James Barrett

James Barrett

he/him

Understanding pyrenoid biology and evolution to aid engineering in plants

Publications

Below is a selection of my publications, you can find a comprehensive list on my Google Scholar profile.

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

Published in Nature Plants, 2024

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

Predicting Rubisco-Linker Condensation from Titration in the Dilute Phase

Published in Physical Review Letters, 2024

Abstract
The condensation of Rubisco holoenzymes and linker proteins into “pyrenoids,” a crucial supercharger of photosynthesis in algae, is qualitatively understood in terms of “sticker-and-spacer” theory. We derive semianalytical partition sums for small Rubisco-linker aggregates, which enable the calculation of both dilute-phase titration curves and dimerization diagrams. By fitting the titration curves to surface plasmon resonance and single-molecule fluorescence microscopy data, we extract the molecular properties needed to predict dimerization diagrams. We use these to estimate typical concentrations for condensation, and successfully compare these to microscopy observations.

Description
In this contribution, the York Physics of Pyrenoids Project (YP3) consortium joined forces to parametrise and experimentally validate a model for Rubisco dimerisation, and by proxy liquid-liquid phase separation. Using affinity measurements of variants of EPYC1 with different numbers of stickers for Rubisco using single molecular dilute binding assays (SMDBAs), based on Slimfield microscopy, as well as Surface Plasmon Resonance measurements a binding energy between EPYC1 and Rubisco could be described. By extending this binding energy for use in a statistical physics approach developed by Charley Schaefer to predict dimerization concentrations, a critical concentration for liquid-liquid phase separation (LLPS) could be predicted. Strikingly, the predicted values and observed concentrations compare execptionally well.

Cite: Payne-Dwyer, A. et al. (2024). "Predicting Rubisco-Linker Condensation from Titration in the Dilute Phase". Physical review letters, 132 (21), p.218401. https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.132.218401

Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii

Published in bioRxiv, 2024

Abstract
Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

Description
In this study René Inckemann, Tanguy Chotel and colleagues in the lab of Tobias Erb developed a complete expression toolkit for the chloroplast in Chlamydomonas, taking a synthetic biology approach based on a Modular Cloning (MoClo) framework. Prior to this work, no systematic approach had been taken to understand heterologous expression in the Chlamydomonas chloroplast. René and Tanguy developed a high throughput approach to generate and characterize transplastomic lines that were generated by particle bombardment with huge number of synthetic and naturally occurring expression parts. I contributed to this study in the development of fluorescent protein reporters for the chloroplast (mScarlet-I, mCherry, mVenus and mCerulean). We also used vectors developed by René in our recent expression of the Chlorella pyrenoid linker protein (CsLinker) in Chlamydomonas, which you can read about here.

Cite: Inckemann, R. et al. (2024). "Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii" bioRxiv. (2024) https://www.biorxiv.org/content/10.1101/2024.05.08.593163v2