EP1532266A2 - Procede d'obtention de cetocarotinoides dans des fruits de plantes - Google Patents

Procede d'obtention de cetocarotinoides dans des fruits de plantes

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Publication number
EP1532266A2
EP1532266A2 EP03792349A EP03792349A EP1532266A2 EP 1532266 A2 EP1532266 A2 EP 1532266A2 EP 03792349 A EP03792349 A EP 03792349A EP 03792349 A EP03792349 A EP 03792349A EP 1532266 A2 EP1532266 A2 EP 1532266A2
Authority
EP
European Patent Office
Prior art keywords
ketolase
plant
seq
genetically modified
nucleic acid
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.)
Withdrawn
Application number
EP03792349A
Other languages
German (de)
English (en)
Inventor
Christel Renate Schopfer
Ralf Flachmann
Karin Herbers
Irene Kunze
Matt Sauer
Martin Klebsattel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SunGene GmbH
Original Assignee
SunGene GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from DE10238978A external-priority patent/DE10238978A1/de
Priority claimed from DE10238979A external-priority patent/DE10238979A1/de
Priority claimed from DE2002138980 external-priority patent/DE10238980A1/de
Priority claimed from DE2002153112 external-priority patent/DE10253112A1/de
Priority claimed from DE2002158971 external-priority patent/DE10258971A1/de
Application filed by SunGene GmbH filed Critical SunGene GmbH
Publication of EP1532266A2 publication Critical patent/EP1532266A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/179—Colouring agents, e.g. pigmenting or dyeing agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A23L33/155—Vitamins A or D
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
    • A23L5/44—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives using carotenoids or xanthophylls
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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
    • C12N15/825—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 pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Definitions

  • the present invention relates to a process for the production of ketocarotenoids by cultivating genetically modified plants which have a ketolase activity in fruits, the genetically modified plants, and their use as food or feed and for the production of ketocarotenoid extracts.
  • Ketocarotenoids are synthesized de novo in bacteria, algae, fungi and plants.
  • ketocarotenoids and especially astaxanthin are used as pigmenting aids in animal nutrition, especially in trout, salmon and shrimp farming.
  • Natural ketocarotenoids such as natural astaxanthin
  • WO 98/18910 describes the synthesis of ketocarotenoids in nectaries of tobacco flowers by introducing a ketolase gene into tobacco.
  • WO 01/20011 describes a DNA construct for the production of ketocarotenoids, in particular astaxanthin, in seeds of oilseed plants such as oilseed rape, sunflower, soybean and mustard using a seed-specific promoter and a ketolase from Haematococcus.
  • the methods disclosed in the prior art provide genetically modified plants which contain ketocarotenoids in specific tissues, but have the disadvantage that the level of the ketocarotenoids and the purity, in particular astaxanthin, are not yet satisfactory ,
  • the object of the invention was therefore to provide an alternative process for the production of ketocarotenoids by cultivating plants, or to provide further transgenic plants which produce ketocarotenoids which have optimized properties, such as a higher ketocarotenoid content , and do not have the described disadvantage of the prior art.
  • ketocarotenoids Accordingly, a method for producing ketocarotenoids has been found by cultivating genetically modified plants that have ketolase activity in fruits.
  • Ketolase activity means the enzyme activity of a ketolase.
  • a ketolase is understood to mean a protein which has the enzymatic activity of introducing a keto group on the optionally substituted ⁇ -ionone ring of carotenoids.
  • ketolase is understood to mean a protein which has the enzymatic activity of converting ⁇ -carotene into cantaxanthin.
  • ketolase activity is understood to mean the amount of ⁇ -carotene converted or the amount of canthaxanthin formed in a certain time by the protein ketolase.
  • genetically modified plants which express a ketolase in fruits are used in order to have ketoase activity in the fruits of the genetically modified plants.
  • Genetically modified plants which contain at least one nucleic acid, coding for a ketolase, are therefore preferably used in the method according to the invention.
  • No plants are known which have a ketolase activity as a wild type in fruits.
  • the preferred plants described below in fruits as wild type have no ketolase activity.
  • the ketolase activity in fruits of the genetically modified plants is caused by the genetic modification of the parent plant.
  • the genetically modified plant according to the invention thus has a ketolase activity in fruits in comparison to the genetically unmodified starting plant and is therefore preferably able to express a ketolase in fruits.
  • parent plant or wild type is understood to mean the corresponding non-genetically modified parent plant.
  • genetically modified plant is preferably understood to mean a plant which is genetically modified in comparison with the starting plant.
  • plant can mean the starting plant (wild type) or a genetically modified plant according to the invention or both.
  • the gene expression of a nucleic acid encoding a ketolase is caused in the fruits of the plants preferably by introducing nucleic acids encoding ketolases into the starting plant.
  • the invention therefore relates in particular to the method described above, characterized in that genetically modified plants are used, into which, starting from a starting plant, at least one nucleic acid coding for a ketolase has been introduced.
  • any ketolase gene ie any nucleic acid encoding a ketolase, can be used for this.
  • nucleic acids mentioned in the description can be, for example, an RNA, DNA or cDNA sequence.
  • nucleic acid sequences which have already been processed such as the corresponding cDNAs
  • nucleic acids encoding a ketolase and the corresponding ketolases which can be used in the method according to the invention or in the genetically modified plants according to the invention described below are, for example, sequences from
  • Haematoccus pluvialis especially from Haematoccus pluvialis Flotow em. Wille (Accession No. X86782; nucleic acid: SEQ ID No. 1, protein SEQ ID No. 2),
  • Agrobacterium aurantiacum (Accession No. D58420; nucleic acid: SEQ. ID. No. 5, protein SEQ ID No. 6),
  • Paracoccus marcusii (Accession No. Y15112; nucleic acid:
  • Synechocystis sp. Strain PC6803 (Accession No. S76617, NP442491; nucleic acid: SEQ ID No. 11, protein SEQ ID N ⁇ . 12).
  • Bradyrhizobium sp. (Accession No. AF218415, BAB 74888; nucleic acid: SEQ ID No. 13, protein SEQ ID No. 14).
