WO2013022353A1 - Procédés permettant d'accroître l'assimilation de co2 et la production d'huile dans des organismes photosynthétiques - Google Patents

Procédés permettant d'accroître l'assimilation de co2 et la production d'huile dans des organismes photosynthétiques Download PDF

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WO2013022353A1
WO2013022353A1 PCT/NZ2012/000138 NZ2012000138W WO2013022353A1 WO 2013022353 A1 WO2013022353 A1 WO 2013022353A1 NZ 2012000138 W NZ2012000138 W NZ 2012000138W WO 2013022353 A1 WO2013022353 A1 WO 2013022353A1
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Prior art keywords
photosynthetic
plant
promoter
oleosin
cell
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PCT/NZ2012/000138
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English (en)
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Nicholas John Roberts
Richard William Scott
Somrutai Winichayakul
Marissa Roldan
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Agresearch Limited
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Priority to NZ620832A priority Critical patent/NZ620832B2/en
Priority to AU2012294956A priority patent/AU2012294956B2/en
Priority to BR112014002748A priority patent/BR112014002748A2/pt
Priority to MX2014001441A priority patent/MX354499B/es
Priority to CA2844239A priority patent/CA2844239C/fr
Publication of WO2013022353A1 publication Critical patent/WO2013022353A1/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
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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 plant in addition to the increased rate of CO 2 assimilation also has at least one of: a) an increased rate of photosynthesis, and b) increased water use efficiency, and c) an increased growth rate.
  • the plant has all of a) to c).
  • the increase in biomass is in the range 2% to 100%, preferably 4% to 90%, preferably 6% to 80%, preferably 8% to 70%, preferably 10% to 60% relative to a control plant.
  • Preferred plants include those from the following genera; Oryza, Glycine, Hordeum, Secale, Avena, Pennisetum, Setaria, Panicum, Eleusine, Solanum, Brassica, Helianthus and Carthamus.
  • the plant accumulates at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably 150%, more preferably 200%, more preferably 250%, more preferably 300%, more preferably 350%, more preferably 400%, more preferably 450%, more preferably 500%, more total lipid in its non-photosynthetic tissues/organs than does a control plant.
  • the plant produce oil in its non-photosynthetic tissues/organs in the range 3x to 15x, more preferably 4x to 14x, , more preferably 5x to 13x , more preferably 6x to 12x , more preferably 7x to 1 lx , more preferably 8x to lOx more than a control plant.
  • Suitable control plants include non-transformed or wild-type versions of plant of the same variety and/or species as the transformed plant used in the method of the invention. Suitable control plants also include plants of the same variety and or species as the transformed plant that are transformed with a control construct. Suitable control plants also include plants that have not been transformed with a
  • the non-photosynthetic tissue/organ is selected from below ground tissue/organs of the plant.
  • the below ground tissue/organ is selected from root, tuber, bulb, corm and rhizome.
  • the non-photosynthetic tissue/organ is selected from root, tuber, bulb, corm, rhizome, and endosperm.
  • the non-photosynthetic tissue/organ is root.
  • the method includes the step of transforming the plant with the polynucleotide encoding a modified oleosin including at least one artificially introduced cysteine. Promoters
  • the polynucleotide is operably linked to a promoter polynucleotide.
  • the promoter is capable of driving expression of the polynucleotide in the non- photosynthetic tissues of the plant.
  • the promoter is a constitutive promoter.
  • the promoter is a non-photosynthetic tissue preferred promoter.
  • the promoter is a root preferred promoter.
  • the promoter is a root specific promoter.
  • the promoter is a tuber preferred promoter.
  • the promoter is a tuber specific promoter.
  • the promoter is a bulb preferred promoter.
  • the promoter is a bulb specific promoter. In a further embodiment the promoter is a corm preferred promoter. In a further embodiment the promoter is a corm specific promoter. In a further embodiment the promoter is a rhizome preferred promoter. In a further embodiment the promoter is a rhizome specific promoter. In a further embodiment the promoter is an endosperm preferred promoter. In a further embodiment the promoter is an endosperm specific promoter.
  • Polynucleotide is part of a genetic construct
  • the polynucleotide is transformed as part of a genetic construct.
  • the genetic construct is an expression construct.
  • the expression construct includes the
  • Plant is also transformed with a TAG synthesising enzyme
  • nucleic acid is operably linked to a promoter polynucleotide.
  • the promoter is capable of driving expression of the polynucleotide in the non- photosynthetic tissues of the plant.
  • the promoter is a constitutive promoter.
  • the promoter is a non-photosynthetic tissue preferred promoter.
  • the promoter is a root preferred promoter.
  • the promoter is a root specific promoter.
  • the promoter is a tuber preferred promoter.
  • the promoter is a tuber specific promoter.
  • the promoter is a corm preferred promoter.
  • the promoter is a corm specific promoter.
  • the promoter is a rhizome preferred promoter.
  • the promoter is a rhizome specific promoter. In a further embodiment the promoter is an endosperm preferred promoter. In a further embodiment the promoter is an endosperm specific promoter.
  • TAG triacylglycerol
  • polynucleotide and nucleic acid can be transformed into the cell without a promoter, but expression of either or both of the polynucleotide and nucleic acid could be driven by a promoter, or promoters, endogenous to the plant transformed.
  • the method comprises the additional step of processing the non-photosynthetic tissue/organ of the plant into an animal feedstock.
  • the method comprises the additional step of extracting oil from the non- photosynthetic tissue/organ of the plant. In a further embodiment the method comprises the additional step of processing the non-photosynthetic tissue/organ into an oil fraction.
  • the non-photosynthetic tissue/organ contains at least 100%, more preferably 150%, more preferably 200%, more preferably 250%, more preferably 300%, more preferably 350%, more preferably 400%, more preferably 450%, more preferably 500%, more total lipid than the corresponding non-photosynthetic tissue/organ of a control plant. In one embodiment the non-photosynthetic tissue/organ contains 100% to 900%, more preferably 200% to 800%, more preferably 300% to 700%, more preferably 400% to 600%, more total lipid than the corresponding non-photosynthetic tissue/organ of a control plant.
  • the non-photosynthetic tissus/organ contains at least 2x, more preferably 3x, more preferably 4x, more preferably 5x, more preferably 6x, more preferably 7x, more preferably 8x, more preferably 9x, more preferably lOx, more preferably 1 lx, more preferably 12x, more preferably 13x, more preferably 14x, more preferably 15x, more oil than the corresponding non-photosynthetic tissue/organ of a control plant.
  • the non-photosynthetic tissue/organ contains 3x to 15x, more preferably 4x to 14x, , more preferably 5x to 13x , more preferably 6x to 12x , more preferably 7x to 1 lx , more preferably 8x to lOx more oil than the corresponding non-photosynthetic tissue/organ of a control plant.
  • Suitable control plants include non-transformed or wild-type versions of plant of the same variety and or species as the transformed plant used in the method of the invention. Suitable control plants also include plants of the same variety and or species as the transformed plant that are transformed with a control construct. Suitable control plants also include plants that have not been transformed with a
  • polynucleotide encoding a modified oleosin including at least one artificially introduced cysteine.
  • Suitable control plants also include plants that do not express a modified oleosin including at least one artificially introduced cysteine.
  • a modified oleosin including at least one artificially introduced cysteine Preferably the increased level of oil production is caused by expression of the modified oleosin including at least one artificially introduced cysteine.
  • Animal feed comprising non-photosynthetic tissue/organ of the invention
  • the invention provides an animal feed comprising the non-photosynthetic tissue/organ of the invention.
  • Biofuel feedstock comprising non-photosynthetic tissue/organ of the invention
  • the invention provides a biofuel feedstock comprising the non-photosynthetic tissue/organ of the invention.
  • the modified oleosins may be modified naturally occurring oleosins.
  • the plants from which the unmodified oleosin sequences are derived may be from any plant species that contains oleosins and polynucleotide sequences encoding oleosins.
  • the plant cells, in which the modified oleosins are expressed, may be from any plant species.
  • the plants, in which the modified oleosins are expressed may be from any plant species.
  • the plant cell or plant is derived from a gymnosperm plant species. In a further embodiment the plant cell or plant, is derived from an angiosperm plant species. In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species. In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant species.
  • Preferred plant species are those that produce tubers (modified stems) such as but not limited to Solanum species.
  • Other preferred plant species are those that produce bulbs (below ground storage leaves) such as but not limited to Lilaceae, Amaryllis, Hippeastrum, Narcissus, Iridaceae, and Oxalis species.
  • Other preferred plant species are those that produce corms (swollen underground stems) such as but not limited to Musa, Elocharis, Gladiolus and Colocasia species.
  • Other preferred plant species are those that produce rhizomes (underground storage stem) such as but not limited to Asparagus, Zingiber and Bambuseae species.
  • Other preferred are those that produce substantial endosperm in their seeds, such as but not limited to maize and sorghum.
