The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

The global regulator LaeA plays crucial roles in morphological development and secondary metabolite biosynthesis in filamentous fungi. However, the functions of LaeA in basidiomycetes are less reported. The basidiomycete Pleurotus ostreatus is a well-known fungus used both in medicine and as food that produces polysaccharides and cellulolytic enzymes. In this study, we characterized three LaeA homologs (PoLaeA1, PoLaeA2, and PoLaeA3) in P. ostreatus. PoLaeA1 showed different expression patterns than PoLaeA2 and PoLaeA3 during different developmental stages. Silencing PoLaeA1 decreased the intracellular polysaccharide (IPS) content by approximately 28–30% and reduced intracellular ROS levels compared with those of the WT strain. However, silencing PoLaeA2 and PoLaeA3 decreased cellulase activity by 31–34% and 35–40%, respectively, and reduced the cytosolic Ca²⁺ content, compared with those of the WT strain. Further analysis showed that PoLaeA1 regulated IPS biosynthesis through intracellular ROS levels, whereas PoLaeA2 and PoLaeA3 regulated cellulase activity through intracellular Ca²⁺ signaling. Our results provide new insights into the regulation of polysaccharide biosynthesis and cellulase production in filamentous fungi.

Heat stress (HS) is a crucial environmental challenge for edible fungi. Salicylic acid (SA), a plant defense-related hormone, plays key roles in plant responses to biotic and abiotic stresses.

Heat stress (HS), induced by global climate warming, is one of the major limiting factors in edible fungi production. HS suppresses mycelial growth and fruiting body formation by causing excessive accumulation of intracellular reactive oxygen species (ROS), disrupting the integrity of cell membranes and cell walls, and impairing cellular metabolism. Increasing evidence suggests that the application of exogenous substances (ESs) effectively mitigates HS in edible fungi. Based on the recent literature, this review categorizes ESs into three groups—core signaling molecules, plant growth regulators, and cytoprotective agents—and summarizes their beneficial effects against HS in edible fungi. The underlying mechanisms of ES-mediated alleviation of heat-induced damage primarily involve four pathways: (1) regulation of antioxidant systems; (2) preservation of cell wall and membrane structural integrity; (3) modulation of defense-related gene expression; and (4) regulation of carbon metabolic flux. Current challenges and corresponding strategies are discussed to provide a reference for elucidating the mechanisms by which ESs alleviate HS and to promote their practical application in edible fungi production.

The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The 11α-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the 11α-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The H₂O₂ levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the 11α-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.