EP0770134A2 - Method for producing fatty acids or derivatives thereof from oil plants - Google Patents

Method for producing fatty acids or derivatives thereof from oil plants

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Publication number
EP0770134A2
EP0770134A2 EP95925897A EP95925897A EP0770134A2 EP 0770134 A2 EP0770134 A2 EP 0770134A2 EP 95925897 A EP95925897 A EP 95925897A EP 95925897 A EP95925897 A EP 95925897A EP 0770134 A2 EP0770134 A2 EP 0770134A2
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EP
European Patent Office
Prior art keywords
plant
lipase
gene
promoter
expression cassette
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95925897A
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German (de)
French (fr)
Inventor
Gilbert Alibert
Zéphirin Mouloungui
Alain Boudet
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Institut National Polytechnique de Toulouse INPT
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Institut National Polytechnique de Toulouse INPT
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Publication of EP0770134A2 publication Critical patent/EP0770134A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
    • 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
    • C12N15/8242—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
    • 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
    • C12N15/8242—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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to a method for producing fatty acids or fatty acid derivatives (esters or other derivatives) from oil plants.
  • the process of the invention applies in particular to oleoproteinous plants such as rapeseed, sunflower, soybean, crambé ...
  • the invention can in particular be used to manufacture bio-fuels (diester), lubricants, phytosanitary adjuvants, detergents ... by transformation of the fatty acids produced.
  • oilseed plants as raw material goes through the production of free fatty acids which constitute the raw material for the industries of transformation towards fuels, lubricants ...
  • the lipids accumulated by oilseed plants can be transformed into fatty acid by hydrolysis: two processes are currently used industrially to effect this transformation.
  • a first method consists in hydrolyzing the lipids after extraction by bringing the extracted lipids into hot contact and under pressure with sulfuric methanol or methanolic potash.
  • Another method consists in operating the hydrolysis under similar conditions directly on the ground material of seeds without prior extraction.
  • the well-known shortcomings of these methods are as follows: high cost of implementation, infrastructure heavy industrial, polluting character of effluents, production of glycerol as a by-product without market currently.
  • the present invention proposes to provide a new solution to the problem of the production of fatty acid from oil plants. It aims to provide a solution whose implementation costs are considerably lower compared to known processes (both industrial chemical processes and laboratory enzymatic process).
  • An objective of the invention is thus to provide an industrially exploitable process under mild conditions of temperature and pressure, which benefits from a simple and non-polluting implementation, uses a light infrastructure and does not lead to any sub - troublesome product.
  • Another objective linked to the previous one, is to make it possible to multiply the fatty acid production installations with a view to bringing them closer to the places of cultivation of oilseed plants and thus to achieve savings in transport of the raw material.
  • the process according to the invention for the production of fatty acids or derivatives of fatty acids from oil plants is characterized in that: oil plants are produced PCI7FR95 / 00957
  • transgenics having, on the one hand, at least one gene coding for a lipase enzyme, called the lipase gene, on the other hand, associated with this lipase gene, a promoter allowing expression of said gene either in cellular, extracellular or tissues different from those where the lipids of the plant accumulate, either on exogenous induction,
  • the seeds or fruits containing the lipids of the said plants are collected, - the said seeds or fruits are ground, where appropriate after inductive treatment, so as to bring the lipids and the lipase contained in the said seeds or fruits into contact,
  • the fatty acids from the hydrolysis are extracted or transformed to obtain the desired fatty acid derivatives.
  • the process of the invention is an enzymatic hydrolysis process which benefits from the advantages of this type of process (gentle conditions of implementation, absence of pollution, light and inexpensive installations, absence of annoying by-products).
  • the plant is caused to produce itself the enzyme necessary for the subsequent transformation of the lipids, while preventing this enzyme from coming into contact prematurely with the lipids so as to eliminate any risk of self-degradation of the plant. before harvest.
  • Hydrolysis is then obtained without the addition of exogenous enzyme by bringing the lipids and enzymes produced by the plant into contact.
  • Such a method has a particularly low overall cost of implementation.
  • the crushing and incubation facilities are light and common in the agricultural environment, so these operations can be carried out at the plant harvesting sites.
  • transgenic plants are obtained by initially carrying out the genetic transformation of a natural oleaginous plant, by causing the genetically transformed plant to multiply by the sexual way for the production of transgenic seeds and then using these seeds to obtain descendant transgenic plants.
  • the initial genetic transformation consists, according to a currently well-known process, of producing an expression cassette comprising the lipase gene and the expression promoter of this gene and of introducing this expression cassette into the genome of the plant.
  • the promoter associated with the lipase gene is adapted to avoid premature contact of the enzyme and the lipids; this promoter can be of several types: it can either (1) direct the expression of the gene in compartments different from those where lipids accumulate, or (2) initiate the expression of the gene at the appropriate time by exogenous induction.
  • (1A) either an expression cassette comprising a lipase gene and a promoter controlling the expression of this gene in a cell or tissue compartment different from the lipid accumulation compartment,
  • the promoter used in the expression cassette is of the type which can be controlled exogenously by physical, chemical or biochemical signals, in particular a stress promoter controlling expression on application of a physical trauma to seeds or fruits.
  • the genetic transformation of the plant is carried out by producing an expression cassette comprising a lipase gene and a promoter controlling the expression of a determined protein from the seed, and by introducing this expression cassette into the genome of the plant so as to cause lipase to be expressed in the compartments of the seed where the abovementioned protein accumulates,
  • the promoter of the protein used is advantageously the promoter of napine which allows a massive accumulation of lipase in the protein bodies of the seed, separated from the lipid globules.
  • the embodiment 1B above can be used regardless of the type of oleaginous plant, for example by choosing the constitutive promoter 35S of CaMV (Cauliflower mosaic virus) and the addressing sequence PR- S tobacco in order to direct the excretion of the lipases produced towards the extracellular compartments.
  • the contacting of lipids and lipases is also carried out in this case by simple grinding.
  • the above embodiment (2) can be used regardless of the type of oleaginous plant, for example by choosing the protease inhibitor promoter isolated from the potato, which controls the expression of genes in the event of injuries.
  • the inductive treatment which causes the synthesis of lipases can in this case be an action of shelling of the seeds, carried out before grinding.
  • a gene coding for a non-specific lipase is used, that is to say characterized by a non-specific hydrolytic activity, in order to obtain a total hydrolysis of the lipids accumulated by the plant and to avoid parasitic saponification reactions.
  • lipases with specific hydrolytic activity in order to favor a given type of hydrolysis (for example: monoacylglycerollipase of Peni cylli um camembertii only carrying out the hydrolysis of one of the three bonds of glycerol with fatty acids, for the production of diacylglycerols).
  • non-specific lipase genes characterized by the following sequences or by sequences analogous to the following sequences (the arrows mark the coding part): SEQUENCE I ⁇ G'ATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCC
  • Sequence I corresponds to a Rhizopus ni veus cDNA
  • sequence II can be isolated from the genome of Pseudomonas aeruginosa
  • sequence III from Pseudomonas fluorescens
  • sequence IV from Pseudomonas sp
  • sequence V from Geotrichu candidum
  • sequence VI from Candida cylindracea.
  • lipase gene / promoter of expression of this gene into the genome of the oleaginous plant can be carried out by any known protocol.
  • this expression cassette can be introduced into the genome of somatic cells of the plant by a transfer using the bacterium Agrobacterium um tumefaciens.
  • This introduction into said somatic cells of the plant can also be carried out by another known technique, in particular by electroporation, by biolistics or by microinjection.
  • the extraction of fatty acids from hydrolysis is then carried out by any known method, in particular by liquid / liquid extraction using an apolar solvent such as chloroform or hexane.
  • the fatty acids resulting from hydrolysis can be methylated in situ by contacting with ethanol in acid catalysis under ultrasound in order to transform them into methyl esters, the latter being extracted by a liquid / liquid extraction using a solvent apolar.
  • the present application is intended, as a new product, any oleaginous plant or seed of oleaginous plant, of a variety which cannot be protected by a plant variety certificate, which comprises in its genome an expression cassette having at least one gene coding for a lipase enzyme, associated with a promoter allowing expression of said gene in compartments cellular, extracellular or tissue different from the lipid compartments of the plant or seed.
  • the promoter associated with the lipase gene can be a promoter with specific cellular or tissue expression.
  • This promoter can also be a constitutive promoter, in which case the lipase gene is provided with an addressing sequence towards cellular or extracellular compartments different from the lipid accumulation compartments.
  • the present application also relates to any oleaginous plant or seed of oleaginous plant, of a variety which cannot be protected by a plant variety certificate, which comprises in its genome an expression cassette having at least one gene coding for a lipase enzyme, associated to a promoter allowing expression of said gene on exogenous induction, in particular by stress.
  • Rape seeds (Brassica napus, Var Tapidor) are used, which are obtained commercially.
  • the seeds are sown in the greenhouse and grown under standard conditions. The health of the plants is rigorously monitored.
  • the genetic construction selected involves the promoter of napine, the cDNA of the lipase from Rhizopus ni veus and the terator NOS.
  • the napin promoter directs the expression of this protein in a protein compartment of the seed different from that in which the lipids accumulate.
  • the whole is introduced into the plasmid pRTI containing the selection gene pa t used to screen the transformants in order to form the construct pRTIL.
  • the expression cassette thus obtained is introduced into a plasmid designated "Blue-Script” (trade name) in order to carry out the amplification thereof.
  • the amplified expression cassette is then extracted from “Blue-Script” and introduced into a plasmid designated pRT1 which was prepared by grafting the promoter designated CaMV35S onto the 5 'end of the "pat” gene encoding phosphinothricin acetyl transferase (selection gene) and the NOS terminator on the 3 'end.
  • a plasmid designated pRTIL containing the targeted expression cassette is obtained. c) Gene transfer and production of transgenic plants
  • microspores isolated in a) (10 ° microspores ml- ') are suspended in the medium of Brewbaker and Kwack (JL Brewbaker and BH Kwack, 1963, Am. J. Bot. 50, p. 859-865) containing 13 % sucrose and adjusted to pH 5.9. 50 ⁇ g per ml of the plasmid pRTIL are added to the medium and electrical pulses of 400 V / cm for 10 ms are applied to the suspension using an electroporator "Jouan" (registered trademark) (TRX, GHT) delivering pulses in square wave.
  • the culture medium Containing 100 mg / 1 of phosphinothricin is added to the microspores.
  • the microspores are cultured in the dark for 24 h at 35 ° C. and then at 25 ° C. After 2 weeks of culture an equal volume of new medium is added and the microspores placed in the light under photoperiod 16 h day / 8 h dark.
  • the regenerated plants resistant to phosphinothricin are analyzed in "southern" so as to verify the presence in their genome of the sequence coding for lipase.
  • the chromosomal stock of the selected plants is doubled by colchicine (0.1 g / 1 plus a few drops of teepol) and the fertile diploid plants produced are self-fertilized.
  • Lipase activity is sought on a limited number of seeds. Plants with seeds with the highest lipase activity are retained and the seeds used for propagation plants until a sufficient seed stock is obtained to carry out the experiments on hydrolysis of the lipids of the seed by endogenous lipases.
  • the seeds are ground, the ground material placed in an incubator maintained at a constant temperature of 40 °.
  • the ground material is subjected to permanent agitation so as to increase the contact between the lipids and the lipase.
  • the fatty acids are extracted with chloroform.
  • the chloroform is evaporated and the fatty acids recovered.

Abstract

A method for producing fatty acids or derivatives thereof from oil plants by producing transgenic oil plants that have at least one gene coding for a lipase, i.e. a lipase gene, and a promoter combined with the lipase gene and enabling the expression thereof either in compartments other than the lipid accumulation compartments or by exogenous induction, collecting seeds or fruits containing lipids of the plants, crushing them, optionally after inducer processing, to contact the lipids with the lipase, incubating the resulting material to cause enzymatic hydrolysis of the lipids, and extracting the fatty acids or fatty acid derivatives.

Description

PROCEDE DE PRODUCTION D'ACIDES GRAS OU DERIVES A PARTIR DE PLANTES OLEAGINEUSES PROCESS FOR PRODUCING FATTY ACIDS OR DERIVATIVES FROM OLEAGINOUS PLANTS
L'invention concerne un procédé de production d'acides gras ou dérivés d'acides gras (esters ou autres dérivés) à partir de plantes oléagineuses. Le procédé de l'invention s'applique en particulier à des plantes oléoprotéagineuses telles que colza, tournesol, soja, crambé... L'invention peut notamment être utilisée pour fabriquer des bio-carburants (diester), lubrifiants, adjuvants phytosanitaires, détergents... par transformation des acides gras produits.The invention relates to a method for producing fatty acids or fatty acid derivatives (esters or other derivatives) from oil plants. The process of the invention applies in particular to oleoproteinous plants such as rapeseed, sunflower, soybean, crambé ... The invention can in particular be used to manufacture bio-fuels (diester), lubricants, phytosanitary adjuvants, detergents ... by transformation of the fatty acids produced.
On a tenté depuis 1970 de substituer aux produits dérivés du pétrole (notamment carburants) des produits obtenus à partir de matière végétale afin de réduire la dépendance vis-à-vis des pays producteurs de pétrole et d'élargir les débouchés des produits agricoles. La filière utilisant les plantes oléagineuses comme matière première passe par la production d'acides gras libres qui constituent la matière première pour les industries de transformation vers les carburants, lubrifiants... Les lipides accumulés par les plantes oléagineuses peuvent être transformés en acide gras par hydrolyse : deux procédés sont actuellement exploités industriellement pour opérer cette transformation.An attempt has been made since 1970 to substitute products obtained from petroleum products (notably fuels) for products obtained from vegetable matter in order to reduce dependence on petroleum producing countries and to widen outlets for agricultural products. The sector using oilseed plants as raw material goes through the production of free fatty acids which constitute the raw material for the industries of transformation towards fuels, lubricants ... The lipids accumulated by oilseed plants can be transformed into fatty acid by hydrolysis: two processes are currently used industrially to effect this transformation.
Un premier procédé consiste à hydrolyser les lipides après extraction en mettant en contact à chaud et sous pression les lipides extraits avec du méthanol sulfurique ou de la potasse méthanolique. Un autre procédé consiste à opérer l'hydrolyse dans des conditions similaires directement sur le broyât de graines sans extraction préalable. On pourra par exemple se reporter à la référence suivante pour plus de détails sur ces procédés qui sont les seuls exploités dans l'industrie pour hydrolyser les huiles végétales : K.J. Harrington et C. d'Arcy-Evans, Ind. Eng. Chem. Prod. Res. Dev. 1985, 24, 314-318. Les défauts bien connus de ces procédés sont les suivants : coût de mise en oeuvre élevé, infrastructure industrielle lourde, caractère polluant des effluents, production de glycérol comme sous-produit sans marché actuellement. Ces procédés sont mis en oeuvre faute de techniques de remplacement. Par ailleurs, des expérimentations ont été conduites en laboratoire pour réaliser l'hydrolyse des lipides par voie enzymatique en mélangeant une lipase avec les lipides extraits des graines (G.P. McNeill et al., JAOCS, Vol. 68 n° 1 janvier 1991, p. 1-5 ; S.M. Kim et J.S. Rhee, JAOCS Vol. 68 n° 7 juillet 1991, p. 499-503 ; C. Gancet, In Heterogeneous Catalysis And Fine Chemicals II 1991, Guisnet Editors, p. 93-104). Toutefois, ces essais sont restés au stade du laboratoire car la technique est incompatible avec une exploitation industrielle en raison des quantités d'enzyme nécessaires et du coût de celle-ci.A first method consists in hydrolyzing the lipids after extraction by bringing the extracted lipids into hot contact and under pressure with sulfuric methanol or methanolic potash. Another method consists in operating the hydrolysis under similar conditions directly on the ground material of seeds without prior extraction. We can for example refer to the following reference for more details on these processes which are the only ones used in the industry for hydrolyzing vegetable oils: KJ Harrington and C. d'Arcy-Evans, Ind. Eng. Chem. Prod. Res. Dev. 1985, 24, 314-318. The well-known shortcomings of these methods are as follows: high cost of implementation, infrastructure heavy industrial, polluting character of effluents, production of glycerol as a by-product without market currently. These methods are implemented in the absence of replacement techniques. In addition, experiments were carried out in the laboratory to carry out the hydrolysis of lipids by the enzymatic route by mixing a lipase with the lipids extracted from the seeds (GP McNeill et al., JAOCS, Vol. 68 no. January 1, 1991, p. 1-5; SM Kim and JS Rhee, JAOCS Vol. 68 No. 7 July 1991, p. 499-503; C. Gancet, In Heterogeneous Catalysis And Fine Chemicals II 1991, Guisnet Editors, p. 93-104). However, these tests remained at the laboratory stage because the technique is incompatible with industrial exploitation due to the quantities of enzyme required and the cost thereof.
La présente invention se propose de fournir une nouvelle solution au problème de la production d'acide gras à partir de plantes oléagineuses. Elle vise à fournir une solution dont les coûts de mise en oeuvre sont considérablement abaissés par rapport aux procédés connus (aussi bien procédés chimiques industriels que procédé enzymatique de laboratoire).The present invention proposes to provide a new solution to the problem of the production of fatty acid from oil plants. It aims to provide a solution whose implementation costs are considerably lower compared to known processes (both industrial chemical processes and laboratory enzymatic process).
