Killer yeasts: expanding frontiers in the age of synthetic biology

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.

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Alanine-scanning of the yeast killer toxin K2 reveals key residues for activity, gain-of-function variants, and supports prediction of precursor processing and 3D structure

Prins,

Marinus,

Dafni

et al. 2024

Preprint

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Yeast killer toxins (YKTs) are antimicrobial proteins secreted by yeast with potential applications ranging from food preservation to therapeutic agents in human health. However, the practical use of many YTKs is limited by specific pH requirements, low temperature stability, low production yields, and narrow target specificity. While protein engineering could potentially overcome these challenges, progress is hindered by a lack of detailed knowledge about sequence-function relationships and structural data for these often multi-step processed proteins. In this study, we focused on the YKT K2, encoded by the M2 dsRNA satellite virus in Saccharomyces cerevisiae. Using alanine scanning mutagenesis of the full open reading frame and structure predictions combined with molecular dynamics simulations, we generated a comprehensive sequence-function map, refined the model for the proteolytic processing of the K2 precursor, and predicted the mature toxin structure. Our findings also demonstrate that K2 can be engineered towards enhanced toxicity and altered target specificity through single-site mutations. Furthermore, we identified structural homology between K2 and the SMK toxin from the yeast Millerozyma farinosa. Our cost-effective workflow provides a platform to broadly map YKT sequence-structure-function relationships, facilitating the engineering towards toxin-based technologies. The workflow could also serve as a template to resolve the processing and conformations of other proteins within the secretory pathway, a dynamic multi-step process that is challenging to structurally capture by purification and solving structures of intermediates.

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Killer yeasts: expanding frontiers in the age of synthetic biology

Cited by 3 publications

References 94 publications

Biocontrol yeast Kurtzmaniella quercitrusa BS-AY-S1 controls postharvest green mold of citrus fruit by producing metabolites with antifungal activity

Biocontrol yeast Kurtzmaniella quercitrusa BS-AY-S1 controls postharvest green mold of citrus fruit by producing metabolites with antifungal activity

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

Alanine-scanning of the yeast killer toxin K2 reveals key residues for activity, gain-of-function variants, and supports prediction of precursor processing and 3D structure

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