Improvement of Enfumafungin Production by Hormonema carpetanum via Mutagenesis and Process Optimization: Mutant Generation and Preliminary Transcriptomic Analysis

Enfumafungin, a triterpenoid antifungal compound, serves as the key starting material for the synthesis of ibrexafungerp and SCY-247. However, its low fermentation yield and high production cost constrain its supply, limiting drug accessibility and the ability to meet clinical demand. Consequently, enhancing enfumafungin biosynthesis is crucial for achieving large-scale production. In this study, physical and chemical mutagenesis treatments were adopted for Hormonema carpetanum ATCC 74360, and a genetically stable high-producing mutant, H. carpetanum ENF-5-29, was generated. This mutant produced 817 mg/L enfumafungin in flasks, a 30-fold increase over that of wild-type ATCC 74360. Comparative transcriptome analysis revealed that the proposed biosynthetic pathway from glucose to enfumafungin presented the most significant upregulation in the mutant. The fermentation medium and conditions were subsequently optimized to further enhance enfumafungin production. Transcriptome analysis revealed improved yields under low-temperature culture, likely due to the upregulated expression of key genes in the mevalonate pathway and the enfumafungin biosynthetic gene cluster (BGC). Together, these transcriptome findings provide molecular insight into enhanced enfumafungin production. Furthermore, batch fermentation of ENF-5-29 in a 5 L bioreactor achieved a yield of 1,621 mg/L, the highest yield reported to date. Our results demonstrate the efficacy of mutagenesis for H. carpetanum improvement and pinpoint key targets for future genetic engineering aimed at enhancing enfumafungin biosynthesis. Overall design: RNA-seq profiling of wildtype ATCC 74360 and mutant ENF-5-29 at stationary phase of fermentation.