Un objectif de l'invention est ainsi de fournir un procédé exploitable sur le plan industriel dans des conditions douces de température et de pression, qui bénéficie d'une mise en oeuvre simple et non polluante, utilise une infrastructure légère et ne conduit à aucun sous-produit gênant.An objective of the invention is thus to provide an industrially exploitable process under mild conditions of temperature and pressure, which benefits from a simple and non-polluting implementation, uses a light infrastructure and does not lead to any sub - troublesome product.
Un autre objectif, lié au précédent, est de permettre de multiplier les installations de production d'acides gras en vue de les rapprocher des lieux de culture des plantes oléagineuses et de réaliser ainsi des économies de transport de la matière première.Another objective, linked to the previous one, is to make it possible to multiply the fatty acid production installations with a view to bringing them closer to the places of cultivation of oilseed plants and thus to achieve savings in transport of the raw material.
A cet effet, le procédé conforme à l'invention pour la production d'acides gras ou dérivés d'acides gras à partir de plantes oléagineuses se caractérise en ce que : on produit des plantes oléagineuses PCI7FR95/00957To this end, the process according to the invention for the production of fatty acids or derivatives of fatty acids from oil plants is characterized in that: oil plants are produced PCI7FR95 / 00957
transgéniques possédant, d'une part, au moins un gène codant pour une enzyme lipase, dit gène de lipase, d'autre part, associé à ce gène de lipase, un promoteur permettant une expression dudit gène soit dans des compartiments cellulaires, extracellulaires ou tissulaires différents de ceux où s'accumulent les lipides de la plante, soit sur induction exogène,transgenics having, on the one hand, at least one gene coding for a lipase enzyme, called the lipase gene, on the other hand, associated with this lipase gene, a promoter allowing expression of said gene either in cellular, extracellular or tissues different from those where the lipids of the plant accumulate, either on exogenous induction,
- on recueille les graines ou fruits contenant les lipides desdites plantes, - on broie lesdites graines ou fruits, le cas échéant après traitement inducteur, de façon à mettre en contact les lipides et la lipase contenue dans lesdites graines ou fruits,- the seeds or fruits containing the lipids of the said plants are collected, - the said seeds or fruits are ground, where appropriate after inductive treatment, so as to bring the lipids and the lipase contained in the said seeds or fruits into contact,
- on laisse incuber l'ensemble pour engendrer une hydrolyse enzymatique des lipides du broy t sous l'action catalytique de la lipase contenue dans ledit broyât,the whole is incubated to generate an enzymatic hydrolysis of the lipids of the ground material under the catalytic action of the lipase contained in the said ground material,
- on extrait les acides gras issus de l'hydrolyse ou on les transforme pour obtenir les dérivés d'acides gras recherchés.- The fatty acids from the hydrolysis are extracted or transformed to obtain the desired fatty acid derivatives.
Ainsi le procédé de l'invention est un procédé d'hydrolyse enzymatique qui bénéficie des avantages de ce type de procédé (conditions douces de mise en oeuvre, absence de pollution, installations légères et peu coûteuses, absence de sous-produits gênants). Dans ce procédé, on amène la plante à produire elle-même l'enzyme nécessaire à la transformation ultérieure des lipides, en évitant que cette enzyme ne vienne prématurément au contact des lipides de façon à écarter tout risque d'auto- dégradation de la plante avant la récolte. L'hydrolyse est ensuite obtenue sans addition d'enzyme exogène en opérant la mise en contact des lipides et des enzymes produits par la plante. Un tel procédé présente un coût global de mise en oeuvre particulièrement réduit. Les installations de broyage et d'incubation sont légères et courantes dans le milieu agricole, de sorte que ces opérations peuvent être exécutées sur les sites de récolte des plantes.Thus, the process of the invention is an enzymatic hydrolysis process which benefits from the advantages of this type of process (gentle conditions of implementation, absence of pollution, light and inexpensive installations, absence of annoying by-products). In this process, the plant is caused to produce itself the enzyme necessary for the subsequent transformation of the lipids, while preventing this enzyme from coming into contact prematurely with the lipids so as to eliminate any risk of self-degradation of the plant. before harvest. Hydrolysis is then obtained without the addition of exogenous enzyme by bringing the lipids and enzymes produced by the plant into contact. Such a method has a particularly low overall cost of implementation. The crushing and incubation facilities are light and common in the agricultural environment, so these operations can be carried out at the plant harvesting sites.
La production des plantes transgéniques est obtenue en réalisant initialement la transformation génétique d'une plante oléagineuse naturelle, en amenant la plante génétiquement transformée à se multiplier par la voie sexuée pour la production de semences transgéniques et en utilisant ensuite ces semences pour l'obtention de plantes transgéniques descendantes.The production of transgenic plants is obtained by initially carrying out the genetic transformation of a natural oleaginous plant, by causing the genetically transformed plant to multiply by the sexual way for the production of transgenic seeds and then using these seeds to obtain descendant transgenic plants.
La transformation génétique initiale consiste, selon un processus actuellement bien connu, à réaliser une cassette d'expression comprenant le gène de lipase et le promoteur d'expression de ce gène et à introduire cette cassette d'expression dans le génome de la plante.The initial genetic transformation consists, according to a currently well-known process, of producing an expression cassette comprising the lipase gene and the expression promoter of this gene and of introducing this expression cassette into the genome of the plant.
Une des caractéristiques essentielles du procédé de l'invention est que le promoteur associé au gène de lipase est adapté pour éviter une mise en contact prématurée de l'enzyme et des lipides ; ce promoteur peut être de plusieurs types : il peut soit (1) diriger l'expression du gène dans des compartiments différents de ceux où s'accumulent les lipides, soit (2) initier l'expression du gène au moment opportun par induction exogène.One of the essential characteristics of the process of the invention is that the promoter associated with the lipase gene is adapted to avoid premature contact of the enzyme and the lipids; this promoter can be of several types: it can either (1) direct the expression of the gene in compartments different from those where lipids accumulate, or (2) initiate the expression of the gene at the appropriate time by exogenous induction.
Dans le premier cas, deux types de cassettes d'expression peuvent être utilisés :In the first case, two types of expression cassettes can be used:
(1A) soit une cassette d'expression comprenant un gène de lipase et un promoteur contrôlant l'expression de ce gène dans un compartiment cellulaire ou tissulaire différent du compartiment d'accumulation des lipides,(1A) either an expression cassette comprising a lipase gene and a promoter controlling the expression of this gene in a cell or tissue compartment different from the lipid accumulation compartment,
(1B) soit une cassette d'expression comprenant un promoteur constitutif et un gène de lipase muni d'une séquence d'adressage vers des compartiments cellulaires ou extracellulaires différents de ceux où s'accumulent les lipides.(1B) or an expression cassette comprising a constitutive promoter and a lipase gene provided with an addressing sequence towards cellular or extracellular compartments different from those in which the lipids accumulate.
Dans le second cas (2), le promoteur utilisé dans la cassette d'expression est du type contrôlable de façon exogène par des signaux physiques, chimiques ou biochimiques, en particulier promoteur de stress commandant l'expression sur application d'un traumatisme physique sur les graines ou fruits.In the second case (2), the promoter used in the expression cassette is of the type which can be controlled exogenously by physical, chemical or biochemical signals, in particular a stress promoter controlling expression on application of a physical trauma to seeds or fruits.
Par exemple pour produire des acides gras à partir de plantes oléoprotéagineuses, on peut utiliser le mode de mise en oeuvre 1A ci-dessus évoqué : - la transformation génétique de la plante est effectuée en réalisant une cassette d'expression comprenant un gène de lipase et un promoteur contrôlant l'expression d'une protéine déterminée de la graine, et en introduisant cette cassette d'expression dans le génome de la plante de façon à faire exprimer la lipase dans les compartiments de la graine où s'accumulent la protéine précitée,For example, to produce fatty acids from oleoproteinous plants, one can use the mode of implementation 1A mentioned above: the genetic transformation of the plant is carried out by producing an expression cassette comprising a lipase gene and a promoter controlling the expression of a determined protein from the seed, and by introducing this expression cassette into the genome of the plant so as to cause lipase to be expressed in the compartments of the seed where the abovementioned protein accumulates,
- la mise en contact des lipides et de la lipase est effectuée par simple broyage. Pour le colza, le promoteur de la protéine utilisé est avantageusement le promoteur de la napine qui permet une accumulation massive de lipase dans les corps protéiques de la graine, séparés des globules lipidiques.- bringing the lipids and the lipase into contact is carried out by simple grinding. For rapeseed, the promoter of the protein used is advantageously the promoter of napine which allows a massive accumulation of lipase in the protein bodies of the seed, separated from the lipid globules.
Le mode de mise en oeuvre 1B ci-dessus peut être utilisé quel que soit le type de plante oléagineuse, par exemple en choisissant le promoteur constitutif 35S du CaMV (Virus de la mosaïque du chou-fleur) et la séquence d'adressage PR-S du tabac afin de diriger l'excrétion des lipases produites vers les compartiments extracellulaires. La mise en contact des lipides et des lipases est également effectuée dans ce cas par simple broyage.The embodiment 1B above can be used regardless of the type of oleaginous plant, for example by choosing the constitutive promoter 35S of CaMV (Cauliflower mosaic virus) and the addressing sequence PR- S tobacco in order to direct the excretion of the lipases produced towards the extracellular compartments. The contacting of lipids and lipases is also carried out in this case by simple grinding.
