Continuous carbon source supply is essential for high rifamycin productivity of Amycolatopsis mediterranei in nitrate-stimulated fermentation revealed by a metabolomic study

• Nitrate supplementation in Amycolatopsis mediterranei U32 fermentation significantly depletes key central carbon metabolites, including glucose-6-phosphate, UDP-glucose, and acetyl-CoA, indicating a carbon source limitation. • Glucose supplementation during the logarithmic growth phase markedly enhances rifamycin SV production, with yields increasing up to 354.3% when added at multiple time points in mid- and late-exponential phases. • Metabolomic and qPCR analyses confirm that glucose replenishment compensates for carbon deficiency and upregulates genes involved in the AHBA synthesis pathway, a key precursor for rifamycin. • This study provides the first systematic metabolomic insight into the nitrate-stimulating effect (NSE), linking carbon metabolism to antibiotic productivity and offering a practical strategy for industrial fermentation optimization.