Propanol had been widely used as a precursor for erythromycin synthesis in industrial production. However, the knowledge on the exact metabolic fate of propanol was still unclear. In the present study, the metabolic fate of propanol in industrial erythromycin-producing strain Saccharopolyspora erythraea E3 was explored via C-13 labeling experiments. An unexpected pathway in which propanol was channeled into tricarboxylic acid cycle was uncovered, resulting in uneconomic catabolism of propanol. By deleting the sucC gene, which encodes succinyl-CoA synthetase that catalyse a reaction in the unexpected propanol utilization pathway, a novel strain E3-Delta sucC was constructed. The strain E3-Delta sucC showed a significant enhancement in erythromycin production in the chemically defined medium compared to E3 (786.61 vs 392.94 mg/L). Isotopically nonstationary C-13 metabolic flux analysis were employed to characterize the metabolic differences between Saccharopolyspora erythraea E3 and E3-Delta sucC. The results showed that compared with the starting strain E3, the fluxes of pentose phosphate pathway in E3-Delta sucC increased by almost 200%. The flux of the metabolic reaction catalyzed by succinyl-CoA synthetase in E3-Delta sucC was almost zero, while the glyoxylate bypass flux significantly increased. These new insights into the precursor utilization of antibiotic biosynthesis by rational metabolic engineering in Saccharopolyspora erythraea provided the new vision in increasing industrial production of secondary metabolites. (C) 2021 Elsevier Inc. All rights reserved.