Integrated Transcriptomic and Metabolomic Analyses Reveal Acyl-CoA Dehydrogenase-Mediated Primordium Formation and Metabolite Synthesis in Cordyceps militaris

Cordyceps militaris is an edible entomopathogenic fungus with substantial nutritional and medicinal value. However, the molecular mechanisms underlying the vegetative mycelia-to-primordia (PR) transition and bioactive metabolite synthesis are still poorly defined. Deletion of the acyl-CoA dehydrogenase gene (ACAD) caused malformed, stunted PR-like structures together with excessive aerial mycelial growth, severely disrupting PR initiation and fruiting-body elongation in C. militaris. Transcriptomic, untargeted metabolomic and multi-omics analyses were performed on wild-type (WT) and ACAD-knockout strains at the mycelial and PR stages to explore gene expression reprogramming and bioactive nucleoside accumulation during the developmental transition and under ACAD deficiency. In total, 2014, 448, and 838 differentially expressed genes (DEGs) were identified in WT PR vs. WT mycelia, ΔACAD mycelia vs. WT mycelia, and ΔACAD PR vs. WT PR, demonstrating extensive transcriptional reprogramming during the development switch and upon ACAD deletion. KEGG enrichment and WGCNA analyses showed that ACAD deficiency significantly suppresses multiple reproductive-development pathways, including cell cycle progression, purine biosynthesis, the TCA cycle, MAPK signaling, redox homeostasis, fatty acid and amino acid metabolism. Consistently, ACAD deletion markedly increased intracellular ROS level and substantially reduced cellular ATP levels in mycelia. Several crucial genes modulated by ACAD were identified, including GTP cyclohydrolase I, histone lysine N-methyltransferase ASHR1, adenine phosphoribosyl transferase 1, glutathione-S-transferase F3 and CoA-ligase CCL8. Multi-omics integration demonstrated tightly coordinated transcriptional and metabolic regulation dependent on ACAD function. ACAD deficiency significantly disturbed amino acid, lipid, and nucleotide metabolism and reduced the abundance of amino acid derivatives, lipid compounds and purine nucleoside precursors in mycelia. Particularly, adenosine analogs serve as key precursors of the core bioactive nucleoside cordycepin. Key DEGs, including peroxisomal (S)-2-hydroxyacid oxidase, isocitrate lyase 2 and L-ascorbate peroxidase 5, were linked to these metabolic shifts. Our findings demonstrate that ACAD serves as an essential regulator that interconnects carbon metabolism, redox homeostasis, lipid, amino acid and purine metabolism to promote PR formation and secondary metabolite synthesis in C. militaris.