Metabolomic Interrogation of Substrate-Driven Carbon/Nitrogen Flux Governing Nutrient Biosynthesis and Antioxidant Capacity in Phallus rubrovolvatus Mycelia
The mycelium of Phallus rubrovolvatus contains abundant metabolites with broad application prospects in functional foods and pharmaceuticals. However, how carbon and nitrogen substrate availability regulate the accumulation dynamics of mycelial metabolites remains under-explored. In this study, the supply levels of carbon (glucose) and nitrogen (peptone) were optimized to select high-biomass mycelia. Integrating nutritional activity assays with metabolomics and redundancy analysis, the regulatory mechanisms governing carbon–nitrogen metabolic flux were deciphered. The results demonstrated that mycelial nutritional content and antioxidant capacity exhibited a progressive upward trend under three distinct modes: carbon-driven, nitrogen-driven, and carbon–nitrogen synergistic-driven regimes. In the optimal carbon–nitrogen synergistic-driven group, the contents of total soluble sugars, reducing sugars, flavonoids, total phenolics, and soluble proteins increased by 72.06%, 160.80%, 52.47%, 113.69%, and 8.21%, respectively, compared with the control group. Meanwhile, the scavenging rates of superoxide anion, hydroxyl, ABTS, and DPPH free radicals increased by 14.12%, 24.09%, 14.77%, and 66.47%, respectively, compared with the control group. Differentially accumulated metabolites were significantly enriched in amino acid metabolism, energy metabolism, and secondary metabolite biosynthesis pathways. Carbon and nitrogen substrates reshaped intracellular metabolic flux, cooperatively regulating nutrient synthesis and intracellular redox equilibrium. This work reveals a cascade-linking relationship in which nutrient supply triggers metabolic remodeling, regulates oxidative balance, and drives pathway response. It provides theoretical support for the precision fermentation and industrial upgrade of P. rubrovolvatus, while offering a valuable reference paradigm for the high-value exploitation of other rare edible and medicinal fungi.