  • Haematococcus pluvialis (Accession NO: AF534876, AAN03484; nucleic acid: SEQ ID NO: 37, protein: SEQ ID NO: 38)
  • Paracoccus sp. MBIC1143 (Accession NO: D58420, P54972; nucleic acid: SEQ ID NO: 39, protein: SEQ ID NO: 40)
  • Brevundimonas aurantiaca (Accession NO: AY166610, AAN86030; Nucleic acid: SEQ ID NO: 41, Protein: SEQ ID NO: 42)
  • Nodularia spu igena NSOR10 (Accession NO: AY210783, AA064399; nucleic acid: SEQ ID NO: 43, protein: SEQ ID NO: 44)
  • Nostoc punctiforme ATCC 29133 (Accession NO: NZ_AABC01000195, ZP_00111258; nucleic acid: SEQ ID NO: 45, protein: SEQ ID NO: 46)
  • Nostoc punctiforme ATCC 29133 (Accession NO: NZ_AABC01000196; nucleic acid: SEQ ID NO: 47, protein: SEQ ID NO: 48)
  • ketolases and ketolase genes which can be used in the process according to the invention can be obtained, for example, from different organisms, the genome sequence of which is known, by comparing the identity of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the databases described above Sequences and in particular with the sequences SEQ ID NO. 2 and / or SEQ ID NO. 16 easy to find.
  • ketolases and ketolase genes can also be derived from the nucleic acid sequences described above, in particular from the sequences SEQ ID. No 1 and / or SEQ ID NO. 15 from different organisms, the genomic sequence of which is not known, can easily be found by hybridization techniques in a manner known per se. x
  • the hybridization can take place under moderate (low stringency) or preferably under stringent (high stringency) conditions.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with 2X SSC at 50 ° C) and those with high stringency (with 0.2X SSC at 50 ° C, preferably at 65 ° C) (20X SSC: 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).
  • the temperature during the washing step can be raised from moderate conditions at room temperature, 22 ° C, to stringent conditions at 65 ° C.
  • Both parameters, salt concentration and temperature, can be varied at the same time, one of the two parameters can also be kept constant and only the other can be varied.
  • denaturing agents such as Formamide or SDS can be used.
  • the hybridization is preferably carried out at 42 ° C.
  • nucleic acids are encoded which encode a protein containing the amino acid sequence SEQ ID NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, preferably at least 70 %, more preferably at least 80%, particularly preferably at least 90% at the amino acid level with the sequence SEQ ID NO. 2 and has the enzymatic property of a ketolase.
  • This can be a natural ketolase sequence that can be found as described above by comparing the identity of the sequences from other organisms or an artificial ketolase sequence that starts from the sequence SEQ ID NO. 2 has been modified by artificial variation, for example by substitution, insertion or deletion of amino acids.
  • nucleic acids are encoded which encode a protein containing the amino acid sequence SEQ ID NO. 16 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70 %, more preferably at least 80%, particularly preferably at least 90% at the amino acid level with the sequence SEQ ID NO. 16 and has the enzymatic property of a ketolase.
  • This can be a natural ketolase sequence that can be found as described above by comparing the identity of the sequences from other organisms or an artificial ketolase sequence that starts from the sequence SEQ ID NO. 16 was modified by artificial variation, for example by substitution, insertion or deletion of amino acids.
  • substitution is to be understood as meaning the replacement of one or more amino acids by one or more amino acids. So-called conservative exchanges are preferably carried out, in which the replaced amino acid has a similar property to the original amino acid, for example exchange of Glu by Asp, Gin by Asn, Val by Ile, Leu by Ile, Ser by Thr.
  • Deletion is the replacement of an amino acid with a direct link.
  • Preferred positions for deletions are the termini of the polypeptide and the links between the individual protein domains.
  • Inserts are insertions of amino acids into the polypeptide chain, whereby a direct bond is formally replaced by one or more amino acids.
  • Identity between two proteins is understood to mean the identity of the amino acids over the respective total protein length, in particular the identity that is obtained by comparison using the laser gene software from DNASTAR, inc. Madison, Wisconsin (USA) using the clustal method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5 (2): 151-1) is calculated using the following parameters: x
  • Gap penalty 10 Gap length penalty 10
  • a protein is accordingly understood which, when its sequence is compared with the sequence SEQ ID NO. 2 or 16, in particular according to the above program algorithm with the above parameter set, has an identity of at least 20%.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • Those codons which are frequently used in accordance with the plant-specific codon usage are preferably used for this.
  • the codon usage can easily be determined on the basis of computer evaluations of other known genes of the organisms concerned.
  • a nucleic acid containing the sequence SEQ ID NO is brought. 1, in the plant.
  • nucleic acid containing the sequence SEQ ID NO is brought. 15, in the plant.
  • ketolase genes can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can, for example, in a known manner, according to the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, page
  • genetically modified plants are used which have the highest expression rate of a ketolase in fruits.
  • the gene expression of the ketolase takes place under the control of a fruit-specific promoter.
  • the nucleic acids described above, as described in detail below are introduced into the plant in a nucleic acid construct, functionally linked to a fruit-specific promoter.
  • plants are preferably understood to mean plants which have chromoplasts as wild type in fruits.
  • Further preferred plants have, as wild type in the fruit, carotenoids, in particular ⁇ -carotene, zeaxanthin, neoxanthine, violaxanthin or lutein.
  • carotenoids in particular ⁇ -carotene, zeaxanthin, neoxanthine, violaxanthin or lutein.
  • Further preferred plants have a hydroxylase activity as wild type in the fruit.
  • Hydroxylase activity means the enzyme activity of a hydroxylase.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity of introducing a hydroxyl group on the optionally substituted ⁇ -ionone ring of carotenoids.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity to convert ⁇ -carotene into zeaxanthin or canthaxanthin into astaxanthin.
  • hydroxylase activity is understood to mean the amount of ⁇ -carotene or canthaxanthin converted or the amount of zeaxanthin or astaxanthin formed in a certain time by the protein hydroxylase.
  • plants are cultivated which, in addition to the wild type, have an increased hydroxylase activity and / or ⁇ -cyclase activity.
  • Hydroxylase activity means the enzyme activity of a hydroxylase.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity of introducing a hydroxyl group on the optionally substituted ⁇ -ionone ring of carotenoids.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity to convert ⁇ -carotene into zeaxanthin or cantaxanthin into astaxanthin.
