WO2017196790A1 - Composants algaux du mécanisme de concentration de carbone de pyrénoïde - Google Patents

Composants algaux du mécanisme de concentration de carbone de pyrénoïde Download PDF

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WO2017196790A1
WO2017196790A1 PCT/US2017/031669 US2017031669W WO2017196790A1 WO 2017196790 A1 WO2017196790 A1 WO 2017196790A1 US 2017031669 W US2017031669 W US 2017031669W WO 2017196790 A1 WO2017196790 A1 WO 2017196790A1
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epycl
rubisco
pyrenoid
amino acid
protein
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PCT/US2017/031669
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Luke C.M. MACKINDER
Moritz T. MEYER
Tabea METTLER-ALTMANN
Leif PALLESEN
Mark Stitt
Howard GRIFFITHS
Martin C. JONIKAS
Original Assignee
Mackinder Luke C M
Meyer Moritz T
Mettler-Altmann Tabea
Pallesen Leif
Mark Stitt
Griffiths Howard
Jonikas Martin C
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Publication of WO2017196790A1 publication Critical patent/WO2017196790A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/08—Fusion polypeptide containing a localisation/targetting motif containing a chloroplast localisation signal
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2

Definitions

  • the invention relates to molecules (e.g., algal polypeptides and the nucleic acids encoding them), their transport into the pyrenoid, and their involvement in metabolic reactions such as photosynthesis.
  • molecules e.g., algal polypeptides and the nucleic acids encoding them
  • photosynthesis in chloroplasts is improved by a carbon concentrating mechanism that includes a pyrenoid.
  • Components of the pyrenoid's carbon concentrating mechanism are identified. They are used, inter alia, to manipulate metabolic processes, thereby providing improvements in photo- synthesis, increased biomass production and crop yield in photosynthetic organisms, and novel processes and products resulting therefrom.
  • Carbon fixation or carbon assimilation which converts inorganic carbon (CO2) into organic compounds, can occur through photosynthesis by cyanobacteria, algae, and land plants. They grow by fixing inorganic carbon and synthesizing organic compounds using energy from the sun.
  • Carboxysomes in cyanobacteria and the pyrenoid in algae are packed with ribulose-l,5-biphosphate carboxylase/oxygenase (RuBisCO), which is the enzyme (EC 4.1.1.39) catalyzing the rate-limiting step in the Calvin-Benson cycle.
  • RuBisCO ribulose-l,5-biphosphate carboxylase/oxygenase
  • Carboxysomes and the pyrenoid increase the local concentration of carbon dioxide around RuBisCO to fix carbon dioxide at a higher rate.
  • Photosynthesis occurs in two stages. In the first, light-dependent (or light) reactions capture the energy of light and store the energy as ATP and NADPH. During the second, the light-independent (or dark) reactions use these products to capture and reduce carbon dioxide. Since water is used as the electron donor in oxygenic photosynthesis, CO2 + 2H2O + photons ⁇ [CH2O] + O2 + H2O. Water is a reactant in the first stage and a product in the second stage. Thus, canceling one water molecule on each side of the equation, gives CO2 + H2O + photons ⁇ [CH2O] + O2.
  • the light-independent reactions use RuBisCO to convert carbon dioxide into a three-carbon sugar in a process called the Calvin-Benson cycle.
  • This simple sugar is an intermediate in the synthesis of carbohydrates and other organic compounds, a precursor for lipid and amino acid biosynthesis, and a source of energy for cellular respiration.
  • RuBisCO is one of many enzymes in the Calvin-Benson cycle. During carbon fixation, ribulose-l,5-bisphosphate and carbon dioxide are this enzyme's substrates. RuBisCO also catalyzes a reaction between ribulose- 1,5-bisphosphate and molecular oxygen instead of carbon dioxide. Only 3- 10 carbon dioxide molecules are fixed per molecule of enzyme each second. Under most conditions, the speed of RuBisCO responds positively to an increase in the concentration of carbon dioxide when the amount of light is not limiting. Enzyme side activities can lead to useless or inhibitory byproducts (e.g., xylulose-l,5-bisphosphate).
  • the products of the oxygenase reaction are phosphoglycolate and 3-phosphoglycerate.
  • Phosphoglycolate is recycled through a sequence of reactions called photorespiration, where two molecules of phosphoglycolate are converted into one molecule of 3-phosphoglycerate, which can reenter the Calvin-Benson cycle, and the release (loss) of carbon dioxide and some nitrogen into the environment.
  • Photorespiration uses energy but does not produce sugars.
  • RuBisCO's slow enzymatic activity and its inability to prevent reacting with oxygen greatly reduces the photosynthetic capacity of many land plants.
  • Some plants, many algae, and cyanobacteria have addressed this problem by increasing the concentration of CO2 around RuBisCO through carbon concentrating mechanisms, which include C 4 carbon fixation, crassulacean acid metabolism, and use of carboxysomes and the pyrenoid.
  • RuBisCO is the most abundant protein in leaves, accounting for up to 50% of soluble leaf protein in C3 plants (20-30% of total leaf nitrogen) and 30% of soluble leaf protein in C 4 plants (5-9% of total leaf nitrogen). Thus, it would be difficult to increase production of this rate-limiting enzyme. Given its importance in the biosphere, increasing RuBisCO activity by genetic engineering of pyrenoid components and transfer of its carbon concentrating mechanism addresses long-felt needs in the fields of synthetic biology, food security, energy independence, adaptation to climate change, and mitigation of its long-term effects.
  • the inventors describe below their identification of components of the pyrenoid, essential proteins transported into the pyrenoid, and manipulation of metabolic processes with such proteins or variants thereof. Products, compositions, and processes for using and making them are described as different aspects of the invention. They provide alternatives to the transfer of C 4 carbon fixation, crassulacean acid metabolism, or carboxysomes into land plants. Further advantages and improvements are described below or would be apparent from the disclosure herein.
  • EPYC1 or a variant protein thereof is provided.
  • the protein comprises a polypeptide domain having (i) a length of at least 56-63 contiguous amino acids and (ii) one or more variations from the EPYC1 amino acid sequence (SEQ ID NO: 1); wherein said variations are 1-15 amino acid substitutions, 1-7 amino acid deletions, 1-7 amino acid insertions, or a combination thereof as compared to SEQ ID NO: 1.