Le mode de mise en oeuvre (2) ci-dessus peut être utilisé quel que soit le type de plante oléagineuse, par exemple en choisissant le promoteur inhibiteur de protease isolé de la pomme de terre, qui commande l'expression des gènes en cas de blessures. Le traitement inducteur qui provoque la synthèse des lipases peut être dans ce cas une action de décorticage des graines, réalisée avant le broyage. De préférence, quel que soit le mode de mise en oeuvre choisi, on utilise un gène codant pour une lipase non spécifique, c'est-à-dire caractérisée par une activité hydrolytique non spécifique, afin d'obtenir une hydrolyse totale des lipides accumulés par la plante et d'éviter les réactions parasites de saponification. Il est toutefois possible, pour certaines utilisations des acides gras, de faire produire à la plante des lipases à activité hydrolytique spécifique afin de favoriser un type donné d'hydrolyse (par exemple : monoacylglycérollipase du Peni cylli um camembertii réalisant uniquement l'hydrolyse d'une des trois liaisons du glycérol avec les acides gras, en vue de la fabrication de diacylglycérols) . On peut en particulier utiliser des gènes de lipase non spécifique, caractérisé par les séquences suivantes ou par des séquences analogues aux séquences suivantes (les flèches délimitent la partie codante) : SEQUENCE I ι G'ATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCCThe above embodiment (2) can be used regardless of the type of oleaginous plant, for example by choosing the protease inhibitor promoter isolated from the potato, which controls the expression of genes in the event of injuries. The inductive treatment which causes the synthesis of lipases can in this case be an action of shelling of the seeds, carried out before grinding. Preferably, whatever the mode of implementation chosen, a gene coding for a non-specific lipase is used, that is to say characterized by a non-specific hydrolytic activity, in order to obtain a total hydrolysis of the lipids accumulated by the plant and to avoid parasitic saponification reactions. It is however possible, for certain uses of fatty acids, to make the plant produce lipases with specific hydrolytic activity in order to favor a given type of hydrolysis (for example: monoacylglycerollipase of Peni cylli um camembertii only carrying out the hydrolysis of one of the three bonds of glycerol with fatty acids, for the production of diacylglycerols). It is possible in particular to use non-specific lipase genes, characterized by the following sequences or by sequences analogous to the following sequences (the arrows mark the coding part): SEQUENCE I ι G'ATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCC
51 TCCTATCAGC CTCTCTAGCT CTACCAACAG CGCCTCTGAT GGTGGTAAGG51 TCCTATCAGC CTCTCTAGCT CTACCAACAG CGCCTCTGAT GGTGGTAAGG
101 TTGTTGCTGC TACTACTGCT CAGATCCAAG AGTTCACCAA GTATGCTGGT101 TTGTTGCTGC TACTACTGCT CAGATCCAAG AGTTCACCAA GTATGCTGGT
151 ATCGCTGCCA CTGCCTACTG TCGTTCTGTT GTCCCTGGTA ACAAGTGGGA151 ATCGCTGCCA CTGCCTACTG TCGTTCTGTT GTCCCTGGTA ACAAGTGGGA
201 TTGTGTCCAA TGTCAAAAGT GGGTTCCTGA TGGCAAGATC ATCACTACCT201 TTGTGTCCAA TGTCAAAAGT GGGTTCCTGA TGGCAAGATC ATCACTACCT
251 TTACCTCCTT GCTTTCCGAT ACAAATGGTT ACGTCTTGAG AAGTGATAAA251 TTACCTCCTT GCTTTCCGAT ACAAATGGTT ACGTCTTGAG AAGTGATAAA
301 CAAAAGACCA TTTATCTTGT TTTCCGTGGT ACCAACTCCT TCAGAAGTGC301 CAAAAGACCA TTTATCTTGT TTTCCGTGGT ACCAACTCCT TCAGAAGTGC
351 CATCACTGAT ATCGTCTTCA ACTTTTCTGA CTACAAGCCT GTCAAGGGCG351 CATCACTGAT ATCGTCTTCA ACTTTTCTGA CTACAAGCCT GTCAAGGGCG
401 CCAAAGTTCA TGCTGGTTTC CTTTCCTCTT ATGAGCAAGT TGTCAATGAC401 CCAAAGTTCA TGCTGGTTTC CTTTCCTCTT ATGAGCAAGT TGTCAATGAC
451 TATTTCCCTG TCGTCCAAGA ACAATTGACC GCCCACCCTA CTTATAAGGT451 TATTTCCCTG TCGTCCAAGA ACAATTGACC GCCCACCCTA CTTATAAGGT
501 CATCGTTACC GGTCACTCAC TCGGTGGTGC ACAAGCTTTG CTTGCCGGTA501 CATCGTTACC GGTCACTCAC TCGGTGGTGC ACAAGCTTTG CTTGCCGGTA
551 TGGATCTCTA CCAACGTGAA CCAAGATTGT CTCCCAAGAA TTTGAGCATC551 TGGATCTCTA CCAACGTGAA CCAAGATTGT CTCCCAAGAA TTTGAGCATC
601 TTCACTGTCG GTGGTCCTCG TGTTGGTAAC CCCACCTTTG CTTACTATGT601 TTCACTGTCG GTGGTCCTCG TGTTGGTAAC CCCACCTTTG CTTACTATGT
651 TGAATCCACC GGTATCCCTT TCCAACGTAC CGTTCACAAG AGAGATATCG651 TGAATCCACC GGTATCCCTT TCCAACGTAC CGTTCACAAG AGAGATATCG
701 TTCCTCACGT TCCTCCTCAA TCCTTCGGAT TCCTTCATCC CGGTGTTGAA701 TTCCTCACGT TCCTCCTCAA TCCTTCGGAT TCCTTCATCC CGGTGTTGAA
751 TCTTGGATGA AGTCTGGTAC TTCCAACGTT CAAATCTGTA CTTCTGAAAT751 TCTTGGATGA AGTCTGGTAC TTCCAACGTT CAAATCTGTA CTTCTGAAAT
801 TGAAACCAAG GATTGCAGTA ACTCTATCGT TCCTTTCACC TCTATCCTTG801 TGAAACCAAG GATTGCAGTA ACTCTATCGT TCCTTTCACC TCTATCCTTG
851 ACCACTTGAG TTACTTTGAT ATCAACGAAG GAAGCTGTTT GTAAAACACT851 ACCACTTGAG TTACTTTGAT ATCAACGAAG GAAGCTGTTT GTAAAACACT
901 TGACGTGTTA CTCTAATTTT ATAATAAAAT TAAGTTTTTA TACAAT S EQUE NCE I I901 TGACGTGTTA CTCTAATTTT ATAATAAAAT TAAGTTTTTA TACAAT S EQUE NCE II
1 GTCGACCATT TCAGCCTGTT TTGCTCGCAA AACGACGCCG CGGGCGTGCG 51 CTACCGCACA CTCCGTCGCT GGGCGTTGTG CGGGGAAGAT TCAAACGAGC1 GTCGACCATT TCAGCCTGTT TTGCTCGCAA AACGACGCCG CGGGCGTGCG 51 CTACCGCACA CTCCGTCGCT GGGCGTTGTG CGGGGAAGAT TCAAACGAGC
101 GTTTCGCGCC GTAACAACCC GCTCTCTTCC GCTCTGCCAC GCAGGTTATG101 GTTTCGCGCC GTAACAACCC GCTCTCTTCC GCTCTGCCAC GCAGGTTATG
151 ACCGGCCGCC AGGAAGCCGC GGATTTCCTG GCCTGGAGGA AAAAAGCCGA151 ACCGGCCGCC AGGAAGCCGC GGATTTCCTG GCCTGGAGGA AAAAAGCCGA
201 AGCTGGCACG GTTCCTGGCG CAAGGGACAG CGAAGCGGTT CTCCCGGAAG 251 GATTCGGGCG ATGGCTGGCA GGACGCGCCC CTCGGCCCCA TCAACCTGAG201 AGCTGGCACG GTTCCTGGCG CAAGGGACAG CGAAGCGGTT CTCCCGGAAG 251 GATTCGGGCG ATGGCTGGCA GGACGCGCCC CTCGGCCCCA TCAACCTGAG
301 ATGAGAACAA C TGAAGAAG AAGTCTCTGC TCCCCCTCGG CCTGGCCATC301 ATGAGAACAA C TGAAGAAG AAGTCTCTGC TCCCCCTCGG CCTGGCCATC
351 GGTCTCGCCT CTCTCGCTGC CAGCCCTCTG ATCCAGGCCA GCACCTACAC351 GGTCTCGCCT CTCTCGCTGC CAGCCCTCTG ATCCAGGCCA GCACCTACAC
401 CCAGACCAAA TACCCCATCG TGCTGGCCCA CGGCATGCTC GGCTTCGACA 451 ACATCCTCGG GGTCGACTAC TGGTTCGGCA TTCCCAGCGC CTTGCGCCGT401 CCAGACCAAA TACCCCATCG TGCTGGCCCA CGGCATGCTC GGCTTCGACA 451 ACATCCTCGG GGTCGACTAC TGGTTCGGCA TTCCCAGCGC CTTGCGCCGT
501 GACGGTGCCC AGGTCTACGT CACCGAAGTC AGCCAGTTGG ACACCTCGGA501 GACGGTGCCC AGGTCTACGT CACCGAAGTC AGCCAGTTGG ACACCTCGGA
551 AGTCCGCGGC GAGCAGTTGC TGCAACAGGT GGAGGAAATC GTCGCCCTCA551 AGTCCGCGGC GAGCAGTTGC TGCAACAGGT GGAGGAAATC GTCGCCCTCA
601 GCGGCCAGCC CAAGGTCAAC CTGATCGGCC ACAGCCACGG CGGGCCGACC 651 ATCCGCTACG TCGCCGCCGT ACGTCCCGAC CTGATCGCTT CCGCCACCAG601 GCGGCCAGCC CAAGGTCAAC CTGATCGGCC ACAGCCACGG CGGGCCGACC 651 ATCCGCTACG TCGCCGCCGT ACGTCCCGAC CTGATCGCTT CCGCCACCAG
701 CGTCGGCGCC CCGCACAAGG GTTCGGACAC CGCCGACTTC CTGCGCCAGA701 CGTCGGCGCC CCGCACAAGG GTTCGGACAC CGCCGACTTC CTGCGCCAGA
751 TCCCACCGGG TTCGGCCGGC GAGGCAGTCC TCTCCGGGCT GGTCAACAGC751 TCCCACCGGG TTCGGCCGGC GAGGCAGTCC TCTCCGGGCT GGTCAACAGC
801 CTCGGCGCGC TGATCAGCTT CCTTTCCAGC GGCAGCACCG GTACGCAGAA 851 TTCACTGGGC TCGCJGGAGT CGCTGAACAG CGAGGGTGCC GCGCGCTTCA801 CTCGGCGCGC TGATCAGCTT CCTTTCCAGC GGCAGCACCG GTACGCAGAA 851 TTCACTGGGC TCGCJGGAGT CGCTGAACAG CGAGGGTGCC GCGCGCTTCA
901 ACGCCAAGTA CCCGCAGGGC ATCCCCACCT CGGCCTGCGG CGAAGGCGCC901 ACGCCAAGTA CCCGCAGGGC ATCCCCACCT CGGCCTGCGG CGAAGGCGCC
951 TACAAGGTCA ACGGCGTGAG CTATTACTCC TGGAGCGGTT CCTCGCCGCT951 TACAAGGTCA ACGGCGTGAG CTATTACTCC TGGAGCGGTT CCTCGCCGCT
1001 GACCAACTTC CTCGATCCGA GCGACGCCTT CCTCGGCGCC TCGTCGCTGA 1051 CCTTCAAGAA CGGCACCGCC AACGACGGCC TGGTCGGCAC CTGCAGTTCG1001 GACCAACTTC CTCGATCCGA GCGACGCCTT CCTCGGCGCC TCGTCGCTGA 1051 CCTTCAAGAA CGGCACCGCC AACGACGGCC TGGTCGGCAC CTGCAGTTCG
1101 CACCTGGGCA TGGTGATCCG CGACAACTAC CGGATGAACC ACCTGGACGA1101 CACCTGGGCA TGGTGATCCG CGACAACTAC CGGATGAACC ACCTGGACGA
1151 GGTGAACCAG GTCTTCGGCC TCACCAGCCT GTTCGAGACC AGCCCGGTCA1151 GGTGAACCAG GTCTTCGGCC TCACCAGCCT GTTCGAGACC AGCCCGGTCA
1201 GCGTCTACCG CCAGCACGCC AACCGCCTGA AGAACGCCAG CCTGTAff SEQUENCE III1201 GCGTCTACCG CCAGCACGCC AACCGCCTGA AGAACGCCAG CCTGTAff SEQUENCE III
ι :GGGTGCATGC CAGCTCCCAC CGGACACCTG GCCCGTCGCT GAAAC-STGTTι: GGGTGCATGC CAGCTCCCAC CGGACACCTG GCCCGTCGCT GAAAC-STGTT
51 TTCGCTTTCT CTACAAATCC AACAACAGAG AGGCACTACC 'ATGGGTATCT 101 TTGACTATAA AAACCTTGGC ACCGAGGGTT CCAAAACGTT GTTCGCCGAT51 TTCGCTTTCT CTACAAATCC AACAACAGAG AGGCACTACC 'ATGGGTATCT 101 TTGACTATAA AAACCTTGGC ACCGAGGGTT CCAAAACGTT GTTCGCCGAT
151 GCCATGGCGA TCACGTTGTA TTCCTATCAC AACCTGGATA ACGGCTTTGC151 GCCATGGCGA TCACGTTGTA TTCCTATCAC AACCTGGATA ACGGCTTTGC
201 CGTGGGCTAC CAGCACAACG GGTTGGGCTT GGGGCTACCG GCCACGCTGG201 CGTGGGCTAC CAGCACAACG GGTTGGGCTT GGGGCTACCG GCCACGCTGG
251 TCGGTGCGCT GCTCGGCAGC ACGGATTCCC AGGGCGTGAT CCCTGGCATC251 TCGGTGCGCT GCTCGGCAGC ACGGATTCCC AGGGCGTGAT CCCTGGCATC
301 CCGTGGAACC CGGATTCAGA AAAAGCCGCC CTTGAGGCGG TGCAGAAAGC 351 CGGTTGGACA CCGATCAGCG CCAGTGCCCT GGGCTACGCC GGCAAGGTCG301 CCGTGGAACC CGGATTCAGA AAAAGCCGCC CTTGAGGCGG TGCAGAAAGC 351 CGGTTGGACA CCGATCAGCG CCAGTGCCCT GGGCTACGCC GGCAAGGTCG
401 ATGCACGTGG CACCTTCTTT GGGGAAAAAG CCGGCTACAC CACGGCCCAG401 ATGCACGTGG CACCTTCTTT GGGGAAAAAG CCGGCTACAC CACGGCCCAG
451 GTCGAGGTAC TCGGCAAATA CGATGACGCC GGCAAGCTGC TCGAAATCGG451 GTCGAGGTAC TCGGCAAATA CGATGACGCC GGCAAGCTGC TCGAAATCGG
501 CATCGGTTTT CGTGGCACTT CGGGGCCACG GGAAACCTTG ATCAGCGACT501 CATCGGTTTT CGTGGCACTT CGGGGCCACG GGAAACCTTG ATCAGCGACT
551 CGATCGGCGA CTTGATCAGC GATCTGCTCG CGGCCCTGGG GCCCAAGGAT 601 TACGCGAAAA ACTACGCCGG CGAAGCCTTC GGCGGCTTGC TCAAGAATGT551 CGATCGGCGA CTTGATCAGC GATCTGCTCG CGGCCCTGGG GCCCAAGGAT 601 TACGCGAAAA ACTACGCCGG CGAAGCCTTC GGCGGCTTGC TCAAGAATGT
651 TGCCGACTAC GCCGGTGCCC ATGGCCTGAC CGGCAAGGAC GTGGTGGTCA651 TGCCGACTAC GCCGGTGCCC ATGGCCTGAC CGGCAAGGAC GTGGTGGTCA
701 GCGGCCACAG CCTGGGCGGG CTGGCGGTCA ACAGCATGGC GGACTTGAGC701 GCGGCCACAG CCTGGGCGGG CTGGCGGTCA ACAGCATGGC GGACTTGAGC
751 AACTACAAAT GGGCGGGGTT CTACAAGGAC GCCAACTATG TTGCCTATGC751 AACTACAAAT GGGCGGGGTT CTACAAGGAC GCCAACTATG TTGCCTATGC
801 CTCGCCGACC CAGAGTGCCG GCGACAAGGT GCTCAATATC GGTTACGAAA 851 ACGACCCGGT GTTCCGCGCG CTGGACGGCT CGTCGTTTAA CCTGTCGTCG801 CTCGCCGACC CAGAGTGCCG GCGACAAGGT GCTCAATATC GGTTACGAAA 851 ACGACCCGGT GTTCCGCGCG CTGGACGGCT CGTCGTTTAA CCTGTCGTCG
901 CTGGGCGTGC ACGACAAACC CCACGAGTCC ACCACCGATA ACATCGTCAG901 CTGGGCGTGC ACGACAAACC CCACGAGTCC ACCACCGATA ACATCGTCAG
951 CTTCAACGAC CACTACGCCT CGACGCTGTG GAATGTGCTG CCGTTTTCCA951 CTTCAACGAC CACTACGCCT CGACGCTGTG GAATGTGCTG CCGTTTTCCA
1001 TCGTCAACCT GCCCACCTGG GTCTCGCATT TGCCGACGGC GTACGGCGAT1001 TCGTCAACCT GCCCACCTGG GTCTCGCATT TGCCGACGGC GTACGGCGAT
1051 GGCATGACGC GCATCCTCGA GTCCGGCTTC TACGACCAGA TGACCCGTGA 1101 CTCCACGGTG ATTGTTGCCA ACCTGTCCGA TCCGGCGCGG GCCAACACCT1051 GGCATGACGC GCATCCTCGA GTCCGGCTTC TACGACCAGA TGACCCGTGA 1101 CTCCACGGTG ATTGTTGCCA ACCTGTCCGA TCCGGCGCGG GCCAACACCT
1151 GGGTGCAGGA CCTCAACCGC AATGCCGAGC CCCACAAGGG CAACACGTTC1151 GGGTGCAGGA CCTCAACCGC AATGCCGAGC CCCACAAGGG CAACACGTTC
1201 ATCATCGGCA GCGACGGCAA CGACCTGATC CAGGGCGGCA ACGGTGCGGA1201 ATCATCGGCA GCGACGGCAA CGACCTGATC CAGGGCGGCA ACGGTGCGGA
1251 CTTTATCGAG GGTGGCAAAG GCAACGACAC GATCCGCGAC AACAGCGGGC1251 CTTTATCGAG GGTGGCAAAG GCAACGACAC GATCCGCGAC AACAGCGGGC
1301 ACAACACCTT TTTGTTCAGC GGCCACTTTG GCAATGATCG CGTGATTGGC 1351 TACCAGCCCA CCGACAAACT GGTGTTCAAG GACGTGCAAG GAAGCACCGA1301 ACAACACCTT TTTGTTCAGC GGCCACTTTG GCAATGATCG CGTGATTGGC 1351 TACCAGCCCA CCGACAAACT GGTGTTCAAG GACGTGCAAG GAAGCACCGA
1401 CCTGCGTGAC CACGCGAAGG TGGTCGGCGC CGATACGGTG CTTACGTTTG1401 CCTGCGTGAC CACGCGAAGG TGGTCGGCGC CGATACGGTG CTTACGTTTG
1451 GGGCCGACTC GGTGACGCTG GTCGGCGTGG GGCATGGCGG GCTGTGGACG 1501 GAGGGCGTGG TGATCGGCTG A TACTCACG CAACCGATCA GTGCCAGTGC1451 GGGCCGACTC GGTGACGCTG GTCGGCGTGG GGCATGGCGG GCTGTGGACG 1501 GAGGGCGTGG TGATCGGCTG A TACTCACG CAACCGATCA GTGCCAGTGC
1551 TGCCCCCGCC AGCCACCGCC CCAATTGGGC CGGTGGGGGT AGCCATAGCC1551 TGCCCCCGCC AGCCACCGCC CCAATTGGGC CGGTGGGGGT AGCCATAGCC
SEQUENCE IVSEQUENCE IV
1 GGGCGATGGC TGGCAGGACG CGCCCCTCGG CCCCATCAAC CTGAGATGAG1 GGGCGATGGC TGGCAGGACG CGCCCCTCGG CCCCATCAAC CTGAGATGAG
51 AACAACATGA AGAAGAAGTC TCTGCTCCCC CTCGGCCTGG CCATCGGCCT f 101 CGCCTCTCTC GCTGCCAGCC CTCTGATCCA GGCCTAGCACC TACACCCAGA51 AACAACATGA AGAAGAAGTC TCTGCTCCCC CTCGGCCTGG CCATCGGCCT f 101 CGCCTCTCTC GCTGCCAGCC CTCTGATCCA GGCC T AGCACC TACACCCAGA
151 CCAAATACCC CATCGTGCTG GCCCACGGCA TGCTCGGCTT CGACAATATC151 CCAAATACCC CATCGTGCTG GCCCACGGCA TGCTCGGCTT CGACAATATC
201 CTCGGGGTCG ACTACTGGTT CGGCATTCCC AGCGCCTTGC GCCGTGACGG201 CTCGGGGTCG ACTACTGGTT CGGCATTCCC AGCGCCTTGC GCCGTGACGG
251 TGCCCAGGTC TACGTCACCG AAGTCAGCCA GTTGGACACC TCGGAAGTCC251 TGCCCAGGTC TACGTCACCG AAGTCAGCCA GTTGGACACC TCGGAAGTCC
