James Barrett

James Barrett

he/him

Understanding pyrenoid biology and evolution to aid engineering in plants

Posts by Collection

portfolio

presentations

protocols

Linker purification protocol

Published:

Protocol for the soluble purification of linker proteins from algal pyrenoids. Used for the purification of Chlamydomonas (EPYC1) and Chlorella (CsLinker) linker proteins, as well as a host of other soluble algal pyrenoid proteins.

Golden gate assembly protocol

Published:

Protocol for golden gate assembly used in the Mackinder lab. Kindly typed up by visiting rotation student Mel Ludwig.

Rubisco purification protocol (15Q)

Published:

Protocol for the high quality purification of Rubisco, applied to green algae, seaweed and plants, yielding active, intact Rubisco holoenzyme complexes. This protocol was developed from an original protocol that included sucrose gradient centrifugation, which was limiting for the throughput of the purification. In this protocol, a 15Q ion exchange column is used. Lower quality ion exchange resins (e.g. HiTrap Q XL) are not appropriate for this.

Atto NHS ester protein labelling

Published:

Protocol for fluorescent labelling of purified proteins using Atto NHS ester dyes. This protocol has been used successfully for a range of proteins at concentrations down to ~0.4 mg mL-1. The addition of bicarbonate immediately prior to labelling improves reaction efficiency by increasing the pH into the optimal range for NHS ester chemistry.

publications

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

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

A modular high-throughput approach for advancing synthetic biology in the chloroplast of Chlamydomonas

Published in Nature Plants, 2025

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). "A modular high-throughput approach for advancing synthetic biology in the chloroplast of Chlamydomonas" Nat. Plants. (2025) https://www.nature.com/articles/s41477-025-02126-2

Pyrenoid Structure, Function, Evolution, and Characterization Across Diverse Lineages

Published in Annual Review of Plant Biology, 2026

Abstract
Pyrenoids are eukaryotic CO2-fixing organelles that are evolutionarily diverse, globally abundant, and critical to global carbon cycling. Despite being described over 200 years ago, the vast majority of our molecular understanding of pyrenoids has emerged only in the past decade. Here, we review the recent advances in characterizing pyrenoid structure, function, and evolutionary variation across lineages containing primary, secondary, and tertiary plastids of both red and green origins. We outline experimental frameworks that can be used to answer key questions about these enigmatic organelles. We discuss the utility of pyrenoids as model biomolecular condensates for investigating fundamental properties of liquid–liquid phase separation. Finally, we summarize how understanding convergently evolved pyrenoids across diverse lineages may be used to advance efforts to engineer functional pyrenoids into crop plants to enhance CO2 fixation for yield improvements and carbon dioxide removal.

Cite: *Barrett, J., *Nam, O., *Naduthodi, M.I.S., Mackinder L.C.M.. "Pyrenoid Structure, Function, Evolution, and Characterization Across Diverse Lineages." Annual Review Plant Biology. (2026). https://www.annualreviews.org/content/journals/10.1146/annurev-arplant-070225-034846

Sticker number modulates pyrenoid condensate assembly to support algal fitness

Published in bioRxiv, 2026

Abstract
The valency of intrinsically disordered proteins underpins liquid-liquid phase separation (LLPS), yet how this parameter shapes condensate function and cellular fitness remains poorly understood. Here we exploit the algal pyrenoid–a minimal, two component LLPS system–to directly link condensate properties to physiological performance. Pyrenoid assembly is driven by a disordered, multivalent Linker protein that binds Rubisco at symmetry-related surface sites, with the number of binding motifs (“stickers”) varying across species. Using Chlamydomonas reinhardtii, we systematically tuned sticker number from two to nine and examined effects on Rubisco condensation, pyrenoid architecture and CO2 fixation. Three stickers were sufficient for condensation in vitro, but at least four were required for pyrenoid assembly in vivo. Cryo-electron tomography and single-molecule tracking revealed that increasing sticker number enhances Rubisco packing and mobility, while time-resolved imaging and competition assays demonstrated that sticker number governs the kinetics of pyrenoid formation and determines cellular fitness under fluctuating carbon conditions. Our findings establish sticker number as an evolutionary tuning parameter that balances condensate formation, dynamics, and function, providing a quantitative framework for linking the molecular grammar of phase separation to biological fitness. Description

Cite: *Kumar, G., *Barrett, J., *Van der Stappen, P., et. al., "Sticker number modulates pyrenoid condensate assembly to support algal fitness." bioRxiv. (2026). https://www.biorxiv.org/content/10.64898/2026.01.27.701992v1

