Serotonin G Protein-Coupled Receptor-Based Biosensing Modalities in Yeast.

Serotonin is a key neurotransmitter involved in numerous physiological processes and serves as an important precursor for manufacturing bioactive indoleamines and alkaloids used in the treatment of human pathologies. In humans, serotonin sensing and signaling can occur by 12 G protein-coupled receptors (GPCRs) coupled to Gα proteins. In yeast, human serotonin GPCRs coupled to Gα proteins have previously been shown to function as whole-cell biosensors of serotonin. However, systematic characterization of serotonin biosensing modalities between variant serotonin GPCRs and application thereof for high-resolution serotonin quantification is still awaiting. To systematically assess GPCR signaling in response to serotonin, we characterized reporter gene expression at two different pHs of a 144-sized library encoding all 12 human serotonin GPCRs in combination with 12 different Gα proteins engineered in yeast. From this screen, we observed changes in the biosensor sensitivities of >4 orders of magnitude. Furthermore, adopting optimal biosensing designs and pH conditions enabled high-resolution high-performance liquid chromatography-validated sensing of serotonin produced in yeast. Lastly, we used the yeast platform to characterize 19 serotonin GPCR polymorphisms found in human populations. While major differences in signaling were observed among the individual polymorphisms when studied in yeast, a cross-comparison of selected variants in mammalian cells showed both similar and disparate results. Taken together, our study highlights serotonin biosensing modalities of relevance to both biotechnological and potential human health applications.

A Reporter System for Cytosolic Protein Aggregates in Yeast.

Protein misfolding and aggregation are linked to neurodegenerative diseases of mammals and suboptimal protein expression within biotechnology. Tools for monitoring protein aggregates are therefore useful for studying disease-related aggregation and for improving soluble protein expression in heterologous hosts for biotechnology purposes. In this work, we developed a promoter-reporter system for aggregated protein on the basis of the yeast native response to misfolded protein. To this end, we first studied the proteome of yeast in response to the expression of folded soluble and aggregation-prone protein baits and identified genes encoding proteins related to protein folding and the response to heat stress as well as the ubiquitin-proteasome system that are over-represented in cells expressing an aggregation-prone protein. From these data, we created and validated promoter-reporter constructs and further engineered the best performing promoters by increasing the copy number of upstream activating sequences and optimization of culture conditions. Our best promoter-reporter has an output dynamic range of approximately 12-fold upon expression of the aggregation-prone protein and responded to increasing levels of aggregated protein. Finally, we demonstrate that the system can discriminate between yeast cells expressing different prion precursor proteins and select the cells expressing folded soluble protein from mixed populations. Our reporter system is thus a simple tool for diagnosing protein aggregates in living cells and should be applicable for the health and biotechnology industries.