301 GCGGCGAGCA GTTGCTGCAA CAGGTGGAGG AAATCGTCGC CCTCAGCGGC 351 CAGCCCAAGG TCAACCTGAT CGGCCACAGC CACGGCGGGC CGACCATCCG301 GCGGCGAGCA GTTGCTGCAA CAGGTGGAGG AAATCGTCGC CCTCAGCGGC 351 CAGCCCAAGG TCAACCTGAT CGGCCACAGC CACGGCGGGC CGACCATCCG
401 CTACGTCGCC GCCGTACGTC CCGACCTGAT GCCTTCCGCC ACCAGCGTCG401 CTACGTCGCC GCCGTACGTC CCGACCTGAT GCCTTCCGCC ACCAGCGTCG
451 GCGCCCCGCA CAAGGGTTCG GACACCGCCG ACTTCCTGCG CCAGATCCCA451 GCGCCCCGCA CAAGGGTTCG GACACCGCCG ACTTCCTGCG CCAGATCCCA
501 CCGGGTTCGG CCGGCGAGGC AGTCCTCTCC GGGCTGGTCA ACAGCCTCGG501 CCGGGTTCGG CCGGCGAGGC AGTCCTCTCC GGGCTGGTCA ACAGCCTCGG
551 CGCGCTGATC AGCTTCCTTT CCAGCGGCAG CGCCGGTACG CAGAATTCAC 601 TGGGCTCGCT GGAGTCGCTG AACAGCGAGG GGGCCGCGCG CTTCAACGCC551 CGCGCTGATC AGCTTCCTTT CCAGCGGCAG CGCCGGTACG CAGAATTCAC 601 TGGGCTCGCT GGAGTCGCTG AACAGCGAGG GGGCCGCGCG CTTCAACGCC
651 AAGTACCCGC AGGGCATCCC CACCTCGGCC TGCGGCGAAG GCGCCTACAA651 AAGTACCCGC AGGGCATCCC CACCTCGGCC TGCGGCGAAG GCGCCTACAA
701 GGTCAACGGC GTGÂGCTATT ACTCCTGGAG CGGTTCCTCG CCGCTGACCA701 GGTCAACGGC GTGÂGCTATT ACTCCTGGAG CGGTTCCTCG CCGCTGACCA
751 ACTTCCTCGA TCCGAGCGAC GCCTTCCTCG GCGCCTCGTC GCTGACCTTC 8OΓ—fiflTϋ7-τACGGCA_CσGOOAnCGA CGGCCTGGTC GUCSCCTGCA GTTCGCRCCT 851 GGGCATGGTG ATCCGCGACA ACTACCGGAT GAACCACCTG GACGAGGTGA751 ACTTCCTCGA TCCGAGCGAC GCCTTCCTCG GCGCCTCGTC GCTGACCTTC 8OΓ - fiflTϋ7-τACGGCA _ CσGOOAnCGA CGGCCTGGTC GUCSCCTGCA GTTCGCRCCT 851 GGGCATGGTG ATCCGCGACA ACTACGGGA
901 ACCAGGTCTT CGGCCTCACC AGCCTGTTCG AGACCAGCCC GGTCAGCGTC901 ACCAGGTCTT CGGCCTCACC AGCCTGTTCG AGACCAGCCC GGTCAGCGTC
951 TACCGCCAGC ACGCCAACCG CCTGAAGAAC GCCAGCCTGT AGGACCCCGG951 TACCGCCAGC ACGCCAACCG CCTGAAGAAC GCCAGCCTGT AGGACCCCGG
10Q1 CCGGGGCCTC GGCCCCGGCC CTTTCCCGGA AGCCCCCTCG CGTGAAGAAA10Q1 CCGGGGCCTC GGCCCCGGCC CTTTCCCGGA AGCCCCCTCG CGTGAAGAAA
1051 ATCCTCCTGC TGATTCCACT GGCGTTCGCC GCCAGCCTGG CCTGGTTCGT SE QUENCE V1051 ATCCTCCTGC TGATTCCACT GGCGTTCGCC GCCAGCCTGG CCTGGTTCGT SE QUENCE V
1 'CAGGCCCCCA CGGCCGTTCT TAATGGCAAC GAGGTCATCT CTGGTGTCCT1 'CAGGCCCCCA CGGCCGTTCT TAATGGCAAC GAGGTCATCT CTGGTGTCCT
51 TGGGGGCAAG GTTGATACCT TTAAGGGAAT TCCATTTGCT GACCCTCCTG 101 TTGGTGACTT GCGGTTCAAG CACCCCCAGC CTTTCACTGG ATCCTACCAG51 TGGGGGCAAG GTTGATACCT TTAAGGGAAT TCCATTTGCT GACCCTCCTG 101 TTGGTGACTT GCGGTTCAAG CACCCCCAGC CTTTCACTGG ATCCTACCAG
151 GGTCTTAAGG CCAACGACTT CAGCTCTGCT TGTATGCAGC TTGATCCTGG151 GGTCTTAAGG CCAACGACTT CAGCTCTGCT TGTATGCAGC TTGATCCTGG
201 CAATGCCATT TCTTGGCTTG ACAAAGTCGT GGGCTTGGGA AAGATTCTTC201 CAATGCCATT TCTTGGCTTG ACAAAGTCGT GGGCTTGGGA AAGATTCTTC
251 CTGATAACCT TAGAGGCCCT CTTTATGACA TGGCCCAGGG TAGTGTCTCC251 CTGATAACCT TAGAGGCCCT CTTTATGACA TGGCCCAGGG TAGTGTCTCC
301 ATGAATGAGG ACTGTCTCTA CCTTAACGTT TTCCGCCCTG CTGGCACCAA 351 GCCTGATGCT AAGCTCCCCG TCATGGTTTG GATTTACGGT GGTGCCTTTG301 ATGAATGAGG ACTGTCTCTA CCTTAACGTT TTCCGCCCTG CTGGCACCAA 351 GCCTGATGCT AAGCTCCCCG TCATGGTTTG GATTTACGGT GGTGCCTTTG
401 TGTTTGGTTC TTCTGCTTCT TACCCTGGTA ACGGCTACGT CAAGGAGAGT401 TGTTTGGTTC TTCTGCTTCT TACCCTGGTA ACGGCTACGT CAAGGAGAGT
451 GTGGAAATGG GCCAGCCTGT TGTGTTTGTT TCCATCAACT ACCGTACCGG451 GTGGAAATGG GCCAGCCTGT TGTGTTTGTT TCCATCAACT ACCGTACCGG
501 CCCCTATGGA TTCCTGGGTG GTGATGCCAT CACCGCTGAG GGTAACACCA501 CCCCTATGGA TTCCTGGGTG GTGATGCCAT CACCGCTGAG GGTAACACCA
551 ACGCTGGTCT GCACGACCAG CGCAAGGGTC TCGAGTGGGT TAGCGACAAC 601 ATTGCCAACT TTGGTGGTGA TCCCGACAAG GTCATGATTT TCGGTGAGTC551 ACGCTGGTCT GCACGACCAG CGCAAGGGTC TCGAGTGGGT TAGCGACAAC 601 ATTGCCAACT TTGGTGGTGA TCCCGACAAG GTCATGATTT TCGGTGAGTC
651 CGCTGGTGCC ATGAGTGTTG CTCACCAGCT TGTTGCCTAC GGTGGTGACA651 CGCTGGTGCC ATGAGTGTTG CTCACCAGCT TGTTGCCTAC GGTGGTGACA
701 ACACCTACAA CGGAAAGAAG CTTTTCCACT CTGCCATTCT TCAGTCTGGC701 ACACCTACAA CGGAAAGAAG CTTTTCCACT CTGCCATTCT TCAGTCTGGC
751 GGTCCTCTTC CTTACTTTGA CTCTACTTCT GTTGGTCCCG AGAGTGCCTA751 GGTCCTCTTC CTTACTTTGA CTCTACTTCT GTTGGTCCCG AGAGTGCCTA
801 CAGCAGATTT GCTCAGTATG CCGGATGTGA TGCCAGCGCC AGTGACAATG 851 AAACTCTGGC TTGTCTCCGC AGCAAGTCCA GCGATGTCTT GCACAGTGCC801 CAGCAGATTT GCTCAGTATG CCGGATGTGA TGCCAGCGCC AGTGACAATG 851 AAACTCTGGC TTGTCTCCGC AGCAAGTCCA GCGATGTCTT GCACAGTGCC
901 CAGAACTCGT ACGATCTCAA GGACCTGTTT GGCCTGCTCC CTCAATTCCT901 CAGAACTCGT ACGATCTCAA GGACCTGTTT GGCCTGCTCC CTCAATTCCT
951 TGGATTTGGT CCCAGACCCG ACGGCAACAT TATTCCCGAT GCCGCTTATG951 TGGATTTGGT CCCAGACCCG ACGGCAACAT TATTCCCGAT GCCGCTTATG
1001 AGCTCTACCG CAGCGGTAGA TACGCCAAGG TTCCCTACAT TACTGGTAAC1001 AGCTCTACCG CAGCGGTAGA TACGCCAAGG TTCCCTACAT TACTGGTAAC
1051 CAGGAGGATG AGGGTACTAT TCTTGCCCCC GTTGCTATTA ATGCTACCAC1051 CAGGAGGATG AGGGTACTAT TCTTGCCCCC GTTGCTATTA ATGCTACCAC
1101 GACTCCCCAT GTTAAGAAGT GGTTGAAGTA CATTTGTAGC GAGGCTTCTG1101 GACTCCCCAT GTTAAGAAGT GGTTGAAGTA CATTTGTAGC GAGGCTTCTG
1151 ACGCTTCGCT TGATCGTGTT TTGTCGCTCT ACCCCGGCTC TTGGTCGGAG1151 ACGCTTCGCT TGATCGTGTT TTGTCGCTCT ACCCCGGCTC TTGGTCGGAG
1201 GGTGCGCCAT TCCGCACTGG TATTCTTAAT GCTCTGACCC CTCAGTTCAA1201 GGTGCGCCAT TCCGCACTGG TATTCTTAAT GCTCTGACCC CTCAGTTCAA
1251 GCGCATTGCT GCCATTTTCA CTGATTTGCT GTTCCAGTCT CCTCGTCGTG1251 GCGCATTGCT GCCATTTTCA CTGATTTGCT GTTCCAGTCT CCTCGTCGTG
1301 TTATGCTTAA CGCTACCAAG GACGTCAACC GCTGGACTTA CCTTGCCACC 1351 CAGCTCCATA ACCTCGTTCC ATTTTTGGGT ACTTTCCATG GTAGTGATCT1301 TTATGCTTAA CGCTACCAAG GACGTCAACC GCTGGACTTA CCTTGCCACC 1351 CAGCTCCATA ACCTCGTTCC ATTTTTGGGT ACTTTCCATG GTAGTGATCT
1401 TCTTTTCCAA TACTACGTGG ACCTTGGCCC ATCTTCTGCT TACCGCCGCT1401 TCTTTTCCAA TACTACGTGG ACCTTGGCCC ATCTTCTGCT TACCGCCGCT
1451 ACTTTATCTC GTTTGCCAAC CACCACGACC CCAACGTTGG CACCAACCTG 1501 AAACAGTGGG ATATGTACAC TGATGCAGGC A GGAGATGC TTCAGATTCA1451 ACTTTATCTC GTTTGCCAAC CACCACGACC CCAACGTTGG CACCAACCTG 1501 AAACAGTGGG ATATGTACAC TGATGCAGGC TO GGAGATGC TTCAGATTCA
1551 TATGGTTGGT AACTCTATGA GAACTGACGA CTTTAGAATC GAGGGAATCT1551 TATGGTTGGT AACTCTATGA GAACTGACGA CTTTAGAATC GAGGGAATCT
1601 CGAACTTTGA GTCTGACGTT ACTCTCTTCG GTTAAfT 1601 CGAACTTTGA GTCTGACGTT ACTCTCTTCG GTTAAf T
SEQUENCE VISEQUENCE VI
1 ATGGAGCTCG CTCTTGCGCT CCTGCTCATT GCCTCGGTGG CTGCTGCCCC 51 CACCGCCACG CTCGCCAACG GCGACACCAT CACCGGTCTC AACGCCATCA1 ATGGAGCTCG CTCTTGCGCT CCTGCTCATT GCCTCGGTGG CTGCTGCCCC 51 CACCGCCACG CTCGCCAACG GCGACACCAT CACCGGTCTC AACGCCATCA
101 TCAACGAGGC GTTCCTCGGC ATTCCCTTTG CCGAGCCGCC GGTGGGCAAC101 TCAACGAGGC GTTCCTCGGC ATTCCCTTTG CCGAGCCGCC GGTGGGCAAC
151 CTCCGCTTCA AGGACCCCGT GCCGTACTCC GGCTCGCTCG ATGGCCAGAA151 CTCCGCTTCA AGGACCCCGT GCCGTACTCC GGCTCGCTCG ATGGCCAGAA
201 GTTCACGCTG TACGGCCCGC TGTGCATGCA GCAGAACCCC GAGGGCACCT201 GTTCACGCTG TACGGCCCGC TGTGCATGCA GCAGAACCCC GAGGGCACCT
251 ACGAGGAGAA CCTCCCCAAG GCAGCGCTCG ACTTGGTGAT GCAGTCCAAG 301 GTGTTTGAGG CGGTGCTGCC GCTGAGCGAG GACTGTCTCA CCATCAACGT251 ACGAGGAGAA CCTCCCCAAG GCAGCGCTCG ACTTGGTGAT GCAGTCCAAG 301 GTGTTTGAGG CGGTGCTGCC GCTGAGCGAG GACTGTCTCA CCATCAACGT
351 GGTGCGGCCG CCGGGCACCA AGGCGGGTGC CAACCTCCCG GTGATGCTCT351 GGTGCGGCCG CCGGGCACCA AGGCGGGTGC CAACCTCCCG GTGATGCTCT
401 GGATCTTTGG CGGCGGGTTT GAGGTGGGTG GCACCAGCAC CTTCCCTCCC401 GGATCTTTGG CGGCGGGTTT GAGGTGGGTG GCACCAGCAC CTTCCCTCCC
451 GCCCAGATGA TCACCAAGAG CATTGCCATG GGCAAGCCCA TCATCCACGT451 GCCCAGATGA TCACCAAGAG CATTGCCATG GGCAAGCCCA TCATCCACGT
501 GAGCGTCAAC TACCGCGTGT CGTCGTGGGG GTTCTTGGCT GGCGACGAGA 551 TCAAGGCCGA GGGCAGTGCC AACGCCGGTT TGAAGGACCA GCGCTTGGGC501 GAGCGTCAAC TACCGCGTGT CGTCGTGGGG GTTCTTGGCT GGCGACGAGA 551 TCAAGGCCGA GGGCAGTGCC AACGCCGGTT TGAAGGACCA GCGCTTGGGC
601 ATGCAGTGGG TGGCGGACAA CATTGCGGCG TTTGëCGGCG ACCCGACCAA601 ATGCAGTGGG TGGCGGACAA CATTGCGGCG TTTGëCGGCG ACCCGACCAA
651 GGTGACCATC TTTGGCGAGC TGGCGGGCAG CATGTCGGTC ATGTGCCACA651 GGTGACCATC TTTGGCGAGC TGGCGGGCAG CATGTCGGTC ATGTGCCACA
701 TTCTCTGGAA CGACGGCGAC AACACGTACA AGGGCAAGCC GCTCTTCCGC701 TTCTCTGGAA CGACGGCGAC AACACGTACA AGGGCAAGCC GCTCTTCCGC
751 GCGGGCATCA TGCAGCTGGG GGCCATGGTG CCGCTGGACG CCGTGGACGG 801 CATCTACGGC AACGAGATCT TTGACCTCTT GGCGTCGAAC GCGGGCTGCG751 GCGGGCATCA TGCAGCTGGG GGCCATGGTG CCGCTGGACG CCGTGGACGG 801 CATCTACGGC AACGAGATCT TTGACCTCTT GGCGTCGAAC GCGGGCTGCG
851 GCAGCGCCAG CGACAAGCTT GCGTGCTTGC GCGGTGTGCT GAGCGACACG851 GCAGCGCCAG CGACAAGCTT GCGTGCTTGC GCGGTGTGCT GAGCGACACG
901 TTGGAGGACG CCACCAACAA CACCCCTGGG TTCTTGGCGT ACTCCTCGTT 951 GCGGTTGCTG TACCTCCCCC GGCCCGACGG CGTGAACATC ACCGACGACA901 TTGGAGGACG CCACCAACAA CACCCCTGGG TTCTTGGCGT ACTCCTCGTT 951 GCGGTTGCTG TACCTCCCCC GGCCCGACGG CGTGAACATC ACCGACGACA
1001 TGTACGCCTT GGTGCGCGAG GGCAAGTATG CCAACATCCC TGTGATCATC1001 TGTACGCCTT GGTGCGCGAG GGCAAGTATG CCAACATCCC TGTGATCATC
1051 GGCGACCAGA ACGACGAGGG CACCTTCTTT GGCACCCTGC TGTTGAACGT 1101 GACCACGGAT GCCCAGGCCC GCGAGTACTT CAAGCAGCTG TTTGTCCACG1051 GGCGACCAGA ACGACGAGGG CACCTTCTTT GGCACCCTGC TGTTGAACGT 1101 GACCACGGAT GCCCAGGCCC GCGAGTACTT CAAGCAGCTG TTTGTCCACG
1151 CCAGCGACGC GGAGATCGAC ACGTTGATGA CGGCGTACCC CGGCGACATC1151 CCAGCGACGC GGAGATCGAC ACGTTGATGA CGGCGTACCC CGGCGACATC
1201 ACCCAGGGCC TGCCGTTCGA CACGGGTATT CTCAACGCCC TCACCCCGCA1201 ACCCAGGGCC TGCCGTTCGA CACGGGTATT CTCAACGCCC TCACCCCGCA
1251 GTTCAAGAGA ATCCTGGCGG TGCTCGGCGA CCTTGGCTTT ACGCTTGCTC1251 GTTCAAGAGA ATCCTGGCGG TGCTCGGCGA CCTTGGCTTT ACGCTTGCTC
1301 GTCGCTACTT CCTCAACCAC TACACCGGCG GCACCAAGTA CTCATTCCTC 1351 CTGAAGCAGC TCCTGGGCTT GCCGGTGCTC GGAACGTTCC ACTCCAACGA1301 GTCGCTACTT CCTCAACCAC TACACCGGCG GCACCAAGTA CTCATTCCTC 1351 CTGAAGCAGC TCCTGGGCTT GCCGGTGCTC GGAACGTTCC ACTCCAACGA
1401 CATTGTCTTC CAGGACTACT TGTTGGGCAG CGGCTCGCTC ATCTACAACA1401 CATTGTCTTC CAGGACTACT TGTTGGGCAG CGGCTCGCTC ATCTACAACA
1451 ACGCGTTCAT TGCGTTTGCC ACGGACTTGG ACCCCAACAC CGCGGGGTTG1451 ACGCGTTCAT TGCGTTTGCC ACGGACTTGG ACCCCAACAC CGCGGGGTTG
1501 TTGGTGAAGT GGCCCGAGTA CACCAGCAGC CTGCAGCTGG GCAACAACTT1501 TTGGTGAAGT GGCCCGAGTA CACCAGCAGC CTGCAGCTGG GCAACAACTT
1551 GATGATGATC AACGCCTTGG GCTTGTACAC CGGCAAGGAC AACTTCCGCA 1601 CCGCCGGCTA CGACGCGTTG TTCTCCAACC CGCCGCTGTT CTTTGTGTAA 1551 GATGATGATC AACGCCTTGG GCTTGTACAC CGGCAAGGAC AACTTCCGCA 1601 CCGCCGGCTA CGACGCGTTG TTCTCCAACC CGCCGCTGTT CTTTGTGTAA
La séquence I correspond à un cDNA de Rhizopus ni veus, la séquence II peut être isolée à partir du génome de Pseudomonas aeruginosa, la séquence III à partir de Pseudomonas fluorescens, la séquence IV à partir de Pseudomonas sp, la séquence V à partir de Geotrichu candidum, la séquence VI à partir de Candida cylindracea .Sequence I corresponds to a Rhizopus ni veus cDNA, sequence II can be isolated from the genome of Pseudomonas aeruginosa, sequence III from Pseudomonas fluorescens, sequence IV from Pseudomonas sp, sequence V from Geotrichu candidum, sequence VI from Candida cylindracea.
Par ailleurs, l'introduction de la cassette d'expression : gène de lipase/promoteur d'expression de ce gène, dans le génome de la plante oléagineuse peut être réalisée par tout protocole connu.Furthermore, the introduction of the expression cassette: lipase gene / promoter of expression of this gene, into the genome of the oleaginous plant can be carried out by any known protocol.
Par exemple, selon le protocole le plus courant actuellement, cette cassette d'expression peut être introduite dans le génome de cellules somatiques de la plante par un transfert à l'aide de la bactérie Agrobacteri um tumefaciens . Cette introduction dans lesdites cellules somatiques de la plante peut également être réalisée par une autre technique connue, notamment par électroporation, par biolistique ou par microinjection.For example, according to the most current protocol currently, this expression cassette can be introduced into the genome of somatic cells of the plant by a transfer using the bacterium Agrobacterium um tumefaciens. This introduction into said somatic cells of the plant can also be carried out by another known technique, in particular by electroporation, by biolistics or by microinjection.
Il est également possible d'introduire la cassette d'expression dans le génome de microspores de la plante par électroporation ou biolistique.It is also possible to introduce the expression cassette into the genome of microspores of the plant by electroporation or biolistics.
