An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity， global food security， and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO fixing enzyme ribulose-1，5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts. Here we refashion metabolism by expressing a phosphoribulokinase-neomycin phosphotransferase fusion protein to produce a high-fidelity， high-throughput Rubisco-directed evolution (RDE2) screen that negates false-positive selection. Successive evolution rounds using the plant-like -Rubisco from the cyanobacterium BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate， efficiency， and specificity. Structural analysis revealed the amino acids clustered in an unexplored subunit interface of the holoenzyme. To study its effect on plant growth， the -Rubisco was transformed into tobacco by chloroplast transformation. As previously seen for PCC6301 Rubisco， the specialized folding and assembly requirements of -Rubisco hinder its heterologous expression in leaf chloroplasts. Our findings suggest that the ongoing efforts to improve crop photosynthesis by integrating components of a cyanobacteria CO-concentrating mechanism will necessitate co-introduction of the ancillary molecular components required for Rubisco biogenesis.