  • Preferred plants incude those from the following genera: Brassica, Solanum, Raphanus, Allium, Foeniculum, Lilaceae, Amaryllis, Hippeastrum, Narcissus, Iridaceae, Oxalis, Musa, Eleocharis, Gladiolus, Colocasia, Asparagus, Zingiber, and Bambuseae.
  • a preferred Brassica species is Brassica rapa var. rapa (turnip)
  • Preferred Solanum species are those which produce tubers.
  • a preferred Solanum species is Solanum tuberosum (potato)
  • Raphanus species include Raphanus raphanistrum, Raphanus caudatu, and Raphanus sativus.
  • a preferred Raphanus species is Raphanus sativus (radish)
  • Preferred Allium species include: Allium cepa (onion, shallot), Allium flstulosum (bunching onion), Allium schoenoprasum (chives), Allium tuberosum (Chinese chives), Allium ampeloprasum (leek, kurrat, great-headed garlic, pearl onion), Allium sativum (garlic) and Allium chinense (rakkyo).
  • a preferred A Ilium species is Allium cepa (onion)
  • Preferred Musa species include: Musa acuminata and Musa balbisiana.
  • a preferred Musa species is Musa acuminata (banana, plantains)
  • a preferred Zingiber species is Zingiber officinale (ginger)
  • a preferred Colocasia species is Colocasia esculenta (taro).
  • Another preferred genera is Zea.
  • a preferred Zea species is Zea mays.
  • Another preferred genera is Sorghum.
  • a preferred Sorghum species is Sorghum bicolor.
  • the plants may be selected from forage legumes including, alfalfa, clover; leucaena; grain legumes including, beans, lentils, lupins, peas, peanuts, soy bean; bloom legumes including lupin, pharmaceutical or industrial legumes; and fallow or green manure legume species.
  • a particularly preferred genus is Trifolium.
  • Preferred Trifolium species include Trifolium repens; Trifolium arvense; Trifolium affine; and Trifolium occidentale.
  • a particularly preferred Trifolium species is Trifolium repens.
  • Medicago Another preferred genus is Medicago.
  • Preferred Medicago species include Medicago sativa and Medicago truncatula.
  • a particularly preferred Medicago species is Medicago sativa, commonly known as alfalfa.
  • Mucana Another preferred genus is Mucana.
  • Preferred Mucana species include Mucana pruniens.
  • a particularly preferred Mucana species is Mucana pruniens commonly known as velvetbean.
  • Another preferred genus is Arachis.
  • a particularly preferred Arachis species is Arachis glabrata
  • Pisum Another preferred genus is Pisum.
  • a preferred Pisum species is Pisum sativum commonly known as pea.
  • Lotus Another preferred genus is Lotus.
  • Preferred Lotus species include Lotus corniculatus, Lotus
  • oils seed crops including but not limited to the following genera: Brassica, Carthumus, Helianthus, Zea and Sesamum.
  • a preferred oil seed genera is Brassica.
  • a preferred oil seed species is Brassica napus.
  • a preferred oil seed genera is Brassica.
  • a preferred oil seed species is Brassica oleraceae.
  • a preferred oil seed genera is Carthamus.
  • a preferred oil seed species is Carthamus tinctorius.
  • a preferred oil seed genera is Helianthus.
  • a preferred oil seed species is Helianthus annuus.
  • a preferred oil seed genera is Zea.
  • a preferred oil seed species is Zea mays.
  • a preferred oil seed genera is Sesamum.
  • a preferred oil seed species is Sesamum indicum.
  • a preferred silage genera is Zea.
  • a preferred silage species is Zea mays.
  • a preferred grain producing genera is Hordeum.
  • a preferred grain producing species is Hordeum vulgare.
  • a preferred grazing genera is Lolium.
  • a preferred grazing species is Lolium perenne.
  • a preferred grazing genera is Lolium.
  • a preferred grazing species is Lolium arundinaceum.
  • a preferred grazing genera is Trifolium.
  • a preferred grazing species is Trifolium repens.
  • a preferred grazing genera is Hordeum.
  • a preferred grazing species is Hordeum vulgare.
  • Preferred plants also include forage, or animal feedstock plants. Such plants include but are not limited to the following genera: Miscanthus, Saccharum, Panicum.
  • a preferred bio fuel genera is Miscanthus.
  • a preferred bio fuel species is Miscanthus giganteus.
  • a preferred bio fuel genera is Saccharum.
  • a preferred bio fuel species is Saccharum qfficinarum.
  • a preferred biofuel genera is Panicum.
  • a preferred biofuel species is Panicum virgatum. DETAILED DESCRIPTION OF THE INVENTION
  • lipids On a weight for weight basis lipids have approximately double the energy content of either proteins or carbohydrates.
  • the bulk of the world's lipids are produced by plants and the densest form of lipid is as a triacyl glycerol (TAG).
  • TAG triacyl glycerol
  • Dicotyledonous plants can accumulate up to approximately 60% of their seed weight as TAG which is subsequently used as an energy source for germination. As such there have been a number of efforts targeted at using seeds rich in oils to sustainably produce sufficient lipids for both animal and biofuel feed stock.
  • OBs oil bodies
  • OBs oil bodies
  • OBs consist of a TAG core surrounded by a phospholipid monolayer embedded with proteinaceous emulsifiers. The latter make up 0.5-3.5% of the OB; of this, 80-90% is oleosin with the remainder predominantly consisting of the calcium binding (caloleosin) and sterol binding (steroleosin) proteins (Lin and Tzen, 2004).
  • oleosins The emulsification properties of oleosins derives from their three functional domains which consist of an amphipathic N-terminal arm, a highly conserved central hydrophobic core ( ⁇ 72 residues) and a C-terminal amphipathic arm. Similarly, both caloleosin and steroleosin possess hydrophilic N and C-terminal arms and their own conserved hydrophobic core.
  • lipid profile of ruminant animal feed in turn influences the lipid profile of meat and dairy products (Demeyer and Doreau, 1999).
  • Different plants have different lipid profiles; by selectively feeding animals only plants with the desired lipid profile it is possible to positively influence the lipid profile of downstream meat and dairy products.
  • the final lipid make up of the meat and milk is not only influenced by the dietary lipids but is also heavily influenced by biohydrogenation (Jenkins and McGuire 2006; Firkins et al., 2006; Lock and Bauman, 2004).
  • Biohydrogenation is the hydrogenation of non-reduced compounds (such as unsaturated fats) by the biota present in the rumen. Biohydrogenation can be prevented/delayed by encapsulating the lipids in a protein or proteins that provide resistance to microbial degradation (Jenkins and Bridges 2007). The prevention of
  • Oleosins are comparatively small (15 to 24 kDa) proteins which allow the OBs to become tightly packed discrete organelles without coalescing as the cells desiccate or undergo freezing conditions (Leprince et al, 1998; Siloto et al, 2006; Slack et al , 1980; Shimada et a/.2008).
  • the topology of oleosin is attributed to its physical properties which includes a folded hydrophobic core flanked by hydrophilic domains. This arrangement confers an amphipathic nature to oleosin resulting in the hydrophobic domain being embedded in the phospholipid monolayer (Tzen et al., 1992) while the flanking hydrophilic domains are exposed to the aqueous environment of the cytoplasm.
  • oleosins do not contain cysteines
  • Preferred oleosins for use in the invention are those which contain a central domain of approximately 70 non-polar amino acid residues (including a proline knot) uninterrupted by any charged residues, flanked by two hydrophilic arms.
  • oleosin as used herein also includes steroleosin and caloleosin Steroleosins
  • Steroleosins comprises an N-terminal anchoring segment comprising two amphipathic ohelices 912 residues in each helix) connected by a hydrophobic anchoring region of 14 residues.
  • the soluble dehydrogenase domain contains a NADP+- binding subdomain and a sterol-binding subdomain.
  • the apparent distinction between steroleosins-A and -B occurs in their diverse sterol-binding subdomains (Lin and Tzen, 2004).
  • Steroleosins have a proline knob in their hydrophobic domain and contains a sterol-binding dehydrogenase in one of their hydrophilic arms.
  • Steroleosin Z. mays NM_001 159142.1 58 NP_001152614.1 59
  • acyl chains Although a portion of the newly synthesized acyl chains is then used for lipid biosynthesis within the plastid (the prokaryotic pathway), a major portion is exported into the cytosol for glycerolipid assembly at the endoplasmic reticulum (ER) or other sites (the eukaryotic pathway). In addition, some of the extraplastidial glycerolipids return to the plastid, which results in considerable intermixing between the plastid and ER lipid pools (Ohlrogge and Jaworski 1997).
  • acyl- ACP thioesterases of which there are two main types : one thioesterase relatively specific for 18: 1 - ACP and a second more specific for saturated acyl-ACPs.
  • Fatty acids that have been released from ACPs by thioesterases leave the plastid and enter into the eukaryotic lipid pathway, where they are primarily esterified to glycerolipids on the ER.
