Plecomacrolides, such as concanamycin and bafilomycin, are potent and specific inhibitors of vacuolar-type ATPase. Concanamycins are 18-membered macrolides with promising therapeutic potential against multiple diseases, including viral infection, osteoporosis, and cancer. Due to the complexity of their total synthesis, the production of concanamycins is only achieved through microbial fermentation. However, the low titers of concanamycin A and its analogs in the native producing strains are a significant bottleneck for scale-up, robust structure-activity relationship studies, and drug development. To address this challenge, we designed a library of engineered Streptomyces strains for the overproduction of concanamycins by combining the overexpression of target regulatory genes with the optimization of fermentation media. Integration of two endogenous regulators from the concanamycin biosynthetic gene cluster (cms) and one heterologous regulatory gene from the bafilomycin biosynthetic gene cluster into the attB site significantly increased production of concanamycin A and its low abundant analog concanamycin B in Streptomyces eitanensis. The highest titers reported to date were observed in the engineered S. eitanensis DHS10676, which produced over 900 mg/L of concanamycin A and 300 mg/L of concanamycin B. Heterologous overexpression of the identified target regulatory genes across a panel of Streptomyces spp., harboring a putative concanamycin biosynthetic gene cluster confirmed its identity, and significantly improved concanamycin A production in all tested strains. Strain engineering, optimization of fermentation, and extraction purification protocols enabled swift access to these structurally complex plecomacrolides for semi-synthetic medicinal chemistry-based approaches. Together, this work established a platform for robust overproduction of concanamycin analogs across species.

中文翻译：

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使用合理的代谢工程策略优化伴刀豆霉素的生产。


多倍大环内酯类药物，如康卡霉素和巴弗洛霉素，是空泡型 ATP 酶的有效和特异性抑制剂。伴刀豆霉素是 18 元大环内酯类药物，对多种疾病（包括病毒感染、骨质疏松症和癌症）具有很好的治疗潜力。由于它们总合成的复杂性，伴刀豆霉素的生产只能通过微生物发酵来实现。然而，康卡霉素 A 及其类似物在天然生产菌株中的低滴度是放大、稳健的构效关系研究和药物开发的重要瓶颈。为了应对这一挑战，我们通过将靶标调节基因的过表达与发酵培养基的优化相结合，设计了一个工程化链霉菌菌株库，用于伴刀豆霉素的过量生产。将来自伴刀菌素生物合成基因簇 （cms） 的两个内源调节因子和来自巴弗洛霉素生物合成基因簇的一个异源调节基因整合到 attB 位点，显著增加了链霉菌中伴刀菌素 A 及其低丰度类似物伴刀霉素 B 的产生。迄今为止报道的最高滴度是在工程化的 S. eitanensis DHS10676中观察到的，其产生超过 900 mg/L 的康卡霉素 A 和 300 mg/L 的康卡霉素 B。在一组链霉菌属中鉴定的靶标调节基因异源过表达，含有假定的康卡霉素生物合成基因簇，证实了其身份，并显着改善了所有测试菌株中锥刀菌素 A 的产生。 菌株工程、发酵优化和提取纯化方案使这些结构复杂的多大环内酯类药物能够快速用于基于半合成药物化学的方法。总之，这项工作为跨物种康卡霉素类似物的稳健超生产建立了一个平台。