A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage

Abstract
CO₂ fixation is commonly limited by inefficiency of the CO₂-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO₂ in phase-separated condensates called pyrenoids, which complete up to one-third of global CO₂ fixation. Condensation of Rubisco in pyrenoids is dependent on interaction with disordered linker proteins that show little conservation between species. We developed a sequence-independent bioinformatic pipeline to identify linker proteins in green algae. We report the linker from Chlorella and demonstrate that it binds a conserved site on the Rubisco large subunit. We show that the Chlorella linker phase separates Chlamydomonas Rubisco and that despite their separation by ∼800 million years of evolution, the Chlorella linker can support the formation of a functional pyrenoid in Chlamydomonas. This cross-species reactivity extends to plants, with the Chlorella linker able to drive condensation of some native plant Rubiscos in vitro and in planta. Our results represent an exciting frontier for pyrenoid engineering in plants, which is modelled to increase crop yields.

Description
Since the discovery of the protein EPYC1, which is the molecular glue of the Chlamydomonas pyrenoid, in 2016 by Mackinder et al., it was conceived that similar mechanisms to phase separate carbon fixation in pyrenoids must exist elsewhere. Considerable weight was granted to this hypothesis in 2023 when Zhen Guo Oh, Warren Ang and colleagues in Oliver Mueller-Cajar’s lab at NTU discovered an analagous protein, PYCO1, in the red algal diatom Phaeodactylum tricornutum. The hypothesis that there are EPYC1 and PYCO1 analogues in other pyrenoids was the motivating factor to build a bioinformatic tool to aid in the identification of candidate proteins. The Fast Linker Identification Pipeline for Pyrenoids (FLIPPer) developed in this study works on the basis of identifying proteins with properties essential to the function of EPYC1 and PYCO1, in a sequence-independent manner. FLIPPer filters for proteins that have low sequence complexity, contain structural elements consistent with those of EPYC1/PYCO1, identifies tandem repeats using XSTREAM, and filters for disorder using metapredict. This approach achieves high selectivity (~0.1% of input sequences) across genomes, and allows for more intensive characterisations of the candidate sequences.

In this study, we used FLIPPer to identify the pyrenoid linker protein in the green alga Chlorella sorokiniana. We were intererested in Chlorella, as despite their highly similar cellular and pyrenoid appearances, Chlorella and Chlamydomonas diverged ~800 million years ago. The linker protein from Chlorella bears little sequence homology to that of Chlamydomonas (EPYC1) nor Phaeodactylum (PYCO1), suggesting they are of independent origin. In line with this, a structural characterisation of the binding site of the Chlorella linker to Rubisco indicated a novel binding interface. Excitingly, the Chlorella linker was found to utilise the large subunit of Rubisco to underpin its function in cross-linking Rubiscos in the pyrenoid matrix. In contrast to the small subunit binding site of EPYC1, the Chlorella binding site is highly conserved across the green lineage, owing to its plastid encoding. In this study we demonstrate that the conservation of this binding site permits a level of promiscuity to the Chlorella linker, allowing it to cross-link and phase separate non-cognate Rubiscos. This behaviour extends to Solanaceae plant Rubiscos, providing an exciting prospect fast-tracking engineering of pyrenoid machinery in important crop plants (Tomato, Potato, Pepper etc.).

Cite: Barrett, J., Naduthodi, M.I.S., Mao, Y. et al. "A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage." Nat. Plants. (2024). https://www.nature.com/articles/s41477-024-01812-x