红色糖多孢菌SACE_2701-2702转运系统的功能表征及对红霉素合成的影响

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

摘要:

对抗生素生产菌而言，提高产物外排可以缓解产物对于生产菌的毒性，同时也是提高抗生素产量的重要工程策略。本研究旨在阐明红色糖多孢菌中红霉素外排转运系统的分子机制，并探索通过强化外排能力提升红霉素产量的工程改造策略。以红色糖多孢菌(Saccharopolyspora erythraea)中负责红霉素外排的ABC转运系统SACE_2701-2702为研究对象，对SACE_2701-2702蛋白的序列结构分析表明，此系统为典型的I型ABC转运系统，跨膜蛋白SACE_2702多富集在红色糖多孢菌的膜蛋白组分中。在红霉素低产野生型菌株S. erythraea NRRL23338及高产工业菌株Ser0中分别对该系统进行过表达，获得重组菌株Se-2702与S0-2702。摇瓶发酵7 d后，Se-2702红霉素效价为31.2 mg/L，较对照菌株提升168.0%，外排比例提高24.4%；S0-2702红霉素效价达926.9 mg/L，较对照菌株提升15.6%。外排系统的过表达显著增强了低产菌株对于高浓度红霉素的耐受能力，赋予了工程菌株在发酵后期的生长优势。本研究证明了定向增强SACE_2701-2702介导的红霉素主动外排可有效解除产物对于生产菌的毒性胁迫，且在不同的菌株遗传背景下均可实现红霉素产量的提升，为红霉素生产菌株的理性代谢工程改造提供了通用策略。

Abstract:

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.