Dans le protocole fourni plus loin à titre d'exemple, on a décrit la technique d'électroporation pour l'introduction de la cassette dans les microspores du colza.In the protocol provided below by way of example, the electroporation technique for the introduction of the cassette into the rapeseed microspores has been described.
On pourra se reporter au document suivant "P.J.J. Hooykaas et R.A. Schilperoort, TIBS août 1985, p. 305-309" pour plus de détail sur la technique de transfert à l'aide de la bactérie Agrobacteri um tumefaciens. On rappelle que cette technique consiste à introduire la cassette d'expression concernée dans le plasmide Ti de la bactérie notamment par choc thermique, puis à mettre en contact la bactérie avec des disques de feuilles de la plante, à laisser incuber l'ensemble jusqu'à obtenir le transfert de la cassette d'expression dans le génome des cellules des disques foliaires, et à cultiver ces disques foliaires sur une succession de milieux jusqu'à régénération des plantes transgéniques. On pourra se reporter au document suivant "J.A. Russell et al., In Vitro Cell. Dev. Biol., 1992, 28P, p. 97-105" pour plus de détail sur la technique de biolistique. On rappelle que cette technique consiste à fixer le plas ide contenant la cassette d'expression sur des microbilles d'or ou de tungstène, à projeter ces microbilles à l'aide d'un canon à particules sur les cellules de la plante à transformer, et à cultiver ces cellules jusqu'à régénération des plantes transgéniques. On pourra se reporter au document suivantReference may be made to the following document "PJJ Hooykaas and RA Schilperoort, TIBS August 1985, pp. 305-309" for more details on the transfer technique using the bacterium Agrobacteri um tumefaciens. It is recalled that this technique consists in introducing the expression cassette concerned into the Ti plasmid of the bacterium in particular by thermal shock, then in bringing the bacteria into contact with discs of leaves of the plant, in allowing the whole to incubate until to obtain the transfer of the expression cassette into the genome of the cells of the leaf discs, and to cultivate these leaf discs on a succession of media until regeneration of the transgenic plants. Reference may be made to the following document "JA Russell et al., In Vitro Cell. Dev. Biol., 1992, 28P, p. 97-105" for more details on the biolistic technique. It is recalled that this technique consists in fixing the plas ide containing the expression cassette on gold or tungsten microbeads, in projecting these microbeads using a particle gun onto the cells of the plant to be transformed, and to cultivate these cells until regeneration of the transgenic plants. We can refer to the following document
"Crossway A et Al, 1986, Mol. Gen. Genêt 202, 179-185" pour plus de détail sur la technique de micro-injection. On rappelle que cette technique consiste à injecter dans des protoplastes ou de très jeunes embryons le plasmide contenant la cassette d'expression à l'aide de micro¬ seringues, et à cultiver les protoplastes jusqu'à régénération des plantes transgéniques."Crossway A et Al, 1986, Mol. Gen. Genêt 202, 179-185" for more details on the micro-injection technique. It will be recalled that this technique consists in injecting the plasmid containing the expression cassette into protoplasts or very young embryos using micro-syringes, and cultivating the protoplasts until regeneration of the transgenic plants.
Après mise au contact par broyage de la lipase et des lipides, on laisse incuber l'ensemble pour réaliser l'hydrolyse enzymatique. Cette incubation est réalisée dans des conditions classiques, en particulier entre 20° C et 60° C, pendant le temps nécessaire à l'obtention d'une hydrolyse totale ou quasi-totale.After bringing into contact by grinding the lipase and the lipids, the whole is left to incubate in order to carry out the enzymatic hydrolysis. This incubation is carried out under conventional conditions, in particular between 20 ° C. and 60 ° C., for the time necessary to obtain total or almost total hydrolysis.
L'extraction des acides gras issus de l'hydrolyse est ensuite opérée par tout procédé connu, en particulier par une extraction liquide/liquide utilisant un solvant apolaire tel que le chloroforme ou l'hexane.The extraction of fatty acids from hydrolysis is then carried out by any known method, in particular by liquid / liquid extraction using an apolar solvent such as chloroform or hexane.
Selon un autre mode de mise en oeuvre, il est possible de réaliser in situ une transformation des acides gras pour obtenir des dérivés d'acides gras qui sont ensuite extraits. Par exemple, les acides gras issus de l'hydrolyse peuvent être méthylés in situ par mise en contact avec du éthanol en catalyse acide sous ultrasons en vue de les transformer en esters méthyliques, ces derniers étant extraits par une extraction liquide/liquide utilisant un solvant apolaire.According to another embodiment, it is possible to carry out in situ a transformation of the fatty acids to obtain fatty acid derivatives which are then extracted. For example, the fatty acids resulting from hydrolysis can be methylated in situ by contacting with ethanol in acid catalysis under ultrasound in order to transform them into methyl esters, the latter being extracted by a liquid / liquid extraction using a solvent apolar.
La présente demande vise, en tant que produit nouveau, toute plante oléagineuse ou semence de plante oléagineuse, d'une variété non protegeable par un certificat d'obtention végétale, qui comprend dans son génome une cassette d'expression possédant au moins un gène codant pour une enzyme lipase, associé à un promoteur permettant une expression dudit gène dans des compartiments cellulaires, extracellulaires ou tissulaires différents des compartiments lipidiques de la plante ou de la semence.The present application is intended, as a new product, any oleaginous plant or seed of oleaginous plant, of a variety which cannot be protected by a plant variety certificate, which comprises in its genome an expression cassette having at least one gene coding for a lipase enzyme, associated with a promoter allowing expression of said gene in compartments cellular, extracellular or tissue different from the lipid compartments of the plant or seed.
Le promoteur associé au gène de lipase peut être un promoteur à expression cellulaire ou tissulaire spécifique. Ce promoteur peut également être un promoteur constitutif, auquel cas le gène de lipase est muni d'une séquence d'adressage vers des compartiments cellulaires ou extracellulaires différents des compartiments d'accumulation des lipides. La présente demande vise également toute plante oléagineuse ou semence de plante oléagineuse, d'une variété non protegeable par un certificat d'obtention végétale, qui comprend dans son génome une cassette d'expression possédant au moins un gène codant pour une enzyme lipase, associé à un promoteur permettant une expression dudit gène sur induction exogène, en particulier par un stress.The promoter associated with the lipase gene can be a promoter with specific cellular or tissue expression. This promoter can also be a constitutive promoter, in which case the lipase gene is provided with an addressing sequence towards cellular or extracellular compartments different from the lipid accumulation compartments. The present application also relates to any oleaginous plant or seed of oleaginous plant, of a variety which cannot be protected by a plant variety certificate, which comprises in its genome an expression cassette having at least one gene coding for a lipase enzyme, associated to a promoter allowing expression of said gene on exogenous induction, in particular by stress.
La description qui suit en référence au dessin fournit à titre d'exemple un protocole de mise en oeuvre du procédé de l'invention ; la figure 1 du dessin schématise la préparation du matériel génétique à transférer.The description which follows with reference to the drawing provides by way of example a protocol for implementing the method of the invention; Figure 1 of the drawing shows schematically the preparation of the genetic material to be transferred.
1/ PROTOCOLE D'OBTENTION DE PLANTES TRANSGENIQUES DE COLZA EXPRIMANT UN GENE DE LIPASE a) Matériel végétal1 / PROTOCOL FOR THE PRODUCTION OF TRANSGENIC COLZA PLANTS EXPRESSING A LIPASE GENE a) Plant material
On utilise des graines de colza ( Brassica napus, Var Tapidor) qui sont obtenues dans le commerce.Rape seeds (Brassica napus, Var Tapidor) are used, which are obtained commercially.
Les graines sont semées en serre et cultivées dans des conditions standard. L'état sanitaire des plantes est rigoureusement surveillé.The seeds are sown in the greenhouse and grown under standard conditions. The health of the plants is rigorously monitored.
Les jeunes bourgeons (taille inférieure à 3,5 mm) sont prélevés, stérilisés dans l'hypochlorite de sodium pendant 30 minutes et les microspores sont extraites des anthères par broyage au aring Blendor dans le milieu de Huang et al (Huang et al. 1990, Plant. Cell. Rep. 8, 594-597). Après filtration sur un tamis métallique de 50 μm de vide de maille, les microspores sont récupérées par centrifugation 5 minutes à lOOxg (Protocole décrit dans : Jardinaud et al., 1993, Plant. Sci. 93, 177-184). b) Construction génétiqueYoung buds (size less than 3.5 mm) are removed, sterilized in hypochlorite from sodium for 30 minutes and the microspores are extracted from the anthers by Blendor aring grinding in the medium of Huang et al (Huang et al. 1990, Plant. Cell. Rep. 8, 594-597). After filtration through a metal sieve of 50 μm of mesh vacuum, the microspores are recovered by centrifugation for 5 minutes at 100 × g (Protocol described in: Jardinaud et al., 1993, Plant. Sci. 93, 177-184). b) Genetic construction
La construction génétique retenue met en oeuvre le promoteur de la napine, le cDNA de la lipase de Rhizopus ni veus et le ter inateur NOS. Le promoteur de la napine dirige l'expression de cette protéine dans un compartiment protéique de la graine différent de celui où s'accumulent les lipides. L'ensemble est introduit dans le plasmide pRTI contenant le gène de sélection pa t utilisé pour cribler les transformants en vue de former la construction pRTIL.The genetic construction selected involves the promoter of napine, the cDNA of the lipase from Rhizopus ni veus and the terator NOS. The napin promoter directs the expression of this protein in a protein compartment of the seed different from that in which the lipids accumulate. The whole is introduced into the plasmid pRTI containing the selection gene pa t used to screen the transformants in order to form the construct pRTIL.
Le détail de la construction est donné à la figure 1 du dessin. Le promoteur napine désigné "Prom. Nap" a été isolé par l'équipe du Professeur Rask (Stalberg K. et al, 1993, Plant Molecular Biology, 23 : 671-683). Le cDNA de la lipase de Rhizopus ni veus, désigné "Lip", a été isolé par le Central Research Institute (Kugimiya et al., 1992, Biosci. Biotech. Biochem. 56, 716-719). Un codon d'initiation, désigné "start codon", compatible avec l'extrémité 5' du cDNA (disponible sur le marché) est greffé sur cette extrémité. Sur l'extrémité 3' de l'ensemble obtenu, désigné "lip", on greffe un terminateur désigné NOS extrait du plasmide pRT1 ci-après évoqué et, sur l'extrémité 5', le promoteur Nap.The detail of the construction is given in Figure 1 of the drawing. The napine promoter designated "Prom. Nap" was isolated by the team of Professor Rask (Stalberg K. et al, 1993, Plant Molecular Biology, 23: 671-683). The Rhizopus ni veus lipase cDNA, designated "Lip", was isolated by the Central Research Institute (Kugimiya et al., 1992, Biosci. Biotech. Biochem. 56, 716-719). An initiation codon, designated "start codon", compatible with the 5 ′ end of cDNA (available on the market) is grafted onto this end. On the 3 'end of the assembly obtained, designated "lip", a terminator designated NOS extracted from the pRT1 plasmid mentioned below is grafted, and on the 5' end, the Nap promoter.
La cassette d'expression ainsi obtenue est introduite dans un plasmide désigné "Blue-Script" (nom commercial) en vue d'en réaliser l'amplification. La cassette d'expression amplifiée est ensuite extraite de "Blue-Script" et introduite dans un plasmide désigné pRT1 qui a été préparé en greffant le promoteur désigné CaMV35S sur l'extrémité 5' du gène "pat" codant pour la phosphinothricine acétyl transférase (gène de sélection) et le terminateur NOS sur l'extrémité 3'.The expression cassette thus obtained is introduced into a plasmid designated "Blue-Script" (trade name) in order to carry out the amplification thereof. The amplified expression cassette is then extracted from "Blue-Script" and introduced into a plasmid designated pRT1 which was prepared by grafting the promoter designated CaMV35S onto the 5 'end of the "pat" gene encoding phosphinothricin acetyl transferase (selection gene) and the NOS terminator on the 3 'end.
On obtient un plasmide désigné pRTIL contenant la cassette d'expression visée. c) Transfert de gène et production des plantes transgéniquesA plasmid designated pRTIL containing the targeted expression cassette is obtained. c) Gene transfer and production of transgenic plants
Les microspores isolées en a) (10° microspores ml-') sont mises en suspension dans le milieu de Brewbaker et Kwack (J.L. Brewbaker et B.H. Kwack, 1963, Am. J. Bot. 50, p. 859-865) contenant 13 % de saccharose et ajusté à pH 5,9. 50μg par ml du plasmide pRTIL sont ajoutés au milieu et des impulsions électriques de 400 V/cm pendant 10 ms sont appliquées à la suspension à l'aide d'un électroporateur "Jouan" (marque déposée) (TRX, GHT) délivrant des impulsions en vague carrée. Après 20 minutes de repos, le milieu de culture (Huang et al.) contenant 100 mg/1 de phosphinothricine est ajouté aux microspores. Les microspores sont cultivées à l'obscurité 24 h à 35° C puis à 25° C. Après 2 semaines de culture un volume égal de milieu neuf est ajouté et les microspores placées à la lumière sous photopériode 16 h jour / 8 h obscurité.The microspores isolated in a) (10 ° microspores ml- ') are suspended in the medium of Brewbaker and Kwack (JL Brewbaker and BH Kwack, 1963, Am. J. Bot. 50, p. 859-865) containing 13 % sucrose and adjusted to pH 5.9. 50 μg per ml of the plasmid pRTIL are added to the medium and electrical pulses of 400 V / cm for 10 ms are applied to the suspension using an electroporator "Jouan" (registered trademark) (TRX, GHT) delivering pulses in square wave. After 20 minutes of rest, the culture medium (Huang et al.) Containing 100 mg / 1 of phosphinothricin is added to the microspores. The microspores are cultured in the dark for 24 h at 35 ° C. and then at 25 ° C. After 2 weeks of culture an equal volume of new medium is added and the microspores placed in the light under photoperiod 16 h day / 8 h dark.
Après environ 1 mois les embryons sont transférés en milieu B5 (Gamborg et al., 1979, Exp. Cell. Res. 50, 151-158) contenant G3A3 1 ml/g, 20g/l saccharose et solidifié par 8/1 de gélifiant "Bacto Agar" (marque déposée) .After about 1 month the embryos are transferred to medium B5 (Gamborg et al., 1979, Exp. Cell. Res. 50, 151-158) containing G3A3 1 ml / g, 20g / l sucrose and solidified with 8/1 gelling agent "Bacto Agar" (registered trademark).
Les plantes régénérées résistantes à la phosphinothricine sont analysées en "southern" de façon à vérifier la présence dans leur génome de la séquence codant pour la lipase. Le stock chromosomique des plantes retenues est doublé par la colchicine (0,1 g/1 plus quelques gouttes de teepol) et les plantes diploïdes fertiles produites sont autofécondées. Sur un nombre restreint de graines, on recherche l'activité de la lipase. Les plantes présentant des graines dont l'activité lipase est la plus élevée sont retenues et les graines utilisées pour la multiplication des plantes jusqu'à obtention d'un stock de graines suffisant pour réaliser les expériences d'hydrolyse des lipides de la graine par les lipases endogènes.The regenerated plants resistant to phosphinothricin are analyzed in "southern" so as to verify the presence in their genome of the sequence coding for lipase. The chromosomal stock of the selected plants is doubled by colchicine (0.1 g / 1 plus a few drops of teepol) and the fertile diploid plants produced are self-fertilized. Lipase activity is sought on a limited number of seeds. Plants with seeds with the highest lipase activity are retained and the seeds used for propagation plants until a sufficient seed stock is obtained to carry out the experiments on hydrolysis of the lipids of the seed by endogenous lipases.
2/ HYDROLYSE ENZYMATIQUE DES LIPIDES DES GRAINES DE COLZA OBTENUES2 / ENZYMATIC HYDROLYSIS OF LIPIDS FROM COLZA SEEDS OBTAINED
Les graines sont broyées, le broyât placé dans un incubateur maintenu à la température constante de 40°. Le broyât est soumis à une agitation permanente de façon à augmenter le contact entre les lipides et la lipase.The seeds are ground, the ground material placed in an incubator maintained at a constant temperature of 40 °. The ground material is subjected to permanent agitation so as to increase the contact between the lipids and the lipase.
Après 48 h d'hydrolyse, les acides gras sont extraits par le chloroforme. Le chloroforme est évaporé et les acides gras récupérés. After 48 h of hydrolysis, the fatty acids are extracted with chloroform. The chloroform is evaporated and the fatty acids recovered.

