Reprogramming Enfumafungin Biosynthesis for High-Titer Production in the Endophytic Fungus Hormonema carpetanum ATCC 74360

Enfumafungin is the essential precursor for the FDA-approved antifungal agent Ibrexafungerp, yet its production is hindered by low yields, restrictive solid-state fermentation, and the genetic intractability of its producer strain, Hormonema carpetanum ATCC 74360. Here, we developed a CRISPR/Cas9-mediated platform that halved the genetic iteration cycle to less than 15 days. By rewiring the biosynthetic cluster through promoter engineering, we successfully transitioned production to scalable liquid fermentation, achieving an initial 21.6 mg/L. Then, the mevalonate pathway genes (tHMGR, ERG9, and ERG1) were overexpressed, which successfully boosted the titer to 250.1 mg/L. Following medium optimization, the engineered strain HC07 reached a record-breaking 592.4 mg/L in 250 mL shake flasks, the highest yield achieved via metabolic engineering to date. This study provides a robust microbial platform and a streamlined genetic toolbox, paving the way for the sustainable biomanufacturing of next-generation triterpenoid antifungals.