The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacteria CO2concentrating mechanism, could enhance photosynthetic CO2assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration intoNicotiana benthamianachloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependentEscherichia colifailed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed inArabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2concentrating mechanism components to improve plant photosynthesis.