Claims

REVENDICATIONS 1/ - Procédé de production d'acides gras ou de dérivés d'acides gras à partir de plantes oléagineuses, caractérisé en ce que : - on produit des plantes oléagineuses transgéniques possédant, d'une part, au moins un gène codant pour une enzyme lipase, dit gène de lipase, d'autre part, associé à ce gène de lipase, un promoteur permettant une expression dudit gène soit dans des compartiments cellulaires, extracellulaires ou tissulaires différents de ceux où s'accumulent les lipides de la plante, soit sur induction exogène,- on recueille les graines ou fruits contenant les lipides desdites plantes, - on broie ledites graines ou fruits, le cas échéant après traitement inducteur, de façon à mettre en contact les lipides et la lipase contenue dans lesdites graines ou fruits,- on laisse incuber l'ensemble pour engendrer une hydrolyse enzymatique des lipides du broyât sous l'action catalytique de la lipase contenue dans ledit broyât,- on extrait les acides gras issus de l'hydrolyse ou on les transforme pour obtenir les dérivés d'acides gras recherchés.2/ - Procédé selon la revendication 1 , dans lequel on produit les plantes transgéniques en effectuant une transformation génétique d'une plante oléagineuse naturelle, en amenant la plante génétiquement transformée à se multiplier par la voie sexuée pour la production de semences transgéniques et en utilisant lesdites semences pour l'obtention de plantes transgéniques descendantes.3/ - Procédé selon la revendication 2 pour la production d'acides gras à partir de plantes oléoprotéagineuses, caractérisé en ce que :- la transformation génétique de la plante est effectuée en réalisant une cassette d'expression comprenant un gène de lipase et un promoteur contrôlant l'expression d'une protéine déterminée de la graine, et en introduisant cette cassette d'expression dans le génome de la plante de façon à faire exprimer la lipase dans les compartiments de la graine où s'accumulent la protéine précitée,- la mise en contact des lipides et de la lipase est effectuée par simple broyage.4/ - Procédé selon la revendication 3 pour la production d'acides gras à partir de colza, caractérisé en ce que la transformation génétique est effectuée à partir d'une cassette d'expression comprenant un gène de lipase et le promoteur de la napine.5/ - Procédé selon la revendication 2, caractérisé en ce que : . la transformation génétique de la plante est effectuée en réalisant une cassette d'expression comprenant un gène de lipase et un promoteur contrôlable de façon exogène, et en introduisant cette cassette d'expression dans le génome de la plante, . un traitement inducteur est appliqué aux graines et fruits avant broyage de façon à provoquer la synthèse de la lipase.6/ - Procédé selon la revendication 5, caractérisé en ce que : . la transformation génétique est effectuée à partir d'une cassette d'expression comprenant un gène de lipase et un promoteur de stress,. le traitement inducteur est constitué par un traumatisme physique appliqué sur les graines ou fruits avant le broyage.7/ - Procédé selon la revendication 2, caractérisé en ce que :. la transformation génétique de la plante est effectuée en réalisant une cassette d'expression comprenant un promoteur constitutif et un gène de lipase muni d'une séquence d'adressage vers des compartiments cellulaires ou extracellulaires différents de ceux où s'accumulent les lipides, . la mise en contact des lipides et de la lipase est effectuée par simple broyage.8/ - Procédé selon l'une des revendications 1 à 7, caractérisé en ce qu'on produit des plantes transgéniques possédant un gène de lipase caractérisée par une activité hydrolytique non spécifique.9/ - Procédé selon la revendication 8, caractérisé en ce qu'on produit des plantes transgéniques possédant un gène de lipase caractérisé par une séquence identique ou analogue à l'une des séquences suivantes (les flèches délimitent la partie codante) : SEQUENCE I1 G'ATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCC51 TCCTATCAGC CTCTCTAGCT CTACCAACAG CGCCTCTGAT GGTGGTAAGG101 TTGTTGCTGC TACTACTGCT CAGATCCAAG AGTTCACCAA GTATGCTGGT151 ATCGCTGCCA CTGCCTACTG TCGTTCTGTT GTCCCTGGTA ACAAGTGGGA201 TTGTGTCCAA TGTCAAAAGT GGGTTCCTGA TGGCAAGATC ATCACTACCT251 TTACCTCCTT GCTTTCCGAT ACAAATGGTT ACGTCTTGAG AAGTGATAAA301 CAAAAGACCA TTTATCTTGT TTTCCGTGGT ACCAACTCCT TCAGAAGTGC351 CATCACTGAT ATCGTCTTCA ACTTTTCTGA CTACAAGCCT GTCAAGGGCG401 CCAAAGTTCA TGCTGGTTTC CTTTCCTCTT ATGAGCAAGT TGTCAATGAC451 TATTTCCCTG TCGTCCAAGA ACAATTGACC GCCCACCCTA CTTATAAGGT501 CATCGTTACC GGTCACTCAC TCGGTGGTGC ACAAGCTTTG CTTGCCGGTA551 TGGATCTCTA CCAACGTGAA CCAAGATTGT CTCCCAAGAA TTTGAGCATC601 TTCACTGTCG GTGGTCCTCG TGTTGGTAAC CCCACCTTTG CTTACTATGT651 TGAATCCACC GGTATCCCTT TCCAACGTAC CGTTCACAAG AGAGATATCG701 TTCCTCACGT TCCTCCTCAA TCCTTCGGAT TCCTTCATCC CGGTGTTGAA751 TCTTGGATGA AGTCTGGTAC TTCCAACGTT CAAATCTGTA CTTCTGAAAT801 TGAAACCAAG GATTGCAGTA ACTCTATCGT TCCTTTCACC TCTATCCTTG851 ACCACTTGAG TTACTTTGAT ATCAACGAAG GAAGCTGTTT GTAAAACACT901 TGACGTGTTA CTCTAATTTT ATAATAAAAT TAAGTTTTTA TACAAT SEQUENCE II CLAIMS 1 / - Process for the production of fatty acids or fatty acid derivatives from oleaginous plants, characterized in that: - transgenic oleaginous plants are produced having, on the one hand, at least one gene coding for a enzyme lipase, called lipase gene, on the other hand, associated with this lipase gene, a promoter allowing expression of said gene either in cellular, extracellular or tissue compartments different from those where the lipids of the plant accumulate, or on exogenous induction, - the seeds or fruits containing the lipids of the said plants are collected, - the said seeds or fruits are ground, if necessary after inducing treatment, so as to bring the lipids and the lipase contained in said seeds or fruits into contact, - the whole is incubated to generate an enzymatic hydrolysis of the lipids of the ground product under the catalytic action of the lipase contained in said ground material, - the acid is extracted es fat from hydrolysis or they are transformed to obtain the desired fatty acid derivatives. 2 / - Method according to claim 1, in which transgenic plants are produced by carrying out a genetic transformation of a natural oil plant, into causing the genetically transformed plant to multiply sexually for the production of transgenic seeds and using said seeds to obtain descendant transgenic plants.3 / - Process according to claim 2 for the production of fatty acids from oleoproteinous plants, characterized in that: - the genetic transformation of the plant is carried out by producing an expression cassette comprising a lipase gene and a promoter controlling the expression of a determined protein from the seed, and by introducing this cassette of expression in the genome of the plant so as to express lipase in the compartments of the gr groin where the abovementioned protein accumulates, - the contacting of the lipids and the lipase is carried out by simple grinding. 4 / - Method according to claim 3 for the production of fatty acids from rapeseed, characterized in that the genetic transformation is carried out using an expression cassette comprising a lipase gene and the napin promoter. 5 / - Method according to claim 2, characterized in that:. the genetic transformation of the plant is carried out by producing an expression cassette comprising a lipase gene and an exogenously controllable promoter, and by introducing this expression cassette into the genome of the plant,. an inducing treatment is applied to the seeds and fruits before grinding so as to cause the synthesis of lipase. 6 / - Method according to claim 5, characterized in that:. the genetic transformation is carried out using an expression cassette comprising a lipase gene and a stress promoter. the inductive treatment consists of a physical trauma applied to the seeds or fruits before grinding. 7 / - Method according to claim 2, characterized in that:. the genetic transformation of the plant is carried out by producing an expression cassette comprising a constitutive promoter and a lipase gene provided with an addressing sequence towards cellular or extracellular compartments different from those in which the lipids accumulate,. contacting the lipids and the lipase is carried out by simple grinding. 8 / - Method according to one of claims 1 to 7, characterized in that transgenic plants are produced having a lipase gene characterized by hydrolytic activity non-specific. 9 / - Method according to claim 8, characterized in that transgenic plants are produced having a lipase gene characterized by a sequence identical or analogous to one of the following sequences (the arrows delimit the coding part): SEQUENCE I1 G'ATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCC51 TCCTATCAGC CTCTCTAGCT CTACCAACAG CGCCTCTGAT GGTGGTAAGG101 TTGTTGCTGC TACTACTGCT CAGATCCAAG AGTTCACCAA GTATGCTGGT151 ATCGCTGCCA CTGCCTACTG TCGTTCTGTT GTCCCTGGTA ACAAGTGGGA201 TTGTGTCCAA TGTCAAAAGT GGGTTCCTGA TGGCAAGATC ATCACTACCT251 TTACCTCCTT GCTTTCCGAT ACAAATGGTT ACGTCTTGAG AAGTGATAAA301 CAAAAGACCA TTTATCTTGT TTTCCGTGGT ACCAACTCCT TCAGAAGTGC351 CATCACTGAT ATCGTCTTCA CADTC TCTGA CTACAAGCCT GTCAAGGGCG401 CCAAAGTTCA TGCTGGTTTC CTTTCCTCTT ATGAGCAAGT TGTCAATGAC451 TATTTCCCTG TCGTCCAAGA ACAATTGACC GCCCACCCTA CTTATAAGGT501 CATCGTTACC GGTCACTCAC TCGGTGGTGC ACAAGCTTTG CTTGCCGGTA551 TGGATCTCTA CCAACGTGAA CCAAGATTGT CTCCCAAGAA TTTGAGCATC601 TTCACTGTCG GTGGTCCTCG TGTTGGTAAC CCCACCTTTG CTTACTATGT651 TGAATCCACC GGTATCCCTT TCCAACGTAC CGTTCACAAG AGAGATATCG701 TTCCTCACGT TCCTCCTCAA TCCTTCGGAT TCCTTCATCC CGGTGTTGAA751 TCTTGGATGA AGTCTGGTAC TTCCAACGTT CAAATCTGTA CTTCTGAAAT801 TGAAACCAAG GATTGCAGTA ACTCTATCGT TCCTTTCACC TCTATCCTTG851 ACCACTTGAG TTACTTTGAT ATCAACGAAG GAAGCTGTTT GTAAAACACT901 TGACGTGTTA CTCTAATTTT ATAATAAAAT TAAGTTTTTA TACAAT SEQUENCE II
1 .GTCGACCATT TCAGCCTGTT TTGCTCGCAA AACGACGCCG CGGGCGTGCG1 .GTCGACCATT TCAGCCTGTT TTGCTCGCAA AACGACGCCG CGGGCGTGCG
51 CTACCGCACA CTCCGTCGCT GGGCGTTGTG CGGGGAAGAT TCAAACGAGC 101 GTTTCGCGCC GTAACAACCC GCTCTCTTCC GCTCTGCCAC GCAGGTTATG51 CTACCGCACA CTCCGTCGCT GGGCGTTGTG CGGGGAAGAT TCAAACGAGC 101 GTTTCGCGCC GTAACAACCC GCTCTCTTCC GCTCTGCCAC GCAGGTTATG
151 ACCGGCCGCC AGGAAGCCGC GGATTTCCTG GCCTGGAGGA AAAAAGCCGA151 ACCGGCCGCC AGGAAGCCGC GGATTTCCTG GCCTGGAGGA AAAAAGCCGA
201 AGCTGGCACG GTTCCTGGCG CAAGGGACAG CGAAGCGGTT CTCCCGGAAG201 AGCTGGCACG GTTCCTGGCG CAAGGGACAG CGAAGCGGTT CTCCCGGAAG
251 GATTCGGGCG ATGGCTGGCA GGACGCGCCC CTCGGCCCCA TCAACCTGAG251 GATTCGGGCG ATGGCTGGCA GGACGCGCCC CTCGGCCCCA TCAACCTGAG
301 ATGAGAACAA C TGAAGAAG AAGTCTCTGC TCCCCCTCGG CCTGGCCATC 351 GGTCTCGCCT CTCTCGCTGC CAGCCCTCTG ATCCAGGCCA GCACCTACAC301 ATGAGAACAA C TGAAGAAG AAGTCTCTGC TCCCCCTCGG CCTGGCCATC 351 GGTCTCGCCT CTCTCGCTGC CAGCCCTCTG ATCCAGGCCA GCACCTACAC
401 CCAGACCAAA TACCCCATCG TGCTGGCCCA CGGCATGCTC GGCTTCGACA401 CCAGACCAAA TACCCCATCG TGCTGGCCCA CGGCATGCTC GGCTTCGACA
451 ACATCCTCGG GGTCGACTAC TGGTTCGGCA TTCCCAGCGC CTTGCGCCGT451 ACATCCTCGG GGTCGACTAC TGGTTCGGCA TTCCCAGCGC CTTGCGCCGT
501 GACGGTGCCC AGGTCTACGT CACCGAAGTC AGCCAGTTGG ACACCTCGGA501 GACGGTGCCC AGGTCTACGT CACCGAAGTC AGCCAGTTGG ACACCTCGGA
551 AGTCCGCGGC GAGCAGTTGC TGCAACAGGT GGAGGAAATC GTCGCCCTCA 601 GCGGCCAGCC CAAGGTCAAC CTGATCGGCC ACAGCCACGG CGGGCCGACC551 AGTCCGCGGC GAGCAGTTGC TGCAACAGGT GGAGGAAATC GTCGCCCTCA 601 GCGGCCAGCC CAAGGTCAAC CTGATCGGCC ACAGCCACGG CGGGCCGACC
651 ATCCGCTACG TCGCCGCCGT ACGTCCCGAC CTGATCGCTT CCGCCACCAG651 ATCCGCTACG TCGCCGCCGT ACGTCCCGAC CTGATCGCTT CCGCCACCAG
701 CGTCGGCGCC CCGCACAAGG GTTCGGACAC CGCCGACTTC CTGCGCCAGA701 CGTCGGCGCC CCGCACAAGG GTTCGGACAC CGCCGACTTC CTGCGCCAGA
751 TCCCACCGGG TTCGGCCGGC GAGGCAGTCC TCTCCGGGCT GGTCAACAGC751 TCCCACCGGG TTCGGCCGGC GAGGCAGTCC TCTCCGGGCT GGTCAACAGC
