Reconstitution of the Liquid Liquid Phase Separation Underlying the Microalgal Rubisco Supercharger

Abstract

Global transformation of carbon dioxide to biomass is almost entirely dependent on the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Paradoxically this critical catalyst displays a low carboxylation velocity and is prone to confuse CO2 with oxygen. To mitigate these properties the chemo- and photosynthetic organisms relying on Rubisco activity have evolved elaborate machineries to assist this catalyst. Almost all eukaryotic microalgae utilize CO2- concentrating mechanisms (CCMs) that function as carbon dioxide superchargers for Rubisco. The heartpiece of this CCM is a liquid-like Rubisco-containing membraneless organelle known as the pyrenoid. Recently a putative Rubisco linker protein comprised of four highly conserved 60-residue repeats was identified in the green alga Chlamydomonas reinhardtii. We report a reconstituted system using pure components that recapitulates properties of the pyrenoid in vitro. Addition of the linker protein to Rubisco results in a salt sensitive liquid-liquid phase separation. The two proteins rapidly demix from the bulk solution to form dense droplets that fuse and can be harvested by centrifugation. The droplet composition is dynamic and both components exchange rapidly with the bulk solution. Highly homologous Rubisco enzymes from diverse organisms such as higher plants and cyanobacteria show a great variability in their tendency to demix with the algal linker. Linker protein variants containing fewer repeats can phase separate with Rubisco, but require higher protein concentrations. Our data permits first conclusions regarding the interaction of Rubisco and the linker protein to be drawn. The ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to inform and guide synthetic biology ambitions aiming to engineer microalgal CCMs into crop plants.