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Proteins from Flammulina filiformis were prepared by sodium chloride extraction and fractionated by ammonium sulfate precipitation with increasing saturation degrees to obtain the protein fractions Ffsp-30, Ffsp-50, Ffsp-70, Ffsp-90, and Ffp-90. Among these protein fractions, Ffsp-50 possessed the most significant cytotoxic effect against three human gastrointestinal cancer cell lines, viz. HT-29, SGC-7901, and HepG2. SDS-PAGE and MALDI-TOF/TOF MS/MS analyses revealed that flammutoxin (FTX) was present as a dominating protein in Ffsp-50, which was further evidenced by HPLC-MS/MS determination. Furthermore, native FTX was purified from Ffsp-50 with a molecular weight of 26.78 kDa, exhibiting notable cytotoxicity against gastrointestinal cancer cell lines. Both Ffsp-50 and FTX exposure could enhance intercellular reactive oxygen species (ROS) generation and induce significant apoptosis in HepG2 cells. FTX was identified to be relatively conserved in basidiomycetes according to phylogenetic analysis, and its expression was highly upregulated in the primordium as well as the pileus of the fruiting body from the elongation and maturation stages, as compared with that in mycelium. Taken together, FTX could remarkably inhibit cell growth and induce ROS and apoptosis in HepG2 cells, potentially participating in the growth and development of the fruiting body. These findings from our investigation provided insight into the antigastrointestinal cancer activity of FTX, which could serve as a biological source of health-promoting and biomedical applications. [ABSTRACT FROM AUTHOR]

Background: A high concentration of CO2 will stagnate the development of the newly formed primordia of Hypsizygus marmoreus, hinder the development of the mushroom cap, thereby inhibiting the normal differentiation of the fruiting body. Moreover, in the previous experiment, our research group obtained the mutant strain HY68 of H. marmoreus, which can maintain normal fruiting under the condition of high concentration of CO2. Our study aimed to evaluate the CO2 tolerance ability of the mutant strain HY68, in comparison with the starting strain HY61 and the control strain HY62. We analyzed the mycelial growth of these strains under various conditions, including different temperatures, pH levels, carbon sources, and nitrogen sources, and measured the activity of the cellulose enzyme. Additionally, we identified and predicted β-glucosidase-related genes in HY68 and analyzed their gene and protein structures. Results: Our results indicate that HY68 showed superior CO2 tolerance compared to the other strains tested, with an optimal growth temperature of 25 °C and pH of 7, and maltose and beef paste as the ideal carbon and nitrogen sources, respectively. Enzyme activity assays revealed a positive correlation between β-glucosidase activity and CO2 tolerance, with Gene14147 identified as the most closely related gene to this activity. Inbred strains of HY68 showed trait segregation for CO2 tolerance. Conclusions: Both HY68 and its self-bred offspring could tolerate CO2 stress. The fruiting period of the strains resistant to CO2 stress was shorter than that of the strains not tolerant to CO2 stress. The activity of β-GC and the ability to tolerate CO2 were more closely related to the growth efficiency of fruiting bodies. This study lays the foundation for understanding how CO2 regulates the growth of edible fungi, which is conducive to the innovation of edible fungus breeding methods. The application of the new strain HY68 is beneficial to the research of energy-saving production in factory cultivation. [ABSTRACT FROM AUTHOR]

β-1, 6-glucan synthase is a key enzyme of β-1, 6-glucan synthesis, which plays a vital role in the cell wall cross-linking of fungi. However, the role of the β-1, 6-glucan synthase gene in the development of the fruiting body and the stress response of macrofungi is largely unknown. In this study, four overexpression transformants of the β-1, 6-glucan synthase gene (FfGS6) were successfully obtained, and gene function was studied in Flammulina filiformis. The overexpression of FfGS6 can increase the width of mycelium cells and improve the tolerance ability under mechanical injury and oxidative stress. Moreover, FfGS6 gene expression fluctuated in up-regulation during the recovery process of mycelium injury but showed a negative correlation with H2O2 concentration. Fruiting body phenotype tests showed that mycelia's recovery ability after scratching improved when the FfGS6 gene was overexpressed. However, primordia formation and the stipe elongation ability were significantly inhibited. Our findings indicate that FfGS6 is involved in regulating mycelial cell morphology, the mycelial stress response, and fruit body development in F. filiformis. [ABSTRACT FROM AUTHOR]