The extracellular β-glucosidase BGL2 has two variants with different molecular sizes and hydrolytic activities in the stipe or pilei of Coprinopsis cinerea

Two variants of extracellular β-glucosidase (BGL2) were purified from the stipe and pilei of Coprinopsis cinerea. In the stipe, BGL2 was a monomeric protein with an apparent molecular mass of approximately 220 kDa, representing a mature full-length peptide of BGL2. However, in the pilei, the apparent molecular mass of BGL2 was only approximately 120 kDa, consisting of the 60 kDa N-terminal fragment and 55 kDa C-terminal fragment. The hydrolytic activities of BGL2 purified from the pilei were higher than those of BGL2 purified from the stipe. No mRNA splice variants of bgl2 were detected. Therefore, the different variants of BGL2 in the stipe and pilei were not formed by differential RNA splicing. Furthermore, in vitro experiments showed that full-length BGL2 could be cleaved by endogenous proteases from pilei or commercial trypsin at a similar site to form an oligomeric protein consisting of the N-terminal fragment and C-terminal fragment similar to BGL2 from pilei. The hydrolytic activity of BGL2 increased after cleavage by those proteases in vitro. We conclude that the 120 kDa variant of BGL2 in the pilei of C. cinerea is formed by posttranslational proteolytic cleavage. Posttranslational proteolytic cleavage is an efficient way to regulate the activity of BGL2 to adapt to the needs of different physiological functions in the elongation stipe and expansion pilei of C. cinerea.

Production of Recombinant Laccase From Coprinopsis cinerea and Its Effect in Mediator Promoted Lignin Oxidation at Neutral pH

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Cytoplasmic Lipases—A Novel Class of Fungal Defense Proteins Against Nematodes

Fungi are an attractive food source for predators such as fungivorous nematodes. Several fungal defense proteins and their protective mechanisms against nematodes have been described. Many of these proteins are lectins which are stored in the cytoplasm of the fungal cells and bind to specific glycan epitopes in the digestive tract of the nematode upon ingestion. Here, we studied two novel nematotoxic proteins with lipase domains from the model mushroom Coprinopsis cinerea. These cytoplasmically localized proteins were found to be induced in the vegetative mycelium of C. cinerea upon challenge with fungivorous nematode Aphelenchus avenae. The proteins showed nematotoxicity when heterologously expressed in E. coli and fed to several bacterivorous nematodes. Site-specific mutagenesis of predicted catalytic residues eliminated the in-vitro lipase activity of the proteins and significantly reduced their nematotoxicity, indicating the importance of the lipase activity for the nematotoxicity of these proteins. Our results suggest that cytoplasmic lipases constitute a novel class of fungal defense proteins against predatory nematodes. These findings improve our understanding of fungal defense mechanisms against predators and may find applications in the control of parasitic nematodes in agriculture and medicine.

Democratization of fungal highway columns as a tool to investigate bacteria associated with soil fungi

ABSTRACT Bacteria–fungi interactions (BFIs) are essential in ecosystem functioning. These interactions are modulated not only by local nutritional conditions but also by the physicochemical constraints and 3D structure of the environmental niche. In soils, the unsaturated and complex nature of the substrate restricts the dispersal and activity of bacteria. Under unsaturated conditions, some bacteria engage with filamentous fungi in an interaction (fungal highways) in which they use fungal hyphae to disperse. Based on a previous experimental device to enrich pairs of organisms engaging in this interaction in soils, we present here the design and validation of a modified version of this sampling system constructed using additive printing. The 3D printed devices were tested using a novel application in which a target fungus, the common coprophilous fungus Coprinopsis cinerea, was used as bait to recruit and identify bacterial partners using its mycelium for dispersal. Bacteria of the genera Pseudomonas, Sphingobacterium and Stenotrophomonas were highly enriched in association with C. cinerea. Developing and producing these new easy-to-use tools to investigate how bacteria overcome dispersal limitations in cooperation with fungi is important to unravel the mechanisms by which BFIs affect processes at an ecosystem scale in soils and other unsaturated environments.

Necrotising fungal scleritis with full-thickness scleral melt and circumferential progression: a novel debridement approach

A 44-year-old Asian Indian woman presented with a history of pain and redness in the left eye for 3 weeks. Scleral congestion with a nodular swelling was present inferotemporally. Raised C reactive protein and positive antinuclear and perinuclear antineutrophil cytoplasmic antibodies suggested autoimmune scleritis. The patient was therefore managed with corticosteroids. Nevertheless, the development of severe pain associated with a scleral abscess led to a revised diagnosis of infectious scleritis. Corticosteroids therapy was halted and urgent debridement was performed. Microbiology confirmed fungal scleritis due to Coprinopsis cinerea. Multiple full-thickness circumferential debridements with antifungal therapy resulted in satisfactory anatomical and visual outcomes. This case presented a unique challenge, since laboratory results were misleading, and corticosteroids resulted in a fulminant clinical course. Therefore, aggressive circumferential debridement was performed to achieve the elimination of a rare fungal aetiology of scleritis, which has not been reported previously to cause human infection.

Fungal Pretreatments on Non-Sterile Solid Digestate to Enhance Methane Yield and the Sustainability of Anaerobic Digestion

Fungi can run feedstock pretreatment to improve the hydrolysis and utilization of recalcitrant lignocellulose-rich biomass during anaerobic digestion (AD). In this study, three fungal strains (Coprinopsis cinerea MUT 6385, Cyclocybe aegerita MUT 5639, Cephalotrichum stemonitis MUT 6326) were inoculated in the non-sterile solid fraction of digestate, with the aim to further (re)use it as a feedstock for AD. The application of fungal pretreatments induced changes in the plant cell wall polymers, and different profiles were observed among strains. Significant increases (p < 0.05) in the cumulative biogas and methane yields with respect to the untreated control were observed. The most effective pretreatment was carried out for 20 days with C. stemonitis, causing the highest hemicellulose, lignin, and cellulose reduction (59.3%, 9.6%, and 8.2%, respectively); the cumulative biogas and methane production showed a 182% and 214% increase, respectively, compared to the untreated control. The increase in AD yields was ascribable both to the addition of fungal biomass, which acted as an organic feedstock, and to the lignocellulose transformation due to fungal activity during pretreatments. The developed technologies have the potential to enhance the anaerobic degradability of solid digestate and untap its biogas potential for a further digestion step, thus allowing an improvement in the environmental and economic sustainability of the AD process and the better management of its by-products.