| Summary: | In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO<sub>2</sub>-concentrating mechanisms (CCMs) into terrestrial C<sub>3</sub> plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO<sub>3</sub><sup>−</sup>) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO<sub>2</sub> and HCO<sub>3</sub><sup>−</sup>) or active HCO<sub>3</sub><sup>−</sup> transport can be of great value in the process of introducing CCM components into terrestrial C<sub>3</sub> plants. In this study, we generated a <i>Saccharomyces cerevisiae</i> CA knock-out (<i>ΔNCE103</i> or <i>ΔCA)</i> that has a high-CO<sub>2</sub>-dependent phenotype (5% (<i>v</i>/<i>v</i>) CO<sub>2</sub> in air). CAs produce HCO<sub>3</sub><sup>−</sup> for anaplerotic pathways in <i>S. cerevisiae</i>; therefore, the unavailability of HCO<sub>3</sub><sup>−</sup> for neutral lipid biosynthesis is a limitation for the growth of <i>ΔCA</i> in ambient levels of CO<sub>2</sub> (0.04% (<i>v</i>/<i>v</i>) CO<sub>2</sub> in air). <i>ΔCA</i> can be complemented for growth at ambient levels of CO<sub>2</sub> by expressing a CA from human red blood cells. <i>ΔCA</i> was also successfully complemented for growth at ambient levels of CO<sub>2</sub> through the expression of CAs from <i>Chlamydomonas reinhardtii</i> and <i>Arabidopsis thaliana</i>. The <i>ΔCA</i> 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 <i>ΔCA</i> strains can be probed using the Wilbur–Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new <i>ΔCA-</i>based screening system are also discussed.
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