A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C-3 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 C-3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (Delta NCE103 or Delta CA) 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 Delta CA in ambient levels of CO2 (0.04% (v/v) CO2 in air). Delta CA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. Delta CA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The Delta CA 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 Delta CA strains can be probed using the Wilbur-Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new Delta CA-based screening system are also discussed.