Ashwani Rai, Robert DiMario, Remmy Kasili, Michael Groszmann, Asaph Cousins, David Donze, Jim Moroney

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO₂-concentrating mechanisms (CCMs) into terrestrial C₃ plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO₃⁻) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO₂ and HCO₃⁻) or active HCO₃⁻ transport can be of great value in the process of introducing CCM components into terrestrial C₃ plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO₂-dependent phenotype (5% (v/v) CO₂ in air). CAs produce HCO₃⁻ for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO₃⁻ for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO₂ (0.04% (v/v) CO₂ in air).  ΔCA can be complemented for growth at ambient levels of CO₂ by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO₂ through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur–Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.