Structure-function studies of the CO2 uptake complex in cyanobacteria

Increase of CO2 atmospheric level has led to an intense search for solutions to mitigate the problem. A natural pathway exists in photosynthesis where CO2 is fixed into biomass. Cyanobacteria, which constitute a large phylum of natural oxygenic photosynthetic bacteria, have a huge potential for bioengineering. This includes candidates for use in diverse CO2 capture and storage projects and the potential to redirect its energy for the production of valuable compounds. Our aim in this project is to understand the structure-function of Cup (CO2 uptake) proteins in the NDH-13,4 complex, part of the cyanobacterial CO2 concentrating mechanism (CCM). Cyanobacteria have five CCM systems responsible for increasing inorganic carbon (Ci) concentration inside of the cell, an effort to raise CO2 level close to Rubisco, the main enzyme responsible for carbon fixation. Among them, two CO2 uptake systems are specialized NDH-1 complexes, NDH-13 (NdhF3/NdhD3/CupA/CupS) and NDH-14 (NdhF4/NdhD4/CupB), which have very little known about their mechanism. CO2 is usually regulated in living organisms by carbonic anhydrases (CAs), enzymes that catalyzes the interconversion of CO2 and HCO3-. The hypothesis is that Cup, in the NDH-13,4 complex, is involved in this (non)reversible reaction, possibly coupled with release of a proton across the membrane. We constructed a double knockout mutant, where no NDH-13,4 is produced by the bacteria, and introduced the whole cupA operon under Rubisco promoter control in Synechocystis 6803. Physiological analysis of Ci depleted mutants with chlorophyll fluorescence traces and O2 evolution dependent on Ci, show a high Ci requirement feature on the double knockout mutants. However, the strain expressing constitutively NDH-13 lost its high CO2-requiring phenotype, displaying restored cell CO2 uptake. In parallel, we also analyzed Synechococcus 7942 mutants with no functional CO2 uptake systems, later complemented with only chpX (cupB). These systems were used in studies directed to analyze the effect of point mutations replacing amino acids (His/Cys) of CupA/CupB proteins to evaluate their potential role on CO2 uptake in cyanobacteria