Fungal Biology and Biotechnology最新文献

Background: 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.

Results: We analyzed 110 Aspergillus clinical isolates for antifungal susceptibility. Among A. fumigatus strains, 8.6% exhibited azole resistance, with half of these harboring the tandem repeat mutation TR34/L98H in the cyp51A gene. One resistant strain carried the TR46/Y121F/T289A mutation combination. Minimum inhibitory concentration testing revealed that 8.6% of A. fumigatus isolates were resistant to amphotericin B We identified one resistant isolate without known resistance mutations and characterized it through whole-genome sequencing and transcriptomic analysis. Novel mutations S437F in cyp51A and P298T in hmg1 were discovered; however, functional validation using CRISPR-Cas9 demonstrated these mutations do not directly confer resistance. Instead, transcriptomic profiling revealed significant upregulation of ergosterol biosynthesis pathway genes and efflux transporter genes, suggesting alternative resistance mechanisms. Among non-A. fumigatus species, all A. niger isolates displayed resistance to itraconazole and posaconazole.

Conclusions: This comprehensive regional surveillance reveals concerning patterns of antifungal resistance among major human pathogenic fungi in Zhejiang Province, China. The identification of both classical resistance mutations and novel transcription-mediated resistance mechanisms highlights the complex and evolving nature of antifungal resistance. These findings emphasize the critical need for continued surveillance programs and rapid resistance detection methods to inform clinical treatment strategies and improve patient outcomes.

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 CO₂ 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 CO₂- and N₂-based headspaces in otherwise identical bicarbonate-buffered cultures showed consistently higher hydrogen production under N₂. 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 N₂ 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 OD₇₅₀-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.

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.

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.

Background: Since decades, fungi are leveraged in biotechnology to produce high-value compounds used in multiple economic sectors. Strain and process optimisation is based on a comprehensive understanding of the production organism on the cellular and molecular level. Among three antifungal protein families in fungi, consisting of small cysteine-stabilised proteins, it has been shown that, for some family members, bioactivities are also associated with additional functions in their hosts, e.g., carbon metabolism, autophagy, or asexual development. These proteins are interesting as alternative source of novel antifungal drugs. However, their potential impact on biotechnological production is not yet elucidated.

Results: In this study, we introduce the antifungal bubble protein "AgBP", from Aspergillus giganteus and further elucidate the reservoir of bioactive proteins in fungi. We used NCBI PSI-BLAST and subsequent phylogenetic and structural analyses of the Antifungal Protein (AFP), Bubble Protein (BP), and Neosartorya fischeri antifungal protein 2 (NFAP2) family members. We could identify further putative members: 165 AFP-, 102 BP-, and 219 NFAP2-like proteins. Six of the AFP and all 219 NFAP2 family members are not yet assigned on InterPro. All proteins were exclusively identified in fungi. To our best knowledge, this is the first study to report this group of bioactive proteins is shared among the two divisions of Ascomycetes and Basidiomycetes. Phylogenetic tree analyses demonstrate restricted taxonomic distribution within single genera. Furthermore, the comparison of the tertiary structures of all members of the three AFP families clearly separates them from each other and from non-fungal small cysteine-stabilised antifungal proteins.

Conclusion: We hypothesise that the three protein families represent a distinct superfamily of evolutionary related proteins. We further hypothesise that these proteins could be categorised as secondary metabolite like molecules of ribosomal origin. For brevity, we named this superfamily BPF (bioactive proteins from fungi). The distribution of BPF members is presumably driven by horizontal gene transfer. Furthermore, we hypothesise that BPF members likely serve rather different biological roles than merely acting as antimicrobials. Their hypothetical classification as potential secondary metabolite like proteins in combination with their occurrence among several biotechnologically relevant fungal genera, e.g., Aspergillus, Penicillium, Trichoderma, Schizophyllum, etc., emphasises their potential relevance for genetic and metabolic engineering.

