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Micafungin, an echinocandin antifungal agent, has been extensively utilized for the treatment of invasive fungal infections. It is semi-synthesized from the lipohexapeptide FR901379, a natural product from the filamentous fungus Coleophoma empetri. The genetic manipulation of C. empetri has been significantly hindered by the scarcity of both effective neutral sites and selectable markers, which has impeded the systematic metabolic engineering of the industrial strain. Here, a visualizable and marker-free gene editing platform was developed in the industrial strain C. empetri MEFC09. Firstly, the biosynthesis gene cluster of melanin in C. empetri MEFC09 was characterized by gene knockoutin vivo, which comprises a core polyketide synthase gene cemelA and a transcriptional regulator cemelR. Based on this, two neutral sites were designed at the genomic loci of cemelA and cemelR, both demonstrating high-intensity expression of the luciferase reporter. Furthermore, a CRISPR/Cas9-mediated marker-free gene editing platform was developed. When the protoplast concentration was adjusted to 104 cells/mL, a positive transformation rate of 12.4% was achieved on antibiotic-free screening plates. Finally, the transcriptional activator McfJ was overexpressed at the neutral site cemelR using the visualizable and marker-free gene editing platform. It substantially increased the FR901379 titer to 1807.8 mg/L, corresponding to a 3.3-fold improvement over the parental strain. This genetic manipulation system is poised to serve as a versatile platform for accelerating metabolic engineering and functional genomics investigations in industrial strains C. empetri. It will also shed light on the development of genetic manipulation platforms in other filamentous fungi. • Two visualizable neutral sites were characterized in Coleophoma empetri. • The CRISPR/Cas9-mediated marker-free gene editing platform was developed. • The transcriptional activator McfJ was efficiently expressed at the neutral site.
