1 Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montréal, QC, H4B1R6, Canada.
2 Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, 78712, USA.
3 DEVCOM Army Research Laboratory-South, Austin, 78712, TX, USA.
4 Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, 78712, USA. jgollihar2@houstonmethodist.org.
5 DEVCOM Army Research Laboratory-South, Austin, 78712, TX, USA. jgollihar2@houstonmethodist.org.
6 Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute, Houston Methodist Hospital, Houston, TX, USA. jgollihar2@houstonmethodist.org.
7 Department of Biology, Centre for Applied Synthetic Biology

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.