Dataset for "Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts"
Description
The ATP-driven bicarbonate transporter BCT1, a four-component complex in the cyanobacteria CO2 concentrating mechanism (CCM), could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space (IMS) protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane (IEM). CmpC and CmpD were targeted to the stroma by RecA and recruited to the IEM by CmpB. Despite successful targeting, expression of this complex in CO2-dependent E. coli provided no bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms which were always active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CCM components to improve plant photosynthesis.
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Funding
The Realizing Increased Photosynthetic Efficiency (RIPE) project was funded by the Bill & Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the UK Government’s Department for International Development
OPP1172157