Engineering Yeast to Produce Cholesterol Enables Construction of Yeast-Based Opioid Biosensors

Biosensors, detecting devices with a biological component, are powerful screening tools. A biosensor for opioids is needed to accelerate the development of opioid-producing yeast strains and to aid in the search for novel opioids with fewer side effects. As opioid receptors are G protein-coupled receptors (GPCRs), we applied the established strategy of making yeast-based biosensors by coupling GPCRs to the pheromone response pathway. However, previous studies have shown that opioid receptors, like many other human GPCRs, require cholesterol as opposed to the fungal sterol ergosterol for optimal function.

By engineering yeast to produce cholesterol and introducing opioid receptors, we successfully generated fifteen opioid biosensors. The biosensors, based on mu, kappa and delta opioid receptors from humans and throughout Vertebrata, all showed some activity against an array of nine agonists with EC50s as low as 2.5 nM. Remarkably, agonist specificities were similar to those of human receptors in native environments, demonstrating functional conservation between species and retention of specificity in yeast. Opioid receptor function was highly dependent on cholesterol, demonstrated by a 21x increase in the DAMGO sensitivity of human mu opioid receptor-based biosensors. We used our platform to study how receptor function is modified by adding signal sequences, deleting conserved terminal motifs, and introducing missense mutations found in uncommon human alleles. Our cholesterol biosensor background should broadly improve functional expression of human GPCRs in yeast, aiding in ongoing receptor deorphanization efforts.