A synthetic sensor to report oxygen dynamics in yeast

Perceiving the environmental availability of oxygen is essential to most organisms on Earth. Plants and animals exploit the Cys/Arg branch of the N-degron pathway to attune selective proteolysis to oxygen availability, while this does not occur in the yeast Saccharomyces cerevisiae. We put forward that this was due to the lack in its proteome of key regulatory enzymes to oxidize N-terminal Cys to Cys-sulfinic acid. Indeed, we found that expressing thiol oxidase enzymes such as Plant Cysteine Oxidases (PCO) or the Cysteamine Dioxygenase (ADO) enzyme was enough to switch on the Cys/Arg N-degron pathway in yeast. Following-up we used the Dual Luciferase Oxygen Reporter (DLOR),a ratiometric construct based on the ubiquitin split system to monitor the activity of the pathway. The firefly luciferase module exposes a Cys N-degron typical of plant ERF-VII factors and its turnover was made O2-conditional by the sole co-expression of PCO or ADO. DLOR acts as a proxy of oxygen dynamics in cultures, which can be deployed towards the investigation of the activity of thiol oxidases from different organisms, screen for chemical inhibitors, and identify novel substrate proteins in living cells.