[3S04e] 11. The Society for Biotechnology - Part 3

5:40 PM - 6:00 PM JST(8:40 AM - 9:00 AM UTC)

[3SBT-17] Discovery and implications of new skeletal proteins encasing the Rubisco condensate in the diatom chloroplast

○Yusuke Matsuda Matsuda¹ (1. Kwansei Gakuin University (Japan))

Keywords:

diatom,photosynthesis,CO2-concentrating mechanism,pyrenoid

Marine diatom is a group of eukaryotic algae that accounts for up to 20% of annual global primary production. This massive productivity by diatoms is sustained by the function of their uniquely structured secondary chloroplast. Diatom chloroplasts possess globe shaped thylakoid membranes layered at the interior of the chloroplast envelope, which is comprised of outer-most girdle lamellae and inner stroma thylakoid membranes. At the central part of this layered thylakoid system, there is an endo-plastidic organelle, pyrenoid. Pyrenoid is known to be a phase-separated condensate of CO₂-fixing enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). As unique features of the diatom pyrenoid, a couple of layers of thylakoid membranes traverse along the axis of the pyrenoid core, that is denoted as the pyrenoid-penetrating thylakoid (PPT). The PPT lumen possesses a specifically localized θ-Type carbonic anhydrase (CA), which is critical a critical factor for the operation of diatom CO₂-concentrating mechanism (CCM). We call this pyrenoid structure possessing the PPT with luminal θ-CA the “CO₂-evolving machinery” that supply concentrated CO₂ to Rubisco condensate. Our approach to feed diatoms cells with artificial photo-amino acids (pAA) followed by in situ photo-crosslinking of protein containing pAA revealed several new proteins that should be associated with Rubisco enzyme. A GFP-tagging localization of one of these proteins clearly showed the location surrounding the periphery of the pyrenoid. As this protein did not show any sequence homology to any known proteins, we denoted this protein as Pyrenoid Shell (PyShell). PyShell genes occur 2 in the marine diatoms Phaeodactylum tricornitum and 6 in Thalassiosira peudonana. Single particle analysis by Cryo-electron microscopy showed purified TpPyShell structure that forms lattice sheets and the same structure was observed in vivo by cryo-electron tomogram that surrounds the pyrenoid. Genome editing knock-out of the two major TpPyShell1 and TpPyShell2 genes in T. pseudonana resulted in a clear high-CO₂ requiring phenotype. The Cryo-ET analysis of KO mutants showed the disappearance of PyShell and PPT and pyrenoid structure taking fragmented spherical shapes. These PyShell KO mutants showed a null CCM phenotype that requires saturate photosynthesis more than 10 mM dissolved inorganic carbon that is an equivalent amount in seawater under 5 times more than current atmospheric pCO₂. PyShell gene sequence occur all known diatom genome and expressed in world oceans. These data indicate that diatom chloroplast structure and function and thus productivity are sustained by the novel skeletal protein structure of PyShell.