Mengmeng Xu

Extracellular vesicles (EVs) from the edible mushroom Pleurotus tuber-regium (PTR) were investigated with respect to their environmental responsiveness, molecular features, and preliminary functional properties. PTR-EVs were characterized by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. Proteomic analysis revealed enrichment of ribosomal and proteasomal proteins, redox-related enzymes, and vesicle trafficking components, suggesting non-random molecular representation. Small RNA sequencing identified abundant novel miRNAs with predicted targets involved in nitrogen metabolism, cell wall remodeling, redox regulation, and ubiquitin-mediated proteolysis. Among the tested factors, temperature showed the strongest association with vesicle production, with particle concentration increasing from 1.22 × 10⁹ to 7.31 × 10⁹ particles/mL at 34 °C, approximately six-fold higher than at 30 °C. Transcriptomic profiling showed coordinated repression of cell wall-associated genes and redox enzymes, together with induction of endoplasmic reticulum proteostasis pathways, consistent with stress-associated changes in the cellular context of vesicle release. Ultrasonicated PTR-EVs exhibited enhanced DPPH and ABTS radical-scavenging activities in chemical assays, with DPPH increasing from 59.52% to 71.73% and ABTS from 38.25% to 40.51%. Encapsulation efficiencies reached 32.67% ± 1.3% for proanthocyanidins and 46.01% ± 0.5% for curcumin. PTR-EVs showed the best short-term stability at pH 7 and 4 °C, supporting their further evaluation as an edible fungal vesicle platform for food-related nanoscale delivery.

Ganoderma lucidum polysaccharides (GLPs) are natural compounds with a broad spectrum of biological activities. β-1,3-glucosyltransferase (GL20535) plays an important role in polysaccharide synthesis by catalyzing the transfer of UDP-glucose to extend sugar chains, but its underlying mechanism remains unclear. In this study, the regulatory mechanism of GL20535 in polysaccharide synthesis was elucidated by overexpressing and silencing gl20535 in G. lucidum. Overexpression of gl20535 resulted in maximum increases of 18.08%, 79.04%, and 18.01% in intracellular polysaccharide (IPS), extracellular polysaccharide (EPS), and β-1,3-glucan contents, respectively. In contrast, silencing gl20535 resulted in maximum reductions of 16.97%, 30.20%, and 23.56% in IPS, EPS, and β-1,3-glucan contents, respectively. These phenomena in the overexpression strains were attributed to gl20535-mediated promotion of UDP-glucose synthesis in the sugar donor pathway and upregulation of the expression of glycoside hydrolase genes. The opposite trend was observed in the silenced strains. In mycelial growth studies, neither overexpression nor silencing of gl20535 affected biomass and cell wall thickness. Furthermore, the GL20535 isozyme gene gl24465 remained unaffected in gl20535-overexpressed strains but was upregulated in gl20535-silenced strains, suggesting a compensatory regulatory relationship. These findings reveal the regulatory role of GL20535 on gene expression in the GLPs synthesis pathway and deepen our understanding of GL20535 function in the polysaccharide network of edible and medicinal fungi.

Background: Selenium (Se) pollution poses serious threats to terrestrial ecosystems. Mushrooms are important sources of Se with the potential for bioremediation. Pre-eminent Se resources must possess the ability to tolerate high levels of Se. To obtain Se-accumulating fungi, we isolated selenite-tolerance-enhanced Ganoderma lucidum JNUSE-200 through adaptive evolution. Methods: The molecular mechanism responsible for selenite tolerance and accumulation was explored in G. lucidum JNUSE-200 by comparing it with the original strain, G. lucidum CGMCC 5.26, using a combination of physiological and transcriptomic approaches. Results: G. lucidum JNUSE-200 demonstrated tolerance to 200 mg/kg selenite in liquid culture and exhibited normal growth, whereas G. lucidum CGMCC 5.26 experienced reduced growth, red coloration, and an unpleasant odor as a result of exposure to selenite at the same concentration. In this study, G. lucidum JNUSE-200 developed a triple defense mechanism against high-level selenite toxicity, and the key genes responsible for improved selenite tolerance were identified. Conclusions: The present study offers novel insights into the molecular responses of fungi towards selenite, providing theoretical guidance for the breeding and cultivation of Se-accumulating varieties. Moreover, it significantly enhances the capacity of the bio-manufacturing industry and contributes to the development of beneficial applications in environmental biotechnology through fungal selenite transformation bioprocesses.

Polysaccharides are crucial dietary supplements and traditional pharmacological components of Ganoderma lucidum; however, the mechanisms responsible for high polysaccharide yields in G. lucidum remain unclear. Therefore, we investigated the mechanisms underlying the high yield of polysaccharides in submerged cultures of G. lucidum using transcriptomic and proteomic analyses. Several glycoside hydrolase (GH) genes and proteins, which are associated with the degradation of fungal cell walls, were significantly upregulated under high polysaccharide yield conditions. They mainly belonged to the GH3, GH5, GH16, GH17, GH18, GH55, GH79, GH128, GH152, and GH154 families. Additionally, the results suggested that the cell wall polysaccharide could be degraded by GHs, which is beneficial for extracting more intracellular polysaccharides from cultured mycelia. Furthermore, some of the degraded polysaccharides were released into the culture broth, which is beneficial for obtaining more extracellular polysaccharides. Our findings provide new insights into the mechanisms underlying the roles that GH family genes play to regulate high polysaccharide yields in G. lucidum.

Selenium biofortification of edible and medicinal mushrooms is an effective way to produce selenium-enriched food supplements. Ganoderma lucidum is the typical one with excellent biological activity. This study investigated G. lucidum growth and bioactive metabolites alterations during liquid culture with different concentrations of selenite. Low selenium levels did not affect growth and mycelia morphology, whereas high selenium levels negatively influenced growth, dramatically decreased biomass, caused nucleic acid and protein leakage, damaged cell walls and membranes, and resulted in indicators such as degraded cells, a red color, and an unpleasant odor. Through headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis, ten volatile Se compounds were identified in G. lucidum with 200 ppm selenite, which led to an odor change, whereas only three with 50 ppm selenite. SeMet was the major selenoamino acid in the 50 ppm selenite group by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS), but more MeSeCys was produced with 200 ppm selenite. Polysaccharide yields were promoted and inhibited with 50 and 200 ppm selenite, respectively. These results provide comprehensive insights into the effects of selenite on G. lucidum in liquid culture and are beneficial for functional selenium-enriched mushroom production and improving nutritive values.