Morphological switching in response to environmental stimuli is a well-known phenomenon in fungi, leading to diverse morphotypes. Microscopic observation remains a widely used approach to study these phenotypes, but variation in sample preparation and operator skill can limit the scale of sample processing or introduce operator bias. Although several image-based cell detection tools have been developed, most are tailored to specific applications or limited to a particular taxon. To address the need for a tool applicable to the polymorphic, yeast-like fungus Aureobasidium pullulans, and with potential applicability to other taxa, we developed TU_MyCo-Vision, an Ultralytics YOLO (You Only Look Once) based object detection tool for identifying 13 fungal morphotypes in bright-field microscopic images. Identification of 13 fungal morphotypes, including variation of vacuolated single cells, cells with granular cytoplasmic appearance, and diverse hyphal forms, is achieved by integrating a YOLOv11m-based object detector trained on a custom dataset of 1,504 annotated images and a standalone graphical user interface that enables downstream data analysis and visualization of results. The best-performing model (Zulu_s3) achieved a mean precision of 73.4%, a recall of 66.5%, a mean average precision at 50% IoU (mAP@50) of 73.5%, and a mean average precision at varying IoU thresholds between 50 and 90% IoU (mAP@50-95) of 54.5% across all 13 classes. The single-group analysis pipeline was validated on a 90-image test set, generating six quantitative summaries that capture the distribution and co-occurrence of fungal morphotypes, including absolute counts, relative and mean relative abundance plots, stacked bar plots and clustered heatmaps. Multi-group evaluation on previously unseen datasets comprising Candida albicans, Komagataella phaffi, and Aspergillus niger spores demonstrated that these morphotype profiles can be compared across biologically distinct genera, highlighting the tool's potential applicability for studying fungal morphological diversity.

Background: Ambrosia beetles rely on obligate fungal partners for survival, and these associations are emerging as model systems for examining the development and evolution of fungal-animal mutualism. However, genetic tools for the manipulation of such mutualistic fungi remain largely lacking, and consequently the genetic basis employed by these fungi to establish associations with their beetle partners remains almost completely unexplored. Here, we provide methods for protoplast generation and transformation of several major filamentous fungal partners of Xyleborus ambrosia beetles including Raffaela arxii, R. fusca, Harringtonia aguacate, and Graphium ambrosium using reporter constructs driven by the H. lauricola gpd promoter sequence. In addition, we developed inducible expression systems for the beetle symbiont, but plant pathogen, H. lauricola, responsible for laurel wilt disease, and show that these constructs are also functional in G. ambrosium. This work demonstrates the utility of H. lauricola genetic parts in transforming diverse groups of fungi and offers a toolbox for their genetic dissection.

Results: Fungal strains showed sensitivity to hygromycin, and protoplasts derived from the fungi were transformed with plasmids expressing the hygromycin selective marker (HPH) along with either green- or red-fluorescent proteins (GFP/RFP), with expression driven by the constitutive H. lauricola gpd promoter. Transformed strains for the fungal species described above showing expression of either GFP or RFP were obtained. A series of inducible plasmids using the H. lauricola alcA_p-RFP-(ethanol) and glaA_p-RFP-(maltose) inducible promoters were also tested and validated.

Conclusions: Our data provide methods and tools for genetic manipulation of a wide range of ambrosia beetle fungal symbionts, including marking cells with fluorescent proteins and use of inducible expression systems. Intriguingly, the method/plasmids developed did not result in transformation of the related Xyleborus fungal symbiont, Neocosmospora affinis. The fluorescent strains developed can be used to monitor symbiotic colonization of the beetle host, fungal development in host galleries, host preferences, and a range of other applications, and promoters may be used for further knockout and expression studies.

