Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns

Heterologous synthesis of a biophysical CO₂-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C₃ plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO₂ concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C₃ plant chloroplast is functional bicarbonate (HCO₃⁻) transporters and vectorial CO₂-to-HCO₃⁻ converters. Engineering strategies aim to locate these transporters and conversion systems to the C₃ chloroplast, enabling elevation of HCO₃⁻ concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C₃ plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO₃⁻ transporter, and CO₂-to-HCO₃⁻ converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes.