The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

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Photodynamic inactivation (PDI) has received increasing attention as a promising approach to combat Candida albicans infections. This study aimed to evaluate the synergistic effect of a new BODIPY (4,4-difluoro-boradiazaindacene) derivative and hydrogen peroxide on C. albicans. BDP-4L in combination with H 2 O 2 demonstrated enhanced photokilling efficacy. In suspended cultures of C. albicans, the maximum decrease was 6.20 log and 2.56 log for PDI using BDP-4L (2.5 μM) with or without H 2 O 2 , respectively. For mature C. albicans biofilms, 20 μM BDP-4L plus H 2 O 2 eradicated C. albicans, causing an over 6.7 log count reduction in biofilm-associated cells, while only a reduction of ~ 1 log count was observed when H 2 O 2 was omitted. Scanning electron microscopy analysis and LIVE/DEAD assays suggested that PDI using BDP-4L plus H 2 O 2 induced more damage to the cell membrane. Correspondingly, amplification of nucleic acids release was observed in biofilms treated with the combined PDI. Additionally, we also discovered that the addition of hydrogen peroxide potentiated the generation of 1 O 2 in PDI using the singlet oxygen sensor green probe. Collectively, BDP-4L combined with H 2 O 2 presents a promising approach in the treatment of C. albicans infections.