Engineering Rubisco condensation in chloroplasts to manipulate plant photosynthesis

Summary Although Rubisco is the most abundant enzyme globally, it is inefficient for carbon fixation because of its low turnover rate and limited ability to distinguish CO 2 and O 2 , especially under high O 2 conditions. To address these limitations, phytoplankton, including cyanobacteria and algae, have evolved CO 2 ‐concentrating mechanisms (CCM) that involve compartmentalizing Rubisco within specific structures, such as carboxysomes in cyanobacteria or pyrenoids in algae. Engineering plant chloroplasts to establish similar structures for compartmentalizing Rubisco has attracted increasing interest for improving photosynthesis and carbon assimilation in crop plants. Here, we present a method to effectively induce the condensation of endogenous Rubisco within tobacco ( Nicotiana tabacum ) chloroplasts by genetically fusing superfolder green fluorescent protein (sfGFP) to the tobacco Rubisco large subunit (RbcL). By leveraging the intrinsic oligomerization feature of sfGFP, we successfully created pyrenoid‐like Rubisco condensates that display dynamic, liquid‐like properties within chloroplasts without affecting Rubisco assembly and catalytic function. The transgenic tobacco plants demonstrated comparable autotrophic growth rates and full life cycles in ambient air relative to the wild‐type plants. Our study offers a promising strategy for modulating endogenous Rubisco assembly and spatial organization in plant chloroplasts via phase separation, which provides the foundation for generating synthetic organelle‐like structures for carbon fixation, such as carboxysomes and pyrenoids, to optimize photosynthetic efficiency.