  • hydroxyase activity is understood to mean the amount of ⁇ -carotene or cantaxanthin converted or the amount of zeaxanthin or astaxanthin formed in a certain time by the protein hydroxylase.
  • the amount of ⁇ -carotene or cantaxantin or the amount of zeaxanthin or astaxanthin formed is increased in a certain time by the protein hydroxylase compared to the wild type.
  • This increase in hydroxylase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the hydroxylase Wild type activity.
  • ⁇ -cyclase activity means the enzyme activity of a ⁇ -cyclase.
  • a ß-cyclase is understood to be a protein which has the enzymatic activity to convert a terminal, linear residue of lycopene into a ß-ionone ring.
  • a ⁇ -cyclase is understood to be a protein which has the enzymatic activity to convert ⁇ -carotene into ⁇ -carotene.
  • ⁇ -cyclase activity is understood to mean the amount of ⁇ -carotene converted or the amount of ⁇ -carotene formed in a certain time by the protein ß-cyclase.
  • the amount of ⁇ -carotene converted or the amount of ⁇ -carotene formed is increased by the protein ß-cyclase in a certain time compared to the wild type.
  • This increase in the ⁇ -cyclase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the ⁇ - Wild-type cyclase activity.
  • wild type is understood to mean the corresponding non-genetically modified starting plant.
  • wild type is used to increase the hydroxylase activity, to increase the ⁇ -cyclase activity and to increase the ketocarotenoid content in each case understood a reference plant.
  • This reference plant is preferably Lycopersicon esculentum.
  • hydroxylase activity in genetically modified plants according to the invention and in wild-type or reference plants is preferably determined under the following conditions:
  • the activity of the hydroxylase is according to Bouvier et al. (Biochim. Biophys. Acta 1391 (1998), 320-328) in vi tro. Ferredoxin, ferredoxin-NADP oxidoreductase, catalase, NADPH and beta-carotene with mono- and digalact psylglycerides are added to a certain amount of plant extract.
  • the hydroxylase activity is particularly preferably determined under the following conditions according to Bouvier, Keller, d'Harginue and Camara (xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper 5 fruits (Capsicum annuum L .; Biochim. Biophys Acta 1391 (1998) 320-328):
  • the in vitro assay is carried out in a volume of 0.250 ml volume.
  • the batch contains 50 mM potassium phosphate (pH 7.6),
  • reaction mixture 15 plant extract in different volumes.
  • the reaction mixture is incubated for 2 hours at 30C.
  • the reaction products are extracted with organic solvent such as acetone or chloroform / methanol (2: 1) and determined by means of HPLC.
  • ⁇ -cyclase activity in genetically modified plants according to the invention and in wild-type or reference plants is preferably carried out under the following conditions:
  • the activity of the ⁇ -cyclase is determined in vitro according to Fräser and Sandmann (Bio-25 formerly Biophys. Res. Comm. 185 (1) (1992) 9-15). Potassium phosphate as a buffer (pH 7.6), lycopene as a substrate, paprika stromal protein, NADP +, NADPH and ATP are added to a certain amount of plant extract.
  • the hydroxylase activity is particularly preferably determined under the following conditions according to Bouvier, d'Harlingue and Camara (Molecular Analysis of carotenoid cyclae inhibition; Aren. Biochem. Biophys. 346 (1) (1997) 53-64):
  • NADP / NADPH and ATP are in
  • the hydroxylase activity and / or ⁇ -cyclase activity can be increased in various ways, for example by switching off inhibitory regulatory mechanisms at the expression and protein level or by increasing the gene expression of nucleic acids encoding a hydroxylase and / or of nucleic acids encoding a ⁇ -cyclase the wild type.
  • the increase in the gene expression of the nucleic acids encoding a hydroxylase and / or the increase in the gene expression of the nucleic acid encoding a ⁇ -cyclase compared to the wild type can likewise be carried out in various ways, for example by inducing the hydroxylase gene and / or ⁇ -cyclase gene by activators or by introducing one or more hydroxylase gene copies and / or ⁇ -cyclase gene copies, ie by introducing at least one nucleic acid encoding a hydroxylase and / or at least one nucleic acid encoding an ⁇ -cyclase into the plant.
  • Increasing the gene expression of a nucleic acid encoding a hydroxylase and / or ⁇ -cyclase is also understood according to the invention to mean the manipulation of the expression of the plants' own endogenous hydroxylase and / or ⁇ -cyclase.
  • an altered or increased expression of an endogenous hydroxylase and / or ⁇ -cyclase gene can be achieved in that a regulator protein which does not occur in the non-transformed plant interacts with the promoter of this gene.
  • a regulator protein which does not occur in the non-transformed plant interacts with the promoter of this gene.
  • Such a regulator can represent a chimeric protein which consists of a DNA binding domain and a transcription activator domain, as described for example in WO 96/06166.
  • the gene expression of a nucleic acid encoding a hydroxylase is increased and / or the gene expression of a nucleic acid encoding a ⁇ -cyclase is increased by introducing at least one nucleic acid encoding a hydroxylase and / or by introducing at least one nucleic acid encoding one ß-cyclase into the plant.
  • any hydroxylase gene or each ⁇ -cyclase gene that is to say any nucleic acid which codes for a hydroxylase and any nucleic acid which codes for a ⁇ -cyclase, can be used for this purpose.
  • genomic hydroxylase or. ⁇ -cyclase nucleic acid sequences from eukaryotic sources which contain introns are preferably already processed in the event that the host plant is unable or cannot be able to express the corresponding hydroxylase or ⁇ -cyclase Nucleic acid sequences, how to use the corresponding cDNAs.
  • hydroxylase genes are nucleic acids
  • hydroxylase is the hydroxylase from tomato (nucleic acid: SEQ. ID. No. 55; protein: SEQ. ID. No. 56)
  • b-cyclase genes are nucleic acids encoding a b-cyclase from tomato (Accession X86452). (Nucleic acid: SEQ ID NO; 53, protein: SEQ ID NO: 54), and b-cyclase genes of the following accession numbers:
  • AAF18989 lycopene beta-cyclase [Daucus carota] ZP_001140 hypothetical protein [Prochlorococcus marinus str.
