Development of GPCR-based yeast biosensors towards biomedical applications

Doktorsavhandling, 2025

Biosensors, i.e. systems utilizing biological components such as antibodies or enzymes for sensing, have broad applicability in research, industry, and healthcare. Over the past three decades, the yeast Saccharomyces cerevisiae has emerged as a biosensor platform, often utilizing the cells´ internal signaling pathways for signal transduction and the transcriptional machinery for production of measurable outputs. G-protein coupled receptor (GPCR)-based yeast biosensors are prominent examples of this, leveraging the yeast mating pathway for signal transduction upon binding of a ligand to the GPCR at the yeast cell surface. These yeast-based biosensors (YBBs) have facilitated the deorphanization of human GPCRs, identification of GPCR-specific designer drugs, and the development of point-of-care (POC) YBBs applicable outside laboratory settings. In this thesis, I present our efforts to further advance GPCR-based YBBs towards biomedical applications and expand the available toolkit for their development.

In the first project, YBB outputs were evaluated as these affect the ease-of-use, time-of-detection, and sensitivity of a sensor. Specifically, two colorimetric outputs - prodeoxyviolacein and deoxyviolacein - were benchmarked against the previously established lycopene across different conditions, thereby broadening the range of available colorimetric outputs.

In the second and third project, we focused on the investigation of key residues in fungal GPCRs. These receptors have shown potential both as therapeutic targets for treatment of fungal infection and as YBB sensing elements for detection of fungal infection. To this end, an untargeted mutant library of the Ste2 mating receptor from the pathogenic fungus Candidozyma auris was screened, identifying amino acid substitutions that improved the sensor sensitivity and response curve metrics. Additionally, a designed mutant library for S. cerevisiae Ste2, targeting the orthosteric site, was screened and sequenced. Substitutions generating loss-of-function, constitutive activity, or no change in receptor activation were identified.

Finally, a YBB was constructed and optimized for evaluation of ligands of the human free fatty acid 2 receptor (FFA2R), a GPCR implicated in metabolic and inflammatory disease.

Collectively, the results presented in this thesis advance our understanding of fungal mating GPCR structure-function relationships and expand the repertoire of YBB outputs, contributing towards new possibilities in diagnostic, therapeutic, and screening applications for YBBs.