Ergosterol synthase reduces ochratoxin A synthesis in Aspergillus carbonarius via oxidative stress pathway

Ochratoxin A (OTA), a carcinogenic mycotoxin produced by Aspergillus and Penicillium species that contaminates food crops and threatens public health. Although ergosterol and its synthetic enzymes are important antifungal targets, their regulatory roles and mechanisms in OTA production remain unclear. Therefore, elucidating the roles of ergosterol synthase genes erg3 (C-5 sterol desaturase) and erg24 (C-14 sterol reductase) in oxidative stress response and OTA biosynthesis in Aspergillus carbonarius is of critical importance. Herein, we employed homologous recombination to knockout and overexpress ergosterol synthase gene erg3 and erg24 in A. carbonarius. We identified two homologous erg3 (erg3-1 and erg3-5) and one erg24 in A. carbonarius. Δerg24 significantly reduced ergosterol levels whereas Δerg3-1 markedly increased it. Notably, only mutant with erg24 knockout, including single (Δerg24), double (Δerg3-5Δerg24) and triple knockout (Δerg3-1Δerg3-5Δerg24) strains showed significantly reduced OTA production, colony diameter, conidial formation and germination rates. Furthermore, transcriptomic analysis revealed that Δerg24 significantly downregulated expression of OTA biosynthetic genes pks and hal, while genes associated with antioxidant defense mechanisms regulating ROS levels were upregulated. Enzyme assay confirmed that catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) were enhanced, accompanied by reduced ROS levels. This study provides new insights into the regulatory role of ergosterol synthase in OTA synthesis and potential targets for developing innovative antifungal strategies.