of HCAs from plant cell wall-derived and natural substrates. The obtained results substantiate the previous SF classification as a useful tool to predict the substrate specificity of FAEs, which eases the selection of FAE candidates for industrial applications.

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. This article is protected by copyright. All rights reserved.

The fungal members of Carbohydrate Esterase family 1 (CE1) from the CAZy database include both acetyl xylan esterases (AXEs) and feruloyl esterases (FAEs). AXEs and FAEs are essential auxiliary enzymes to unlock the full potential of feedstock. They are being used in many biotechnology applications including food and feed, pulp and paper, and biomass valorization. AXEs catalyze the hydrolysis of acetyl group from xylan, while FAEs release ferulic and other hydroxycinnamic acids from xylan and pectin. Previously, we reported a phylogenetic analysis for the fungal members of CE1, establishing five subfamilies (CE1_SF1–SF5). Currently, the characterized AXEs are in the subfamily CE1_SF1, whereas CE1_SF2 contains mainly characterized FAEs. These two subfamilies are more related to each other than to the other subfamilies and are predicted to have evolved from a common ancestor, but target substrates with a different molecular structure. In this study, four ascomycete enzymes from CE1_SF1 and SF2 were heterologously produced in Pichia pastoris and characterized with respect to their biochemical properties and substrate preference toward different model and plant biomass substrates. The selected enzymes from CE1_SF1 only exhibited AXE activity, whereas the one from CE1_SF2 possessed dual FAE/AXE activity. This dual activity enzyme also showed broad substrate specificity toward model substrates for FAE activity and efficiently released both acetic acid and ferulic acid (∼50%) from wheat arabinoxylan and wheat bran which was pre-treated with a commercial xylanase. These fungal AXEs and FAEs also showed promising biochemical properties, e.g., high stability over a wide pH range and retaining more than 80% of their residual activity at pH 6.0–9.0. These newly characterized fungal AXEs and FAEs from CE1 have high potential for biotechnological applications. In particular as an additional ingredient for enzyme cocktails to remove the ester-linked decorations which enables access for the backbone degrading enzymes. Among these novel enzymes, the dual FAE/AXE activity enzyme also supports the evolutionary relationship of CE1_SF1 and SF2.