  • ZP_001050 hypothetical protein [Prochlorococcus marinus subsp. pastoris str. CCMP1378]
  • ZP_001046 hypothetical protein [Prochlorococcus marinus subsp. pastoris str. CCMP1378]
  • ZP_001134 hypothetical protein [Prochlorococcus marinus str.
  • ZP_001150 hypothetical protein [Synechococcus sp. WH 8102] AAF10377 lycopene cyclase [Deinococcus radiodurans] BAA29250 393aa long hypothetical protein [Pyrococcus horikoshii]
  • AAF78200 lycopene cyclase [Bradyrhizobium sp. ORS278] BAB79602 crtY [Pantoea agglomerans pv. Milletiae] CAA64855 lycopene cyclase [Streptomyces griseus] AAA21262 dycopene cyclase [Pantoea agglomerans] C37802 crtY protein - Erwinia uredovora BAB79602 crtYans [Pantoea millglomer. AAA64980 lycopene cyclase [Pantoea agglomerans]
  • CAA67331 lycopene cyclase [Narcissus pseudonarcissus]
  • a particularly preferred ⁇ -cyclase is also the chromoplast-specific b-cyclase from tomato (AAG21133) (nucleic acid: SEQ. ID. No. 57; protein: SEQ. ID. No. 58)
  • the preferred transgenic plants according to the invention therefore have at least one further hydroxylase gene and / or ⁇ -cyclase gene compared to the wild type.
  • the genetically modified plant has, for example, at least one exogenous nucleic acid, coding for a hydroxylase or at least two endogenous nucleic acids, coding for a hydroxylase and / or at least one exogenous nucleic acid, coding for a ⁇ -cyclase or at least two endogenous nucleic acids, coding for one ⁇ -cyclase.
  • nucleic acids encoding proteins are preferably used which contain the amino acid sequence SEQ ID NO: 52 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 30 %, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ. ID. NO: 52, and which have the enzymatic property of a hydroxylase.
  • hydroxylases and hydroxylase genes can be obtained, for example, from various organisms whose genomic sequence is known, as described above, by comparing the amino acids or the corresponding ones with homology back-translated nucleic acid sequences from databases with the SeQ ID. NO: 52 easy to find.
  • hydroxylases and hydroxylase genes can also be found, for example, based on the sequence
  • SEQ ID NO: 51 from various organisms whose genomic sequence is not known, as described above, can easily be found by hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the hydroxylase of the sequence SEQ ID NO: 52.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons that are frequently used in accordance with the plant-specific codon usage are preferably used for this.
  • the codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ is brought. ID. NO: 51 in the organism.
  • the ⁇ -cyclase genes used are preferably nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 54 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 35 at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 54, and which have the enzymatic property of a ⁇ -cyclase.
  • ⁇ -cyclases and ⁇ -cyclase genes can be obtained, for example, from various organisms whose genomic sequence is known, as described above, by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SEQ ID NO:
  • ⁇ -cyclases and ⁇ -cyclase genes can also be easily found, for example, starting from the sequence SEQ ID NO: 53 from various organisms whose genomic sequence is not known, using hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the ⁇ -cyclase of the sequence SEQ. ID. NO: 54.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons that are frequently used in accordance with the plant-specific codon usage are preferably used for this.
  • the codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ is brought. ID. NO: 53 in the organism.
  • hydroxylase genes or ⁇ -cyclase genes can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can, for example, be known
  • Particularly preferred plants are plants selected from the plant genera Actinophloeus, Aglaeonema, pineapple, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Caliptocalix, Capsicum, Carica, Celastrus, Citrullus, Citrus, Convallaria Cotoneaster, Crataegus, Cucumis, Cucurbita, Cuscuta, Cycas, Cyphomandra, Dioscorea, Diospyrus, Dura, Elaeagnus, Elaeis, Erythroxylon, Euonymus, Ficus, Fortunella, Fragaria, Gardinia, Gonocaryum, Gossypium, Guava, Guilielma, Guilielma, Guilielma, Guilielma, Guilielma, Guilielma, Guilielma, Guilielma, Guilielma
  • the ketolase activity in genetically modified plants according to the invention is determined in accordance with the method of Frazer et al. , (J. Biol. Chem. 272 (10): 6128-6135, 1997).
  • the ketolase activity in plant extracts is determined with the substrates beta-carotene and canthaxanthin in the presence of lipid (soy lecithin) and detergent (sodium cholate).
  • Substrate / product ratios from the ketolase assays are determined by means of HPLC.
  • the cultivation step of the genetically modified plants is preferably followed by harvesting the plants and isolating ketocarotenoids from the fruits of the plants.
  • the transgenic plants are raised in a manner known per se
  • Ketocarotenoids are isolated from the harvested fruits in a manner known per se, for example by drying and subsequent extraction and, if appropriate, further chemical or physical purification processes, such as, for example, precipitation methods, crystallography, thermal separation processes, such as rectification processes or physical separation processes, such as chromatography. Ketocarotenoids are isolated from the fruit, for example, preferably by organic solvents such as acetone, hexane, ether or tert. Methyl butyl ether.
  • ketocarotenoids are described, for example, in Egger and Kleinig (Phytochemistry (1967) 6, 437-440) and Egger (Phytochemistry (1965) 4, 609-618).
  • ketocarotenoids are preferably selected from the group astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin and adonixanthin.
  • ketocarotenoid is astaxanthin.
  • the transgenic plants are preferably produced by transforming the starting plants, using a nucleic acid construct which contains at least one, preferably also more than one of the above-described nucleic acids which are functionally linked to one or more regulatory signals which ensure transcription and translation in plants ,
  • nucleic acid constructs in which the coding nucleic acid sequence is functionally linked to one or more regulatory signals which ensure transcription and translation in plants, are also called expression cassettes below.
  • the regulation signals preferably contain one or more 15 promoters which ensure transcription and translation in plants.
  • the expression cassettes contain regulatory signals, that is, regulatory nucleic acid sequences which express the expression of the
  • an expression cassette comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and optionally further regulatory elements which are associated with
  • An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and possibly other regulatory elements such that each of the regulatory
  • 30 elements can perform its function as intended in the expression of the coding sequence.
