Screening a 681-membered yeast collection for the secretion of proteins with antifungal activity

Fungal pathogens pose a threat to human health and food security. Few antifungals are available and resistance to all has been reported. Novel strategies to control plant and human pathogens as well as food spoilers are urgently required. Environmental yeasts provide a functionally diverse, yet underexploited potential for fungal control based on their natural competition via the secretion of proteins and other small molecules such as iron chelators, 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 spoilage yeasts and relevant human and plant pathogens, herein focusing on discovering yeasts that secrete antifungal proteins. The workflow includes the classification of the secreted molecules and cross-comparison of their antifungal capacity using an independent synthetic calibrant. Our workflow delivered a collection of 681 yeasts of which 212 isolates (31 %) displayed antagonism against at least one target strain. While 57.5 % of the active yeasts showed iron-depended antagonism, likely due to pulcherrimin-like iron chelators, 31.7 % secreted antifungal proteins. Those yeast candidates clustered within twelve OTUs, showed narrow and broad target spectra, and several showed a broad pH and temperature activity profile. Given the tools for yeast biotechnology and protein engineering available, our collection can serve as a rich starting point for genetic and molecular characterization of the various antifungal phenotypes, their mode of action and their future exploitation.