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

ABSTRACTSynthetic 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 {beta}-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. One-sentence summaryInspired by the light-driven conformational transitions of orange carotenoid proteins of cyanobacteria, we generated a molecular device able to switch its dimeric state in response to blue light.