Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes

Improving the photosynthetic enzyme Rubisco is a key target for enhancing C₃ crop 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-null <i>Nicotiana tabacum</i> platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (<i>rbcS</i>) 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 (<i>k</i>_cat,air<i>/K</i>_c,air) under ambient conditions, driven by a significantly faster turnover rate (<i>k</i>_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 C₃ crops.