  • SEQ ID NO: 1 the EPYC1 amino acid sequence
  • the latter can be used to identify a similar sequence (SEQ ID NO: 2) in another green alga.
  • the polypeptide domain may comprise at least residues 1-51, residues 52-114, residues 115-174, residues 175-235, residues 236-291, or residues 292-317 of SEQ ID NO: 1, which may or may not have been mutated from the amino acid sequence of SEQ ID NO : 1 by 1-15 amino acid substitutions, 1-7 amino acid deletions, 1-7 amino acid insertions, or a combination thereof.
  • the polypeptide domain may or may not comprise residues 1-51, residues 52-114, residues 115-174, residues 175-235, residues 236-291, or residues 292-317 of SEQ ID NO: 1.
  • Fusion proteins may comprise the polypeptide domains and heterologous domains.
  • EPYC1 or a variant protein may be used to bind RuBisCO in a cell, wherein the cell does not contain a native copy of EPYC1 or an ortholog thereof.
  • the cell or an organism from which the cell is obtained may be transformed with a nucleic acid (e.g., expression vector) encoding EPYC1 or the variant protein.
  • the nucleic acid is manipulated to enable transformation and/or expression before transformation, then a functional characteristic is assessed (e.g., quantitative measurement or qualitative determination) for a change in RuBisCO enzymatic activity or other cellular function after transformation.
  • EPYC1 or a variant protein may be used to bind RuBisCO in a green alga, wherein the green alga contains an inactivated or silenced native copy of EPYC1 or an ortholog thereof.
  • the green alga may be transformed with a nucleic acid (e.g., expression vector) encoding EPYC1 or the variant protein.
  • the nucleic acid is manipulated to enable transformation and/or expression before transformation, then a functional characteristic is assessed (e.g., quantitative measurement or qualitative determination) for a change in RuBisCO enzymatic activity or other cellular function after transformation.
  • EPYC1 or a variant protein may be used to bind RuBisCO in a green alga, wherein the green alga contains at least one native copy of EPYC1 or an ortholog thereof.
  • the green alga may be transformed with a nucleic acid (e.g., expression vector) encoding EPYC1 or the variant protein.
  • the nucleic acid is manipulated to enable transformation and/or expression before transformation, then a functional characteristic is assessed (e.g., quantitative measurement or qualitative determination) for a change in RuBisCO enzymatic activity or other cellular function after transformation.
  • EPYC1 or a variant protein may be used to bind RuBisCO in a cell containing a pyrenoid, wherein expression of said EPYC1 or variant protein at least increases EPYC1 expression to promote RuBisCO localization to the pyrenoid or increases the size of the pyrenoid.
  • a heterologous domain is at least ten, at least 20, at least 40, at least 80, at least 160, at least 320, at least 640, or at least 1280 contiguous amino acids that are not in the amino acid sequence of EPYC1.
  • the heterologous domain may have an enzymatic activity, or another cellular function such as a substrate or binding partner for another protein.
  • the heterologous domain may be identical or similar in amino acid sequence to another molecular component of the carbon concentrating mechanism, or be a functional equivalent thereof.
  • the fusion protein is expressed in a host cell that does not express a host protein containing the heterologous domain, EPYCl, and/or the variant protein.
  • the host cell expressing the fusion protein may also express a host protein containing the heterologous domain, EPYCl, and/or the variant protein.
  • the fusion protein could comprise (i) EPYCl or a variant protein linked to (ii) a heterologous domain that is not present in EPYCl; the heterologous domain is translated before or after the EPYCl or variant protein (i.e., one is N- or C-terminal to the other in the fusion protein).
  • the host cell expressing the fusion protein may or may not have an active, mutated, inactivated, or silenced copy of a host protein containing the heterologous domain, EPYCl, and/or the variant protein.
  • the heterologous domain has a finite length (e.g., not more than 10,000, not more than 5000, or not more than 2000 contiguous amino acids).
  • a nucleic acid or expression vector encoding the aforementioned protein may be transformed into a cell or organism.
  • the cell may be obtained from the transformed organism.
  • the cell or organism may or may not be photosynthetic; the cell or organism may or may not have a C3 metabolic pathway, C 4 metabolic pathway, crassulacean acid metabolic pathway, carbon concentrating mechanism, or carboxysome.
  • the cell may be derived from or the organism may be a cyanobacterium, red or green alga, or embryophyte.
  • the embryophyte may be a C3 or C 4 plant, such as Arabidopsis, soybean, a rice, tobacco, a wheat, maize, or a grass (e.g., sugar cane).
  • the cell or organism may or may not produce or be a source for biomass, dietary protein, oils, and/or grains (e.g., a cereal or legume).
  • Expression of EPYCl or a variant protein in a cell may at least increase the rate of production of biomass, reduce the requirement for fertilizer and/or irrigation, increase CO2 assimilation per unit of RuBisCO, decrease the rate of oxygenation of ribulose-l,5-biphosphate catalyzed by RuBisCO, enhance RuBisCO's catalytic rate, or any combination thereof.
  • Expression of EPYCl or a variant protein in a cell may cause at least some of the RuBisCO to cluster such that the mean center-to-center distance between molecules of RuBisCO is decreased as compared to the absence of EPYCl or variant protein expression in the cell.
  • FIG. 1 EPYCl is an abundant pyrenoid protein.
  • FIG. 1A TEM images of Chlamydomonas reinhardtii whole cells and pyrenoid-enriched pellet fraction from cells grown at low CO2. Arrow in the panel labeled "Whole cell” indicates the pyrenoid; three arrows in the panel labeled “Pellet” indicate pyrenoid-like structures. Scale bar: 2 pm.
  • FIG. IB Mass- spectrometric analysis of 366 proteins in pyrenoid-enriched pellet fractions from low- and high-C02 grown cells (mean of 4 biological replicates).
  • RbcL, RBCS, EPYCl and RCA1 are abundant in low CO2 pellets (determined by intensity-based absolute quantification (iBAQ); y-axis). Additionally, these proteins showed increased abundance in low CO2 compared to high CO2 pellets (determined by label-free quantification (LFQ); x-axis).
