CONCURRENT AND CONVERGENT EVOLUTION OF A PROMISCUOUS MECHANISM TO PHASE SEPARATE EUKARYOTIC CARBON FIXATION

Eukaryotic algae perform approximately one third of global CO2 fixation in organelles called pyrenoids which supercharge fixation and are found across vast evolutionary taxa. Transplanting algal pyrenoids into pyrenoid-less crop plants to supercharge their CO2 fixation offers a promising strategy to increase yields. It has long been known that algal pyrenoids are densely packed with the CO2-fixing enzyme Rubisco. More recently it has emerged that Rubisco packaging and formation of the pyrenoid is underpinned by a dynamic process called liquid-liquid phase separation (LLPS) that is critical to pyrenoid function. Understanding of pyrenoid assembly and evolution outside of model species has been limited by the lack of sequence conservation of the proteins that drive LLPS of Rubisco across algal lineages, termed ‘linker’ proteins. To address this knowledge gap, a sequence-independent Fast Linker Identification Pipeline for Pyrenoids (FLIPPer) was developed to identify analogous linker proteins in unexplored pyrenoids. Assessment of the time-calibrated phylogenetic distribution of these linker proteins allowed identification of a shared timepoint for the convergent evolution of pyrenoids that coincided with plant-driven atmospheric oxygenation at the end of the Paleozoic Era. Detailed characterisation of a linker protein identified in the green microalga Chlorella sorokiniana (CsLinker) subsequently demonstrated its role as a bona fide pyrenoid linker protein. Structurally, it was shown that LLPS of Chlorella Rubisco is underpinned by multivalent binding of CsLinker to a highly conserved interface on the Rubisco large subunit which enables CsLinker to promiscuously phase separate other Rubiscos. This promiscuity enables CsLinker to functionally replace the native linker protein in the pyrenoid of Chlamydomonas, despite sharing no sequence homology with the native sequence. Excitingly, the promiscuity of CsLinker also extends to certain plant Rubiscos, where it demonstrated both in vitro and in planta Rubisco condensation. CsLinker therefore presents exciting future opportunities for engineering pyrenoids in crop plants.