  • nucleic acid constructs, expression cassettes and vectors for plants and methods for producing transgenic plants and the transgenic plants themselves are described below by way of example.
  • sequences preferred but not limited to the operative linkage are targeting sequences to ensure
  • any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette.
  • Constant promoter means those promoters that have a
  • a vegetable one is preferably used
  • Promoter or a promoter derived from a plant virus is particularly preferred.
  • the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140 : 281-288; Gardner et al. (1986) Plant Mol Biol
  • Another suitable constitutive promoter is the pds Prooter (Pecker et al. (1992) Proc. Natl. Acad. Be USA 89: 4962-4966) or the "Rubisco small subunit (SSU)" promoter (US 4,962,028) , the LeguminB promoter (GenBank Acc.No. X0S677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995 ) Plant Mol Biol 29: 637-649), the Ubiquitin 1 promoter (Christensen et al.
  • the expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the ketolase gene in the plant is controlled at a specific point in time can.
  • a chemically inducible promoter such as the PRPl promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a promoter inducible by tetracycline (Gatz et al.
  • Promoters which are induced by biotic or abiotic stress are also preferred, for example the pathogen-inducible promoter of the PRPL gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp70 or hsp80 Promoter from tomato (US Pat. No. 5,187,267), the cold-inducible alpha-amylase promoter from the potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinII promoter (EP375091).
  • the pathogen-inducible promoter of the PRPL gene Ward et al. (1993) Plant Mol Biol 22: 361-366
  • the heat-inducible hsp70 or hsp80 Promoter from tomato US Pat. No. 5,187,267
  • the cold-inducible alpha-amylase promoter from the potato WO 96/12814
  • Pathogen-inducible promoters include those of genes induced by pathogen attack such as genes from PR proteins, SAR proteins, b-1, 3-glucanase, chitinase etc. (e.g. Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes, et al. (1992) The Plant
  • wound-inducible promoters such as that of the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wunl and wun2 gene (US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin (McGurl et al. (1992) Science 225: 1570-1573), the WIPl gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Ekelkamp et al. (1993) FEBS Letters 323: 73-76), the MPI gene (Corderok et al. (1994) The Plant J 6 ( 2): 141-150) and the like.
  • promoters are, for example, fruit ripening-specific promoters, such as, for example, the fruit ripening-specific promoter from tomato (WO 94/21794, EP 409 625).
  • Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.
  • promoters are particularly preferred which ensure expression in tissues or parts of plants in which, for example, the biosynthesis of ketocarotenoids or their precursors takes place.
  • promoters with specificities for the anthers, ovaries, petals, sepals, flowers, leaves, stems, roots and fruits and combinations thereof are preferred.
  • Tuber-, storage root- or root-specific promoters are, for example, the patatin class I promoter (B33) or the potato cathepsin D inhibitor promoter.
  • Leaf-specific promoters are, for example, the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al (1989) EMBO J 8: 2445-2451).
  • Flower-specific promoters are, for example, the phytoene synthesis promoter (WO 92/16635) or the promoter of the P-rr gene (WO
  • Anther-specific promoters are, for example, the 5126 promoter (US 5,689,049, US 5,689,051), the gl ⁇ b-1 promoter or the g-zein promoter.
  • Fruit-specific promoters are, for example
  • the cucumisin promoter (Yamagata, H., Yonesu, K., Hirata, A. and Aizono, Y., TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene J. Biol. Chem. 277 (13), 11582-11590 (2002), SEQ ID NO. 19, the promoter of the endogalacturonase gene (Redondo-Nevado, J., Medina-Escobar, N., Caballero-Repullo, JL and Munoz-Blanco, J.
  • constitutive and in particular fruit-specific promoters are particularly preferred.
  • the present invention therefore relates in particular to a nucleic acid construct containing functionally linked a fruit-specific promoter, particularly preferably a fruit-specific promoter described above, and a nucleic acid encoding a ketolase.
  • An expression cassette is preferably produced by fusing a suitable promoter with a nucleic acid described above, encoding a ketolase and preferably a nucleic acid inserted between promoter and nucleic acid sequence, which codes for a plastid-specific transit peptide, and a polyadenylation signal in accordance with current standards Recombination and cloning techniques, as described, for example, in T. Maniatis, EF Fritsch and J.
  • nucleic acids encoding a plastic transit peptide ensure localization in plastids and in particular in chromoplasts.
  • Expression cassettes the nucleic acid sequence of which codes for a ketolase fusion protein, can also be used, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
  • Preferred transit peptides are preferred for the chromoplasts, which are cleaved enzymatically from the ketolase part after translocation of the ketolase into the chromoplasts.
  • Nucleic acid sequences of three cassettes of the plastid transit peptide of plastid transketolase from tobacco in three reading frames are particularly preferred as Kpnl / BamHI fragments with an ATG codon in the Ncol interface:
  • a plastid transit peptide examples include the transit peptide of the plastid isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabisopsis thaliana and the transit peptide of the small subunit of the ribulose bisphosphate carboxylase (rbcS) from pea (Guerineau, F, Woolston, S Brooks, L, Mullineaux, P (1988) An expression cassette for targeting foreign proteins into the chloroplstas. Nucl. Acids Res. 16: 11380).
  • IPP-2 plastid isopentenyl pyrophosphate isomerase-2
  • rbcS ribulose bisphosphate carboxylase
  • nucleic acids according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural nucleic acid constituents, and can consist of different heterologous gene segments from different organisms.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp within the regulatory areas, often less than 60 bp, but at least 5 bp.
  • the promoter can be native or homologous as well as foreign or heterologous to the host plant his.
  • the expression cassette preferably contains in the 5 '-3' transcription direction the promoter, a coding nucleic acid sequence or a nucleic acid construct and a region for the transcriptional termination. Different termination areas are interchangeable.
  • a terminator is the 35S terminator (Guerineau et al. (1988) Nucl Acids Res. 16: 11380), the nos terminator (Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • Suitable methods for the transformation of plants are the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - the so-called particle bombardment method, the electroporation, the incubation of dry embryos in DNA-containing solution, the micro- injection and Agrobacterium-mediated gene transfer described above.
  • the methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993), 128- 143 and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225).