  • TAG biosynthesis The only committed step in TAG biosynthesis is the last one, i.e. the addition of a third fatty acid to an existing diacylglycerol, thus generating TAG.
  • this step is predominantly (but not exclusively) performed by one of five (predominantly ER localised) TAG synthesising enzymes including: acyl CoA: diacylglycerol acyltransferase (DGATl); an unrelated acyl CoA: diacylglycerol acyl transferase
  • DGATl diacylglycerol acyltransferasel
  • DGAT2 diacylglycerol acyl transferase2
  • PDAT phosphatidylcholine-sterol O-acyltransferase
  • cytosolic soluble form of DGAT soluble DGAT or DGAT3
  • the SnRKl proteins are a class of Ser/Thr protein kinases that have been increasingly implicated in the global regulation of carbon metabolism in plants, e.g. the inactivation of sucrose phosphate synthase by phosphorylation (Halford & Hardie 1998).
  • Zou et al. (2008) went on to demonstrate that the obliteration of a potential SnRKl phosphorylation site in DGATl by single point mutation (Serl97Ala of TmDGATl) led to the accumulation of significantly higher levels of TAG in the seed. This mutation increased activity by 38-80%, which led to a 20-50% increase in oil content on a per seed basis in Arabidopsis.
  • Phospholipid:DGA acyltransferase forms TAG from a molecule of phospholipid and a molecule of diacyglycerol.
  • PDAT is quite active when expressed in yeast but does not appreciably increase TAG yields when expressed in plant seeds.
  • PDAT and a proposed DAG:DAG transacylase are neutral lipid synthesizing enzymes that produce TAG, but are not considered part of the Kennedy Pathway.
  • a combination of wax ester synthase and DGAT enzyme (WS/DGAT) has been found in all neutral lipid producing prokaryotes studied so far.
  • WS/DAGAT has extraordinary broad activity on a variety of unusual fatty acids, alcohols and even thiols.
  • This enzyme has a putative membrane-spanning region but shows no sequence homology to the DGATl and DGAT2 families from eukaryotes or the WE synthase from jojoba (Jojoba is the only eukaryote that has been found to accumulate wax ester).
  • LCAT Lecithin-Cholesterol AcylTransferase
  • ACAT Acyl-coenzyme:Cholesterol AcylTransferase
  • DGATl In applications requiring the increase of neutral lipids evidence suggests that the higher activity and broader specificity of DGATl relative to DGAT2 is preferential. Where a specific fatty acid is preferred, such as a long-chain PUFA, DGATl is still applicable, provided it accepts the fatty acid of choice. Plants generally incorporate long chain PUFAs in the sn-2 position. It is not known whether this is due to high activity of LP AT or low activity of DGATl on this substrate. For the improved specificity for PUFAs, a DGAT2 that prefers these fatty acids may be preferable, or the properties of DGATl could be altered using directed evolution or an equivalent procedure.
  • TAG synthesising enzymes suitable for use in the methods and compositions of the invention, from members of several plant species are provided in Table 2 below.
  • sequences both polynucleotide and polypeptide are provided in the Sequence Listing.
  • DGATl Z. mays EU039830 78 ABV91586 79 DGAT2 A. thaliana NM_115011 80 NP 566952 81
  • the inventions also contemplates use of modified TAG synthesizing enzymes, that are modified (for example in their sequence by substitutions, insertions or additions an the like) to alter their specificity and or activity.
  • TAG accumulation in leaves A recent field survey of 302 angiosperm species in the north-central USA found that 24% have conspicuous cytosolic oil droplets in leaves, with usually one large oil droplet per mesophyll cell (Lersten et al., 2006 [from Slocombe et al 2009]).
  • the role of cytosolic leaf TAG is thought to be involved in carbon storage and/or membrane lipid re-modelling (for review see Slocombe et al., 2009). Indeed, in senescing leaves, plastidial fatty acids are partitioned into TAG prior for further mobilization, and DGAT1 is thought to be instrumental in this process (Kaup et al., 2002).
  • TAG TAG biosynthesis
  • mutation of TGD1 or CTS resulting in the prevention of lipid remobilisation
  • LEC1 , LEC2 and WRI1 transcriptional factors involved in storage oil and protein accumulation in developing seeds.
  • TAG and other neutral lipid synthesizing enzymes relies on the presence of sufficient substrate, in the expanding and or mature leaf this is assumed to be provided by the plastid (chloroplast in the case of the leaf) which synthesises lipids for membranes.
  • Slocombe et al. (2009) concluded that recycled membrane fatty acids may be able to be redirected to TAG by expressing the seed-programme in senescing tissue or by a block in fatty acid breakdown.
  • Scott et al (2007) claimed that the co-expression of a triacylglyceride synthesising enzyme and polyoleosin (two or more oleosin units fused in a tandem head-to-tail arrangement) would enable the storage of lipid in a plant cell.
  • Cookson et al (2009) claimed that producing a single oleosin and a TAG synthesising enzyme within vegetative portions of a plant would lead to increased number of oil bodies and TAG in the vegetative tissue. Using either of these techniques leads to a maximum increase in lipid content (not necessarily in the form of TAG) of up to approximately 50%. Furthermore this level begins to decline as the leaves mature; typically in leaves greater than 2 weeks old (unpublished data).
  • Leaf senescence is a highly controlled sequence of events leading ultimately to the death of cells, tissues and finally the whole organ. This entails regulated recruitment of nutrients together with their translocation from the senescing tissue to other tissues that are still growing and developing.
  • the chloroplast is the first organelle of mesophyll cells to show symptoms of senescence and although breakdown of thylakoid membranes is initiated early in the leaf senescence cascade, the chloroplast envelope remains relatively intact until the very late stages of senescence.
  • DGATl is up-regulated during senescence of Arabidopsis leaves and this is temporally correlated with increased levels of TAG- containing fatty acids commonly found in chloroplast galactolipids.
  • modified oleosins for use in the methods of the invention are modified to contain at least one artificially introduced cysteine residue.
  • the engineered oleosins contain at least two cysteines.
  • Such methods include site directed mutagenesis (US 6,448,048) in which the polynucleotide encoding an oleosin is modified to introduce a cysteine into the encoded oleosin protein.
  • polynucleotide encoding the modified oleosins may be synthesised in its entirety.
  • the introduced cysteine may be an additional amino acid (i.e. an insertion) or may replace an existing amino acid (i.e. a replacement).
  • the introduced cysteine replaces an existing amino acid.
  • the replaced amino acid is a charged residue.
  • the charged residue is predicted to be in the hydrophilic domains and therefore likely to be located on the surface of the oil body.
  • hydrophilic, and hydrophobic regions/arms of the oleosin can be easily identified by those skilled in the art using standard methodology (for example: Kyte and Doolitle (1982).
  • the modified oleosins for use in the methods of the invention are preferably range in molecular weight from 5 to 50 kDa, more preferably, 10 to 40kDa, more preferably 15 to 25 kDa.
  • the modified oleosins for use in the methods of the invention are preferably in the size range 100 to 300 amino acids, more preferably 110 to 260 amino acids, more preferably 120 to 250 amino acids, more preferably 130 to 240 amino acids, more preferably 140 to 230 amino acids.
  • the modified oleosins comprise an N-terminal hydrophilic region, two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region.
  • the modified oleosins can be divided almost equally their length into four parts which correspond to the N-terminal hydrophilic region (or arm), the two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region (or arm).
  • modified oleosin is attributed to its physical properties which include a folded hydrophobic core flanked by hydrophilic domains.
  • modified oleosins can be formed into oil bodies when combined with triacylglycerol (TAG) and phospholipid.
  • TAG triacylglycerol
  • topology confers an amphipathic nature to modified oleosin resulting in the hydrophobic domain being embedded in the phospholipid monolayer of the oil body while the flanking hydrophilic domains are exposed to the aqueous environment outside the oil body, such as in the cytoplasm.
  • the modified oleosin for use in the method of the invention comprises a sequence with at least 70% identity the hydrophobic domain of any of the oleosin protein sequences referred to in Table 1 above.
  • the modified oleosin for use in the method of the invention comprises a sequence with at least 70% identity to any of the protein sequences referred to in Table 1 above.
  • the modified oleosin is essentially the same as any of the oleosins referred to in Table 1 above, apart from the additional artificially introduced cysteine or cysteines.
  • the modified oleosin of the invention or used in the method of the invention comprises a sequence with at least 70% identity to the oleosin sequence of SEQ ID NO: 16.
  • the modified oleosin has the same amino acid sequence as that of SEQ ID NO: 16, apart from the additional artificially introduced cysteine or cysteines. In further embodiment the modified oleosin is has the amino acid sequence of any one of SEQ ID NO: 16 to 20.
