Characterising cyanobacterial and algal bicarbonate transporters using heterologous systems; exploring relationships between sequence, structure and function

Abstract

Marine phytoplankton contribute significantly to global primary production, with marine cyanobacteria accounting for up to 25% of this productivity. A major factor underlying this is their carbon dioxide concentrating mechanisms (CCMs). Bicarbonate (HCO3-) transporters facilitate accumulation of intracellular HCO3- which diffuses into micro-compartments (carboxysomes) where the enzyme Rubisco is housed. Within the carboxysomes, carbonic anhydrase (CA) interconverts HCO3- and CO2, increasing the CO2 concentration available for Rubisco carboxylation. Considering the critical role HCO3- transporters play in the CCM, much remains unknown about the properties, mechanism and regulation of these proteins. This thesis addresses some of these knowledge gaps. The introduction of CCMs to chloroplasts of C3 crop plants is a focus of research to improve crop productivity as a potential strategy to circumvent looming food shortages expected as global population increases. Modelling suggests this could boost Rubisco carboxylation, increasing photosynthetic efficiency, thereby improving crop productivity. The expression of functional HCO3- transporters in the chloroplast inner envelope membrane (IEM) could support a 7-16% increase in crop productivity resulting from increased CO2 supply to Rubisco. This thesis focuses on screening members of five putative HCO3- transporter families from aquatic phytoplankton species. The primary aim of this work was to functionally identify and characterise previously undescribed sub-classes of HCO3- transporters, thereby expanding options for the secondary aim of successful integration of HCO3- transporters into C3 crop plants. An E. coli complementation system (CAfree), which lacks CA, was used to identify and characterise previously undescribed HCO3- transporters. CAfree is unable to grow at ambient CO2 unless it expresses a functional inorganic carbon (Ci) transporter or CA capable of supplying cellular HCO3- for anaplerotic pathways. Putative HCO3- transporters assessed for function were from cyanobacteria and unicellular algae (green algae and a diatom). Screening identified two families of HCO3- transporters that were functional in CAfree. Functional transporters, named SbtA2 and LCIA, were characterised using HCO3- uptake assays which measure the influx of H14CO3- in transporter-expressing E. coli. Variations of this assay assessed transporter affinity and the effects of different ions on transporter function. SbtA2 was found to be a medium affinity, high flux transporter with an apparent Cl-/SO42--dependence, whereas LCIA likely functions as a low-flux channel. SbtA2 was targeted to the chloroplast IEM of Nicotiana tabacum. Transgenic plants were assessed but showed little difference in growth and photosynthetic parameters relative to wild type plants. Transporters deemed non-functional in the E. coli system were subjected to directed evolution experiments, where CAfree expressing transport proteins were grown under sub-saturating CO2 conditions. An evolved BicA2 transporter with an "always-active" phenotype facilitated CAfree growth in air. The gene contained a single point mutation resulting in a single amino acid substitution. Sequence analysis of functional BicA members led to the identification of a native BicA2 member that was functional in E. coli. Characterisation of BicA2 in E. coli revealed low affinity for HCO3-, dependence on high Na+ concentrations, and variable HCO3- flux among homologues. These results demonstrated the identification and characterisation of previously undescribed HCO3- transporters and the potential to evolve transport proteins to become functional HCO3- transporters. This has significant implications by broadening the numbers of transporters available for use in biotechnological applications, like improving photosynthesis in C3 plants, as well as adding to the knowledge of how transporters may function in general. Show more