Diatom pyrenoids are encased in a protein shell that enables efficient CO₂fixation

Abstract

Pyrenoids are subcompartments of algal chloroplasts that concentrate Rubisco enzymes and their CO2 substrate, thereby increasing the efficiency of carbon fixation. Diatoms perform up to 20% of global CO2 fixation, but their pyrenoids remain poorly characterized at a molecular level. Here, we used in vivo photo-crosslinking to catalogue components of diatom pyrenoids and identified a pyrenoid shell (PyShell) protein, which we localized to the pyrenoid periphery of both the pennate diatom, Pheaodactylum tricornutum, and the centric diatom, Thalassiosira pseudonana. In situ cryo-electron tomography (cryo-ET) revealed that the pyrenoids of both diatom species are encased in a lattice-like protein sheath. Disruption of PyShell expression in T. pseudonana resulted in the absence of this protein sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with impaired growth and reduced carbon fixation efficiency under standard atmospheric conditions. Pyrenoids in mutant cells were fragmented and lacked the thylakoid membranes that normally traverse the Rubisco matrix, demonstrating how the PyShell plays a guiding role in establishing pyrenoid architecture. Recombinant PyShell proteins self-assembled into helical tubes, enabling us to determine a 3.0 Å-resolution PyShell structure. We then fit this in vitro structure into an in situ subtomogram average of the pyrenoid's protein sheath, yielding a putative atomic model of the PyShell within diatom cells. The structure and function of the diatom PyShell provides a new molecular view of how CO2 is assimilated in the ocean, a crucial biome that is on the front lines of climate change.

A carboxysome‐based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead

“…A similar concept has been suggested for encapsulins which self-assemble and have been demonstrated to encapsulate specific cargo (Giessen & Silver, 2017). The recent identification of encapsulated pyrenoids provides further models for novel Rubisco compartmentalisation systems (Nam et al, 2023;Shimakawa et al, 2023). Collectively, these examples demonstrate that novel synthetic biology approaches could be considered to transplant a faster Rubisco into plants whilst also providing a localised and optimal CO 2 concentration for the enzyme to operate within.…”

“…Surrounding this Rubisco matrix is a starch sheath that minimises CO 2 leakage (Toyokawa et al, 2020), while tubules also traverse the matrix and are speculated to deliver CO 2 (Engel et al, 2015). In recent analyses of diatom pyrenoids, protein shells have been identified as potential diffusional barriers to substrate entry (Nam et al, 2023;Shimakawa et al, 2023). Similar to carboxysomes, pyrenoid Rubisco phaseseparates with EPYC1 to form a dynamic, flexible matrix (Freeman-Rosenzweig et al, 2017;He et al, 2020He et al, , 2023Wunder et al, 2018), in an interaction mediated by Rubisco small subunit helices (Atkinson et al, 2019;Meyer et al, 2012).…”