A synthetic sensor to report oxygen dynamics in yeast

Sensing the environmental availability of oxygen is essential to most organisms on Earth. Indeed, various mechanisms enable different species to perceive oxygen and regulate its usage accordingly. For instance, plants and animals exploit the cysteine branch of the N-degron pathway to attune selective proteolysis to oxygen availability. This does not occur in the yeast Saccharomyces cerevisiae as its proteome lacks a key regulatory enzyme to oxidize N-terminal cysteine to Cys-sulfinic acid. We therefore developed an orthogonal reporter for oxygen levels, namely the Dual Luciferase Oxygen Reporter (DLOR). This synthetic construct consists of two bioluminescent enzymes, renilla and firefly luciferases, separated by an ubiquitin monomer to ensure post-translational cleavage of the two units. The firefly luciferase is equipped with an N-terminal degron, from the plant transcription factor RAP2.12, which confers oxygen-dependent instability when its N-terminal Cys is oxidized. In the present work, we show that the expression in yeast of Plant Cysteine Oxidase enzymes (PCOs) is sufficient to enable the N-degron dependent proteolysis of firefly luciferase. We successfully correlated the output of the couple DLOR-PCO with different oxygen concentrations, thus confirming the obtaining of an oxygen biosensor in yeast. Moreover, we could also compare the performance of different PCO isoforms, which overall correlate with data previously obtained by in vitro characterisation. Our system holds promising potential for detailed and high-throughput studies of the components of the cysteine branch of the N-degron pathway, crucial for several organisms.