  • Photosynthesis encompasses a complex series of reactions that involve light absorption, production of stored energy and reducing power (the Light Reactions). It also includes a multistep enzymatic pathway that uses these to convert C0 2 and water into carbohydrates (the Calvin cycle, Figure 20). In plants the biophysical and biochemical reactions of photosynthesis occur within a single chloroplast (C3 photosynthesis) but can also be separated into chloroplasts of differing cell types (C4 photosynthesis). Carbon fixation is a redox reaction, photosynthesis provides both the energy to drive this process as well as the electrons required to convert C0 2 to carbohydrate ( Figure 19).
  • the thylakoid membranes contain the multiprotein photosynthetic complexes Photosystems I and II (PSI and PSII) which include the reaction centres responsible for converting light energy into chemical bond energy (via an electron transfer chain).
  • PSDI and PSII multiprotein photosynthetic complexes
  • the photosynthetic electron transfer chain moves electrons from water into the thylakoid lumen to soluble redox-active compounds in the stroma.
  • a byproduct of this process is oxygen.
  • the second part of the photosynthetic cycle is the fixation of C0 2 into sugars (Calvin Cycle, Figure 20); this occurs in the stroma and uses the ATP and NADPH generated from the light reaction.
  • Ribulose biphosphate carboxlase is the key enzyme responsible for photosynthetic carbon assimilation in catalysing the reaction of C0 2 with ribulose 1 ,5biophosphate (RuBP) to form two molecules of D-phosphoglyceric acid (PGA) (Parry et al, 2003). Since Rubisco works very slowly, catalyzing only the reaction of a few molecules per second, large quantities of the enzyme are required; consequently Rubisco makes up 30-50% of the soluble protein in leaves (Bock and Khan, 2004). Genetic modification to increase the catalytic rate of Rubisco would have great importance. Parry et al, (2003) reviewed the progress to date, concluding that there are still many technical barriers to overcome and to date all engineering attempts have failed to produce a better Rubisco.
  • Rubisco In the presence of 0 2 , Rubisco also performs an oxygenase reaction which initiates photorespiratory or C2 cycle ( Figure 21 ) by the formation of phosphoglycolate and 3-phosphoglycerate (3-PGA). The recycling of phosphoglycolate results in an indirect loss of fixed nitrogen and CO 2 from the cell which need to be recovered. Genetic modification to increase the specificity of Rubisco for C0 2 relative to 0 2 and to increase the catalytic rate of Rubisco in crop plants would have great agronomic importance. Parry et al, (2003) reviewed the progress to date, concluding that there are still many technical barriers to overcome and to date all engineering attempts have thus far failed to produce a better Rubisco (Peterhansel et al. 2008). Furthermore, it has been demonstrated that photorespiration is required in C3 plants to protect plants from photoxidation under high light intensity (Kozaki and Takeba 1996).
  • Organisms capable of oxygenic photosynthesis began their evolution in a vastly different atmosphere (Giordano et al. 2005).
  • One of the most dramatic changes has been the rise in the 0 2 :C0 2 ratio, where the competition between these two gasses for the active site of Rubisco has become progressively restrictive to the rate of carbon fixation.
  • plant Rubiscos are considerd more evolutionarily recent than algal Rubiscos and as such they are much more selective for C0 2 over 0 2 .
  • Genetic modifications to increase the specificity of Rubisco for C0 2 relative to 0 2 have failed (Parry, Andralojc et al. 2003).
  • glycolate dehydrogenase EC 1.1.99.14
  • GDH glycolate dehydrogenase
  • GDH in C. reinhardtii is a mitochondrially located, low-CCVresponsive gene (Nakamura et al, 2005).
  • Other GDH homologs include the so-called glycolate oxidase (GOX) of E. coli and other bacteria.
  • GOX glycolate oxidase
  • the GOX complex is composed of three functional subunits, GlcD, GlcE, and GlcF of which GlcD and GlcE share a highly conserved amino acid sequence that includes a putative flavin-binding region.
  • C4 plants avoid the C2 cycle through modifications to their architecture involving two different types of chloroplast containing cells, mesophyll cells and bundle sheath cells which isolates Rubisco in a relatively rich CO2 environment thereby increasing the proportion of carboxylase reactions. This enables these plants to initially use phosphoenolpyruvate to fix carbon, forming 4-carbon organic acids (hence C 4 plants).
  • the C4 metabolism involves fixing inorganic carbon in one cell type (mesophyll), transporting it to a cell type partially shielded from atmospheric oxygen (bundle sheath), and releasing the inorganic carbon near Rubsico in this oxygen deprived environment.
  • C 4 plants demonstrate an unusual anatomy involving two different types of chloroplast containing cells, mesophyll cells and bundle sheath cells.
  • mesophyll cells surround the bundle sheath cells which in turn surround the vascular tissue; the chloroplasts of the mesophyll cells contain all the trasmembrane complexes required for the light reactions of photosynthesis but little or no Rubisco while the bundle sheath cell chloroplasts lack stacked thylakoids and contain little PSII.
  • C 4 plants concentrate C0 2 in the bundle sheath cells effectively suppressing Rubiscos oxygenase activity and eliminating photorespiration.
  • Oxaloacetate is generated from HC0 3 " and phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol of mesophyll cells.
  • the HCO 3 " ion is used since its aqueous equilibrium is favoured over gaseous C0 2 .
  • PEP carboxylase cannot fix oxygen, which has a 3D structure similar to that of CO 2 but not HCO 3 " .
  • oxaloacetate is oxidised to malate or condensed with glutamate to form aspartate and a Keto glutarate.
  • the malate and aspartate are transported into the bundle sheath cells and decarboxylated releasing C0 2 which is then available for Rubisco and incorporation into the Calvin cycle.
  • the agronomic downside of this evolved modification is an increase in leaf fibre resulting in a comparatively poor digestibility of leaves from C4 plants (e.g., maize, sugarcane, numerous tropical grasses and some dicotyledonous plants such as Amaranthus).
  • C4 plants e.g., maize, sugarcane, numerous tropical grasses and some dicotyledonous plants such as Amaranthus.
  • the modification of a C3 plant to emulate the whole C4 process is beyond current biotechnology.
  • attempts to engineer Rubisco to either obliterate oxygenase activity or to decrease the affinity for O 2 have failed (for review see Peterhansel et al. 2008).
  • GDH from E. coli is a heterotrimer, consisting of glcD, glcE and g/cF resulting in plants with a 30% increase in leaf biomass by the end of the growth period (Figure 24).
  • This pathway included a chloroplast CO 2 release step which further reduced RubisCO's oxygenase activity in vivo.
  • energy and reducing equivalents were thought to be saved by the bypass as it no longer results in the release of ammonium and the energy from glycolate oxidation is saved in reducing equivalents and not consumed during the formation of 3 ⁇ 4(3 ⁇ 4 (Maurino and Peterhansel 2010).
  • Peterhansel (201 1) concluded that to truly transform a C3 plant into a C4 plant will require the efficient transfer of multiple genes.
  • Acyl transferases in the plastid in contrast to thioesterases, terminate fatty acid synthesis by transesterifying acyl moieties from ACP to glycerol, and they are an essential part of the prokaryotic lipid pathway leading to plastid glycerolipid assembly.
  • Seed specific promoters An example of a seed specific promoter is found in US 6,342,657; and US 7,081 ,565; and US 7,405,345; and US 7,642,346; and US 7,371,928.
  • Suitable control plants also include plants that do not express a modified oleosin including at least one artificially introduced cysteine.
  • total lipid includes fats, oils, waxes, sterols, glycerol lipids, monoglycerides, diglycerides, phospholipids, monogalactolipids, digalactolipids, phosphatidylcholines,
  • a "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length.
  • polypeptide encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.
  • Polypeptides of the present invention, or used in the methods of the invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
  • a "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.
  • polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species.
  • the polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.
  • Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.
  • Polynucleotide sequence identity can be determined in the following manner.
  • the subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences.
  • Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453).
  • a full implementation of the Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A.
  • EMBOSS The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276- 277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/.
  • the European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.
  • the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps.
  • GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
  • Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 -6 more preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x 10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more preferably less than 1 x 10 -30, more preferably less than 1 10 -40, more preferably less than 1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably less than 1 x 10-100 when compared with any one of the specifically identified sequences.
  • hybridize under stringent conditions refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • a target polynucleotide molecule such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot
  • the ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
  • Tm melting temperature
  • exemplary stringent hybridization conditions are 5 to 10° C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) 0 C.
  • Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l ;26(21):5004-6.
  • Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.
  • Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention.
  • a sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
  • Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention.
  • a skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al, 1990, Science 247, 1306).
  • Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih. gov/blast/) via the tblastx algorithm as previously described.
  • Polypeptide variants may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih. gov/blast/) via the tblastx algorithm as previously described.
  • variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%», more preferably at least 58%), more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more
  • Polypeptide sequence identity can be determined in the following manner.
  • the subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • BLASTP from the BLAST suite of programs, version 2.2.5 [Nov 2002]
  • bl2seq which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • NCBI ftp://ftp.ncbi.nih.gov/blast/.
