Ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco) is the most abundant enzyme on Earth, and responsible for the carboxylation of ribulose-bisphosphate with atmospheric CO2 to form organic matter. However, Rubisco is inefficient due to its slow kinetics and affinity for oxygen resulting in the waste product 2-phosphoglycolate.
As the oxygenation side-reactions lower Rubisco efficiency, some plants and algae have evolved carbon-concentrating mechanisms (CCM) which elevate CO2 concentrations at Rubisco active sites. One such CCM is the pyrenoid, a liquid-liquid phase separated compartment in algae containing proteins required for carbon fixation and intrinsically disordered proteins (IDPs). These IDPs are multivalent and often contain sticker motifs, allowing for network formation between (potentially) a myriad of proteins leading to condensate formation. In the model organism Phaeodactylum tricornutum, a red-lineage diatom with a fully sequenced genome, several such proteins have been identified, such as Pyrenoid Component 1 (PYCO1), Glove1, Glove2, and Hydra. As these proteins were identified through a conserved Rubisco-binding motif, experiments have already been conducted to characterize the behaviour of these proteins in vitro and in vivo (Oh et al. 2023). This study focuses on in vivo experiments, where CRISPR/Cas9 is first used to generate knockouts prior to physiological characterization of the mutants via Rubisco activity assays and fluorescent microscopy experiments. In addition, experiments can be carried out to visualize interactions between all these pyrenoid components, which require cryo-electron microscopy due to the disordered nature of these proteins.