Abstract: In order to investigate the effect of the addition of ammonium sulfate on the physiological metabolism of Saccharopolyspora erythraea,the effects of ammonium sulfate on dry cell weight,erythromycin titer,specific glucose consumption rate,specific erythromycin synthesis rate,and other apparent physiological parameters were first investigated. Furthermore,¹³C-assisted quantitative metabolomics was used to explore its regulation mechanism,and the changes of intracellular organic acids levels and amino acids levels after ammonium sulfate addition were compared. The results showed that the addition of ammonium sulfate to the medium in the late stage of erythromycin fermentation increased the specific erythromycin synthesis rate and specific glucose consumption rate by 30.0% and 47.2% respectively,indicating that the metabolic activity of the stains was improved. Compared with the control group,the erythromycin titer increased from 830 μg/mL to 1126 μg/mL,the proportion of erythromycin A component increased from 802.9 μg/mL to 956.1 μg/mL,the conversion rate of erythromycin C reached 99.2% at 96 h. Quantitative metabolomics results showed that the levels of intracellular organic acids were significantly different after the addition of ammonium sulfate,and the decrease in the concentration of α-ketoglutarate(4.15 μmol/g DCW to 1.05 μmol/g DCW)was conducive to the maintenance of cell growth. Intracellular amino acids of cell synthesis precursors and erythromycin synthesis precursor were improved,for instance,the concentration of proline,serine,aspartic acid,isoleucine was increased by 60.4%,90.0%,204.2%,106.7%,respectively. It greatly deepened the understanding of the metabolic characteristics of erythromycin industrial production strains by using quantitative metabolomics to study the effect of nitrogen sources on intracellular central carbon metabolism during the fermentation of erythromycin,and provided certain guiding significance for the subsequent industrial production.