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Climate change poses significant challenges to global agriculture, with rising temperatures, unpredictable weather patterns, and extreme climatic events threatening crop yields. Plant synthetic biology offers promising solutions to enhance the resilience and adaptability of crops to these changing conditions. However, the slow pace of crop engineering, largely due to the inherently slow growth of plants, hinders progress. Currently, the discovery, development, and approval of novel crop traits take approximately 10 to 15 years, with about 4 years dedicated to proof-ofconcept development and optimization of genetic constructs. Chloroplasts present a promising target for introducing novel traits due to their precise DNA integration, absence of gene silencing, ability to stack transgenes in synthetic operons, predictable gene expression outcomes, and reduced risk of transgene escape. Despite their potential, chloroplast biotechnology is still in its early stage, highlighting a desperate need for advanced prototyping capabilities in plant and chloroplast synthetic biology. To advance chloroplast engineering, we have developed a versatile protocol for creating chloroplast-based cell-free gene expression (CFE) systems from various plant species, including wheat (monocot), spinach, and poplar trees (dicots). These systems are compatible with both conventionally used T7 RNA polymerase and endogenous chloroplast polymerases, facilitating detailed characterization and prototyping of regulatory sequences at transcriptional and translational levels. By analyzing a collection of 23 5’ untranslated regions (UTRs), 10 3’ UTRs, and 6 chloroplast promoters in spinach and wheat extracts, we demonstrated consistent expression patterns across species, indicating cross-species compatibility. Recognizing the need for more complex in vivo prototyping, we established Chlamydomonas reinhardtii as a model chassis for chloroplast synthetic biology. Our automated workflow facilitates the generation, handling, and analysis of thousands of transplastomic strains. This included expanding the selection markers, developing new reporter genes, and characterizing over 140 regulatory elements, including native and synthetic promoters, UTRs, and intercistronic elements. We integrated this system with the Phytobrick cloning standard, demonstrating applications a library-based approach to develop synthetic promoter designs in plastids and a chloroplast-based synthetic photorespiration pathway, which resulted in a twofold increase in biomass production. Together, the advancements in chloroplast cell-free systems and the use of Chlamydomonas reinhardtii as a prototyping chassis provide powerful tools for plant synthetic biology. These innovations enhance our ability of engineering gene expression in the chloroplast and support the development of plants designed to meet the demands of a changing global climate.
