A synthetic switch based on orange carotenoid protein to control blue light responses in chloroplasts

[a collaborative effort with Beatrice Giuntoli (UniPi) and Matteo Ballottari (UniVr)]

Synthetic biology approaches to engineer light‐responsive system are widely used, but their applications in plants are still limited, due to the interference with endogenous photoreceptors. Cyanobacteria, such as Synechocystis spp., possess a soluble carotenoid associated protein named Orange Carotenoid binding Protein (OCP) that, when activated by blue‐green light, undergoes reversible conformational changes that enable photoprotection of the phycobilisomes. Exploiting this system, we developed a new chloroplast‐localized synthetic photoswitch based on a photoreceptor‐associated protein‐fragment complementation assay (PCA). Since Arabidopsis thaliana does not possess the prosthetic group needed for the assembly of the OCP2 protein, we implemented the carotenoid biosynthetic pathway with a bacterial β‐carotene ketolase enzyme (crtW), to generate keto‐carotenoids producing plants. The novel photoswitch was tested and characterized in Arabidopsis protoplasts with experiments aimed to uncover its regulation by light intensity, wavelength, and its conversion dynamics. We believe that this pioneer study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses, such as gene transcription or enzymatic activity, upon exposure to specific light spectra.

A Synthetic Oxygen Sensor for Plants Based on Animal Hypoxia Signaling

[a collaborative effort with Beatrice Giuntoli (UniPi)]

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.

A Ratiometric sensor based on plant N-terminal degrons able to report oxygen dynamics in Saccharomyces cerevisiae

[a collaborative effort with Beatrice Giuntoli (UniPi)]

The ability to perceive oxygen levels is crucial to many organisms because it allows discerning environments compatible with aerobic or anaerobic metabolism, as well as enabling rapid switch between these two energy strategies. Organisms from different taxa dedicate distinct mechanisms to associate oxygen fluctuations with biological responses. Following from this observation, we speculated that orthogonal oxygen sensing devices can be created by transfer of essential modules from one species to another in which they are not conserved. We expressed plant cysteine oxidase (PCOs) enzymes in Saccharomyces cerevisiae, to confer oxygen-conditional degradability to a bioluminescent protein tagged with the Cys-exposing N-degron typical of plant ERF-VII factors. Co-translation of a second luciferase protein, not subjected to oxygen-dependent proteolysis, made the resulting Double Luciferase Oxygen Reporter (DLOR) ratiometric. We show that DLOR acts as a proxy for oxygen dynamics in yeast cultures. Moreover, since DLOR activity was enabled by the PCO sensors, we employed this device to disclose some of their properties, such as the dispensability of nitric oxide for N-terminal cysteine oxidation and the individual performance of Arabidopsis PCO isoforms in vivo. In the future, we propose the synthetic DLOR device as a convenient, eukaryotic cell-based tool to easily screen substrates and inhibitors of cysteine oxidase enzymes in vivo. Replacement of the luminescent proteins with fluorescent proteins will further turn our system into a visual reporter for oxygen dynamics in living cells.