Fungal-based materials are currently studied as renewable and sustainable alternatives to fossil-based materials used in construction, packaging, textile, and fashion industries. The polypore fungus Fomes fomentarius produces a range of lightweight, durable, and flexible materials from diverse plant residual streams. Genetic engineering of this fungus is an important technological objective, both from fundamental perspectives and to optimize performance characteristics of F. fomentarius materials. We provide proof of principle that F. fomentarius can be genetically transformed. Hyphae produced high amounts of protoplasts using standard protocols. These protoplasts were transformed using nourseothricin selection and a nourseothricin N-acetyl transferase resistance marker (natR). Expression of natR was successful using a F. fomentarius translation elongation factor promoter. Stable, multicopy, and non-targeted integration of exogenous cassettes with recipient genome was confirmed by diagnostic PCR and Southern blotting. Preliminary attempts at genome editing based disruption of a putative ku70 encoding gene were, however, unsuccessful. We validated expression of two fluorescent proteins using a Schizophyllum commune H2B promoter using qRT-PCR and fluorescent microscopy. These data confirm functionality of mCherry and eGFP in F. fomentarius and suggest these proteins can be used for future protein localisation studies. Finally, we report DNase activity in F. fomentarius which we hypothesize may be a significant technical challenge for genetic transformation of this fungus. F. fomentarius can be genetically transformed using the promoters, marker, and nourseothricin selection system developed in this study. Expression of both eGFP and mCherry is a significant step towards cell biological and gene functional analyses using fluorescent microscopy. We have laid the foundations for overexpression studies, and, additionally, identified several challenges which need to be overcome for targeted genetic engineering in this important material producing fungus.
Invasive fungal infections caused by Aspergillus fumigatus and other pathogenic fungi represent a major global health threat, with treatment increasingly complicated by the emergence of antifungal drug resistance. Azole antifungals serve as the first-line therapy for aspergillosis, but resistance rates vary substantially across geographical regions. This study aimed to characterize the antifungal resistance landscape among clinical fungal isolates from Hangzhou and Yiwu cities in Zhejiang Province, Eastern China, and to elucidate the molecular mechanisms underlying azole resistance in Aspergillus fumigatus. We analyzed 110 Aspergillus clinical isolates for antifungal susceptibility. Among A. fumigatus strains, 8.6
Abstract Background Anaerobic gut fungi ( Neocallimastigomycota ) are major lignocellulose degraders in herbivore gastrointestinal tracts, but reproducible axenic cultivation remains difficult because conventional serum-bottle systems intrinsically couple CO 2 headspace, bicarbonate buffering, dissolved inorganic carbon availability, and pH control. This study aimed to decouple these variables and determine whether bicarbonate is required beyond its buffering function. Results Comparison of CO 2 - and N 2 -based headspaces in otherwise identical bicarbonate-buffered cultures showed consistently higher hydrogen production under N 2 . Screening of chemically defined non-carbonate buffers demonstrated that stable external pH alone was insufficient to sustain activity, as bicarbonate-free cultures lost metabolic performance after serial transfer. Reintroduction of sodium hydrogen carbonate under N 2 restored activity in a strain-dependent manner, revealing low bicarbonate thresholds that supported reproducible growth-associated metabolism in filamentous taxa. To test whether this effect reflected a general carboxylate requirement, twenty structurally diverse carboxylic acids were screened using Aestipascuomyces dupliciliberans ; none restored the bicarbonate-supported hydrogen production phenotype, although maleic acid increased soluble metabolite accumulation. A dual-buffer strategy combining Bis-Tris with minimal bicarbonate enabled independent pH control and identified maximal metabolic activity at pH 6.4. In addition, an OD 750 -based biomass assay calibrated with activated carbon was established and validated in an ammonium sulfate perturbation experiment, providing a practical framework for biomass estimation and future analyses of nitrogen effects. Conclusions Headspace composition and inorganic carbon availability are key determinants of anaerobic gut fungal cultivation. The results indicate that bicarbonate supports sustained anaerobic gut fungal activity beyond extracellular pH stabilization and cannot be functionally replaced by the tested organic carboxylates. Decoupling gas phase, buffering chemistry, and pH control provides a more interpretable and reproducible cultivation framework for physiological studies and biotechnological application of anaerobic gut fungi.