  • the default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
  • Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs.
  • EMBOSS-needle available at http:/www. ebi.ac.uk/emboss/align
  • GAP Human, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.
  • suitable global sequence alignment programs for calculating polypeptide sequence identity.
  • a preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)
  • Polypeptide variants of the present invention, or used in the methods of the invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp
  • Variant polypeptide sequences preferably exhibit an E value of less than 1 10 -6 more preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x 10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than 1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably 1x10-100 when compared with any one of the specifically identified sequences.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
  • the term "genetic construct” refers to a polynucleotide molecule, usually double- stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule.
  • a genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • the insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA.
  • the genetic construct may be linked to a vector.
  • vector refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell.
  • the vector may be capable of replication in at least one additional host system, such as E. coli.
  • expression construct refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • An expression construct typically comprises in a 5' to 3' direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and a terminator functional in the host cell into which the construct will be transformed.
  • coding region or "open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences.
  • the coding sequence may, in some cases, identified by the presence of a 5' translation start codon and a 3' translation stop codon.
  • a "coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.
  • “Operably- linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
  • the term "noncoding region” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination, mRNA stability, and for regulation of translation efficiency. Terminators are sequences, which terminate transcription, and are found in the 3 ' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
  • promoter refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors. Introns within coding sequences can also regulate transcription and influence post-transcriptional processing (including splicing, capping and polyadenylation).
  • a promoter may be homologous with respect to the polynucleotide to be expressed. This means that the promoter and polynucleotide are found operably linked in nature. Alternatively the promoter may be heterologous with respect to the polynucleotide to be expressed. This means that the promoter and the polynucleotide are not found operably linked in nature.
  • transgene is a polynucleotide that is taken from one organism and introduced into a different organism by transformation.
  • the transgene may be derived from the same species or from a different species as'the species of the organism into which the transgene is introduced.
  • An "inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,
  • Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
  • Host cells may be derived from, for example, bacterial, fungal, yeast, insect, mammalian, algal or plant organisms. Host cells may also be synthetic cells. Preferred host cells are eukaryotic cells. A particularly preferred host cell is a plant cell.
  • a "transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation.
  • the new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
  • polypeptides of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art.
  • such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain
  • polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
  • hybridization probes include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes.
  • Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65 °C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution; washing (three washes of twenty minutes each at 5 °C) in 1.
  • An optional further wash (for twenty minutes) can be conducted under conditions of 0.1 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C.
  • polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.
  • a partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5 'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization- based method,
  • inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference).
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region.
  • standard molecular biology approaches can be utilized (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
  • transgenic plant from a particular species, it may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species.
  • the benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms.
  • down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species.
  • Variants including orthologues may be identified by the methods described.
  • Variant polypeptides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The
  • polynucleotide sequence of a primer useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
  • library screening methods well known to those skilled in the art, may be employed
  • Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular
  • variant sequences of the invention may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
  • An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38 A, Room 8N805, Bethesda, MD 20894 USA.
  • NCBI National Center for Biotechnology Information
  • the NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases.
  • BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
  • BLASTP compares an amino acid query sequence against a protein sequence database.
  • BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database.
  • tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.
  • tBLASTX compares the six- frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
  • the use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
  • the "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm align and identify similar portions of sequences.
  • the hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
  • the BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments.
  • the Expect value (E) indicates the number of hits one can "expect” to see by chance when searching a database of the same size containing random contiguous sequences.
  • the Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • the probability of finding a match by chance in that database is 1 % or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • MAST Motif Alignment and Search Tool
  • PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al, 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences.
  • the PROSITE database www.expasy.org/prosite
  • Prosearch is a tool that can search SWISS- PROT and EMBL databases with a given sequence pattern or signature.
  • polypeptides may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 431 A Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.
  • peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 431 A Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.
  • polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification,).
  • polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms.
  • the genetic constructs of the invention are intended to include expression constructs as herein defined.
  • the invention provides a host cell which comprises a genetic construct or vector of the invention.
  • Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention.
  • the expressed recombinant polypeptide which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification). Methods for producing plant cells and plants comprising constructs and vectors
  • the invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention, or used in the methods of the invention. Plants comprising such cells also form an aspect of the invention.
  • Methods for transforming plant cells, plants and portions thereof with polypeptides are described in Draper et al, 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer- Verlag, Berlin.; and Gelvin et al, 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht.
  • a review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
  • strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed.
  • the expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
  • Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
  • the promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature.
  • Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.
  • Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zein gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.
  • Oleosin (or 01e)_3- l means an oleosin with three engineered cysteines in the N-terminal hydrophilic arm and one engineered cysteine in the C-terminal hydrophilic arm.
  • Figure 6 shows a map of the construct pRShl used for transforming plants.
  • the map shows the arrangement of the oleosins, with artificially introduced cysteines (in this case 01eo_3-3) under the control of the CaMV35s promoter as well as Arabidopsis thaliana DGATl (S205A) also under the control of the CaMV35s promoter.
  • cysteines in this case 01eo_3-3
  • S205A Arabidopsis thaliana DGATl
  • Other oleosin sequences and TAG synthesising enzyme sequences can of course be substituted for 01eo_3-3 and DGATl respectively.
  • Figure 12 shows immunoblot analysis of oleosin (Oleo_0-0, 01eo_l -3, 01eo_3-l , and 01eo_3-3, SEQ ID NOs 11-20) accumulation in the leaves of transgenic Arabidopsis thaliana expressing both DGAT1 (S205A) and a sesame oleosin under the control of CaMV35S promoters.
  • Figure 13 shows immunoblot of recombinant oleosin accumulation (black arrow) in transgenic
  • Figure 14 shows FAMES GC/MS results demonstratinging accumulation of additional lipids (black arrows) in Arabidopsis leaves over expressing DGAT1 (S205A) and 01e_3,3.
  • Figure 16 shows GC/MS results showing total TAG profile of wild type and transgenic Arabidopsis (containing DGAT1 (S205A) and 01e_3,3) 2, 3, 4 and 5 weeks after germination. Black arrows indicate additional TAGs found in transgenic leaves but not wild type.
  • Figure 17 shows FAMES GC/MS results showing total leaf lipid profiles of wild type and transgeneic Trifolium repens (containing DGAT1 (S205A) and 01e_3,3).
  • Figure 26 shows schematic presentation of the influence of continual lipid biosynthesis in the transgenic leaf.
  • Fatty acids are synthesised in the plastid transported to the endoplasmic reticulum, sequentially acylated onto a glycerol backbone via the Kennedy pathway; this culminates in the production of triacylglyceride via over expression of the enzyme DGAT.
  • the 3-phosphoglyceric acid is synthesised by Rubisco (without the Calvin cycle) rather than the transformation of sugars.
  • the subsequent transformation of this to acetyl-CoA results in the release of C0 2 in the chloroplast.
  • This increases the partial pressure of C0 2 relative to 0 2 in the chloroplast thus reducing the proportion of C2 to C3 cycles initiated by Rubisco and increasing the rate of C0 2 assimilation.
  • Figure 27 shows schematic presentation of the catabolism of unprotected TAG produced in the transgenic leaf.
  • the over expression of DGAT leads to the accumulation of TAG which is subsequently degraded by lipases resulting in the release of free fatty acids.
  • Some of these free fatty acids are catabolised by ⁇ - oxidation in the peroxisome while others set up a futile cycle by re-entering the endoplasmic reticulum where they are re-incorporated into TAG.
  • Figure 35 left hand panel shows total quantity (as % of DW) for each major lipid species in roots of wild type (black bars) plants and in roots of plants transformed with DGATl-01e_3,3 (grey bars).
  • Right hand panel shows each major lipid species as a % of total lipids in roots of wild type (black bars) plants and in roots of plants transformed with DGATl-01e_3,3 (grey bars).
  • Figure 36 shows four traces offset and over layed.
  • Oleosin-cysteine proteins mutated to include cysteine residues in both the N- and C- terminal hydrophilic regions described here are designated Ole-1-1, Ole-1-3, Ole-3-1 , and Ole-3-3 (SEQ ID NO 2, 3, 4, and 5 respectively), where the first and the second numeral digits correspond to the number of disulfide bonds in the N- and C- terminus, respectively.
  • the standard oleosin without the cysteine residues was used as a control and was designated as Ole-0-0 (SEQ ID NO 1).
  • coli BL21 Rosetta-Gami containing an oleosin (with or without engineered cysteine residues) coding sequence in the pET29 expression vector.
  • the culture was grown at 37°C, 220rpm, until mid log phase (OD 6 oo0.5 - 0.7); expression was induced by the addition of IPTG to 1 mM final concentration.
  • the induced culture was incubated at 37°C, 220rpm, for a further 2-3 h. Given the properties of modified oleosin the applicants did not attempt to express it in a soluble form but rather chose to extract it from inclusion bodies. Aliquots (l mL) of the culture were transferred to 1.5mL microfuge tubes and the cells pelleted by centrifugation (2655 xg for 5 min at 4°C).