A spatial atlas of the seaweed CO2-fixation machinery reveals a unique Rubisco condensation mechanism

Published in bioRxiv (preprint), 2026

Abstract
Seaweeds (macroalgae) are major primary producers in coastal ecosystems, yet the molecular machinery underlying their CO2-concentrating mechanism (CCM) has remained undescribed. We fluorescently tagged 201 proteins associated with CO2 acquisition in the green seaweed Ulva (sea lettuce), assigning 68 to distinct pyrenoid subdomains. We identify Seaweed Ulva Pyrenoid Assembly 1 (SUPA1) as the core pyrenoid assembly factor and show that it condenses Rubisco via a previously undescribed mechanism: a disorder-to-helix transition upon binding, combined with steric occlusion that limits occupancy to four of the eight available Rubisco binding sites. This work provides the first large-scale spatial protein map of carbon fixation in a multicellular seaweed and reveals a mechanistically distinct route to pyrenoid-based Rubisco condensation within the green lineage.

Description
While pyrenoid-based CCMs have now been characterised at the molecular level in the unicellular model Chlamydomonas reinhardtii and, more recently, in the trebouxiophyte Chlorella and in hornworts, no molecular data existed for any multicellular seaweed prior to this study. Using recently developed genetic tools in Ulva compressa/mutabilis, we carried out a large-scale fluorescent protein-tagging screen, localising 160 of 201 tagged candidates and assigning 68 proteins to pyrenoid subdomains (matrix, periphery or the pyrenoid-traversing thylakoid). Strikingly, none of the core structural components that build the Chlamydomonas pyrenoid — including the Rubisco-condensing linker EPYC1 — have identifiable orthologues in Ulva, indicating that seaweed pyrenoid assembly has evolved independently.

Using our sequence-independent linker-identification pipeline, FLIPPer (originally developed for the Chlorella linker discovery described in our 2024 Nature Plants paper), we identified SUPA1 as the Ulva Rubisco-condensing protein. Cryo-EM, ITC and native mass spectrometry reveal that a single SUPA1 repeat binds across two Rubisco large subunits and one small subunit, undergoing a folding-upon-binding transition to form a short α-helix that was unstructured in isolation. Geometric clashes between adjacent binding sites limit simultaneous occupancy to four of the eight sites available on the Rubisco holoenzyme — a binding mode distinct from both the RbcS-binding EPYC1 (Chlamydomonas) and the RbcL-binding CsLinker (Chlorella), and compensated for by roughly 100-fold higher binding affinity. Together with CRISPR knockouts confirming SUPA1 is required for Rubisco condensation into the pyrenoid, and comparative analysis across the three characterised linker systems, this work extends our understanding of how eukaryotic CO2-concentrating mechanisms have convergently evolved across the green lineage, and establishes Ulva as a tractable multicellular model for pyrenoid engineering.

Cite: Barrett, J., Dégut, C., Sebiani Calvo, A. et al. "A spatial atlas of the seaweed CO2-fixation machinery reveals a unique Rubisco condensation mechanism." bioRxiv (2026). doi: 10.64898/2026.07.16.738900 https://www.biorxiv.org/content/10.64898/2026.07.16.738900v1.full

Molecular organization of the Chlorella sorokiniana pyrenoid

Published in bioRxiv (preprint), 2026

Abstract
Algae operate CO2-concentrating mechanisms (CCMs) that deliver concentrated CO2 to Rubisco packaged within a specialized microcompartment called a pyrenoid. Pyrenoids are globally important biomolecular condensates, but their convergent evolution means that molecular composition and architecture cannot be inferred across clades. Here we characterize the pyrenoid of the trebouxiophyte alga Chlorella sorokiniana. Using cryo-electron tomography, we provide an architectural overview of the pyrenoid and visualize pyrenoid-specific protein complexes. Quantitative proteomics and Rubisco co-immunoprecipitation coupled to mass spectrometry demonstrate that inorganic carbon delivery machinery is conserved across green algae, but pyrenoid structural components are not. In vitro reconstitution supports the role of two previously undescribed proteins: one in assembly of pyrenoid-traversing thylakoids (putative matrix-thylakoid tether; PMTT) and another in tethering starch to the Rubisco matrix (putative matrix-starch tether; PMST). In Nicotiana benthamiana, PMTT localizes to the thylakoid stromal lamellae and PMST to chloroplast starch granules. Our findings provide insight into the molecular logic of pyrenoid assembly, how proteins mediate condensate-membrane and condensate-starch interactions, and expand the pyrenoid engineering toolkit for plants.

