Liquid-liquid phase separation that underlies CO2 fixation by microalgae

The bio-conversion of CO₂ by the Ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) reaches 1014 kilograms per year. The carboxylation of Ribulose 1,5 bisphosphate (RuBP) activity of RuBisCO competes with its oxygenation activity. However, the diffusion of CO₂ in water is much less efficient than that of O₂, and the majority of inorganic carbon is in the form of bicarbonate in the aquatic environments. The success of photosynthetic microorganisms relies in their CO₂ concentration mechanisms (CCM) that induces a flux of inorganic carbon near RuBisCO. Contrary to higher plant, eukaryotic microalgae such as the model green algae Chlamydomonas reinhardtii possess biophysical CCMs which involves a peculiar supramolecular organisation and specific location of RuBisCO as well as bicarbonate transporters and carbonic anhydrases. RuBisCO is found in two distinct locations in C. reinhardtii: either diffused in the chloroplast stroma or condensed in a liquid-liquid separated phase (LLSP) named Pyrenoid. The proportion of diffused vs condensed RuBisCO varies from 60%/40% under condition of high partial pressure of CO₂ (P_CO2) to 20%/80% under low P_CO2. While numerous studies have investigated the carboxylation vs oxygenation activities of isolated RuBisCO or in vivo, the effect of condensation on the enzyme activity has been poorly investigated. The objective of this project is to correlate RuBisCO condensation to its activity. This is declined in two main strategies: 1- in-vitro reconstituted LLPS with recombinant RuBisCO and its scaffold protein EPYC1 and a structure-function investigation, and 2- in-vivo quantification of carboxylation and oxygenation activities under conditions in which RuBisCO is mainly diffused or mainly condensed.