  • Fig. 1C Confocal microscopy of EPYCl-Venus and RBCSl-mCherry co- expressed in wild-type cells. Scale bar: 5 pm.
  • EPYC1 is an essential component of the carbon concentrating mechanism.
  • Fig. 2A EPYC1 protein levels in WT and epycl mutant cells grown at low and high CO2 were probed by Western blotting with anti- EPYC1 antibodies. Anti-tubulin is shown as a loading control.
  • Fig. 2B Growth phenotypes of WT, epycl and three lines complemented with EPYCl . Serial 1 : 10 dilutions of WT, epycl, epycl wEPYCl, epycl v.
  • EPYCl- mCherry and epycl EPYCl-Venus lines were spotted on TP minimal medium and grown at low and high CO2 under 500 pmol photons nr 2 s 1 illumination.
  • FIG. 3 EPYCl is essential for RuBisCO aggregation in the pyrenoid.
  • FIG. 3A Representative TEMs of WT and epycl cells grown at low CO2.
  • Fig. 3A Representative TEMs of WT and epycl cells grown at low CO2.
  • FIG. 3G Representative images of anti-RuBisCO immunogold labeling of WT and epycl cells grown at low CO2. Gold particles were enlarged lOx for visibility.
  • FIG. 4 EPYC1 forms a complex with RuBisCO.
  • Fig. 4A Anti-FLAG co-immunoprecipitations (co-IPs) of WT cells expressing Venus-3xFLAG, EPYCl-Venus-3xFLAG and RBCSl-Venus-3xFLAG are shown.
  • co-IP Anti-FLAG co-immunoprecipitations
  • FT flow-through
  • wash wash
  • 3xFLAG elution FLAG Elu.
  • Boil. Elu. boiling elution
  • EPYC1 consists of four nearly identical repeats.
  • Fig. 4C Each repeat has a highly disordered domain (light shading) and less disordered domain (dark shading) containing a predicted alpha-helix (thick line) rich in charged residues.
  • Fig. 4D Amino acid alignments of the four repeats (SEQ ID NOS: 3-6) are shown. Asterisks indicate residues that are identical in all four repeats.
  • N, S, T and W are polar residues.
  • K and R are basic residues; D and E are acid residues.
  • A, G, L, P and V are nonpolar or hydrophobic residues. A conservative substitution replaces a residue with another in the same class.
  • Figs. 4E-4F Two models illustrate how EPYC1 could bind the RuBisCO holoenzyme in a manner that is compatible with the observed packing of RuBisCO in the pyrenoid.
  • FIG. 4E EPYC1 and RuBisCO could form a co-dependent network. If each EPYC1 can bind four RuBisCO holoenzymes, and each RuBisCO holoenzyme can bind eight EPYCls, eight EPYC1 proteins could connect each RuBisCO to twelve neighboring RuBisCOs.
  • EPYC1 could form a scaffold onto which RuBisCO binds. Both arrangements could expand indefinitely in every direction. For clarity, the spacing between RuBisCO holoenzymes was increased and EPYC1 is depicted in light and dark shading for contrast.
  • Figure 5 An illustration of Chlamydomonas reinhardtii in cross section drawn by Ninghui Shi.
  • FIG. 6 A schematic of the carbon concentrating mechanism (CCM) in Chlamydomonas reinhardtii drawn by Moritz Meyer.
  • Chloroplast proteins encoded in the nuclear genome must be transported back to the chloroplast, and imported through at least two chloroplast membranes. In most, but not all cases, nuclear-encoded chloroplast proteins are translated with a transit peptide that is at the N-terminus of the protein precursor and cleaved after transport across the chloroplast membranes. Sometimes the transit sequence is found on the C-terminus of the protein, or within the functional part of the protein.
  • chloroplast proteins After a polypeptide bound for the chloroplast is synthesized on a cytosolic ribosome, an enzyme specific to chloroplast proteins phosphory- lates many (but not all) of them in their transit peptides. Phosphorylation helps many proteins bind the polypeptide, keeping it from folding prematurely. This is important because it prevents chloroplast proteins from assuming their active form and carrying out their chloroplast functions in the wrong place— the cytosol. At the same time, they have to keep just enough shape so that they can be recognized by the chloroplast. Such chap- erones also help import the polypeptide into the chloroplast.
  • chloroplast proteins bound for the stroma must pass through two protein complexes—the TOC (translocon on the outer chloroplast membrane) complex and the TIC (translocon on the inner chloroplast mem- brane) translocon.
  • the TOC translocon on the outer chloroplast membrane
  • the TIC translocon on the inner chloroplast mem- brane
  • Land plants contain chloroplasts that are generally lens-shaped, 5- 8pm in diameter and l-3pm thick. Greater diversity in chloroplast morphol- ogy exists among algae, which often contain a single chloroplast that can be shaped like a cup (e.g., Chlamydomonas). In some algae, the chloroplast takes up most of the cell, with pockets for the nucleus and other organelles. All chloroplasts have at least three membranes: the outer chloroplast membrane, the inner chloroplast membrane, and the thylakoid system. Chloro- plasts that are the product of secondary endosymbiosis may have additional membranes surrounding these three. Between outer and inner chloroplast membranes is the intermembrane space.
  • the chloroplast stroma a semi-gel-like fluid that makes up much of a chlo- roplast's volume, and in which the thylakoid system floats.
  • Nucleoids of chloroplast DNA, chloroplast ribosomes, the thylakoid system, and many proteins (including RuBisCO) can be found floating around in the stroma of land plants.
  • the Calvin-Benson cycle which fixes carbon dioxide into sugar, takes place in the stroma.
  • Thylakoids are membranous sacs where there is chlorophyll and the light reactions of photosynthesis occur. In most vascular plant chloroplasts, the thylakoids are arranged in stacks called grana, though in certain C 4 plant chloroplasts and some algal chloroplasts, the thylakoids are free floating.
  • Algae contain pyrenoids. They are not found in higher plants.
  • the pyrenoid is a roughly spherical and highly refractive body, which in many algae is a site of starch accumulation.
  • the enzyme RuBisCO is found in the pyrenoid.
