Biology subjects

McCormick, A. J.

Publications and source records attributed to McCormick, A. J..

10 recordsLinked to original sources

bioRxiv2026

CyanOperon: an operon building expansion for the CyanoGate MoClo toolkit

Operons are gene clusters controlled by a single promoter that enable coordinated translation from a single messenger RNA. Here we describe an expansion of the CyanoGate MoClo toolkit to assemble synthetic operons. The versatile CyanOperon system includes two Level 0 acceptor vectors for building interchangeable promoter-ribosome bind site (RBS) combinations and 15 Level 1 acceptor vectors for the hierarchical assembly and expression of up to six genes within a single operon. The system also allows for operon assembly into a self-replicating vector or for chromosomal integration by homologous recombination. To showcase CyanOperon, we assembled the violacein biosynthesis pathway as an operon and demonstrated violacein production in Escherichia coli. We then constructed a 20-part RBS library to examine how spacer length between the Shine-Dalgarno sequence and start codon affects translation in E. coli and the model cyanobacterium Synechocystis sp. PCC 6803. Lastly, we compared the expression of up to three operonic fluorescent markers following chromosomal integration or from a self-replicating vector in E. coli and Synechocystis sp. PCC 6803. The CyanOperon system is publicly available and can be readily integrated with other MoClo systems to accelerate the development of standardized operon assemblies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/707249v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@c9e14corg.highwire.dtl.DTLVardef@1e6566borg.highwire.dtl.DTLVardef@133c63forg.highwire.dtl.DTLVardef@1c5dff_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Biogenesis, engineering and function of membranes in the CO2-fixing pyrenoid

Summary ParagraphApproximately one-third of global CO2 assimilation is performed by the pyrenoid1, a liquid-like organelle found in most algae and some plants2. Specialized membranes are hypothesized to drive CO2 assimilation in the pyrenoid by delivering concentrated CO23,4, but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana. SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO2-limiting conditions, providing evidence for the function of membrane tubules in CO2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

molecular biology↗

bioRxiv2024

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

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 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.

plant biology↗

bioRxiv2023

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated proto-pyrenoid Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well studied pyrenoid, Chlamydomonas reinhardtii (1). Here we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesised as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, whilst also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and opening novel avenues for engineering starch morphology.

plant biology↗

bioRxiv2023

Engineering the highly productive cyanobacterium Synechococcus sp. PCC 11901

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology. On sentence summaryGenetic parts were characterised in Synechococcus sp. PCC 11901, including a tightly regulated inducible promoter system, efficient CRISPRi and a novel markerless Cas12a genome editing approach.

synthetic biology↗

bioRxiv2023

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2- concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules. One-sentence summaryIn Chlamydomonas, the pyrenoid-localized bestrophin-like protein BST4 is a putative ion channel involved in pH regulation of the thylakoid lumen, possibly by mediating bicarbonate transport.

plant biology↗

bioRxiv2020

Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

Photosynthetic CO2 fixation in plants is limited by the inefficiency of the CO2-assimilating enzyme Rubisco (D-ribulose-1,5-bisphosphate carboxylase/ oxygenase)1-3. In plants possessing the C3 pathway, which includes most major staple crops, Rubisco is typically evenly distributed throughout the chloroplast stroma. However, in almost all eukaryotic algae Rubisco aggregates within a microcompartment known as the pyrenoid, in association with a CO2-concentrating mechanism that improves photosynthetic operating efficiency under conditions of low inorganic carbon4. Recent work has shown that the pyrenoid matrix is a phase-separated, liquid-like condensate5. In the alga Chlamydomonas reinhardtii, condensation is mediated by two components: Rubisco and the linker protein EPYC1 (Essential Pyrenoid Component 1)6,7. Here we show that expression of mature EPYC1 and a plant-algal hybrid Rubisco leads to spontaneous condensation of Rubisco into a single phase-separated compartment in Arabidopsis chloroplasts, with liquid-like properties similar to a pyrenoid matrix. The condensate displaces the thylakoid membranes and is enriched in hybrid Rubisco containing the algal Rubisco small subunit required for phase separation. Promisingly, photosynthetic CO2 fixation and growth is not impaired in stable transformants compared to azygous segregants. These observations represent a significant initial step towards enhancing photosynthesis in higher plants by introducing an algal CO2-concentrating mechanism, which is predicted to significantly increase the efficiency of photosynthetic CO2 uptake8,9.

plant biology↗

bioRxiv2020

Chickpea (Cicer arietinum L.) root system architecture adaptation to initial soil moisture improves seed development in dry-down conditions

Soil water deficit (WD) impacts vascular plant phenology, morpho-physiology, and reproduction. Chickpea, which is mainly grown in semi-arid areas, is a good model plant to dissect mechanisms involved in drought resistance. We used a rhizobox-based phenotyping system to simultaneously and non-destructively characterise root system architecture (RSA) dynamics and water use (WU) patterns. We compared the drought-adaptive strategies of Teketay to the drought-sensitive genotype ICC 1882 in high and low initial soil moisture without subsequent irrigation. WD restricted vegetative and reproductive organ biomass for both genotypes. Teketay displayed greater adaptability for RSA dynamics and WU patterns and revealed different drought adaptive strategies depending on initial soil moisture: escape when high, postponement when low. These strategies were manifested in distinct RSA dynamics: in low initial soil moisture, its reduced root growth at the end of the vegetative phase was followed by increased root growth in deeper, wetter soil strata, which facilitated timely WU for seed development and produced better-developed seeds. We demonstrate that RSA adaptation to initial soil moisture is one mechanism by which plants can tolerate WD conditions and ensure reproduction by producing well-developed seeds. Our approach will help in identifying the genetic basis for large plasticity of RSA dynamics which enhances the resilience with which crops can optimally adapt to various drought scenarios. HighlightRoot system architecture and water use patterns change dynamically for distinct drought adaptation strategies in chickpea.

plant biology↗

bioRxiv2020

The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

molecular biology↗

bioRxiv2018

CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and exchange of parts between different labs and biological systems. However, a standardised Modular Cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential in biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. We have generated a suite of parts and acceptor vectors for making i) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and ii) transient multigene expression and repression systems using known and novel replicative vectors. We have tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The system is publicly available and can be readily expanded to accommodate other standardised MoClo parts.

synthetic biology↗