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Improving the photosynthetic enzyme Rubisco is a key target for enhancing C₃crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-nullNicotiana tabacumplatform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (k_cat,air/K_c,air) under ambient conditions, driven by a significantly faster turnover rate (k_cat,air). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C₃crops. 

Rubisco catalyses the first step in carbon fixation and is a strategic target to improve photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits and its biogenesis requires multiple chaperones. We optimised a system to produce tobacco Rubisco in Escherichia coli by co-expressing chaperones in auto-induction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics very similar to that of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO2 affinity than those assembled with other small subunits. Our E. coli expression system will allow probing of features of both subunits of Rubisco that affect its kinetic properties.