  • the pyrenoid is associated with the carbon concentrating mechanism that improves the operating efficiency of carbon assimilation by increasing the CO2 concentration around RuBisCO, and overcomes diffusive limitations in aquatic photosynthesis with inorganic carbon transporters at the plasma membrane and chloroplast envelope and carbonic anhydrases.
  • the carbon concentrating mechanism actively suppresses RuBisCO oxygenase activity and associated photorespiration.
  • RuBisCO usually consists of two protein subunits: the large chain and the small chain.
  • the large-chain gene is encoded by the chloroplast genome in plants. There are typically several small-chain genes in the nuclear genome of plants, and the small chains are imported to the stromal compartment of chloroplasts from the cytosol by crossing the outer chloroplast membrane. Binding sites for RuBisCO's substrate, ribulose 1,5-bisphos- phate, are located in dimerized large chains where amino acid residues from each large chain contribute to the binding sites. A total of eight large chains (or four dimers) and eight small chains assemble into a larger complex.
  • the pyrenoid requires a specific amino acid sequence at two surface-exposed a-helices of the RuBisCO small subunit. Higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid.
  • Chlamydomonas proteins expressed in a land plant e.g., transient expression in Nicotiana tabacum leaves and stable expression in Arabidopsis thaliana
  • Chlamydomonas proteins that are putative components of the carbon concentrating system can be expressed transiently or stably in bacteria (e.g., cyanobacterium), algae (e.g., Chlorophyta such as Chlamydomonas reinhardtii, Volvox carteri, Ostreococcus tauri, and Ulva lactuca), and land plants (e.g., C 4 or C3 plants such as rice, wheat, and soybean).
  • Binary expression vectors carrying the genes that encode each protein component of the carbon concentrating system of algae can be generated by PCR amplification of cDNA or genomic DNA, and subsequent Gateway cloning.
  • Gene expression may be under the control of a constitutive or inducible promoter, slicing signals, 5'- and 3'-untranslated regions and a terminator that are derived from the cauliflower mosaic virus 35S gene, nopaline synthase (NOS), T-DNA, other viruses, and microbes.
  • a constitutive or inducible promoter slicing signals, 5'- and 3'-untranslated regions and a terminator that are derived from the cauliflower mosaic virus 35S gene, nopaline synthase (NOS), T-DNA, other viruses, and microbes.
  • NOS nopaline synthase
  • Stop codons are removed to allow in-frame fusion at the N- or C-terminus with a transit peptide for transport into chloroplasts or other subcellular compartments, an affinity tag (e.g., calmodulin binding protein, FLAG, glutathione-S-trans- ferase, polyhistidine, hemaglutinin, maltose binding protein, c-Myc, strep- tavidin, SUMO, and thioredoxin) for detection or purification, a fluorescent protein to localize the fusion within the chloroplast or pyrenoid, specific protease cleavage sites, and other functional protein domains.
  • an affinity tag e.g., calmodulin binding protein, FLAG, glutathione-S-trans- ferase, polyhistidine, hemaglutinin, maltose binding protein, c-Myc, strep- tavidin, SUMO, and thioredoxin
  • An expression vector can be introduced into a cell by Agrobacterium-mediated gene transfer, agroinfiltration, chemical transfection, electroporation, lipofection, microinjection, or particle gun. See, for example, Barampuram & Zhang, Plant Chromosome Engineering 701: 1-35 (2010).
  • Such genetic engineering reagents and techniques facilitate identification of components of the carbon concentrating mechanism, transfer of one or more of those components into a host cell, modification of the RuBisCO small subunit in a chloroplast-containing cell, inactivation or silencing of carbonic anhydrases in the chloroplast stroma and/or thylakoid lumen, addition or subtraction of certain host proteins, mutation of host gene functions, and improvements of plant functional traits by genetic modification and transfer.
  • Fusion of a heterologous protein domain with EPYC1 can target the heterologous domain to the chloroplast. Fusion between a heterologous protein domain and the RuBisCO small subunit (or its transit peptide) could also be used to target the chloroplast. Localization to the pyrenoid through association with RuBisCO may be obtained by fusing one or more of EPYCl's four repeats with a heterologous protein domain.
  • Chlamydomonas reinhardtii The genetics of Chlamydomonas reinhardtii (see Mussgnug, Appl.
  • Chlorophyta green algae
  • Rhodophyta red algae
  • Cyanophyta cyanobacteria
  • Glaucophyta glaucophyte algae
  • cereals especially maize (Zea mays), rices (e.g., Oryza sativa and Oryza glaberrima), and wheats (e.g., Triticum aestivum and Triticum durum)
  • legumes especially soybean (Glycine
  • Molecular components of the carbon concentrating mechanism may be identified and isolated in green algae generally (e.g., C. globosa and V. globator), but not in cyanobacteria, red algae, and embryo- phytes.
  • Other components of the algal carbon concentrating mechanism may also need to be transferred along with EPYC1 such as, for example, the large subunit of RuBisCO, small subunits RuBisCO, inorganic carbon transporters, carbonic anhydrases, etc.
  • the transfer of the carbon concentrating machinery into a cell from photosynthesizing organisms may be confirmed by measuring photosynthetic efficiency of the genetically modified organism with growth in an artificially low concentration of carbon dioxide.
  • Increased production of biomass, and more efficient use of nitrogen and/or water i.e., a reduced need to fertilize and irrigate crops are some improvements that might be expected even under atmospheric concentration of CO2.
  • Biological carbon-fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn.
  • Approximately one-third of global carbon-fixation occurs in an overlooked algal organelle called the pyrenoid.
  • the pyrenoid contains the C02-fixing enzyme RuBisCO, and enhances carbon-fixation by supplying RuBisCO with a high concentration of CO2. Since the discovery of the pyrenoid over 130 years ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic.
  • EPYC1 Essential Pyrenoid Component 1
  • CCMs carbon concentrating mecha- nisms
  • RuBisCO the most abundant enzyme in the biosphere, fixes CO2 into organic carbon that supports nearly all life on Earth . Over the past three billion years, the enzyme became a victim of its own success, as it drew down the atmospheric CO2 concentration to trace levels and as the oxygen- producing reactions of photosynthesis filled our atmosphere with O2. In today's atmosphere, O2 competes with CO2 at RuBisCO's catalytic active site. This competition results in the production of the toxic compound phos- phoglycolate, which must be metabolized at the expense of energy and the loss of fixed carbon and nitrogen. Carboxylation of ribulose 1,5-bisphos- phate (RuBP) initiates the rate-limiting step of photosynthetic carbon fixation.
