Improving fidaxomicin production through ARTP mutagenesis and fermentation optimization in Actinoplanes deccanensis

Fidaxomicin, a macrolide antibiotic, is widely used to treat Clostridioides difficile infection (CDI). It demonstrats significantly higher clinical efficacy than vancomycin and metronidazole. However, the large-scale industrial production of it remains a significant challenge because of the low fermentation yields. In this study, we chosen the strain OE-R1/WT as the starting strain, in which a pathway-specific positive regulatory factor fadR1 was overexpressed. By using the kanR/gusA dual-reporter system and ARTP mutagenesis, we screened a high-yield strain, PA-13, which produced 757.34 mg/L of fidaxomicin, representing a 5.5-fold increase over OE-R1/WT and having enhanced genetic stability. Furthermore, by overexpressing two methyltransferases within the biosynthetic cluster and supplementing with exogenous DMSO, we further increased the production of fidaxomicin to 929.17 mg/L, while reducing the accumulation of the major by-product to 20.9 %. Finally, through the optimization of fermentation strategies at both the shake flask and 15 L fermenter levels, we achieved a final yield of 3949.05 mg/L in the 15 L fermenter, which represents the highest yield up to date. Our study represents the first successful enhancement of fidaxomicin production in Actinoplanes deccanensis to over 3.9 g/L in a 15 L fermenter, establishing a robust foundation for industrial-scale fermentation. Additionally, it provides significant insights for the development of high-yield strains in other actinomycetes.