Combining Yeast Display and Bacterial Genomic Library for the Unbiased Isolation of Novel Polysaccharide-Binding Peptides

Here, we present a novel yeast surface display-based platform for the discovery of biofilm-associated exopolysaccharide-binding peptides. Unlike conventional synthetic libraries, our approach utilizes the genomic diversity of Burkholderia multivorans strain C1576 through open-reading frame-filtered genomic fragment libraries, thereby enriching for naturally encoded carbohydrate-binding domains. By iterative fluorescence-activated cell sorting, we identified 21 peptides with confirmed binding to two structurally distinct rhamnose-rich polysaccharides: the exopolysaccharide Epol C1576 and the capsular polysaccharide CPS KpB-1. Biophysical characterization revealed that these peptides adopt predominantly α-helical or disordered conformations and undergo structural rearrangements upon polysaccharide binding. Functional assays demonstrated that selected peptides modulate biofilm architecture and bacterial viability in a species-specific manner, although they do not have a direct bactericidal effect against planktonic cells. This proof-of-concept study establishes yeast surface display as a powerful tool for the discovery of biofilm-targeting peptides and provides a basis for development of novel diagnostics and therapeutics to combat biofilm-associated infections.