  • RuBP ribulose 1,5-bisphos- phate
  • CO2/O2 specificity factor Another kinetic constant referred to as the CO2/O2 specificity factor ( ⁇ ) is equal to the catalytic efficiency of carboxylation (V c /K c ) relative to the catalytic efficiency of oxygenation (V 0 /K 0 ).
  • CCMs carbon concentrating mechanisms
  • the pyrenoid is a spherical structure in the chloroplast stroma, discovered over 130 years ago. Pyrenoids have been found in nearly all of the major oceanic eukaryotic primary producers, and mediate approximately 28-44% of global carbon fixation.
  • the pyrenoid typically consists of a matrix surrounded by a starch sheath and traversed by membrane tubules continuous with the photosynthetic thylakoid membranes. The matrix is thought to primarily consist of tightly packed RuBisCO and its chaperone, RuBisCO activase.
  • RuBisCO is instead found in soluble form throughout the chloroplast stroma.
  • the molecular mechanism by which RuBisCO aggregates to form the pyrenoid matrix was previously unknown.
  • RuBisCO holoenzymes could bind each other directly through hydrophobic residues or (b) a linker protein may link RuBisCO holoenzymes together.
  • the second model is based on analogy to the well- characterized prokaryotic carbon concentrating organelle, the ⁇ - carboxysome, where RuBisCO aggregation is mediated by a linker protein consisting of repeats of a domain resembling the RuBisCO small subunit.
  • RuBisCO accumulation in the pyrenoid of the model alga Chlamydomonas reinhardtii is mediated by a disordered repeat protein EPYC1.
  • EPYCl is essential for a functional CCM.
  • the epycl mutant showed defective photoautotrophic growth in low CO2, which was rescued by high CO2 and by reintroducing the EPYC1 gene (Fig. 2B).
  • the epycl mutant retains a number of canonical pyrenoid characteristics, including correct localization in the chloroplast, the presence of a starch sheath under low CO2, and traversing membrane tubules, suggesting that these characteristics are regulated by mechanisms other than EPYCl. Additionally, the epycl mutant shows normal levels of the carbonic anhydrase CAH3, thought to be central to delivering CO2 to RuBisCO in the pyrenoid.
  • EPYCl is required for RuBisCO assembly into the pyrenoid.
  • Our observations of decreased pyrenoid size and apparent matrix density in epycl mutants could be explained by decreased whole-cell levels of RuBisCO.
  • Western blotting revealed no detectable difference in rbcL and RBCS abundance in epycl relative to WT cells or between cells grown at low and high CO2 levels (Fig. 3D). This result led us to hypothesize that the localization of RuBisCO was perturbed in epycl mutants.
  • RuBisCO a large fraction of RuBisCO was found outside the pyrenoid in the epycl mutant.
  • Immunogold-EM confirmed the mislocalization of RuBisCO in epycl .
  • EPYCl and RuBisCO are part of the same complex.
  • EPYCl could promote RuBisCO's localization to the pyrenoid by a physical interaction.
  • EPYCl and RuBisCO are part of the same supramolecular complex in the pyrenoid.
  • EPYCl protein consists of four nearly identical repeats.
  • each repeat consists of a predicted disordered domain and a shorter, less disordered domain containing a predicted alpha helix (Fig. 4C). Given that these repeats cover >80% of the EPYCl protein, it is likely that the RuBisCO binding site(s) are contained within the repeats.
  • EPYCl Two models are proposed for RuBisCO assembly into the pyrenoid matrix by EPYCl. If each repeat of EPYCl binds RuBisCO, EPYCl could link multiple RuBisCO holoenzymes together to form the pyrenoid matrix. Multiple RuBisCO binding sites on EPYCl could arrange RuBisCO into the hexagonal close packed or cubic close packed arrangement observed in recent cryoelectron tomography studies of the Chlamydomonas pyrenoid.
  • EPYC1 and RuBisCO could interact in one of two fundamental ways: (a) they could form a co-dependent network (Fig. 4E) or (b) EPYC1 could form a scaffold onto which RuBisCO binds (Fig. 4F).
  • the 60 amino acid repeat length of EPYC1 is sufficient to span the observed 2- 4.5nm gap between RuBisCO holoenzymes in the pyrenoid, and a stretched out repeat could potentially span the 15nm observed RuBisCO center-to- center distance.
  • a promising candidate for an EPYC1 binding site on RuBisCO would be the two alpha-helices of the small RuBisCO subunit (cf. Meyer et al., Proc. Natl. Acad. Sci. USA 109(47) : 19474-19479, 2012). When these helices are exchanged for higher-plant alpha-helices, pyrenoids fail to form and the CCM does not function, but holoenzyme assembly is normal.
  • Proteins with similar physicochemical properties to EPYC1 are present in a diverse range of eukaryotic algae.
  • the primary sequences of disordered proteins like EPYC1 are known to evolve rapidly compared to structured proteins, but their physicochemical properties are under selective pressure and are evolutionarily maintained.
  • proteins with similar properties were found in most pyrenoid-containing algae (e.g., Micromonas pusilla and Chlorella variabilis were exceptions), and appear to be absent in pyrenoid-less algae (e.g., Chlorella protothecoides, Cyanidioschyzon merolae, Galdieria sulphur aria, and Nannochloropsis gaditana), suggesting that EPYCl-like proteins may play similar roles in pyrenoids across eukaryotic algae. Discussion
  • RuBisCO packaging to form the matrix of the eukaryotic pyrenoid is achieved by a different mech- anism from that used in the well-characterized prokaryotic ⁇ -carboxysome.
  • aggregation of RuBisCO is mediated by the protein CcmM.
  • CcmM contains multiple repeats of a domain resembling the RuBisCO small subunit, and incorporation of these domains into separate RuBisCO holoenzymes is thought to produce a link between RuBisCO holoenzymes.