801 CTCGGCGCGC TGATCAGCTT CCTTTCCAGC GGCAGCACCG GTACGCAGAA 851 TTCACTGGGC TCGCTGGAGT CGCTGAACAG CGAGGGTGCC GCGCGCTTCA801 CTCGGCGCGC TGATCAGCTT CCTTTCCAGC GGCAGCACCG GTACGCAGAA 851 TTCACTGGGC TCGCTGGAGT CGCTGAACAG CGAGGGTGCC GCGCGCTTCA
901 ACGCCAAGTA CCCGCAGGGC ATCCCCACCT CGGCCTGCGG CGAAGGCGCC901 ACGCCAAGTA CCCGCAGGGC ATCCCCACCT CGGCCTGCGG CGAAGGCGCC
951 TACAAGGTCA ACGGCGTGAG CTATTACTCC TGGAGCGGTT CCTCGCCGCT951 TACAAGGTCA ACGGCGTGAG CTATTACTCC TGGAGCGGTT CCTCGCCGCT
1001 GACCAACTTC CTCGATCCGA GCGACGCCTT CCTCGGCGCC TCGTCGCTGA1001 GACCAACTTC CTCGATCCGA GCGACGCCTT CCTCGGCGCC TCGTCGCTGA
1051 CCTTCAAGAA CGGCACCGCC AACGACGGCC TGGTCGGCAC CTGCAGTTCG 1101 CACCTGGGCA TGGTGATCCG CGACAACTAC CGGATGAACC ACCTGGACGA1051 CCTTCAAGAA CGGCACCGCC AACGACGGCC TGGTCGGCAC CTGCAGTTCG 1101 CACCTGGGCA TGGTGATCCG CGACAACTAC CGGATGAACC ACCTGGACGA
1151 GGTGAACCAG GTCTTCGGCC TCACCAGCCT GTTCGAGACC AGCCCGGTCA1151 GGTGAACCAG GTCTTCGGCC TCACCAGCCT GTTCGAGACC AGCCCGGTCA
1201 GCGTCTACCG CCAGCACGCC AACCGCCTGA AGAACGCCAG CCTGTAG' S E QU E N C E I I I1201 GCGTCTACCG CCAGCACGCC AACCGCCTGA AGAACGCCAG CCTGTAG ' WHAT HAPPENS
1 ^ GGGTGCATGC CAGCTCCCAC CGGACACCTG GCCCGTCGCT GAAAC -3TG T T1 ^ GGGTGCATGC CAGCTCCCAC CGGACACCTG GCCCGTCGCT GAAAC -3TG T T
51 T TCGC TTTC T CTACAAATCC AACAACAGAG AGGCACTACC 'ATGGGTATC T 101 TTGACTATAA AAACCTTGGC ACCGAGGGTT CCAAAACGTT GTTCGCCGAT51 T TCGC TTTC T CTACAAATCC AACAACAGAG AGGCACTACC 'ATGGGTATC T 101 TTGACTATAA AAACCTTGGC ACCGAGGGTT CCAAAACGTT GTTCGCCGAT
1 51 GCCATGGCGA TCACGTTGTA T TCCTATCAC AACCTGGATA ACGGCT TTGC1 51 GCCATGGCGA TCACGTTGTA T TCCTATCAC AACCTGGATA ACGGCT TTGC
201 CGTGGGCTAC CAGCACAACG GGTTGGGCTT GGGGCTACCG GCCACGCTGG201 CGTGGGCTAC CAGCACAACG GGTTGGGCTT GGGGCTACCG GCCACGCTGG
251 TCGGTGCGCT GCTCGGCAGC ACGGATTCCC AGGGCGTGAT CCCTGGCATC251 TCGGTGCGCT GCTCGGCAGC ACGGATTCCC AGGGCGTGAT CCCTGGCATC
301 CCGTGGAACC CGGATTCAGA AAAAGCCGCC CTTGAGGCGG TGCAGAAAGC 351 CGGTTGGACA CCGA.TCAGCG CCAGTGCCCT GGGCTACGCC GGCAAGGTCG301 CCGTGGAACC CGGATTCAGA AAAAGCCGCC CTTGAGGCGG TGCAGAAAGC 351 CGGTTGGACA CCGA.TCAGCG CCAGTGCCCT GGGCTACGCC GGCAAGGTCG
401 ATGCACGTGG CACCTTCTTT GGGGAAAAAG CCGGCTACAC CACGGCCCAG401 ATGCACGTGG CACCTTCTTT GGGGAAAAAG CCGGCTACAC CACGGCCCAG
451 GTCGAGGTAC TCGGCAAATA CGATGACGCC GGCAAGCTGC TCGAAATCGG451 GTCGAGGTAC TCGGCAAATA CGATGACGCC GGCAAGCTGC TCGAAATCGG
501 CATCGGTTTT CGTGGCACTT CGGGGCCACG GGAAACCTTG ATCAGCGACT501 CATCGGTTTT CGTGGCACTT CGGGGCCACG GGAAACCTTG ATCAGCGACT
551 CGATCGGCGA CTTGATCAGC GATCTGCTCG CGGCCCTGGG GCCCAAGGAT 601 TACGCGAAAA ACTACGCCGG CGAAGCCTTC GGCGGCTTGC TCAAGAATGT551 CGATCGGCGA CTTGATCAGC GATCTGCTCG CGGCCCTGGG GCCCAAGGAT 601 TACGCGAAAA ACTACGCCGG CGAAGCCTTC GGCGGCTTGC TCAAGAATGT
651 TGCCGACTAC GCCGGTGCCC ATGGCCTGAC CGGCAAGGAC GTGGTGGTCA651 TGCCGACTAC GCCGGTGCCC ATGGCCTGAC CGGCAAGGAC GTGGTGGTCA
701 GCGGCCACAG CCTGGGCGGG CTGGCGGTCA ACAGCATGGC GGACTTGAGC701 GCGGCCACAG CCTGGGCGGG CTGGCGGTCA ACAGCATGGC GGACTTGAGC
751 AACTACAAAT GGGCGGGGTT CTACAAGGAC GCCAACTATG TTGCCTATGC751 AACTACAAAT GGGCGGGGTT CTACAAGGAC GCCAACTATG TTGCCTATGC
801 CTCGCCGACC CAGAGTGCCG GCGACAAGGT GCTCAATATC GGTTACGAAA 851 ACGACCCGGT GTTCCGCGCG CTGGACGGCT CGTCGTTTAA CCTGTCGTCG801 CTCGCCGACC CAGAGTGCCG GCGACAAGGT GCTCAATATC GGTTACGAAA 851 ACGACCCGGT GTTCCGCGCG CTGGACGGCT CGTCGTTTAA CCTGTCGTCG
901 CTGGGCGTGC ACGACAAACC CCACGAGTCC ACCACCGATA ACATCGTCAG901 CTGGGCGTGC ACGACAAACC CCACGAGTCC ACCACCGATA ACATCGTCAG
951 CTTCAACGAC CACTACGCCT CGACGCTGTG GAATGTGCTG CCGTTTTCCA951 CTTCAACGAC CACTACGCCT CGACGCTGTG GAATGTGCTG CCGTTTTCCA
1001 TCGTCAACCT GCCCACCTGG GTCTCGCATT TGCCGACGGC GTACGGCGAT1001 TCGTCAACCT GCCCACCTGG GTCTCGCATT TGCCGACGGC GTACGGCGAT
1051 GGCATGACGC GCATCCTCGA GTCCGGCTTC TACGACCAGA TGACCCGTGA 1101 CTCCACGGTG ATTGTTGCCA ACCTGTCCGA TCCGGCGCGG GCCAACACCT1051 GGCATGACGC GCATCCTCGA GTCCGGCTTC TACGACCAGA TGACCCGTGA 1101 CTCCACGGTG ATTGTTGCCA ACCTGTCCGA TCCGGCGCGG GCCAACACCT
1151 GGGTGCAGGA CCTCAACCGC AATGCCGAGC CCCACAAGGG CAACACGTTC1151 GGGTGCAGGA CCTCAACCGC AATGCCGAGC CCCACAAGGG CAACACGTTC
1201 ATCATCGGCA GCGACGGCAA CGACCTGATC CAGGGCGGCA ACGGTGCGGA1201 ATCATCGGCA GCGACGGCAA CGACCTGATC CAGGGCGGCA ACGGTGCGGA
1251 CTTTATCGAG GGTGGCAAAG GCAACGACAC GATCCGCGAC AACAGCGGGC1251 CTTTATCGAG GGTGGCAAAG GCAACGACAC GATCCGCGAC AACAGCGGGC
1301 ACAACACCTT TTTGTTCAGC GGCCACTTTG GCAATGATCG CGTGATTGGC 1351 TACCAGCCCA CCGACAAACT GGTGTTCAAG GACGTGCAAG GAAGCACCGA1301 ACAACACCTT TTTGTTCAGC GGCCACTTTG GCAATGATCG CGTGATTGGC 1351 TACCAGCCCA CCGACAAACT GGTGTTCAAG GACGTGCAAG GAAGCACCGA
1401 CCTGCGTGAC CACGCGAAGG TGGTCGGCGC CGATACGGTG CTTACGTTTG1401 CCTGCGTGAC CACGCGAAGG TGGTCGGCGC CGATACGGTG CTTACGTTTG
1451 GGGCCGACTC GGTGACGCTG GTCGGCGTGG GGCATGGCGG GCTGTGGACG 1501 GAGGGCGTGG TGATCGGCTG A TACTCACG CAACCGATCA GTGCCAGTGC1451 GGGCCGACTC GGTGACGCTG GTCGGCGTGG GGCATGGCGG GCTGTGGACG 1501 GAGGGCGTGG TGATCGGCTG A TACTCACG CAACCGATCA GTGCCAGTGC
1551 TGCCCCCGCC AGCCACCGCC CCAATTGGGC CGGTGGGGGT AGCCATAGCC1551 TGCCCCCGCC AGCCACCGCC CCAATTGGGC CGGTGGGGGT AGCCATAGCC
SEQUENCE IVSEQUENCE IV
1 GGGCGATGGC TGGCAGGACG CGCCCCTCGG CCCCATCAAC CTGAGATGAG1 GGGCGATGGC TGGCAGGACG CGCCCCTCGG CCCCATCAAC CTGAGATGAG
51 AACAACATGA AGAAGAAGTC TCTGCTCCCC CTCGGCCTGG CCATCGGCCT 101 CGCCTCTCTC GCTGCCAGCC CTCTGATCCA GGCCf'AGCACC TACACCCAGA51 AACAACATGA AGAAGAAGTC TCTGCTCCCC CTCGGCCTGG CCATCGGCCT 101 CGCCTCTCTC GCTGCCAGCC CTCTGATCCA GGCCf'AGCACC TACACCCAGA
151 CCAAATACCC CATCGTGCTG GCCCACGGCA TGCTCGGCTT CGACAATATC151 CCAAATACCC CATCGTGCTG GCCCACGGCA TGCTCGGCTT CGACAATATC
201 CTCGGGGTCG ACTACTGGTT CGGCATTCCC AGCGCCTTGC GCCGTGACGG201 CTCGGGGTCG ACTACTGGTT CGGCATTCCC AGCGCCTTGC GCCGTGACGG
251 TGCCCAGGTC TACGTCACCG AAGTCAGCCA GTTGGACACC TCGGAAGTCC251 TGCCCAGGTC TACGTCACCG AAGTCAGCCA GTTGGACACC TCGGAAGTCC
301 GCGGCGAGCA GTTGCTGCAA CAGGTGGAGG AAATCGTCGC CCTCAGCGGC 351 CAGCCCAAGG TCAACCTGAT CGGCCACAGC CACGGCGGGC CGACCATCCG301 GCGGCGAGCA GTTGCTGCAA CAGGTGGAGG AAATCGTCGC CCTCAGCGGC 351 CAGCCCAAGG TCAACCTGAT CGGCCACAGC CACGGCGGGC CGACCATCCG
401 CTACGTCGCC GCCGTACGTC CCGACCTGAT GCCTTCCGCC ACCAGCGTCG401 CTACGTCGCC GCCGTACGTC CCGACCTGAT GCCTTCCGCC ACCAGCGTCG
451 GCGCCCCGCA CAAGGGTTCG GACACCGCCG ACTTCCTGCG CCAGATCCCA451 GCGCCCCGCA CAAGGGTTCG GACACCGCCG ACTTCCTGCG CCAGATCCCA
501 CCGGGTTCGG CCGGCGAGGC AGTCCTCTCC GGGCTGGTCA ACAGCCTCGG501 CCGGGTTCGG CCGGCGAGGC AGTCCTCTCC GGGCTGGTCA ACAGCCTCGG
551 CGCGCTGATC AGCTTCCTTT CCAGCGGCAG CGCCGGTACG CAGAATTCAC 601 TGGGCTCGCT GGAGTCGCTG AACAGCGAGG GGGCCGCGCG CTTCAACGCC551 CGCGCTGATC AGCTTCCTTT CCAGCGGCAG CGCCGGTACG CAGAATTCAC 601 TGGGCTCGCT GGAGTCGCTG AACAGCGAGG GGGCCGCGCG CTTCAACGCC
651 AAGTACCCGC AGGGCATCCC CACCTCGGCC TGCGGCGAAG GCGCCTACAA651 AAGTACCCGC AGGGCATCCC CACCTCGGCC TGCGGCGAAG GCGCCTACAA
701 GGTCAACGGC GTGAGCTATT ACTCCTGGAG CGGTTCCTCG CCGCTGACCA701 GGTCAACGGC GTGAGCTATT ACTCCTGGAG CGGTTCCTCG CCGCTGACCA
751 ACTTCCTCGA TCCGAGCGAC GCCTTCCTCG GCGCCTCGTC GCTGACCTTC 801~A"AGAACGGC ~CCGCCAACGA CGGCCTGGTC GGCACCTGCA GTTCGCACCT 851 GGGCATGGTG ATCCGCGACA ACTACCGGAT GAACCACCTG GACGAGGTGA751 ACTTCCTCGA TCCGAGCGAC GCCTTCCTCG GCGCCTCGTC GCTGACCTTC 801 ~ A " AGAACGGC ~ CCGCCAACGA CGGCCTGGTC GGCACCTGCA GTTCGCACCT 851 GGGCATGGTG ATCCGCGACA ACTACCGGAT GAACCACCTG GACAG
901 ACCAGGTCTT CGGCCTCACC AGCCTGTTCG AGACCAGCCC GGTCAGCGTC901 ACCAGGTCTT CGGCCTCACC AGCCTGTTCG AGACCAGCCC GGTCAGCGTC
951 TACCGCCAGC ACGCCAACCG CCTGAAGAAC GCCAGCCTGT AGGACCCCGG951 TACCGCCAGC ACGCCAACCG CCTGAAGAAC GCCAGCCTGT AGGACCCCGG
1001 CCGGGGCCTC GGCCCCGGCC CTTTCCCGGA AGCCCCCTCG CGTGAAGAAA1001 CCGGGGCCTC GGCCCCGGCC CTTTCCCGGA AGCCCCCTCG CGTGAAGAAA
1051 ATCCTCCTGC TGATTCCACT GGCGTTCGCC GCCAGCCTGG CCTGGTTCGT S EQUENCE V1051 ATCCTCCTGC TGATTCCACT GGCGTTCGCC GCCAGCCTGG CCTGGTTCGT S EQUENCE V
1 "cAGGCCCCCA CGGCCGTTCT TAATGGCAAC GAGGTCATCT CTGGTGTCCT1 "cAGGCCCCCA CGGCCGTTCT TAATGGCAAC GAGGTCATCT CTGGTGTCCT
51 TGGGGGCAAG GTTGATACCT TTAAGGGAAT TCCATTTGCT GACCCTCCTG 101 TTGGTGACTT GCGGTTCAAG CACCCCCAGC CTTTCACTGG ATCCTACCAG51 TGGGGGCAAG GTTGATACCT TTAAGGGAAT TCCATTTGCT GACCCTCCTG 101 TTGGTGACTT GCGGTTCAAG CACCCCCAGC CTTTCACTGG ATCCTACCAG
151 GGTCTTAAGG CCAACGACTT CAGCTCTGCT TGTATGCAGC TTGATCCTGG151 GGTCTTAAGG CCAACGACTT CAGCTCTGCT TGTATGCAGC TTGATCCTGG
201 CAATGCCATT TCTTGGCTTG ACAAAGTCGT GGGCTTGGGA AAGATTCTTC201 CAATGCCATT TCTTGGCTTG ACAAAGTCGT GGGCTTGGGA AAGATTCTTC
251 CTGATAACCT TAGAGGCCCT CTTTATGACA TGGCCCAGGG TAGTGTCTCC251 CTGATAACCT TAGAGGCCCT CTTTATGACA TGGCCCAGGG TAGTGTCTCC
301 ATGAATGAGG ACTGTCTCTA CCTTAACGTT TTCCGCCCTG CTGGCACCAA 351 GCCTGATGCT AAGCTCCCCG TCATGGTTTG GATTTACGGT GGTGCCTTTG301 ATGAATGAGG ACTGTCTCTA CCTTAACGTT TTCCGCCCTG CTGGCACCAA 351 GCCTGATGCT AAGCTCCCCG TCATGGTTTG GATTTACGGT GGTGCCTTTG
401 TGTTTGGTTC TTCTGCTTCT TACCCTGGTA ACGGCTACGT CAAGGAGAGT401 TGTTTGGTTC TTCTGCTTCT TACCCTGGTA ACGGCTACGT CAAGGAGAGT
451 GTGGAAATGG GCCAGCCTGT TGTGTTTGTT TCCATCAACT ACCGTACCGG451 GTGGAAATGG GCCAGCCTGT TGTGTTTGTT TCCATCAACT ACCGTACCGG
501 CCCCTATGGA TTCCTGGGTG GTGATGCCAT CACCGCTGAG GGTAACACCA501 CCCCTATGGA TTCCTGGGTG GTGATGCCAT CACCGCTGAG GGTAACACCA
551 ACGCTGGTCT GCACGACCAG CGCAAGGGTC TCGAGTGGGT TAGCGACAAC 601 ATTGCCAACT TTGGTGGTGA TCCCGACAAG GTCATGATTT TCGGTGAGTC551 ACGCTGGTCT GCACGACCAG CGCAAGGGTC TCGAGTGGGT TAGCGACAAC 601 ATTGCCAACT TTGGTGGTGA TCCCGACAAG GTCATGATTT TCGGTGAGTC
651 CGCTGGTGCC ATGAGTGTTG CTCACCAGCT TGTTGCCTAC GGTGGTGACA651 CGCTGGTGCC ATGAGTGTTG CTCACCAGCT TGTTGCCTAC GGTGGTGACA
701 ACACCTACAA CGGAAAGAAG CTTTTCCACT CTGCCATTCT TCAGTCTGGC701 ACACCTACAA CGGAAAGAAG CTTTTCCACT CTGCCATTCT TCAGTCTGGC
751 GGTCCTCTTC CTTACTTTGA CTCTACTTCT GTTGGTCCCG AGAGTGCCTA751 GGTCCTCTTC CTTACTTTGA CTCTACTTCT GTTGGTCCCG AGAGTGCCTA
801 CAGCAGATTT GCTCAGTATG CCGGATGTGA TGCCAGCGCC AGTGACAATG 851 AAACTCTGGC TTGTCTCCGC AGCAAGTCCA GCGATGTCTT GCACAGTGCC801 CAGCAGATTT GCTCAGTATG CCGGATGTGA TGCCAGCGCC AGTGACAATG 851 AAACTCTGGC TTGTCTCCGC AGCAAGTCCA GCGATGTCTT GCACAGTGCC
901 CAGAACTCGT ACGATCTCAA GGACCTGTTT GGCCTGCTCC CTCAATTCCT901 CAGAACTCGT ACGATCTCAA GGACCTGTTT GGCCTGCTCC CTCAATTCCT
951 TGGATTTGGT CCCAGACCCG ACGGCAACAT TATTCCCGAT GCCGCTTATG951 TGGATTTGGT CCCAGACCCG ACGGCAACAT TATTCCCGAT GCCGCTTATG
1001 AGCTCTACCG CAGCGGTAGA TACGCCAAGG TTCCCTACAT TACTGGTAAC1001 AGCTCTACCG CAGCGGTAGA TACGCCAAGG TTCCCTACAT TACTGGTAAC
1051 CAGGAGGATG AGGGTACTAT TCTTGCCCCC GTTGCTATTA ATGCTACCAC1051 CAGGAGGATG AGGGTACTAT TCTTGCCCCC GTTGCTATTA ATGCTACCAC
1101 GACTCCCCAT GTTAAGAAGT GGTTGAAGTA CATTTGTAGC GAGGCTTCTG1101 GACTCCCCAT GTTAAGAAGT GGTTGAAGTA CATTTGTAGC GAGGCTTCTG
1151 ACGCTTCGCT TGATCGTGTT TTGTCGCTCT ACCCCGGCTC TTGGTCGGAG1151 ACGCTTCGCT TGATCGTGTT TTGTCGCTCT ACCCCGGCTC TTGGTCGGAG
1201 GGTGCGCCAT TCCGCACTGG TATTCTTAAT GCTCTGACCC CTCAGTTCAA1201 GGTGCGCCAT TCCGCACTGG TATTCTTAAT GCTCTGACCC CTCAGTTCAA
1251 GCGCATTGCT GCCATTTTCA CTGATTTGCT GTTCCAGTCT CCTCGTCGTG1251 GCGCATTGCT GCCATTTTCA CTGATTTGCT GTTCCAGTCT CCTCGTCGTG
1301 TTATGCTTAA CGCTACCAAG GACGTCAACC GCTGGACTTA CCTTGCCACC 1351 CAGCTCCATA ACCTCGTTCC ATTTTTGGGT ACTTTCCATG GTAGTGATCT1301 TTATGCTTAA CGCTACCAAG GACGTCAACC GCTGGACTTA CCTTGCCACC 1351 CAGCTCCATA ACCTCGTTCC ATTTTTGGGT ACTTTCCATG GTAGTGATCT
1401 TCTTTTCCAA TACTACGTGG ACCTTGGCCC ATCTTCTGCT TACCGCCGCT1401 TCTTTTCCAA TACTACGTGG ACCTTGGCCC ATCTTCTGCT TACCGCCGCT
1451 ACTTTATCTC GTTTGCCAAC CACCACGACC CCAACGTTGG CACCAACCTG 1501 AAACAGTGGG ATATGTACAC TGATGCAGGC AAGGAGATGC TTCAGATTCA1451 ACTTTATCTC GTTTGCCAAC CACCACGACC CCAACGTTGG CACCAACCTG 1501 AAACAGTGGG ATATGTACAC TGATGCAGGC AAGGAGATGC TTCAGATTCA
1551 TATGGTTGGT AACTCTATGA GAACTGACGA CTTTAGAATC GAGGGAATCT1551 TATGGTTGGT AACTCTATGA GAACTGACGA CTTTAGAATC GAGGGAATCT
1601 OGAACTTTGA GTCTGACGTT ACTCTCTTCG GTTAA I'1601 OGAACTTTGA GTCTGACGTT ACTCTCTTCG GTTAA I '
SEQUENCE VISEQUENCE VI
1 ATGGAGCTCG CTCTTGCGCT CCTGCTCATT GCCTCGGTGG CTGCTGCCCC 51 CACCGCCACG CTCGCCAACG GCGACACCAT CACCGGTCTC AACGCCATCA1 ATGGAGCTCG CTCTTGCGCT CCTGCTCATT GCCTCGGTGG CTGCTGCCCC 51 CACCGCCACG CTCGCCAACG GCGACACCAT CACCGGTCTC AACGCCATCA