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711) or particularly preferably pSUN2, pSUN3, pSUN4 or pSUN5 (WO 02/00900).
  • Agrobacteria transformed with an expression plasmid can be used in a known manner to transform plants, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the fused expression cassette which expresses a ketolase, is cloned into a vector, for example pBin19 or in particular pSUN2, which is suitable for being transformed into Agrobacterium tumefaciens
  • Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • transgenic plants From the transformed cells of the wounded leaves or leaf pieces, transgenic plants can be regenerated in a known manner, which plants contain a gene encoding the expression cassette for the expression of a nucleic acid encoding a ketolase.
  • an expression cassette is inserted as an insert into a recombinant vector whose vector DNA contains additional functional regulatory signals, for example Contains sequences for replication or integration.
  • additional functional regulatory signals for example Contains sequences for replication or integration.
  • Suitable vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119 (1993).
  • the expression cassettes can be cloned into suitable vectors which enable their multiplication, for example in E. coli.
  • suitable cloning vectors include pJITH7 (Guerineau et al. (1988) Nucl. Acids Res. 16: 11380), pBR332, pUC series, Ml3mp series, pACYC184, pMC1210, pMcl 210 and pCLl920.
  • Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.
  • the expression can be constitutive or preferably specific in the fruit.
  • the invention further relates to a method for producing genetically modified plants, characterized in that a nucleic acid construct containing functionally linked, a fruit-specific promoter and nucleic acids encoding a ketolase is introduced into the genome of the starting plant.
  • the invention further relates to the genetically modified plants which have a ketolase activity in fruits compared to the starting plant.
  • the ketolase activity is achieved in that the genetically modified plant expresses a ketolase in the fruit.
  • the preferred, genetically modified plants therefore contain at least one nucleic acid encoding a keto-glass in fruits.
  • the gene expression of a nucleic acid, coding for a ketolase is caused by introducing nucleic acids, coding for a ketolase, into the starting plant.
  • the invention therefore particularly preferably relates to a genetically modified plant described above, characterized in that, starting from a starting plant, at least one nucleic acid coding for a ketolase has been introduced into the plant.
  • the invention relates in particular to genetically modified plants selected from the plant genera Actinophloeus, Aglaeo-nema, pineapple, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Caliptocalix, Capsicum, Carica, Celastrus, Citrullus, Citrus, Convallaria, Cotoneaster, Crataegus, Cucumis, Cucurbita, Cuscuta, Cycas, Cyphomandra, Dioscorea, Diospyrus, Dura, Elaeagnus, Elaeis, Erythroxylon, Euonymus, Ficus, Fortunella, Fragaria, Gardinia, Gonocaryum, Goss
  • Very particularly preferred plant genera are pineapple, asparagus, capsicum, citrus, cucumis, cucurbita, citrullus, lycopersicum, passiflora, prunus, physalis, solanum, vaccinium and vitis, containing at least one transgenic nucleic acid, encoding a ketolase.
  • the ketolase is expressed in the fruits in preferred transgenic plants, particularly preferably the expression of the ketolase is highest in the fruits.
  • genetically modified plants additionally have an increased hydroxylase activity and / or ⁇ -cyclase activity compared to a wild plant. Further preferred embodiments are described above in the method according to the invention.
  • the present invention further relates to the transgenic plants, their propagation material, and their plant cells, tissue or parts, in particular their fruits.
  • the genetically modified plants can, as described above, be used to produce ketocarotenoids, in particular astaxanthin.
  • Genetically modified plants according to the invention with an increased content of ketocarotenoids which can be consumed by humans and animals can also be used, for example, directly or after processing known per se as food or feed or as feed and food supplements. Furthermore, the genetically modified plants for the production of ketocarotenoids extracts of the plants containing noid and / or for the production of feed and food supplements.
  • the genetically modified plants have an increased ketocarotenoid content compared to the wild type.
  • An increased ketocarotenoid content is generally understood to mean an increased total ketocarotenoid content.
  • ketocarotenoids is also understood to mean, in particular, a changed content of the preferred ketocarotenoids, without the total carotenoid content necessarily having to be increased.
  • the genetically modified plants according to the invention have an increased astaxanthin content compared to the wild type.
  • the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from Licor (sales by MWG Biotech, Ebersbach) according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) , 5463-5467).
  • Example 1 Amplification of a cDNA which contains the entire primary sequence of the ketolase from Haematococcus pluvialis Floow em. Will encodes
  • the cDNA coding for the ketolase from Haematococcus pluvialis was amplified by PCR from Haematococcus pluvialis (strain 40 192.80 from the "Collection of algal cultures of the University of Göttingen") suspension culture.
  • RNA For the preparation of total RNA from a suspension culture of Haematococcus pluvialis (strain 192.80), which has been used for 2 weeks with direct sunlight at room temperature in Haer ⁇ atococcus medium (1 - .2 g / 1 sodium acetate, 2 g / 1 yeast extract, 0.2 g / 1 MgC12x6H20, 0.02 CaCl2x2H20; pH 6.8; after autoclaving, add 400 mg / 1 L-asparagine, 10 mg / 1 FeS04xH20), the cells were harvested, frozen in liquid nitrogen and pulverized in a mortar.
  • Haer ⁇ atococcus medium (1 - .2 g / 1 sodium acetate, 2 g / 1 yeast extract, 0.2 g / 1 MgC12x6H20, 0.02 CaCl2x2H20; pH 6.8; after autoclaving, add 400 mg / 1 L-asparagine, 10 mg / 1
  • RNA For the cDNA synthesis, 2.5 ⁇ g of total RNA were denatured for 10 min at 60 ° C., cooled on ice for 2 min and using a cDNA kit (ready-to-go-you-prime beads, Pharmacia Biotech) according to the manufacturer's instructions rewritten into cDNA using an antisense specific primer (PRI SEQ ID No. 29).
  • the nucleic acid encoding a ketolase from Haematococcus x pluvialis was determined using the polymerase chain reaction
  • PCR from Haematococcus pluvialis using a sense-specific primer (PR2 SEQ ID No. 30) and an antisense-specific primer (PRI SEQ ID No. 29).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the cDNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 29 and SEQ ID No. 30 resulted in an 1155 bp fragment coding for a protein consisting of the entire primary sequence (SEQ ID No. 22).
