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

Abstract

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.

Humanized CB1R and CB2R yeast biosensors enable facile screening of cannabinoid compounds

“…In addition, despite the fact that human GPCRs typically work in yeast at neutral pH [23][24][25] , CB2R showed prohibitively strong constitutive signaling at pH 7.1 (a phenomenon also previously noted with the heterologously expressed serotonin receptor 26 ). When the biosensor was assayed at pH 5.8, we observed greatly reduced background with both AEA and 2-AG.…”

Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

“…Naturally, GPCRs allow for concentration- dependent signal transduction to provide outputs of regulated intensity, and display exceptional diversity to sense various types of ligands, ranging from light rays and small molecules to peptides and larger complex proteins 53,56,68,69 . Genes encoding GPCRs MTNR1A 53 , ADORA2B 53 , ADRA2A 54 , 5HT4b 70 , Mam2 53 , and CXCR4a 71 were chosen accordingly, and individually heterologously expressed and coupled to either native Gpa1 or the chimeric G ɑZ or G ɑi1/2 -subunits in the optimized background strain employing display of CD19 from P FUS1 (strains DIX57-58). The constructed GPCR library covered sensing of small molecules (melatonin, adenosine, adrenaline, serotonin), peptides (P-factor), and complex proteins (CXCL12/SDF-1)( Fig.…”

“…Primers, DNA fragments, amplicons, plasmids, and gDNA were always dissolved in Milli-Q H2O and stored at -20°C. An overview of parts, backbones, constructed plasmids, and plasmids originating from other studies [52][53][54]57,58,67,70,71,84,86 can be found in Suppl. Table 11-12, and primers in Suppl.…”

Reshaping the yeast galactose regulon via GPCR signaling cascade

“…The genetic devices reported here might hold great promise for future metabolic engineering and biosensor applications. 52 , 53 , 54 Given that GPCRs are widely distributed in nature, 55 , 56 the plasticity of our genetic circuits would offer a variety of user-defined expression profiles, which are of significant interest to the synthetic biology and metabolic engineering community.…”