101 TCAACGAGGC GTTCCTCGGC ATTCCCTTTG CCGAGCCGCC GGTGGGCAAC101 TCAACGAGGC GTTCCTCGGC ATTCCCTTTG CCGAGCCGCC GGTGGGCAAC
151 CTCCGCTTCA AGGACCCCGT GCCGTACTCC GGCTCGCTCG ATGGCCAGAA151 CTCCGCTTCA AGGACCCCGT GCCGTACTCC GGCTCGCTCG ATGGCCAGAA
201 GTTCACGCTG TACGGCCCGC TGTGCATGCA GCAGAACCCC GAGGGCACCT201 GTTCACGCTG TACGGCCCGC TGTGCATGCA GCAGAACCCC GAGGGCACCT
251 ACGAGGAGAA CCTCCCCAAG GCAGCGCTCG ACTTGGTGAT GCAGTCCAAG 301 GTGTTTGAGG CGGTGCTGCC GCTGAGCGAG GACTGTCTCA CCATCAACGT251 ACGAGGAGAA CCTCCCCAAG GCAGCGCTCG ACTTGGTGAT GCAGTCCAAG 301 GTGTTTGAGG CGGTGCTGCC GCTGAGCGAG GACTGTCTCA CCATCAACGT
351 GGTGCGGCCG CCGGGCACCA AGGCGGGTGC CAACCTCCCG GTGATGCTCT351 GGTGCGGCCG CCGGGCACCA AGGCGGGTGC CAACCTCCCG GTGATGCTCT
401 GGATCTTTGG CGGCGGGTTT GAGGTGGGTG GCACCAGCAC CTTCCCTCCC401 GGATCTTTGG CGGCGGGTTT GAGGTGGGTG GCACCAGCAC CTTCCCTCCC
451 GCCCAGATGA TCACCAAGAG CATTGCCATG GGCAAGCCCA TCATCCACGT451 GCCCAGATGA TCACCAAGAG CATTGCCATG GGCAAGCCCA TCATCCACGT
501 GAGCGTCAAC TACCGCGTGT CGTCGTGGGG GTTCTTGGCT GGCGACGAGA 551 TCAAGGCCGA GGGCAGTGCC AACGCCGGTT TGAAGGACCA GCGCTTGGGC501 GAGCGTCAAC TACCGCGTGT CGTCGTGGGG GTTCTTGGCT GGCGACGAGA 551 TCAAGGCCGA GGGCAGTGCC AACGCCGGTT TGAAGGACCA GCGCTTGGGC
601 ATGCAGTGGG TGGCGGACAA CATTGCGGCG TTTGGCGGCG ACCCGACCAA601 ATGCAGTGGG TGGCGGACAA CATTGCGGCG TTTGGCGGCG ACCCGACCAA
651 GGTGACCATC TTTGGCGAGC TGGCGGGCAG CATGTCGGTC ATGTGCCACA651 GGTGACCATC TTTGGCGAGC TGGCGGGCAG CATGTCGGTC ATGTGCCACA
701 TTCTCTGGAA CGACGGCGAC AACACGTACA AGGGCAAGCC GCTCTTCCGC701 TTCTCTGGAA CGACGGCGAC AACACGTACA AGGGCAAGCC GCTCTTCCGC
751 GCGGGCATCA TGCAGCTGGG GGCCATGGTG CCGCTGGACG CCGTGGACGG 801 CATCTACGGC AACGAGATCT TTGACCTCTT GGCGTCGAAC GCGGGCTGCG751 GCGGGCATCA TGCAGCTGGG GGCCATGGTG CCGCTGGACG CCGTGGACGG 801 CATCTACGGC AACGAGATCT TTGACCTCTT GGCGTCGAAC GCGGGCTGCG
851 GCAGCGCCAG CGACAAGCTT GCGTGCTTGC GCGGTGTGCT GAGCGACACG851 GCAGCGCCAG CGACAAGCTT GCGTGCTTGC GCGGTGTGCT GAGCGACACG
901 TTGGAGGACG CCACCAACAA CACCCCTGGG TTCTTGGCGT ACTCCTCGTT 951 GCGGTTGCTG TACCTCCCCC GGCCCGACGG CGTGAACATC ACCGACGACA901 TTGGAGGACG CCACCAACAA CACCCCTGGG TTCTTGGCGT ACTCCTCGTT 951 GCGGTTGCTG TACCTCCCCC GGCCCGACGG CGTGAACATC ACCGACGACA
1001 TGTACGCCTT GGTGCGCGAG GGCAAGTATG CCAACATCCC TGTGATCATC1001 TGTACGCCTT GGTGCGCGAG GGCAAGTATG CCAACATCCC TGTGATCATC
1051 GGCGACCAGA ACGACGAGGG CACCTTCTTT GGCACCCTGC TGTTGAACGT 1101 GACCACGGAT GCCCAGGCCC GCGAGTACTT CAAGCAGCTG TTTGTCCACG1051 GGCGACCAGA ACGACGAGGG CACCTTCTTT GGCACCCTGC TGTTGAACGT 1101 GACCACGGAT GCCCAGGCCC GCGAGTACTT CAAGCAGCTG TTTGTCCACG
1151 CCAGCGACGC GGAGATCGAC ACGTTGATGA CGGCGTACCC CGGCGACATC1151 CCAGCGACGC GGAGATCGAC ACGTTGATGA CGGCGTACCC CGGCGACATC
1201 ACCCAGGGCC TGCCGTTCGA CACGGGTATT CTCAACGCCC TCACCCCGCA1201 ACCCAGGGCC TGCCGTTCGA CACGGGTATT CTCAACGCCC TCACCCCGCA
1251 GTTCAAGAGA ATCCTGGCGG TGCTCGGCGA CCTTGGCTTT ACGCTTGCTC1251 GTTCAAGAGA ATCCTGGCGG TGCTCGGCGA CCTTGGCTTT ACGCTTGCTC
1301 GTCGCTACTT CCTCAACCAC TACACCGGCG GCACCAAGTA CTCATTCCTC 1351 CTGAAGCAGC TCCTGGGCTT GCCGGTGCTC GGAACGTTCC ACTCCAACGA1301 GTCGCTACTT CCTCAACCAC TACACCGGCG GCACCAAGTA CTCATTCCTC 1351 CTGAAGCAGC TCCTGGGCTT GCCGGTGCTC GGAACGTTCC ACTCCAACGA
1401 CATTGTCTTC CAGGACTACT TGTTGGGCAG CGGCTCGCTC ATCTACAACA1401 CATTGTCTTC CAGGACTACT TGTTGGGCAG CGGCTCGCTC ATCTACAACA
1451 ACGCGTTCAT TGCGTTTGCC ACGGACTTGG ACCCCAACAC CGCGGGGTTG1451 ACGCGTTCAT TGCGTTTGCC ACGGACTTGG ACCCCAACAC CGCGGGGTTG
1501 TTGGTGAAGT GGCCCGAGTA CACCAGCAGC CTGCAGCTGG GCAACAACTT1501 TTGGTGAAGT GGCCCGAGTA CACCAGCAGC CTGCAGCTGG GCAACAACTT
1551 GATGATGATC AACGCCTTGG GCTTGTACAC CGGCAAGGAC AACTTCCGCA 1601 CCGCCGGCTA CGACGCGTTG TTCTCCAACC CGCCGCTGTT CTTTGTGTAA 1551 GATGATGATC AACGCCTTGG GCTTGTACAC CGGCAAGGAC AACTTCCGCA 1601 CCGCCGGCTA CGACGCGTTG TTCTCCAACC CGCCGCTGTT CTTTGTGTAA
10/ - Procédé selon la revendication 2, dans lequel la transformation génétique de la plante est effectuée en réalisant une cassette d'expression comprenant le gène de lipase et le promoteur associé, et en introduisant cette cassette d'expression dans le génome de cellules somatiques de la plante par un transfert à l'aide de la bactérie Agrobacterium tumefaciens, ou par électroporation, ou par biolistique, ou par microinjection.10 / - Process according to claim 2, in which the genetic transformation of the plant is carried out by producing an expression cassette comprising the lipase gene and the associated promoter, and by introducing this expression cassette into the genome of somatic cells. of the plant by a transfer using the bacterium Agrobacterium tumefaciens, or by electroporation, or by biolistics, or by microinjection.
11/ - Procédé selon la revendication 2, dans lequel la transformation génétique de la plante est effectuée en réalisant une cassette d'expression comprenant le gène de lipase et le promoteur associé, et en introduisant cette cassette d'expression dans le génome de microspores de la plante par électroporation ou biolistique.11 / - Process according to claim 2, in which the genetic transformation of the plant is carried out by producing an expression cassette comprising the lipase gene and the associated promoter, and by introducing this expression cassette into the microspore genome of the plant by electroporation or biolistics.
12/ - Procédé selon l'une des revendications 1 à 11, dans lequel l'incubation pour engendrer l'hydrolyse enzymatique est réalisée à une température comprise entre 20° C et 60° C. 13/ - Procédé selon l'une des revendications 1 à 12, dans lequel l'extraction des acides gras issus de l'hydrolyse est réalisée par une extraction liquide/liquide utilisant un solvant apolaire.12 / - Method according to one of claims 1 to 11, wherein the incubation to generate the enzymatic hydrolysis is carried out at a temperature between 20 ° C and 60 ° C. 13 / - Method according to one of claims 1 to 12, in which the extraction of the fatty acids resulting from the hydrolysis is carried out by a liquid / liquid extraction using an apolar solvent.
14/ - Procédé selon l'une des revendications 1 à 12, dans lequel les acides gras issus de l'hydrolyse sont méthylés in situ par mise en contact avec du méthanol en catalyse acide sous ultrasons en vue de les transformer en esters méthyliques, ces derniers étant extraits par une extraction liquide/liquide utilisant un solvant apolaire.14 / - Method according to one of claims 1 to 12, wherein the fatty acids from the hydrolysis are methylated in situ by contacting with methanol in acid catalysis under ultrasound in order to transform them into methyl esters, these the latter being extracted by a liquid / liquid extraction using an apolar solvent.
15/ - Plante oléagineuse ou semence de plante oléagineuse, d'une variété non protegeable par un certificat d'obtention végétale, caractérisée en ce qu'elle comprend dans son génome une cassette d'expression possédant au moins un gène codant pour une enzyme lipase, associé à un promoteur permettant une expression dudit gène dans des compartiments cellulaires, extracelullaires ou tissulaires différents des compartiments lipidiques de la plante ou de la semence.15 / - Oilseed plant or seed of oilseed plant, of a variety which cannot be protected by a plant variety certificate, characterized in that it comprises in its genome an expression cassette having at least one gene coding for a lipase enzyme , associated with a promoter allowing expression of said gene in cellular, extracellular or tissue compartments different from the lipid compartments of the plant or seed.
16/ - Plante ou semence selon la revendication 15, dans laquelle le promoteur associé au gène de lipase est un promoteur à expression cellulaire ou tissulaire spécifique.16 / - Plant or seed according to claim 15, in which the promoter associated with the lipase gene is a promoter with specific cellular or tissue expression.
17/ - Plante ou semence selon la revendication 15, dans laquelle le gène de lipase est muni d'une séquence d'adressage vers des compartiments cellulaires ou extracellulaires différents des compartiments d'accumulation des lipides, le promoteur étant un promoteur constitutif.17 / - Plant or seed according to claim 15, in which the lipase gene is provided with an addressing sequence towards cellular or extracellular compartments different from the lipid accumulation compartments, the promoter being a constitutive promoter.
18/ - Plante oléagineuse ou semence de plante oléagineuse, d'une variété non protegeable par un certificat d'obtention végétale, caractérisée en ce qu'elle comprend dans son génome une cassette d'expression possédant au moins un gène codant pour une enzyme lipase, associé à un promoteur permettant une expression dudit gène sur induction exogène, en particulier par un stress. 18 / - Oilseed plant or oilseed seed, of a variety not protected by a plant variety certificate, characterized in that it comprises in its genome an expression cassette having at least one gene coding for a lipase enzyme , associated with a promoter allowing expression of said gene on exogenous induction, in particular by stress.
EP95925897A 1994-07-25 1995-07-18 Method for producing fatty acids or derivatives thereof from oil plants Withdrawn EP0770134A2 (en)

Applications Claiming Priority (3)

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FR9409272 1994-07-25
FR9409272A FR2722798B1 (en) 1994-07-25 1994-07-25 1PROCESS FOR THE PRODUCTION OF OLEAGINOUS FATTY ACIDS
PCT/FR1995/000957 WO1996003511A2 (en) 1994-07-25 1995-07-18 Method for producing fatty acids or derivatives thereof from oil plants

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FR2733249B1 (en) 1995-04-20 1997-06-06 Biocem RECOMBINANT DOG GASTRIC LIPASE AND POLYPEPTIDES DERIVED BY PLANTS, PROCESSES FOR OBTAINING SAME AND USES THEREOF
FR2754827B1 (en) * 1996-10-17 1998-12-24 Biocem PANCREATIC LIPASES AND / OR RECOMBINANT COLIPASES AND DERVIED POLYPEPTIDES PRODUCED BY PLANTS, PROCESSES FOR OBTAINING SAME AND USES THEREOF
DE19731990A1 (en) * 1997-07-25 1999-01-28 Studiengesellschaft Kohle Mbh Process for the production and identification of new hydrolases with improved properties
US7166766B1 (en) 2000-04-03 2007-01-23 Total Raffinage Distribution S.A. Method for producing branched fatty acids using genetically modified plants
FR2769320B1 (en) * 1997-10-03 2002-03-29 Total Raffinage Distrib PROCESS FOR PRODUCING BRANCHED FATTY ACIDS USING GENETICALLY MODIFIED PLANTS
ATE313636T1 (en) 1999-02-16 2006-01-15 Senesco Inc DNA CODING FOR PLANT LIPASE, TRANSGENIC PLANTS AND A METHOD FOR CONTROLLING SENSENCE IN PLANTS
GB0022236D0 (en) * 2000-09-11 2000-10-25 Pinet Aylette Partially hydrolysed protein nutrient supplement
MXPA06009411A (en) 2004-02-20 2007-03-29 Pioneer Hi Bred Int Lipases and methods of use.
CA2556927A1 (en) * 2004-02-20 2005-09-09 Pioneer Hi-Bred International, Inc. Methods for enhancing insect resistance in plants
BR112012001131A2 (en) * 2009-07-17 2019-09-24 Korea Advanced Inst Sci & Tech fatty acid alkyl ester production method using microorganisms containing oil producing ability

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WO1991006661A1 (en) * 1989-11-03 1991-05-16 Opta Food Ingredients, Inc. Lipase-catalyzed in situ generation of mono- and di-glycerides
GB8925352D0 (en) * 1989-11-09 1989-12-28 Unilever Plc Fats
IL97645A (en) * 1990-03-23 1997-03-18 Gist Brocades Nv Production of enzymes in seeds and their use
DK162790D0 (en) * 1990-07-06 1990-07-06 Novo Nordisk As PLANT CELL
DK0548228T3 (en) * 1990-09-13 1999-05-10 Novo Nordisk As lipase variants
WO1993003161A1 (en) * 1990-10-22 1993-02-18 Biosource Genetics Corporation Recombinant plant viral nucleic acids

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FR2722798A1 (en) 1996-01-26
AU2984995A (en) 1996-02-22
FR2722798B1 (en) 1996-09-13

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