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) and the clone pGKET02 was obtained.
  • This clone was therefore used for cloning into the expression vector pJITH7 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • the cloning was carried out by isolating the 1027 bp SpHI fragment from pGKET02 and ligation into the SpHI-cut vector pJIT117.
  • the clone that contains the Haematococcus pluvialis ketola gene in the correct orientation as an N-terminal translational fusion with the rbcs transit peptide sequence is called pJKET02.
  • Example 2 Amplification of a cDNA which contains the ketolase from Haematococcus pluvialis Flotow em. Will encoded with an N-terminus shortened by 14 amino acids
  • the cDNA which codes for the ketolase from Haematococcus pluvialis (strain 192.80) with an N-terminus shortened by 14 amino acids, was amplified by PCR from Haematococcus pluvialis suspension culture (strain 192.80 from the "Collection of algal cultures of the University of Göttingen") ,
  • Total RNA was prepared from a suspension culture of Haematococcus pluvialis (strain 192.80) as described in Example 1.
  • the nucleic acid encoding a ketolase from Haema tococcus pluvialis (strain 192.80) with an N-terminus shortened by 14 amino acids was extracted by means of polymerase chain reaction (PCR)
  • the PCR conditions were as follows:
  • the PCR for the amplification of the cDNA which codes for a ketolase protein with an N-terminus shortened by 14 amino acids, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions;
  • PCR amplification with SEQ ID No.29 and SEQ ID No. 31 resulted in an 1111 bp fragment coding for a ketolase protein in which the N-terminal amino acids (position 2-16) are replaced by 30 a single amino acid (leucine).
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) using standard methods and the clone pGKET03 was obtained. Sequencing with the primers T7 and
  • the cloning was carried out by isolating the 985 bp SpHI fragment from pGKET03 and ligation with the SpHI-cut vector pJITH7.
  • the clone that contains the correct Haematococcus pluvialis ketolase with 45 N-terminus shortened by 14 amino acids Containing orientation as an N-terminal translational fusion with the rbcs transit peptide is called pJKET03.
  • Example 3 Amplification of a cDNA which contains the ketolase from Haema-5 tococcus pluvialis Flotow em. Will (tribe 192.80 the ketolase from Haema-5 tococcus pluvialis Flotow em. Will (tribe 192.80 the ketolase from Haema-5 tococcus pluvialis Flotow em. Will (tribe 192.80 the
  • the cDNA coding for the ketolase from Haematococcus pluvialis (strain 192.80) consisting of the entire primary sequence and fused C-terminal myc tag was PCR-analyzed using the plasmid pGKET02 (described in Example 1) and the primer PR15 (SEQ ID No. 32).
  • the PR15 primer SEQ ID No. 32.
  • the nucleic acid encoding a ketolase from Haematococcus 35 pluvialis (strain 192.80) consisting of the entire primary sequence and fused C-terminal myc tag was determined by means of polymerase chain reaction (PCR) from Haematococcus pluvialis using a sense-specific primer (PR2 SEQ ID No. 30 ) and an antisense-specific primer (PR15 SEQ ID No. 32).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the cDNA which codes for a ketolase protein with a fused C-terminal myc tag, was carried out in a 45 50 ⁇ l reaction mixture which contained: - 1 ul of an annealing reaction (prepared as described above) 0.25 mM dNTPs
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 32 and SEQ ID No. 30 resulted in a 1032 bp fragment coding for a protein consisting of the entire primary sequence of the ketolase from Haematococcus pluvialis as a double translational fusion with the rbcS transit peptide at the N-terminus and the myc tag at the C-terminus.
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) using standard methods and the clone pGKET04 was obtained. Sequencing with the primers T7 and SP6 confirmed a sequence SEQ ID no. 22 identical sequence, the 3 'region (position 993-1155) of SEQ ID No. 22 in the amplificate SEQ ID No. 26 was replaced by one in the different sequence from 39 bp. This clone was therefore made for
  • the cloning was carried out by isolating the 1038 bp EcoRI-SpHI fragment from pGKET04 and ligation with the EcoRI-SpHI cut vector pJITH7. The ligation creates a translational fusion between the C-terminus of the rbcS transit peptide sequence and the N-terminus of the ketolase sequence.
  • the clone which contains the Haematococcus pluvialis ketolase with fused C-terminal myc tag in the correct orientation as a translational N-terminal fusion with the rbcs transit peptide is called pJKET4.
  • Example 4 Production of expression vectors for the constitutive expression of the Haematococcus pluvialis ketolase in
  • fragment d35S contains the duplicated 35S promoter (747 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KET03 (985 bp) the primary sequence shortened by 14 N-terminal amino acids coding for the Haematococcus pluvialis Ketolase, fragment term (761 bp) the polyadenylation signal of CaMV.
  • fragment d35S contains the duplicated 35S promoter ((747 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KET04 (1038 bp) the entire primary sequence coding for the Haematococcus pluvialis ketolase with C-terminal myc- Day, fragment term (761 bp) the polyadenylation signal of CaMV.
  • Example 5 Production of expression vectors for the expression of Haematococcus pluvialis ketolase in Lycopersicon esculentum
  • the DNA fragment which contains the AP3 promoter region -902 to +15 from Arabidopsis thaliana, was analyzed by means of PCR
  • genomic DNA isolated from Arabidopsis thaliana according to standard methods
  • primers PR7 and PR10 SEQ ID No. 36
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the AP3 promoter fragment (-902 to +15), was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 922 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods and the plasmid pTAP3 was obtained.
  • Sequencing of the clone pTAP3 confirmed a sequence consisting only of an insertion (a G in position 9765 of the sequence AL132971) and a base exchange (a G instead of an A in position 9726 of the sequence AL132971) from the published AP3 sequence (AL132971, nucleotide region 9298-10200) differs. These nucleotide differences were reproduced in an independent amplification experiment and thus represent the actual nucleotide sequence in the Arabidopsis thaliana plants used.
  • the modified version AP3P was produced by recombinant PCR using the plasmid pTAP3. The region 10200-9771 was amplified with the primers PR7 (SEQ ID No.
