The phase separation underlying the pyrenoid-based microalgal Rubisco supercharger

Abstract

The slow and promiscuous properties of the CO2-fixing enzyme Rubisco constrain photosynthetic efficiency and have prompted the evolution of powerful CO2 concentrating mechanisms (CCMs). In eukaryotic microalgae a key strategy involves sequestration of the enzyme in the pyrenoid, a liquid non-membranous compartment of the chloroplast stroma. Here we show using pure components that two proteins, Rubisco and the linker protein Essential Pyrenoid Component 1 (EPYC1), are both necessary and sufficient to phase separate and form liquid droplets. The phase-separated Rubisco is functional. Droplet composition is dynamic and components rapidly exchange with the bulk solution. Heterologous and chimeric Rubiscos exhibit variability in their tendency to demix with EPYC1. 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.

CO2‐fixing liquid droplets: Towards a dissection of the microalgal pyrenoid

“…In this review, we will focus on the recent realization that the pyrenoid matrix is a non‐membraneous phase‐separated compartment, discuss the reconstitution of this phenomenon and outline key approaches that are now within facile experimental reach.…”

“…E, Centrifugal separation of the dense (P) and light (S) fractions permits quantitation of component ratios, demixing efficiency and the effect of perturbations such as modified ionic strength. Experiments were carried out as detailed in Reference . RbcL, Rubisco large subunit; RbcS, Rubisco small subunit.…”

“…These complications can be avoided by using bottom‐up reconstitution experiments with a defined number of pure components . We consequently characterized an in vitro reconstitution of material resembling the pyrenoid matrix . The two major matrix components, Rubisco and EPYC1 were both necessary and sufficient for the formation of pyrenoid‐like droplets via LLPS.…”

“…Especially at high protein concentrations, the droplets were not limited in size and were far larger than the pyrenoid (~1‐2 μm diameter). This suggests that, in vivo, the size of the compartment is likely component limited …”

A recombineering pipeline to clone large and complex genes in Chlamydomonas

“…To achieve this Chlamydomonas actively transports inorganic carbon across the plasma membrane and chloroplast envelope and delivers it as CO2 to tightly packed Rubisco within the pyrenoid (Wang et al, 2015). The pyrenoid is a liquid-liquid phase separated compartment (Freeman Rosenzweig et al, 2017;Wunder et al, 2018) essential for CCM function in Chlamydomonas (Meyer et al, 2012;Mackinder et al, 2016). Pyrenoids are widely prevalent in eukaryotic algae and it is thought that approximately 30% of global CO2 is fixed within algal pyrenoids (Mackinder et al, 2016).…”