  • EPYCl could regulate
  • RuBisCO partitioning to the pyrenoid or RuBisCO kinetic properties The RuBisCO content of the pyrenoid changes in response to CO2 while total cellular RuBisCO stays constant (Fig. 3D).
  • EPYCl is required for RuBisCO localization to the pyrenoid
  • changes in EPYCl abundance and/or RuBisCO binding affinity could affect RuBisCO partitioning to the pyrenoid.
  • EPYCl was previously found to be upregu- lated at both the transcript and protein levels in response to light and low CO2, and our data further supports this finding (Fig. 2A).
  • EPYCl becomes phosphorylated at multiple sites in response to low CO2, potentially affecting its binding affinity for RuBisCO.
  • Another mode of regulation of EPYCl-RuBisCO binding could be by methylation of RuBisCO: RuBisCO is methylated a multiple residues and in Chlamydomonas the predicted methyltransferase CIA6 is required for RuBisCO localization to the pyrenoid. It is also possible that EPYCl binding to RuBisCO alters the kinetic properties of RuBisCO to fine-tune its performance in the pyrenoid.
  • Wild-type (WT) Chlamydomonas rein- hardtii CC-1690 was maintained at 22°C with 55 pmol photons rrr 2 s 1 light on Tris-acetate-phosphate (TAP) agar (1.4%) plates containing 0.4% Bacto-Yeast extract. It was used for pyrenoid enrichment and proteomics. Chlamydomonas reinhardtii WT strain CMJ030 (CC-4533) and epycl mutant were maintained in the dark or low light ( ⁇ 10 pmol photons rrr 2 s _1 ) on 1.5% agar plates containing TAP with revised or traditional Hutner's trace elements.
  • the epycl mutant was isolated from a collection of high CO2 requiring mutants by a pooled screening approach. A collection of approximately 7500 mutants on 79 plates, each with 96 colonies, was grown in liquid TAP in 96-well plates then pooled by well row, well column, whole plate row and whole plate column to give a total of 38 pools.
  • upstream gDNA-cassette junction cannot be PCR amplified. However, PCR shows the full cassette is intact and that >397bp upstream of the insertion site is also intact. All experiments were performed under photoautotrophic conditions supplemented with high CO2 (3% or 5% v/v CO2 enriched air) or low CO2 (air, ⁇ 0.04% v/v C0 2 ).
  • a 50 mL pre-culture was grown mixotrophi- cally in TAP on a rotatory shaker at 124 rpm and 22°C, under an illumination of 55 pmol photons nr 2 s 1 for three days.
  • a second pre-culture of 500 mL was used to inoculate a 5-liter bioreactor (BIOSTAT®B-DCU, Sartorius Stedim). The absence of contamination was monitored.
  • the pellet was washed three times with 1 ml_, 500 ⁇ _, and 300 ⁇ _ EB before resuspension in lOOpL of 50 mM ammonium bicarbonate. Protein concentrations were measured by Lowry assay using BSA as a standard.
  • Protein digestion and mass spectrometric analysis were prepared and measured by precipitating 20 pg protein per sample in 80% acetone at -20°C overnight.
  • the precipitated proteins were resuspended in 6 M urea and 2 M thiourea (in 50 mM ammonium hydrogen carbonate), reduced by DTT, carbamidomethylated with iodoacetamide, digested with endoproteinase LysC (Roche) and immobilized trypsin (Applied Biosystems, Thermo Fisher Scientific), and subsequently desalted.
  • the resuspended peptides were acidified with 1% acetic acid.
  • Peptides were chromatographically separated by reverse phase separation with a nanoU- PLC (nanoACQUITY UPLC, Waters) using a 10cm x 75pm BEH 130 C18 1.7pm particles (Waters) column for separation and a 2cm x 180pm Symmetry C18 5pm particles (Waters) column for trapping. Peptides were analyzed by a linear trap quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Data processing and data analysis. Raw MS files were processed with MaxQuant (ver. 1.5.2.8).
  • Peak list files were searched against Chlamydo- monas reinhardtii gene model JGIv4 from Phytozome 10.2 (phyto- zome.jgi.doe.gov) including the organelle genome sequences. Maximum precursor and fragment mass deviations were set to 20ppm and 0.5Da. Peptides with at least six amino acids were considered for identification. The search included carbamidomethylation as a fixed modification and variable modifications for oxidation of methionine and protein N-terminal acetylation. The false discovery rate, determined by searching a reverse database, was set at 0.01 for both peptides and proteins. Identification across different replicates and treatments was achieved by enabling the "match between runs" option in MaxQuant within a time window of 2 min.
  • EPYC1 and RbcSl were amplified from gDNA using Phusion Hotstart II polymerase (Thermo Scientific) with the respective EPYCl_ORF_F/R or RBCSl_ORF_F/R primer pairs.
  • strains were also screened on a Tecan Infinite M 1000 PRO. Fluorescence microscopy and RuBisCO-mCherry mislocalization in the epycl mutant. All fluorescence microscopy was performed using a spinning disk confocal microscope (custom adapted Leica DMI6000) with samples imaged on poly-L-lysine coated plates.
  • membranes were stripped (Restore PLUS Western blot stripping buffer, Thermo Scientific) and re-probed with anti-tubulin (1 : 25,000; Sigma) followed by HRP conjugated goat anti-mouse (1 : 10,000; Life Technologies).
  • the anti-EPYCl antibody was raised in rabbit to the C-terminal region of EPYCl (KSKPEIKRTALPA DWRKGL-COOH, SEQ ID NO : 16) by Yenzym Antibodies.
  • Lysate was clarified by centrifugation (13,000g, 20 min, 4°C). Protein content was determined using the Bradford method (Sigma Aldrich). Soluble proteins were separated on 12% (w/v) denaturing polyacrylamide gel. Sample loading was normalized by protein amount (10 pg per lane), and even loading was controlled by staining a gel with identical protein load (GelCode Blue, Life Technologies). After transfer onto a polyvinylidene difluoride membrane (Amersham), RuBisCO was immunodetected with a polyclonal primary antibody raised against RuBisCO (1 : 10,000) followed by a HRP conjugated goat anti-rabbit (1 : 20,000; GE Healthcare).
