Recombinant AnAFP Reveals Genetic Contributors to Antifungal Protein Tolerance and Fungal Development in Aspergillus flavus

ABSTRACT: Aspergillus flavus is an agriculturally important and aflatoxigenic fungus, underscoring the need for alternative antifungal strategies. Cysteine-rich antifungal proteins (AFPs) are promising bioactive molecules, yet their recombinant production and genetic determinants of fungal tolerance remain insufficiently characterized. Here, we investigated AnAFP, an antifungal protein from Aspergillus niger, and evaluated its activity against A. flavus. Bioinformatic analyses predicted an N-terminal signal peptide, a putative intrinsically disordered region, and a mature cysteine-rich domain structurally related to known fungal AFPs. Guided by these features, the predicted mature region of AnAFP was expressed in Escherichia coli and purified through Ni-NTA affinity chromatography, tag cleavage, cation-exchange chromatography, and size-exclusion chromatography. Purified AnAFP inhibited A. flavus growth, and comparison with PgAFP and AfAFP confirmed antifungal activity at micromolar concentrations. To identify genes associated with AFP tolerance, Δado1, Δdef1, and Δadk1 mutants were generated by homologous recombination. All three mutants showed increased sensitivity to AnAFP, PgAFP, and AfAFP relative to the wild-type strain, suggesting that ado1, def1, and adk1 may contribute to AFP tolerance. Deletion of these genes also affected colony growth, conidiation, sclerotial formation, stress responses, and aflatoxin production. These findings establish a recombinant production strategy for AnAFP and provide preliminary evidence linking ado1, def1, and adk1 to AFP sensitivity and fungal physiology more broadly in this pathogenic and aflatoxigenic species.