Abstract
Synthetic biology enables the engineering of biomolecules and cellular pathways but often relies on costly, time-intensive trial-and-error approaches. This work demonstrates that Saccharomyces cerevisiae (yeast) can serve as a cost-effective, translatable, and high-throughput synthetic biology platform by leveraging G protein-coupled receptor (GPCR) signaling. These platforms enable applications in protein biophysics, protease surveillance, biosensor development, and drug discovery.
First, I developed a yeast-based platform to identify functional, protease-sensitive chimeras using the GPCR mating pathway. Screening 91 insertion sites across six pathway proteins identified STE2, FUS3, and mTurquoise2 as tolerant to Hepatitis C peptide integration. Viral protease expression revealed nine cleavage-sensitive chimera sites localized to STE2 and GPA1, establishing a platform for viral protease surveillance and inhibitor screening with potential applications to HIV and SARS-CoV-2. I expanded on this approach to identify bifunctional yeast and human GPCR-reporter chimeras that retained wild-type receptor activity in mammalian cells, demonstrating translational potential.
Second, I developed a yeast complementation assay to characterize Superdark proteins, which share structural similarity with GPCRs but lack defined biological roles and ligands. Among six identified Superdark proteins, TMEM184C emerged as a leading GPCR-like candidate. Leveraging the autophagy-associated yeast homolog HFL1, this assay revealed that only TMEM184C rescued the defective vacuolar phenotype, demonstrating functional conservation across more than one billion years of evolution.
Finally, I increased the functional human GPCR repertoire of our yeast platform DCyFIR through literature-based and error-prone mutagenesis. These approaches enhanced signaling activity, altered Gα coupling, pH-dependent behavior, and rescued previously nonfunctional GPCRs, expanding yeast-based platforms for scalable drug discovery.