Using the green alga, Chlamydomonas reinhardtii, as a chassis to express non-green Rubisco variants

Abstract

Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase), found in photosynthetic organisms from bacteria to plants, carboxylates the substrate ribulose-1, 5-bisphosphate with atmospheric CO2, and as such is the entry point for 90 % of organic carbon globally. In crop species it has low selectivity for CO2 over O2 and a low turnover rate, leading it to become an engineering target to improve photosynthetic efficiency. Catalytically superior variants have been found in non-plant species, and modelling suggests directly replacing the native wheat Rubisco with the variant from the red alga, Griffithsia monilis, could increase carbon gain by 27 %. Incompatibilities with the chaperone proteins needed to assemble functional Rubisco means efforts to express superior non-plant variants in plants have so far been unsuccessful. The research outlined within this thesis took steps to understand the requirements of red-type Rubiscos to fold in green-type systems. First, a genomic investigation of G. monilis was undertaken, including de novo sequencing and assembly of the chloroplast and nuclear genomes, to try to identify convergently evolved chaperone proteins. Secondly, to improve the facility of performing large-scale high-throughput transformations of C. reinhardtii, method development for a novel chloroplast transformation technique using single-walled carbon-nanotubes was undertaken. Alongside this, preliminary investigations to cell wall fingerprinting were performed using a high-throughput antibody-based method. Thirdly, using the chloroplast genome as the engineering platform, Rubisco large subunit chimeras were designed and built to explore the sequence space required to maintain the kinetic performance of red-type Rubisco variants and the interfaces required for correct chaperone interactions in green-type systems. Finally, we successfully expressed the C. reinhardtii Rubisco small subunit from the chloroplast genome, restoring photosynthesis in RbcS deletion strains. This work provides new insights into the promising alga, G. monilis, and contributes to the C. reinhardtii research community, further establishing this organism as a chassis for photosynthetic engineering.