Abstract Background Fungi live in diverse environments requiring tolerance against abiotic and biotic stress and changing atmospheric conditions. Studies with white rot and brown rot species of Polyporales Basidiomycota have demonstrated that aerobic wood decay fungi may adapt to low oxygen and even anoxic conditions, which they undoubtedly encounter in their deadwood habitat. In the white rot fungus Phlebia radiata, oxygen depletion on lignocellulose substrates leads to hypoxia and fermentative metabolism. In this study, we elaborated the atmospheric effect further by subjecting the fungus to oxidative stress on wood substrate under aerobic and low oxygen conditions, with the aim to examine changes in gene expression and metabolic pathways as consequences of the oxidative treatment. Results Overall, 762 genes were significantly differentially expressed (DEGs with absolute Log2FoldChange > 1) 18 h after treatment with hydrogen peroxide according to RNA-Seq data. Half of these (348 genes) were downregulated in low oxygen (< 10% O2) cultures, with 185 genes unique to the condition but one third (121 genes) of unknown function. In aerobic cultures, a different response was observed with less DEGs showing downregulation (236 genes) while a higher number were upregulated (267 genes) and 153 of the upregulated genes were unique to the condition. Among DEGs upregulated under both conditions, small secreted proteins (SSPs) were the most abundant, followed by short-chain dehydrogenase/reductases, aldo-keto reductases, GNAT family acetyltransferases and CAZy carbohydrate active enzymes. Among downregulated DEGs, numerous SSP and CAZy classes, and genes encoding heat-shock proteins and MFS transporters were identified. Closer examination of carbon metabolism genes showed that under both conditions, oxidative treatment led to suppression of pentose catabolic pathway, glycerol metabolism, and formation of ethanol and acetate. Conclusions Oxidative stress caused a substantial change in fungal gene expression, but with different responses depending on the culture atmosphere. This may be explained by contrasting fungal metabolic states before the shock as was observed in extracellular enzyme, aromatic metabolite and redox activity profiles. Surprisingly, oxidative shock caused downregulation of a few heat-shock proteins whereas small secreted proteins were either up- or downregulated, suggesting both sensing and regulative roles for these, functionally yet unknown, diverse fungal proteins.
Abstract Background Pharmaceutical residues are increasingly detected in aquatic environments, and conventional wastewater treatment plants often provide insufficient removal. In this study, we evaluated the capacity of spawn-based pellets of the white-rot fungus Pleurotus ostreatus to remove 11 pharmaceuticals from synthetic wastewater and the secondary effluent from a municipal wastewater treatment plant and conducted a preliminary life cycle assessment (LCA) of the treatment process. Result In synthetic wastewater, living fungal pellets reduced the pharmaceutical concentration by > 30% within 10 min compared to the control. Significant removal of nine compounds, particularly macrolide and sulfonamide antibiotics, was observed. Biosorption contributed to removal but accounted for only a minor fraction. In the secondary effluent, total pharmaceutical concentrations decreased by 51–66% after 2–24 h, with time-dependent removal observed for several compounds. LCA results indicated that fungal treatment combined with incineration of spent biomass had lower environmental impacts than landfilling, largely due to energy recovery. The major contributors to environmental burden were energy demands, particularly due to sterilisation during grain spawn preparation and aeration. Conclusion Use of spawn-based pellets of P. ostreatus is a biologically based treatment technology for pharmaceutical removal. Future work should address transformation products, toxicity, and process optimization to improve sustainability at scale.
