A fungal immunomodulatory protein isolated from Flammulina velutipes (FIP-fve) has structural similarity to the variable region of the immunoglobulin heavy chain. In the present study, the recombinant bioactive FIP-fve protein with a His-tag in N-terminal of recombinant protein was expressed in transetta (DE3) at a high level under the optimized culturing conditions of 0.2 mM IPTG and 28 °C. The efficiency of the purification was improved with additional ultrasonication to the process of lysozyme lysis. The yield of the bioactive FIP-fve protein with 97.1% purity reached 29.1 mg/L with a large quantity for industrial applications. Enzyme-linked immunosorbent assay showed a maximum increase in interleukin-2 (IL-2) and gamma interferon (IFN-γ) for the mice serum group of 5 mg/kg body mass (p < 0.01) with three doses of His-FIP-fve. However, the production of IL-4 had no apparent difference compared to the control.

High-temperature stress is a key factor that reduces the yields of edible fungi. Auricularia heimuer (A. heimuer) is a nutrient-rich edible fungus that is widely cultivated in China. In this study, we analyzed the physiological, transcriptomic, and metabolomic results of A. heimuer (variety “Hei29”) under high-temperature stress. Our findings revealed that high temperatures (30 °C and 35 °C) significantly reduced hyphal growth, increased malondialdehyde content and antioxidant enzyme activity, and enhanced the accumulation of secondary metabolites, such as phenolic compounds and flavonoids. A total of 15 candidate genes potentially responsive to high-temperature stress were identified through transcriptomic analysis, including those involved in regulating antioxidant defense, heat shock response, sugar metabolism, amino acid metabolism, and accumulating secondary metabolites. Metabolomic analysis identified three candidate metabolites potentially responsive to high-temperature stress, including kinetin, flavonoids, and caffeic acid, as well as several metabolic pathways, including nucleotide metabolism, ABC transporters, and cofactor biosynthesis. These mechanisms help mitigate oxidative damage to cellular structures and energy deficits caused by elevated temperatures, enabling the fungus to maintain cellular stability, metabolic function, and growth under heat stress. This study is the first to explore the molecular mechanism of A. heimuer in response to high-temperature stress. The results provide valuable insights into the molecular mechanisms of heat stress tolerance in A. heimuer, highlighting potential targets for developing heat-tolerant strains for industrial application.

Auricularia heimuer is a valuable traditional Chinese fungus used as food and medicine. Hei29 is a strain derived from wild A. heimuer through systematic domestication and selection. It has been the primary A. heimuer variety in Northeast China for 30 years and offers the advantages of high yield, good commercial property, and stable traits. This study used protoplast nucleation on Hei29 to produce two amiable and paired monokaryons, Hei29-D1 and Hei29-D2. The genome of Hei29 was sequenced utilizing the Illumina PE150 and PacBio Sequel sequencing platforms. Hei29-D1 and Hei29-D2 had genomic sizes of 47.54 Mb and 47.49 Mb, GC contents of 56.95% and 56.99%, and an N50 of 2.37 Mb and 4.28 Mb, respectively. Hei29’s genome possessed two phytoene synthase (PSY) protein genes, one of which—PSY encoded by g894—has a transmembrane domain. The phylogenetic tree showed that Hei29 shared the closest evolutionary relationship with Auricularia subglabra TFB-10046 SS5. Collinearity analysis showed that the correlation between the two monokaryons was as high as 90.81%. Cluster analysis revealed that Hei29 contains 12,362 core genes, 223 unique genes in Hei29-D1, and 228 unique genes in Hei29-D2. This study is the first to sequence two related and paired monokaryons from A. heimuer, which is critical for fully understanding the genetic composition and information of the characteristic strain of A. heimuer in Northeast China. It establishes the data and theoretical foundation for gene mining, usage, and molecular breeding. It further promotes the genetic breeding and active substance utilization of A. heimuer.

In this study, the molecular mechanism underlying melanin synthesis within the fermentation broth of Auricularia heimuer (A. heimuer) was explored. The absorbance at 500 nm, melanin yield, and tyrosinase activity in the fermentation broth were assessed across different fermentation durations. Mycelia samples were gathered on the 4th (R1), 8th (R2), and 10th (R3) days of liquid fermentation for transcriptome sequencing. As fermentation progressed, the absorbance, melanin yield, and tyrosinase activity in the broth rose. A total of 5915 differentially expressed genes (DEGs) were detected, with 1136 DEGs between R2 and R1, 3717 between R3 and R1, and 2950 between R3 and R2. Gene Ontology (GO) analysis showed that DEGs were significantly enriched in oxidoreductase activity and ribosome structural constituent terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant enrichment of DEGs in ribosome and amino acid metabolism pathways. Key DEGs, including transcription factors, glycosidases, P450 enzymes, laccases, and glutamate dehydrogenase, were identified during melanin production in the fermentation broth. These DEGs may play crucial roles in melanin synthesis. This study offers a foundation for further exploring the melanin synthesis mechanism in A. heimuer fermentation broth.

The molecular mechanisms of mycelial degeneration during subculturing of Auricularia heimuer strain ‘HWS1908’ were investigated across generations G1 to G20. With successive subculturing, mycelial growth rate and compactness declined, cellulase and laccase activities decreased significantly, whereas antioxidant enzyme activities increased. Comparative transcriptome analysis between G1 and G20 identified 2643 differentially expressed genes (DEGs). Gene Ontology (GO) analysis indicated that the DEGs were significantly enriched in terms associated with protein refolding, response to reactive oxygen species, and ferroxidase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant enrichment of DEGs in pathways including phenylpropanoid biosynthesis, peroxisome, fatty acid degradation, and longevity-regulating pathways. Key DEGs, including transcription factors, glycoside hydrolases, lignin-modifying enzymes, chitin synthases, chitinases, ornithine decarboxylase, and Ras/Rho signaling pathway components (Sos, Ras, Rac1), were identified. These genes may be associated with the progressive decline of mycelial vigor, cell wall integrity, and substrate utilization capacity. These findings provide a basis for further exploration of the molecular mechanisms of strain degeneration in A. heimuer, and a practical recommendation to limit subcultures to within 20 generations for maintaining high vitality.