Description
This study is a direct follow-on from our 2024 Nature Plants paper identifying the Chlorella Rubisco-condensing linker (CsLinker). Here we set out to build a full molecular and architectural picture of the Chlorella pyrenoid, using cryo-electron tomography (cryo-ET) to resolve its native ultrastructure and comparative proteomics/co-immunoprecipitation to identify its protein components.

Cryo-ET revealed a pyrenoid architecture clearly distinct from Chlamydomonas: rather than multiple tubules converging into a reticulated knot, Chlorella has a single pair of appressed pyrenoid-traversing thylakoids (PTTs) that bisect the Rubisco matrix, with small membrane fenestrations connecting the matrix to the surrounding stroma, and a previously undescribed braided membrane morphology along the PTTs. Subtomogram averaging identified a square-lattice protein array on the matrix-facing PTT membrane, consistent in size with the carbonic anhydrase CsCAH3.

Using the Rubisco-binding motif (RBM) of CsLinker as a search query, we identified three further RBM-containing proteins that co-immunoprecipitate with Rubisco: PMTT (a transmembrane protein that localizes to the PTTs and binds thylakoid lipids in vitro, particularly MGDG and SQDG), PMST (a CBM20 starch-binding protein that recruits Rubisco-CsLinker condensates onto starch granules), and RHOP (a rhodanese-domain matrix protein). All three can drive phase separation of Chlorella Rubisco in vitro, though only CsLinker reaches concentrations sufficient to do so efficiently on its own at physiological ratios — PMTT, PMST and RHOP instead appear to be recruited into pre-formed CsLinker-Rubisco condensates. Heterologous expression in Chlamydomonas and Nicotiana benthamiana confirmed domain-appropriate localization (PMTT to thylakoid membranes, PMST to starch), though neither protein could functionally complement the corresponding Chlamydomonas mith1 or saga1 mutants, indicating that despite superficially analogous roles, the two lineages have arrived at pyrenoid assembly through non-interchangeable, convergently evolved solutions. Together with our Ulva and Chlorella-linker work, this further expands the molecular toolkit available for engineering a pyrenoid-based CCM into C3 crop plants.

Cite: Naduthodi, M.I.S., Barrett, J., Pritchard, J. et al. "Molecular organization of the Chlorella sorokiniana pyrenoid." bioRxiv (2026). doi: 10.64898/2026.07.17.739135 https://www.biorxiv.org/content/10.64898/2026.07.17.739135v1.full

software

FLIPPer: Fast Linker Identification Pipeline for Pyrenoids

Published:

Fast Linker Identification Pipeline for Pyrenoids (FLIPPer) was built to identify functional analogs of canonical pyrenoid Linker proteins based on shared feature with EPYC1. This was used to identify the Chlorella sorokiniana Linker protein (CsLinker).

RubiCon: Rubisco Condensation Analysis Toolkit

Published:

RubiCon (Rubisco Condensation) was designed to analyze and quantify phase separation in LAMMPS molecular dynamics simulations of Rubisco and linker proteins. It provides a robust pipeline for detecting condensates, Calculating phase-specific properties (concentration, volume and analyzing dynamics (diffusion).

SEC_Fit

Published:

SEC_Fit is an open-source Python tool designed for the quantitative analysis of analytical Size-Exclusion Chromatography (SEC) data. It allows users to easily fit interactive Gaussian models to overlapping chromatographic peaks, estimate molecular weights based on column calibration standards, and calculate relative species abundance and stoichiometry directly from ÄKTA CSV exports.

Canary

Published:

Open hardware and firmware for continuous, networked gas monitoring in biological growth chambers. CO₂ and O₂ sensing, local touchscreen display, web interface, SD logging, and Grafana Cloud integration. Find the full documentation here

GG-calc: Golden Gate Assembly Calculator

Published:

An interactive pipetting-volume calculator for MoClo Golden Gate digestion–ligation reactions, with a searchable library of acceptor vectors, L0 parts, and end linkers so you don’t need to dig through a spreadsheet to find a sequence.

talks

teaching

Teaching experience 1

Undergraduate course, University 1, Department, 2014

This is a description of a teaching experience. You can use markdown like any other post.

Teaching experience 2

Workshop, University 1, Department, 2015

This is a description of a teaching experience. You can use markdown like any other post.