Antimicrobial peptides (AMPs) are promising candidates for next-generation therapeutics due to their broad-spectrum activity and reduced propensity for resistance, making them valuable in medicine, agriculture, and biotechnology. However, traditional AMP production methods including isolation from natural sources and chemical synthesis are costly, inefficient, and environmentally unsustainable, particularly for longer or post-translationally modified peptides. While heterologous expression has emerged as a scalable and versatile alternative, its success depends strongly on host selection and tailored optimisation strategies. This review examines recent advances in fungal systems as platforms for AMP production. Fungal systems, and particularly yeasts such as Pichia pastoris, offer rapid growth, low-cost fermentation, secretion capacity, and the ability to perform key post-translational modifications (PTMs), making them leading hosts for recombinant AMPs. We outline strain choice and engineering strategies that enhance AMP yield and bioactivity, including promoter and codon optimisation, secretion signal choice, fusion partners, and the construction of tandem or chimeric AMPs. By integrating current methodologies and case studies, this review aims to guide future efforts toward efficient, scalable, and commercially viable AMP manufacturing in fungal hosts, positioning fungal biotechnology as a key enabler in the development of next-generation antimicrobial solutions.

Fungi have evolved two distinct strategies, white-rot and brown-rot, to degrade lignocellulose in plant biomass. White-rot fungi utilize lignin-modifying enzymes (LMEs) to deconstruct lignin and access carbohydrates, whereas brown-rot fungi, which arose from white-rot ancestral lineages, have largely lost LME activities. Instead, brown-rot fungi rely on a small redox metabolite-mediated Fenton system that generates reactive oxygen species (ROS) to rapidly deconstruct lignocellulose and selectively remove carbohydrates. The abandonment of LMEs in brown-rot fungi suggests an intriguing evolutionary strategy to streamline decay machinery, but it remains unclear why fungi evolved this way. Here, we reintroduced LME genes from the white-rot fungus Trametes versicolor into the model brown-rot fungus Gloeophyllum trabeum to create hybrid fungal systems and evaluate LME function within the Fenton-dominant brown-rot context. Analysis of lignocellulose degradation showed that lignin disruption can be partially restored in LME mutants, thereby shifting the typical carbohydrate-selective decay mode of G. trabeum. However, constitutive LME expression also caused pronounced growth defects and reduced the overall rate of lignocellulose degradation, specifically decreasing cellulose and hemicellulose removal. Correlation analysis between genotypes and phenotypes indicates that LMEs, although prevalently used for delignification in fungi, are fundamentally incompatible with the carbohydrate-selective brown-rot system.

The opportunistic pathogenic fungus Nakaseomyces glabratus inhabits diverse host niches with fluctuating nutrient availability. Therefore, efficient control of glycolytic entry is essential, yet the regulatory principles governing hexose phosphorylation in this species remain incompletely understood. Here, we investigated the functional organization of sugar kinases in N. glabratus. Among five predicted sugar kinases, only three (Hxk2, Hxk2b and Glk1) catalyzed phosphorylation of glucose, fructose or mannose, whereas Hxk1 and Glk1b lacked detectable activity. Kinetic analyses revealed a functional specialization, with the hexokinases acting as high-capacity enzymes and Glk1 functioning as a high-affinity, low-capacity kinase optimized for low-sugar concentrations. Despite similar intrinsic kinetics among the hexokinases, Hxk2b emerged as the physiologically dominant enzyme, reflecting differential regulation rather than catalytic properties alone. Both hexokinases, but not Glk1, were strongly inhibited by trehalose-6-phosphate, linking glycolytic entry to trehalose metabolism. Consistent with this, perturbation of trehalose synthesis modulated hexose uptake, revealing that phosphorylation capacity is a major driver of sugar import, while metabolic feedback further constrains uptake. Nuclear localization of sugar kinases and condition-dependent expression patterns indicate additional regulatory layers. Together, our results demonstrate that N. glabratus controls glycolytic entry through a multilayered architecture integrating enzyme specialization, transcriptional tuning, trehalose-6-phosphate-mediated feedback and uptake coupling. This systems-level organization results in robust growth across fluctuating and often sugar-limited host environments.