BACKGROUND & AIMS: Traditional screening methods are powerful tools for exploring large sequence spaces, but they often prioritise target binding—an endpoint that does not always correlate with true functional activity. In this study, we present the first yeast-in-droplet microfluidic platform for function-based screening of disulfide-rich peptide (DRP) libraries, shifting the paradigm from binding affinity to functional performance. DRPs are nature’s molecular specialists, evolved with intricate twisted topologies to perform diverse bioactivities across fungi, insects, plants, and animals—making them ideal scaffolds for molecular design and engineering.
METHODS: Using picoliter-scale water-in-oil droplets as miniaturized “test tubes,” individual DRPs and their functional reporters were encapsulated. We employed yeast secretory expression to ensure correct disulfide folding and structural integrity, while engineering strains to report on functional activity (e.g., protease inhibition, protein-protein interaction inhibition, etc).
CONCLUSION: This platform enabled the rapid enrichment of a potent functional DRPs from libraries of over 100,000 variants in just four selection rounds—surpassing previous droplet-based peptide screens in both scale and efficiency.
SIGNIFICANCE: With established applications in medicine and agriculture, DRPs represent a rich source of bioactive molecules. By integrating custom yeast expression with high-throughput droplet microfluidics, we establish a next-generation platform for the functional engineering of DRPs, enabling also genuine screening of macrocyclic DRPs liberated in solution.