摘要：w. previously demonstrated that w. could hijack the fungal pheromone signaling pathw.y to provide a living yeast biosensor w.ere peptide biomarkers w.re recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, w. demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherw.se, w.uld likely be difficult to detect using binding-based assays. w. characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases- - Saccharomyces cerevisiae, , Candida albicans, , Schizosaccharomyces pombe, , Schizosaccharomyces octosporus, , and Schizosaccharomyces japonicus. . Alanine scanning w.s carried out for the most selective of these-S. S. cerevisiae and C. albicans-w.th -w.th and w.thout the protease. Tw. peptide variants w.re discovered, w.ich show.d diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides w.re then distinguished by utilizing the biosensor strains w.th and w.thout the protease, w.ich selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor w.th a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. w.th further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a w.de variety of peptide sequences beyond the alanine scanning carried out here. In the future, w. envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications.