• cysteine N-degron pathway
• hypoxia
• PCOs
• plant oxygen sensing
• RNAseq
• Saccharomyces cerevisiae
• synthetic biology

In plants, a key mechanism involved in oxygen perception consists in the conditional stabilization of transcription factors belonging to the group VII of Ethylene Response Factors (ERF-VII) under hypoxia. Cysteine exposed at the N-terminus of these transcription factors determine their half-life, following the so called “Cysteine N-degron pathway” (Cys-NDP): dioxygenation of the conserved Cys2 residue of the ERFs-VII will lead to their proteasomal degradation. Such reaction is carried out by enzymes that are sensitive to the oxygen concentration, called Plant Cysteine Oxidases (PCOs) which qualify as oxygen sensors.
The absence of any PCO homolog in Saccharomyces cerevisiae allows us to use synthetic biology to insert orthogonal gene circuits based on the plant oxygen perception system using S. cerevisiae as a host organism. To achieve this aim we engineered a yeast strain able to express PCO4 from Arabidopsis thaliana together with an exogenous reporter based on the N-terminal domain of RAP2.12, an ERF-VII, fused with a luciferase, reconstituting in this manner a synthetic Cys-NDP pathway in yeast.
We evaluated the effect of PCO4 expression on the growth rate and the global regulation of gene expression of yeast, through RNAseq analysis; additionally, we exploited the synthetic Cys-NDP to create an oxygen-dependent transcriptional regulation strategy in yeast.
A successfully reconstituted Cys-NDP in S. cerevisiae can be exploited to better understand plant oxygen perception mechanism and to develop extremely specific systems for gene induction controlled by environmental oxygen levels, to express genes of interest in yeast.