  • Chlorophyll concentration Total pigments were extracted in 100% methanol, and the absorbance of the clarified supernatant (13,000g, 1 min, 4°C) was measured at 470, 652, 665, and 750 nm (UV 300 Unicam, Thermo Spectronic). Concentration of chlorophyll (a+b) was calculated using the equation of Wellburn. Spot tests. WT, epycl, and complemented cell lines were grown in TAP until ⁇ 2xl0 6 cells mL -1 , washed once with Tris-phospate (TP), resuspended in TP to a concentration of 6.6xl0 5 cells mL 1 , then serially diluted 1 : 10 three times. 15 ⁇ _ of each dilution was spotted onto four TP plates and incubated in low or high CO2 with 100 or 500 pmol photons nr 2 s 1 of light for seven days before imaging.
  • TP Tris-phospate
  • Oxygen evolution measurements Apparent affinity for inorganic carbon was determined by oxygen evolution.
  • Photoautotrophically grown liquid cultures were harvested by centrifugation (2,000g, 5 min, 4°C) and re- suspended in 25 mM HEPES-KOH (pH 7.3) to a density of ⁇ 1.5 x 10 8 cells mL -1 , as determined by hemocytometer count.
  • Aliquots of cells (1 mL) were added to a Clark-type oxygen electrode chamber (Rank Brothers, Bottisham, UK) attached to a circulating water bath set to 25°C. The chamber was closed for a light pre-treatment (200-300 pmol photons nr 2 S " 1 illumination for 10-25 min), to allow cells to deplete any internal inorganic carbon pool.
  • fixative glucosealdehyde, final 2.58% was added to cell cultures immediately before harvesting.
  • Cell suspensions containing ⁇ 5 x 10 7 cells in mid-log were pelleted (4,000g, 5 min, 4°C) and fixed in 1 mL tris-minimal medium containing 2.5% glutaraldehyde and 1% H2O2 (30% w/v) for 1 hour on a tube rotator at 4°C. Unless otherwise specified, all following steps were performed at room temperature on a tube rotator. Cells were pelleted (4,000g for 5 min) and washed with ddh O (3X, 5 min).
  • Cells were osmicated for 1 hour in 1 mL 1% (v/v) Os0 4 containing 1.5% (w/v) K 3 [Fe(CN) 6 ] and 2 mM CaCI 2 . Cells were pelleted and washed with ddH20 (4X, as above). Cells were stained for 1 hour in 1 mL 2% (w/v) uranyl acetate. After pelleting and washing with ddH 2 0 (3X), cells were dehydrated in 70%, 95%, and 100% ethanol, and 100% acetonitrile (2X).
  • QFDEEM was performed. Briefly, small samples of pelleted cells were placed on a cushioning material and dropped onto a liquid helium-cooled copper block; the frozen material was transferred to liquid nitrogen and then to an evacuated Balzers apparatus, fractured, etched at -80°C for 2 min, and platinum/carbon rotary- replicated. The replicas were examined with a JEOL electron microscope, model JEM 1400, equipped with an AMTV601 digital camera. The images are photographic negatives; hence, protuberant elements of the fractured/etched surface are more heavily coated with platinum and appear whiter. Immunogold-localization of RuBisCO. Resin embedded material previously used for ultra-structural characterization of the pyrenoid was re-cut and thin sections were mounted on nickel grids.
  • Salt, detergent, and surfactant concentrations were determined empirically to minimize background signal. Binding to primary antibody was done by incubating grids overnight in 1% BSA in HSTBSTT, with 1 : 1,000 dilution of the RuBisCO antibody. Excess antibody was removed by 15 min washes (2X) in HSTBSTT and 15 min washes (2X) in ddH 2 0. Incubation with secondary antibody (15 nm gold particle-conjugated goat anti-rabbit secondary antibody in 1% BSA in HSTBSTT, 1 : 250) was done at RT for 1 hr. Excess secondary antibody was removed by washing as above. Thin sections were prepared and imaged as for Pyrenoid area analysis by transmission electron microscopy, above.
  • Randomization was done as above (see TEM) with scoring capped to ⁇ 25 cells for each treatment.
  • Nonspecific labelling was taken as any particle on a free resin area, i.e. outside a cell.
  • Non-specific density was subtracted from pyrenoid and chloroplast particle density.
  • Fraction of particles in the pyrenoid was calculated as background-adjusted npyrenoid / (npyrenoid + nstroma), where nstroma is the number of particles in the stroma to the exclusion of the pyrenoid and the starch sheath.
  • Fig. 3G particles were enlarged lOx using the image analysis software Fiji. Briefly, images were thresholded to isolate individual gold particles, these were then enlarged lOx, and the new image overlaid on the original image with an opacity of 50%.
  • WT cells expressing pLM005_Venus-3xFLAG, pLM005_EPYCl-Venus-3x FLAG or pLM005_RbcSl-Venus-3xFLAG were grown in 800 mL of TP plus 2 pg mL 1 paromomycin with continual bubbling at low C02 (0.04% CO2) under 150 pmol photons nr 2 s 1 of light until a cell density of ⁇ 2-4 x 10 6 cells mL 1 .
  • Cells were then spun out (2,000g, 4 min, 4°C), washed in 40 mL of ice cold TP, centrifuged then resuspended in a 1 : 1 (v/w) ratio of ice-cold 2xIP buffer (400 mM sorbitol, 100 mM HEPES, 100 mM KOAc, 4 mM Mg(OAc)2-4H 2 0, 2 mM CaC , 2 mM NaF, 0.6 mM Na3V0 4 and 1 Roche com- plete EDTA-free protease inhibitor/ 25 mL) to cell pellet.
  • ice-cold 2xIP buffer 400 mM sorbitol, 100 mM HEPES, 100 mM KOAc, 4 mM Mg(OAc)2-4H 2 0, 2 mM CaC , 2 mM NaF, 0.6 mM Na3V0 4 and 1 Roche com- plete EDTA-free protease inhibitor
  • Chlamydomonas "popcorn" balls approximately 5 mm in diameter. These were stored at -70°C until needed.
