Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Living yeast biosensors previously utilized fungal pheromone signaling pathways for peptide biomarker detection via G-protein-coupled receptors (GPCRs).
• Distinguishing subtle single-amino-acid variations in peptide ligands using binding assays alone is challenging.

Purpose of the Study:

• To reintroduce proteases into the yeast biosensor system to enhance the detection of single-amino-acid changes in peptide biomarkers.
• To characterize the dose-response curves of five fungal GPCRs, peptides, and proteases from different yeast species.
• To validate the biosensor's ability to translate single-amino-acid alterations into significant shifts in peptide activation concentration.

Main Methods:

• Characterized dose-response curves for five fungal GPCRs, peptides, and proteases from *Saccharomyces cerevisiae*, *Candida albicans*, *Schizosaccharomyces pombe*, *S. octosporus*, and *S. japonicus*.
• Performed alanine scanning on selective GPCRs (*S. cerevisiae* and *C. albicans*) with and without protease.
• Utilized biosensor strains with and without protease to distinguish peptide variants with altered protease cleavage.

Main Results:

• Identified two peptide variants (CaPep2A and CaPep2A13A) with diminished protease cleavage.
• Demonstrated that the protease selectively cleaved peptides, altering the apparent concentration for half-maximal activation by over an order of magnitude for CaPep and CaPep13A.
• Confirmed that single-amino-acid changes can be translated into significant apparent shifts in peptide activation concentration using the protease-enhanced yeast biosensor.

Conclusions:

• The living yeast biosensor, augmented with a sequence-specific protease, effectively translates single-amino-acid changes into substantial apparent shifts in peptide activation concentration.
• Further engineering via computational modeling and directed evolution can expand the biosensor's capability to distinguish a wider range of peptide sequences.
• The protease-integrated yeast biosensor holds promise for point-of-care diagnostics, scalable communication languages, and other future applications.