A modular high-throughput approach for advancing synthetic biology in the chloroplast of Chlamydomonas.

Chloroplast synthetic biology holds promise for advancing photosynthetic organisms through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. To that end, we developed an automation workflow that enables the generation, handling and analysis of thousands of transplastomic strains in parallel. Furthermore, we expanded the repertoire of effective selection markers and reporter genes, and we characterized over 140 regulatory parts, including native and synthetic promoters, 5' and 3' untranslated regions, and intercistronic expression elements. We integrated the system with existing molecular cloning standards and demonstrated several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof of concept for prototyping metabolic pathways in plastids by introducing a chloroplast-based synthetic photorespiration pathway, resulting in a threefold increase in biomass production. Overall, our study advances current chloroplast engineering efforts by providing a high-throughput platform and standardized genetic parts for the rapid prototyping and characterization of plastid manipulations with the prospect of high transferability between different chloroplasts, including those of higher plants and crops.