  • Cells were lysed by grinding lg ( ⁇ 500 mg of original cell pellet) of Chlamydomonas popcorn balls by mortar and pestle at liquid nitrogen temperatures, for 10 min. The ground cells were defrosted on ice, then dounced 20 times on ice with a Kontes Glass Duall #21 homogenizer. Membranes were solubilized by incrementally adding an equal volume of ice-cold lxIP buffer plus 2% digitonin (final concentration is 1%), then incubating at 4°C for 40 min with nutation.
  • the lysate was then clarified by spinning for 30 min at full-speed in a table-top centrifuge at 4°C.
  • the supernatant (Input) was then transferred to 225 pL of protein G Dynabeads (Life Technologies) that had been incubated with anti-FLAG M2 antibody (Sigma) according to the manufacturer's instructions, except lxIP buffer was used for the wash steps.
  • the Dynabead-cell lysate was incubated for 2.5 hours on a rotating platform at 4°C, then the supernatant removed (flow-through).
  • the Dynabeads were washed four times with lxIP buffer plus 0.1% digitonin followed by a 30 min elution with 50 ⁇ _ of lxIP buffer plus 0.25% digitonin and 2 pg pL 1 3xFLAG peptide (Sigma; 3xFLAG peptide elution) and a 10 min elution in lx Laemmli buffer with 50 mM beta- mercaptoethanol at 70°C (Boiling elution).
  • EPYC1 sequence analysis To understand the intrinsic disorder of EPYC1, the full-length amino acid sequence was run through several structural disorder prediction programs including VL3, VLTX, and GlobPlot 2. To look for regions of secondary structure, the full-length and repeat region of the EPYC1 amino acid sequence was analysed by PSIPRED v3.3 and Phyre2.
  • EPYCl-RuBisCO interaction model We built a model of the EPYC1- RuBisCO interaction using Blender (blender dot org) based on the following rationale: If each of the four EPYC1 repeats can bind a holoenzyme, the two internal repeats would have different linking properties from the two terminal repeats. If bound to an internal repeat, a holoenzyme would be directly linked through this EPYC1 protein to two other holoenzymes. In contrast, if bound to a terminal repeat, the holoenzyme would only be directly linked through this EPYC1 protein to one other holoenzyme.
  • each EPYC1 repeat would link one RuBisCO holoenzyme to 1.5 other holoenzymes.
  • a holoenzyme likely has 8 binding sites for EPYCl.
  • each holoenzyme would be bound to 12 other nonenzymes by 8 EPYCl proteins, in an arrangement that could expand indefinitely in all directions. A perfect arrangement of this nature would require a stoichiometry of one EPYCl polypeptide for every four RuBisCO small or large subunits.
  • Proteins with >3 repeats, a pi >8, an oscillating disorder profile with a frequency between 40 and 80 amino acids, and no transmembrane domains were classified as potential EPYCl-like RuBisCO linker proteins.
  • stringent parameters we have tried to reduce the number of false positive hits but we realize that our approach has several limitations, including : 1) Missing true linker proteins due to not all of the physicochemical properties of EPYCl being essential for linker function. 2) Incomplete genome assembly of the ban- gated algae. 3) Incorrect gene models resulting in truncated, misspliced and frame-shifted proteins.
  • Pre-established exclusion criteria for TEM image scoring were: (i) only grid areas fully covered with material (54pm 2 ) were considered; (ii) sections through broken cells and cell sections with a cross area ⁇ 12.5pm 2 (a circle with 2pm radius served as a guide) were not scored. Scoring of electron micrographs: images files were renamed with a random number (RAND BETWEEN function in Microsoft Excel), sorted from high to low, and scored blindly. The original filename appearing on the bottom left of each micrograph was masked during the on-screen processing in ImageJ. Randomly selected images were scored by a second experimenter for independent validation. No systematic bias (over- or under-estimation) was measured, and measurements deviated on average only by a couple of percentage points.
  • Two-tailed Student's t-test was used to compare affinities for inorganic carbon of WT and epycl, as well as the mislocalization of RuBisCO by fluorescence microscopy and EM, because this test is robust to non-normal distributions.
  • Welch's t-test was used to compare pyrenoid sizes, because the WT and mutant groups had substantially different standard deviations. Fisher's exact test of independence was used to compare the number of pyrenoids in WT and epycl, as this test is appropriate when there are two nominal variables.

Abstract

La présente invention concerne des algues eucaryotes, qui jouent un rôle fondamental dans la fixation globale du CO2, améliorent la performance de l'enzyme de fixation du carbone RuBisCO en le plaçant dans un organite appelé pyrénoïde où l'activité de l'enzyme est améliorée par le mécanisme de concentration de carbone. Malgré l'omniprésence et l'importance biogéochimique de cet organite, on ne savait pas comment le RuBisCO s'assemblait pour former le pyrénoïde. La présente invention concerne les composants moléculaires du pyrénoïde, leur transport vers le pyrénoïde en tant que composants essentiels de celui-ci, et leur fonction dans le mécanisme de concentration de carbone (CCM). Les protéines de pyrénoïde d'algues et leurs variants peuvent être transférées dans des cellules de bactéries (par exemple, la cyanobactérie), des algues (par exemple, des algues vertes telles que Chlamydomonas), et des embryophytes (par exemple les plantes C3 et C4) pour l'expression stable ou transitoire (en particulier dans les feuilles) pour manipuler l'assemblage du pyrénoïde et ses fonctions biologiques.
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WO2021023982A1 (fr) * 2019-08-02 2021-02-11 The University Court Of The University Of Edinburgh Structures de type pyrénoïde
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Publication number Priority date Publication date Assignee Title
WO2021025962A1 (fr) * 2019-08-02 2021-02-11 Princeton University Motifs protéiques de liaison à rubisco et leurs utilisations
WO2021023982A1 (fr) * 2019-08-02 2021-02-11 The University Court Of The University Of Edinburgh Structures de type pyrénoïde
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CN113293102A (zh) * 2021-04-26 2021-08-24 深圳大学 一种衣藻叶绿体以及叶绿体rna提取的方法
CN113293102B (zh) * 2021-04-26 2023-07-14 深圳大学 一种衣藻叶绿体以及叶绿体rna提取的方法

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