  • EXAMPLE 3 Use of anti-sesame seed oleosin antibodies to bind sesame seed oleosin with artificially introduced cysteines
  • EXAMPLE 4 Creation of artificial oil bodies with E. coli expressed modified oleosins containing at least one artificially introduced cysteine and altering the degree of cross linking Preparation of artificial oil bodies
  • Ole-0-0 Recombinant Ole-0-0, and all variations of the oleosin-cysteines were successfully expressed and located in E. coli inclusion bodies (Figure 9).
  • Ole-0-0 was predominantly present as a monomer (in both inclusion bodies as well as AOBs); this migrated fractionally faster than the 20kDa molecular weight marker (in reducing and non reducing SDS and SDS-UREA PAGE). Also present were two slower migrating immunoreactive bands of approximately 35 and 36 kDa which likely correspond to two forms of dimeric oleosin.
  • Ole-0-0 is not predicted to contain any cysteine residues the overall intensity and ratio of the two apparent dimers was influenced by the presence of reducing agents ( ⁇ - ⁇ @ 5% of the sample loading buffer and 1 OmM DTT).
  • AOBs generated with Ole-1 -1 showed the presence of almost equal portions of monomer and dimer and a small amount of trimer, indicating that the conditions under which the AOBs are formed have some reducing potential.
  • the subsequent addition of GSSG resulted in an increase in the oligomeric portions as well as the appearance of a tetrameric form. While the monomer was the predominant form of Ole-3-3 in the inclusion bodies, a comparatively high percentage was also present in multiple oligomeric forms. The proportion of oligomers declined to a small extent with the addition of reducing agent and slightly more by the addition of both reducing and chaotropic agents.
  • AOB buffer containing Proteinase K [PNK] when appropriate at a 1 : 1 ratio of PNK:total proteins in OB or AOB samples in a 250 ⁇ GC glass insert tubes and covered with a plastic cap.
  • PNK Proteinase K
  • the applicants synthesised individual coding sequences for the sesame seed oleosin (based on GenBank clone AF091840) with different numbers of cysteines in the N- and C-terminal arms.
  • the coding sequence was flanked by a 5' Notl site and a 3' Ndel site.
  • a separate acceptor cassette was synthesised containing an attLl site, a Notl site and Ndel site followed by a nos termination sequence, a forward facing CaMV35s promoter, the Arabidopsis thaliana DGAT1 (S205A) (SEQ ID NOs 11-20 and Figures 1-5) plus its own UBQ10 intron, an attL2 site.
  • the sesame seed oleosins with different numbers of cysteines were individually transferred to the acceptor cassette via the NotI and Ndel sites. Each of these completed cassettes were then transferred to a plant binary vector pRShl , Figure 6 (Winichayakul et al., 2008) via the LR recombination reaction. This placed the oleosin downstream of a CaMV35S promoter (already contained within pRShl) and placed a nos terminator (already contained within pRShl) downstream of the Arabidopsis DGAT1 (S205A) ( Figures 1-5).
  • nucleotide sequences encoding the sesame seed oleosins (with cysteines) and DGAT1 were optimised for expression in Arabidopsis thaliana, this included optimisation of codon frequency, GC content, removal of cryptic splice sites, removal of mRNA instability sequences, removal of potential polyadenylation recognition sites, and addition of tetranucleotide stop codon (Brown et al, 1990; Beelman and Parker, 1995; Rose, 2004; Rose and Beliakoff, 2000; Norris et al., 1993).
  • oleosin sequence used is for example only. Any oleosin or steroleosin or caoleosin sequences could be engineered to contain cross-linking regions.
  • the coding sequences of the complete ORFs (after splicing) were checked against repeat of the original oleosin translated sequence and found to be identical over the oleosin coding regions.
  • Tl seed was collected from the treated plants, germinated and selected by spraying at 14 d and 21 d post- germination with Basta ® .
  • Basta ® resistant Tl plants (71 , 62 and 23 transformants containing the single sesame seed oleosin, and modified oldeosin constructs respectively) were transplanted, allowed to self- fertilise, set seed and the T2 seed was collected.
  • EXAMPLE 6 Extraction and purificiation oil bodies with modified oleosins containing at least one artificially introduced cysteine from the seeds of Arabidopsis thaliana
  • Crude Oil Body Preparations from Arabidopsis thaliana seeds Crude Oil Body Preparations from Arabidopsis thaliana seeds
  • Crude OB preparations were prepared, from seed of plants produced as described in Example 5, by either grinding 200mg seed with a mortar and pestle containing a spatula tip of sand and 750 ⁇ Extraction Buffer (lOmM phosphate buffer, pH 7.5 containing 600mM sucrose) or by homogenising 25mg of seed in 300 ⁇ Extraction Buffer using a Wiggenhauser D-130 Homogenizer. A further 750 ⁇ Extraction Buffer was added and the slurry in the mortar and transferred to a 2mL microfuge tube whereas the homogenizer tip was rinsed in lmL Extraction Buffer and this volume was added to the homogenised seed.
  • Extraction Buffer lOmM phosphate buffer, pH 7.5 containing 600mM sucrose
  • the negative control was a sample extracted from wild type Columbia seed and the positive control was the same extraction method (although grinding was by mortar and pestle) performed on wild type sesame seed. ⁇ of each sample and the negative control were loaded onto the gel, and 5 ⁇ 1 was used for the positive control.
  • the membrane was blocked in a solution of 12.5% skim milk powder in TBST (50 mM Tris pH 7.4, 100 mM NaCl, 0.2 % Tween) for at least 1.5 hours. The membrane was then washed in TBST 3 x 5 mins before incubating with primary antibody (anti-sesame) at 1/1000 in TBST for 1 hour at room temperature. Following 3 further TBST washes, incubation with secondary antibody (anti-rabbit) at 1/5000 was carried out for 1 hour at room temperature. The membrane underwent 3 further washes then the signal was developed using standard chemiluminesence protocol.
  • Figure 1 1 shows the accumulation of sesame seed oleosin units on the oil bodies under the control of the CaMV35S promoter. It can be seen that recombinant oleosin and polyoleosin was found to accumulate in the seeds of Arabidopsis thaliana and was correctly targeted to the oil bodies ( Figure 1 1 ). In addition, it can be seen that in the presence of oxidising agent for 10 minutes the recombinant oleosins containing cysteines formed cross-links as evidenced by the appearance of oligomers and corresponding
  • OB preparations are made up to a total volume of 200 ⁇ using AOB buffer (containing Proteinase [PNK] when appropriate at a 1 : 1 ratio of PNK:total proteins in OB samples in a 250 ⁇ GC glass insert tubes and covered with a plastic cap.
  • AOB buffer containing Proteinase [PNK] when appropriate at a 1 : 1 ratio of PNK:total proteins in OB samples in a 250 ⁇ GC glass insert tubes and covered with a plastic cap.
  • fish oil Vitamax®, Australia
  • the addition of fish oil followed by vortexing enables any TAG that had leaked from the OBs to mix with the added fish oil and be floated by brief centrifugation.
  • TAG that had leaked from the OBs was too small to form a samplable visible layer even after centrifugation, in such a case the maximum volume would have been 6 ⁇ .
  • the very different lipid profiles of fish oil and sesame oil enabled us to easily distinguish the leaked TAG from the added TAG.
  • CI 8:2 the major lipid in sesame seed oil
  • Oil in water emulsions are less stable at elevated temperatures; hence, it is of interest to investigate if modified oleosins with varying numbers in introduced cysteines influence OB and AOB integrity at elevated temperature.
  • the applicants determine the integrity (using the method described above) of OBs (containing different oleosins) in an phosphate buffer (50mM Na-phosphate buffer pH8, lOOmM NaCl) at 95°C. AOBs are heated for 2h. Integrity is determined as above.
  • OB integrity in rumen fluid One of the aims of disulfide was to provide some degree of protection from biohydrogenation by rumen microflora.
  • Assessment of OB stability with rumen fluid can be assessed as follows. OBs are added to an equal volume ⁇ 25 xL) of rumen fluid. Samples are incubated at 39°C for 0, 15, 30, 60, 120 and 240min, at the end of the incubation an equal volume of loading buffer (Invitrogen) is added, mixed and heated at 70°C for lOmin. 15 ⁇ of each sample/loading buffer mix is compared by SDS-PAGE/immunoblot. Integrity is determined as above.
  • TAG biosynthesis is the last one, i.e., the addition of a third fatty acid to an existing diacylglycerol, thus generating TAG.
  • this step is performed by one of three enzymes including: acyl CoA:diacylglycerol acyltransferase (DGAT1), an unrelated acyl CoA:diacylglycerol acyl transferase (DGAT2), and phospholipid:diacylglycerol acyltransferase (Zou et al, 1999; Bouvier-Nave et al, 2000; Dahlqvist et al., 2000; Lardizabal et al., 2001).
