The antifungal capacity of an 681-membered collection of environmental yeast isolates

Fungal pathogens threaten human health and food security, with resistance reported across limited antifungal classes. Novel strategies to control these pathogens and food spoilers are urgently needed. Environmental yeasts provide a functionally diverse, yet underexploited potential for fungal control based on their natural competition via the secretion of iron siderophores, killer toxins (proteins) or other small molecules like volatile organic compounds or biosurfactants. However, there is a lack of standardized workflows to systematically access application- relevant yeast-based compounds and understand their molecular functioning. Towards this goal, we developed a workflow to identify and characterize yeast isolates that are active against relevant human and plant pathogens and spoilage yeasts, herein focusing on discovering yeasts that produce potential killer toxins. The workflow includes the classification of the secreted molecules and cross-comparison of their antifungal capacity using an independent calibrant. Our workflow delivered a collection of 681 yeasts of which 212 isolates (31%) displayed antagonism against at least one of our target strains. While 50% of the active yeasts showed iron-depended antagonism, likely due to siderophore production, more than 25% are potentially secreting a toxic protein. Those killer yeast candidates clustered within ten species, showed target profiles from narrow- to broad spectrum, and several showed a broad pH and temperature activity profile. Given the tools for yeast biotechnology and protein engineering available, our collection offers a foundation for genetic and molecular characterization of antifungal phenotypes, with potential for future exploitation. The scalable workflow can screen other yeast collections or adjust for different antifungal compounds.