Ganoderma lucidum is widely recognized as a medicinal basidiomycete. It was previously reported that the plant hormone methyl jasmonate (MeJA) could induce the biosynthesis of ganoderic acids (GAs), which are the main active ingredients of G. lucidum. However, the regulatory mechanism is still unclear. In this study, integrated proteomics and metabolomics were employed on G. lucidum to globally identify differences in proteins and metabolites under MeJA treatment for 15 min (M15) and 24 h (M24). Our study successfully identified 209 differential abundance proteins (DAPs) in M15 and 202 DAPs in M24. We also identified 154 metabolites by GC–MS and 70 metabolites by LC–MS in M24 that are involved in several metabolic pathways. With an in-depth analysis, we found some DAPs and metabolites that are involved in the oxidoreduction process, secondary metabolism, energy metabolism, transcriptional and translational regulation, and protein synthesis. In particular, our results reveal that MeJA treatment leads to metabolic rearrangement that inhibited the normal glucose metabolism, energy supply, and protein synthesis of cells but promoted secondary metabolites, including GAs. In conclusion, our proteomics and metabolomics data further confirm the promoting effect of MeJA on the biosynthesis of GAs in G. lucidum and will provide a valuable resource for further investigation of the molecular mechanisms of MeJA signal response and GA biosynthesis in G. lucidum and other related species.

Flammulina filiformis, previously known as Asian Flammulina velutipes, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these pyrG mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in F. filiformis, which broadens the application of this advanced tool in Basidiomycetes.

Fungi actively alter their metabolic and synthetic pathways to cope with changing environmental conditions. The mitochondrial pyruvate carrier (MPC) plays a vital role in transporting pyruvate into mitochondria and regulating carbon metabolic homeostasis. However, the mechanism by which MPC regulates carbon metabolism in response to environmental stress in fungi remains unclear. In this study, yeast one-hybrid library screening assays were employed to screen for transcription factors in Ganoderma lucidum that may interact with GlMPC. General control non-derepressible 4 (GCN4), a key transcription factor responding to nitrogen metabolism, was found to interact with the GlMPC promoter region. This interaction was further confirmed by electrophoretic mobility shift assays and chromatin immunoprecipitation quantitative PCR assays, demonstrating that GCN4 is capable of binding to the GlMPC promoter region in vitro and in vivo . Further results indicated that GCN4 could activate GlMPC expression and participate in regulating the tricarboxylic acid (TCA) cycle and ganoderic acid (GA) biosynthesis under low nitrogen conditions in G. lucidum . These findings revealed a specific regulatory mechanism by which GCN4-activated GlMPC regulates the TCA cycle and secondary metabolism under nitrogen limitation conditions in G. lucidum , providing crucial insights into the regulation of carbon and nitrogen metabolism in fungi. IMPORTANCE: Mitochondrial pyruvate carrier (MPC) is a pyruvate transporter that plays a crucial role in regulating the carbon metabolic flow and is considered an essential mechanism for microorganisms to adapt to environmental changes. However, it remains unclear how MPC responds to environmental stress in organisms. General control non-derepressible 4 (GCN4), a key regulator of nitrogen metabolism, plays a pivotal role in the growth and development of fungi. In this study, we report that GCN4 can directly bind to the promoter region and activate the expression of GlMPC, thereby regulating the tricarboxylic acid cycle and secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum . These findings provide significant insights into the regulation of carbon and nitrogen metabolism in fungi, highlighting the critical role of GCN4 in coordinating metabolic adaptation to environmental stresses.