  • DGAT1 acyl CoA:diacylglycerol acyltransferase
  • DGAT2 unrelated acyl CoA:diacylglycerol acyl transferase
  • the total lipids can be extracted using the Folsch method (Folsch et al., 1957 J. Folsch, M. Lees and G.A. Slone-Stanley, A simple method for the determination of total lipid extraction and purification, Journal of Biological
  • Leaves were sampled from plants over expressing the A. thaliana DGAT1 (S205A) and the sesame seed oleosin construct (either Oleo_0-0, or Oleo l-1, or 01eo_l-3, or 01eo_3-l, or 01eo_3-3, SEQ ID NOs 11-20, Figures 1-5) and analysed by SDS-PAGE/immunoblot using the polyclonal anti-sesame seed oleosin antisera. It can be seen that recombinant oleosin was found to accumulate in the leaves of Arabidopsis thaliana leaves (Figure 12).
  • oleosin/modified oleosin protein in the same cell (for example leaf cell) will result in the production of triglyceride oil bodies encapsulated by a phospholipid monolayer embedded with oleosin; this has been demonstrated with un-modified oleosin in yeast (Ting et al., 1997) and seeds (Abell et al., 2004).
  • Oil bodies can be extracted from the leaves of transgenic Arabidopsis thaliana expressing DGATl (S205A) and the sesame seed oleosin construct (either Oleo_0-0, or 01eo_l-l, or 01eo_l-3, or 01eo_3-l, or 01eo_3-3, SEQ ID NOs 11-20, Figures 1-5).
  • the ole-3,3 lines had substantial levels of elevated lipid levels in the form of TAGs when co-expressed with DGATl (S205A) while the lines containing ole-0,0 did not have elevated lipid levels above the DGATl over expressing control.
  • the ole-1,1, ole-1,3 and ole-3,1 showed there was a correlation between the level of lipid accumulation in the leaf and the increase in the number of cysteines engineered into each arm (Table 3).
  • the oleosin presumably needs to have a certain level of negative charge and in the C-terminus this appears to be achieved by K (Lys), hence continuing the strategy of swapping charged or neutral residues with additional cysteines may result in poor stability in terms of preventing coalescence. Furthermore, in the N-terminal hydrophilic region there appears to be too few residues left between the engineered cysteines to enable further substitution of residues whilst maintaining the spacing and oscillation between positive and negatively charged amino acids. Hence, for both N- and C-termini added additional residues (cysteines) rather than substitute existing residues with cysteines. Alternatively, an oleosin with longer hydrophilic arms could have been used.
  • Sublconing strategy was designed to be identical to initial cysteine oleosins, i.e., subcloned into oleoacceptor by Notl/Ndel. This is then recombined by LR reaction into pRSHl (Winichayakul et al., 2008). Essentially places both Arabidopsis DGAT1 (S205A) and oleosin under their own CaMV35s promoters and OCS terminators. Both DGA1 and oleosin clones contain a TJBQIO intron.
  • NetGene2 was used to predict the splicing pattern of 01e_5,6 and 01e_6,7. Both were predicted to have only one donor and acceptor site on the direct strand (both were predicted to have a very high probability of recognition) and no sites on the complementary strand.
  • Transformation of oleosins containing engineered cysteines and DGAT1 into wild type Arabidopsis thaliana Five disulfide-oleosin/DGATl (S205A) gene constructs and one control (construct containing DGAT1 (S205A) but not oleosin) were been transferred to the plant binary vector pRShl (Winichayakul et al, 2008) and transformed into wild type Arabidopsis thaliana using Agrobacterium-mediated transformation.
  • Cotyledons were dissected from seeds using a dissecting microscope. First, the seed coat and endosperm were removed. Cotyledons were separated from the radical with the scalpel by placing the blade between the cotyledons and slicing through the remaining stalk. Cotyledonary explants were harvested onto a sterile filter disk on CR7 media.
  • a 3ul aliquot of Agrobacterium suspension was dispensed to each dissected cotyledon. Plates were sealed and cultured at 25°C under a 16 hour photoperiod. Following a 72 hour period of co- cultivation, transformed cotyledons were transferred to plates containing CR7 medium supplemented with ammonium glufosinate (2.5mg/L) and timentin (300mg/L) and returned to the culture room.
  • explants were transferred to CR5 medium supplemented with ammonium glufosinate (2.5mg/L) and timentin (300mg/L). Regenerating shoots are subcultured three weekly to fresh CR5 media containing selection.
  • plantlets were transferred into tubs containing CR0 medium containing ammonium glufosinate selection. Large clumps of regenerants were divided to individual plantlets at this stage. Whole, rooted plants growing under selection were then potted into sterile peat plugs. Once established in peat plugs plants were then transfer to the greenhouse.
  • FAMES GC/MS results showed the transgeneic Trifolium repens (containing DGATl (S205A) and either 01e_3,3 or 01e_5,6 or Ole 6,7) had elevated total leaf lipid profiles compared to wild type ( Figure 17). There was a general correlation between the highest level of leaf lipid and the highest number of cysteines engineered into the oleosin. FAMES GC/MS results showed the transgeneic Trifolium repens (containing DGATl (S205A) and either 01e_3,3 or 01e_5,6 or Ole 6,7) had elevated C18:l and C18:2 leaf lipid profiles compared to wild type as also seen in Arabidopsis (Figure 18). The highest level of leaf CI 8:1 and CI 8:2 was found in plants transformed with the oleosin containing the highest number of engineered cysteines.
  • Crude oil body (OB) was extracted from 25 mg of seeds in 500 ⁇ L OB buffer (10 mM Sodium phosphate, pH 7.5 containing 600 mM sucrose). After centrifugation at 13,000 x g, the aqueous layer was carefully suck out and the fat pad layer was resuspended in 200 ⁇ iL of OB buffer without disturbing the pellet at the bottom. 20 ⁇ iL of each OB extract was added with 4x loading dye and lOx reducing agent, heated up to 70°C for 5 min and loaded onto 4-12% polyacrylamide gel for immunoblot analysis. The blot was incubated in osesame oleosin antibody (l°Ab) at 1 :750 dilution for one hour, and another one hour in secondary antibody (1 : 10,000).
  • l°Ab osesame oleosin antibody
  • Oleosin is naturally expressed in seeds and not in the leaves. However, since we have co-expressed DGAT1 with oleosin both under the control of CaMV35S promoters it could be anticipated that this would enable detectable levels of oleosin to accumulate in the leaves. Leaves from transformed lines with high expression of recombinant oleosin in the seeds (identified by immunoblot analysis) were analyzed by immunoblot using antibodies raised against the sesame oleosin.
  • Table 5 summarises the number of putative transformants generated and the number of plants expressing recombinant oleosin in the seed and leaf.
  • the seeds from homozygous lines over expressing the oleosin protein in the seeds were germinated to allow growth of 2, 3, 4 or 5 weeks. Sufficient leaf material was harvested for FAMES GC-MS, as well as by GC-MS using a RTX 65-TG Restek column which enable the separation and identification of free fatty acids, diacylglycerides, wax esters, sterol esters and triacylglycerides without derivatization.
  • the sample injector port temperature was maintained at 350 °C, column oven temperature at 200 °C, with a pressure of 131.1 kPa and a purge flow of 3.0 mL.min-1.
  • the mass spectrometric conditions were as follows: ion source temperature was held at 260 °C during the GC-MS runs, the mass spectra were obtained at ionization voltage of 70 eV at an emission current of 60 ⁇ and an interface temperature of 350 °C. Acquisition mode was by scanning at a speed of 5000, 0.25 sec per scan. Chromatograph peaks with mass to charge ratio of 45 m/z to 1090m/z were collected starting at 9 min and ending at 25 min.
  • EXAMPLE 8 Further oleosins, caloleosins and steroleosins engineered to contain additional cysteine residues in the N- and C- terminal hydrophilic arms
  • each protein was aligned with the sesame oleosin (AAD42942) in the original form as well as the forms containing 1 or 3 cysteines per hydrophilic arm (i.e., ole_0,0; ole_l,l ; ole_3,l ; ole_l,3; ole_3,3).
  • the potential glutamic acids and aspartic acids in N-terminus or C-terminus of each of the hydrophilic arms were then highlighted with grey boxes, as were the relevant lysine, arginine and glutamine residues (which were all successfully altered in the sesame oleosin (AAD42942).
  • Table 6 below shows additional oleosin and caoleosins that the applicants have modified to introduce cysteines in the hydrophilic portions.
  • Oleosin Brassica oleraceae (pollen CAA65272.1 90
  • modified sequence can be expressed as described in the examples above to produce oil bodies, emulsions, transgenic host cells, plants etc, and to test the properties of each. It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention.
