[Functional characteristics of the SACE_2701-2702 transport system in Saccharopolyspora erythraea and its impact on...

For antibiotic-producing microorganisms, enhancing product efflux not only alleviates the toxic effects of antibiotics on the cell factory but also represents a powerful metabolic engineering strategy for increasing yields. This study aims to elucidate the molecular mechanism of the erythromycin efflux transporter system in Saccharopolyspora erythraea, and to explore engineering modification strategies for enhancing erythromycin production by strengthening the efflux capacity. In this study, we targeted the ABC transport system SACE_2701-2702, which is responsible for erythromycin export in Saccharopolyspora erythraea. Structural analysis confirmed that SACE_2701-2702 was a canonical type-I ABC transporter, with the transmembrane protein SACE_2702 being enriched in the membrane fraction. SACE_2701-2702 overexpression in both the low-producing wild-type strain NRRL23338 and the high-yield industrial strain Ser0 generated recombinants Se-2702 and S0-2702, respectively. After seven days of shake-flask fermentation, Se-2702 achieved an erythromycin titer of 31.2 mg/L, which represented a 168.0% increase compared with that of the control strain, with a 24.4% rise in the efflux ratio. Strain S0-2702 produced 926.9 mg/L erythromycin, showing a 15.6% increase. Overexpression of the transport system markedly improved tolerance to elevated erythromycin concentrations and conferred a late-fermentation growth advantage. These results demonstrate that targeted enhancement of SACE_2701-2702-mediated erythromycin efflux can effectively relieve product toxicity across different genetic backgrounds and consistently boosts erythromycin production, providing a general strategy for the rational metabolic engineering of erythromycin-producing strains.