  • EXAMPLE 9 Increased biomass production through elevation of chloroplast C0 2 concentration, elevation of C0 2 assimilation rate and elevation of intrinsic Water Use Efficiency in the leaves
  • Example 7 The applicants have used the same strategy in Example 7 (by preventing the catabolism of TAG in the leaf which inturn ensures there is a continual recycling of C(3 ⁇ 4 from pyruvate as it is used by the pyruvate dehydrogenase complex to generate Acetyl-CoA for lipid biosynthesis) to not only increase the CO 2 assimilation rate but also elevate intrinsic Water Use Efficiency. The net effect of this is to elevate the partial pressure of C0 2 compared to O 2 in the chloroplast.
  • Rates of photosynthesis were significantly greater (under photorespiratory conditions) in transgenic plants compared to wild type plants; similarly, rates of photosynthesis were significantly greater (under nonphotorespiratory conditions) in transgenic plants compared to wild type plants.
  • Figure 30, left hand panel The DGATl-01e_3,3 plants had greater increases in photosynthesis when photorespiration was completely removed using a low 0 2 environment compared to wild type ( Figure 30, right hand panel). Thus showing that DGATl-01e_3,3 plants have elevated C0 2 assimilation rates compared to wild type plants.
  • Intrinsic water-use efficiency measurements were significantly greater under nonphotorespiratory conditions than under ambient oxygen concentration for both the WT and T genotypes (Figure 31 , left hand panel). At ambient 0 2 levels the iWUE was consistently higher for plants transformed with DGAT1- 01e_3,3 than wild type plants; this was further demonstrated by the fact that the DGATl-01e_3,3 plants had smaller increases in iWUE when photorespiration was completely removed using a low 0 2 environment ( Figure 31, right hand panel). Thus showing that DGATl-01e_3,3 plants have higher iWUE compared to wild type plants.
  • Roots from 01e_3,3 and wild type plants were extracted using the same procedures described in Example 7. Quantitative FAMES analysis (Figure 35 left panel) showed that the total lipid content of the roots from 01e_3,3 was 8.2% of the DM while the total lipid content of the wild type roots was 1.7% of the DM. FAMES also showed that the lipid profile of the 01e_3,3 roots was not too different from the wild type ( Figure 34 right panel). The most noticeable change was the proportion of CI 8:1 was 4.0% in the wild type roots and rose over four fold to 18.1% of the total fatty acids in the roots of DGATl-01e_3,3 plants.
  • Oleosin disulfide 0,0 nucleotide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 1,1 nucleotide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 1,3 nucleotide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 3,1 nucleotide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 3,3 nucleotide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 0,0 peptide sequence as
  • Oleosin disulfide 1,1 peptide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 1,3 peptide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 3,1 peptide sequence as cloned into pET29b using Ndel and Xhol
  • Oleosin disulfide 3,3 peptide sequence as cloned into pET29b using Ndel and Xhol
  • Nucleotide sequence of Oleosin disulfide 1,1 including Kozac sequence and U BQ10
  • intron as transformed into Arabidopsis thaliana under the control of the CaMV35s promoter.
  • Nucleotide sequence of Oleosin disulfide 5,6 including Kozac sequence and UBQ10
  • Oleoacceptor (contains OCS terminator
  • Oleosin _1,1 and DGAT1 (S205A)
  • Polypeptide A thaliana Oleosin - AAZ23930
  • Polynucleotide Z. mays Oleosin - NM_001153560.1
  • Polypeptide Z. mays Oleosin - NP_001147032.1
  • Polypeptide B oleracea Oleosin - AAD24547.1
  • Polynucleotide Z. mays Steroleosin - NM_001159142.1
  • Polypeptide Z. mays Steroleosin - NP_001152614.1
  • Polypeptide Z. mays Caleosin - NP_001151906
  • Polypeptide A thaliana DGAT1 - NP_179535
  • Polynucleotide Z. mays DGAT1 - EU039830.1
  • Polypeptide Z. mays DGAT1 - ABV91586.1
  • Polypeptide A thaliana DGAT2 - NP_566952.1
  • Polypeptide DGAT3 (soluble DGAT) - AAX62735.1 hypogaea 86 Polynucleotide A. thaliana PDAT - NM_121367
  • Polypeptide Z. mays steroleosin NP_001152614.1
  • Polypeptide Z. mays Modified steroleosin- NP_001152614.1

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Abstract

L'invention porte sur des procédés permettant d'accroître le taux d'assimilation du CO2 dans des cellules et plantes photosynthétiques. L'invention comprend la réduction ou l'inhibition du recyclage de lipides et/ou l'expression d'oléosines modifiées renfermant des résidus de cystéine introduits de façon artificielle dans les cellules et plantes photosynthétiques. L'invention porte également sur des procédés permettant d'accroître la production d'huile dans des plantes, par l'intermédiaire de l'expression d'oléosines modifiées renfermant des résidus de cystéine introduits de façon artificielle dans les tissus/organes non photosynthétiques de plantes. Le procédé comprend également éventuellement l'étape d'extraction de l'huile des tissus/organes non photosynthétiques de la plante ou le traitement des tissus/organes non photosynthétiques riches en huile en matières de base animales ou de biocombustible.
PCT/NZ2012/000138 2011-08-05 2012-08-03 Procédés permettant d'accroître l'assimilation de co2 et la production d'huile dans des organismes photosynthétiques WO2013022353A1 (fr)

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NZ620832A NZ620832B2 (en) 2011-08-05 2012-08-03 Methods for increasing co2 assimilation and oil production in photosynthetic organisms
AU2012294956A AU2012294956B2 (en) 2011-08-05 2012-08-03 Methods for increasing CO2 assimilation and oil production in photosynthetic organisms
BR112014002748A BR112014002748A2 (pt) 2011-08-05 2012-08-03 métodos para aumento de assimilação de co2 e produção de óleo em organismos fotossintéticos
MX2014001441A MX354499B (es) 2011-08-05 2012-08-03 Metodo para aumentar la asimilación de dioxido de carbono (co2) y la producción de aceite en organismos fotosinteticos.
CA2844239A CA2844239C (fr) 2011-08-05 2012-08-03 Procedes permettant d'accroitre l'assimilation de co2 et la production d'huile dans des organismes photosynthetiques

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US9512438B2 (en) 2011-12-27 2016-12-06 Commonwealth Scientific And Industrial Research Organisation Processes for producing hydrocarbon products
US10246718B2 (en) 2011-12-27 2019-04-02 The Commonwealth Scientific And Industrial Research Organisation Processes for producing lipids
US10260021B2 (en) 2006-07-14 2019-04-16 Commonwealth Scientific And Industrial Research Organisation Rice plants and methods of producing rice grain
US10472587B2 (en) 2014-07-07 2019-11-12 Commonwealth Scientific And Industrial Research Organisation Processes for producing industrial products from plant lipids
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US11639507B2 (en) 2011-12-27 2023-05-02 Commonwealth Scientific And Industrial Research Organisation Processes for producing lipids
US11859193B2 (en) 2016-09-02 2024-01-02 Nuseed Global Innovation Ltd. Plants with modified traits

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10260021B2 (en) 2006-07-14 2019-04-16 Commonwealth Scientific And Industrial Research Organisation Rice plants and methods of producing rice grain
US11639507B2 (en) 2011-12-27 2023-05-02 Commonwealth Scientific And Industrial Research Organisation Processes for producing lipids
US9512438B2 (en) 2011-12-27 2016-12-06 Commonwealth Scientific And Industrial Research Organisation Processes for producing hydrocarbon products
US10246718B2 (en) 2011-12-27 2019-04-02 The Commonwealth Scientific And Industrial Research Organisation Processes for producing lipids
US10246641B2 (en) 2011-12-27 2019-04-02 The Commonwealth Scientific And Industrial Research Organisation Processes for producing hydrocarbon products
AU2013205482B2 (en) * 2011-12-27 2016-11-10 Nuseed Global Innovation Ltd Processes for producing lipids
US10472587B2 (en) 2014-07-07 2019-11-12 Commonwealth Scientific And Industrial Research Organisation Processes for producing industrial products from plant lipids
US11365369B2 (en) 2014-07-07 2022-06-21 Commonwealth Scientific And Industrial Research Organisation Processes for producing industrial products from plant lipids
US11814600B2 (en) 2014-07-07 2023-11-14 Nuseed Global Innnovation Ltd. Process for producing industrial products from plant lipids
US11859193B2 (en) 2016-09-02 2024-01-02 Nuseed Global Innovation Ltd. Plants with modified traits
WO2021079297A1 (fr) * 2019-10-25 2021-04-29 Agresearch Limited Procédés d'amélioration d'organismes photosynthétiques
US20220290174A1 (en) * 2019-10-25 2022-09-15 Agresearch Limited Methods for improving photosynthetic organisms
CN114829610A (zh) * 2019-10-25 2022-07-29 农牧研究公司 改进光合生物的方法
EP4048797A4 (fr) * 2019-10-25 2024-01-03 AgResearch Limited Procédés d'amélioration d'organismes photosynthétiques

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