  • the PCR conditions were as follows:
  • the PCR was carried out under the following cycle conditions:
  • the recombinant PCR includes annealing of the amplificates A7 / 9 and A8 / 10, which overlap over a sequence of 25 nucleotides, completion into a double strand and subsequent amplification.
  • the denaturation (5 min at 95 ° C.) and annealing (slow cooling at room temperature to 40 ° C.) of both amplificates A7 / 9 and A8 / 10 was carried out in a 17.6 1 reaction, which contained:
  • the nucleic acid coding for the modified promoter version AP3P was amplified by means of PCR using a sense-specific primer (PR7 SEQ ID No. 28) and an antisense-specific primer (PR10 SEQ ID No. 36).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the AP3P fragment was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 33 and SEQ ID No. 36 resulted in a 778 bp fragment coding for the modified promoter version AP3P.
  • the amplificate was cloned into the cloning vector pCR .1 (Invitrogen) and the clone pTAP3P was obtained. Sequencing with the primers T7 and M13 confirmed a sequence identical to the sequence AL132971, region 10200-9298, the internal region 9285-9526 being deleted. This clone was therefore used for the cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • the cloning was carried out by isolating the 771 bp SacI-HindIII fragment from pTAP3P and ligating into the SacI-HindIII cut vector pJITH7.
  • the clone that contains the AP3P promoter instead of the original d35S promoter is called pJAP3P.
  • the 1027 bp SpHI fragment KET02 (described in Example 1) was cloned into the SpHI-cut vector pJAP3P.
  • the clone that contains the fragment KET02 in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJAP3PKET02.
  • the 1032 bp SpHI-EcoRI fragment KET04 (described in Example 3) was cloned into the SpHI-EcoRI cut vector pJAP3P.
  • the clone that contains the fragment KET04 in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJAP3PKET04.
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled ketolase from Haematococcus pluvialis into L. esculentum was produced using the binary vector pSUN3 (WO02 / 00900).
  • fragment AP3P contains the modified AP3P promoter (771 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KET02 (1027 bp) the entire primary sequence coding for the Haematococcus pluvialis ketolase, fragment term (761 Bp) the polyadenylation signal from CaMV.
  • fragment AP3P contains the modified AP3P promoter (771 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KET04 (1038 bp) the entire primary sequence coding for the Haematococcus pluvialis ketolase with C-terminal myc tag , Fragment term (761 bp) the polyadenylation signal of CaMV.
  • Example 6 Production of transgenic Lycopersicon esculentum plants Transformation and regeneration of tomato plants was carried out according to the published method by Ling and co-workers (Plant Cell Reports (1998), 17: 843-847). For the microtome variety, higher kanamycin concentrations (100 mg / L) were selected. 5
  • the starting explant for the transformation was cotyledons and hypocotyls, seven to ten day old seedlings of the Microtome line.
  • the culture medium according to Murashige and Skoog (1962: Murashige and Skoog, 1962, Physiol. Plant 15,
  • MSZ2 medium MS pH 6, 1 + 3% sucrose, 2 mg / 1 zeatin, 100 mg / 1 kanamycin,
  • the fruit material of the transgenic plants was ground in liquid nitrogen and the powder (about 250 to 500 mg) extracted with 100% acetone (three times 500 ul each). The solvent was evaporated and the carotenoids resuspended in 100 ul acetone.
  • Solvent B 80% methanol, 0.2% ammonium acetate
  • Solvent C 100% t-butyl methyl ether
  • the spectra were determined using a photodiode array detector.
  • the carotenoids were absorbed through their Spectra and their retention times compared to standard samples identified.
  • Table 1 shows the carotenoid profile in tomato fruits of the transgenic tomatoes and control tomato plants produced according to the examples described above. Compared to the genetically unmodified control plant, the genetically modified plants have a ketocarotenoid content and in particular astaxanthin content.
  • Table 2a shows the amounts of carotenoids in ripe fruit of transgenic tomatoes and control plants.
  • the data are mean values of various linines and are given as a percentage of the total carotenoid content.
  • Table 2b shows the amounts of carotenoids in ripening fruits of transgenic tomatoes and control plants.
  • the data are mean values of various linines and are given as a percentage of the total carotenoid content.
  • the DNA encoding the NP196 ketolase from Nostoc punctiform ATCC 29133 was amplified by PCR from Nostoc punctiform ATCC 15 29133 (strain of the "American Type Culture Collection").
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then washed in liquid nitrogen with a mortar.
  • the cell suspension was incubated for 3 hours at 37 ° C. in 100 ⁇ l proteinase K (concentration: 20 mg / ml). Then was
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNP196 was obtained.
  • This clone pNPl96 was therefore used for cloning into the expression vector pJAP3P (described in Example 5).
  • PJAP3P was modified by the 35S terminator through the OCS terminator (octopine synthase) of the Ti plasmid pTil5955 from Agrobacterium tumefaciens (database entry X00493 from position 10 12.541-12.350, Gielen et al. (1984) EMBO J. 3 835-846 ) was replaced.
  • the DNA fragment containing the OCS terminator region was isolated by PCR using the plasmid pHELLSGATE (database entry 15 AJ311874, Wesley et al. (2001) Plant J. 27 581-590, isolated from E. coli by standard methods) as well as the primer OCS-1 (SEQ ID No. 63) and OCS-2 (SEQ ID No. 64).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the octopine synthase (OCS) terminator region (SEQ ID 65), was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 210 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods and the plasmid pOCS was obtained.
  • Sequencing of the clone pOCS confirmed a sequence which corresponds to a sequence section on the Ti plasmid pTil5955 from Agrobacterium tumefaciens (database entry X00493) from positions 12,541 to 12,350.
  • the cloning was carried out by isolating the 210 bp Sall-Xhol fragment from pOCS and ligation into the Sall-Xhol cut vector pJAP3P.
  • This clone is called pJOAP and was therefore used for cloning into the expression vector pJOAP: NP196.
  • the cloning was carried out by isolating the 782 bp Sphl fragment from pNPl96 and ligating into the SphI cut vector pJOAP.
  • the clone that contains the Nostoc punctiforme NPl96 ketolase in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJOAP: NPl96.
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled NPl96 ketolase from Nostoc punctiform ATCC 29133 into L. esculentum was produced using the binary vector pSUN3 (WO02 / 00900).
  • fragment AP3P PROM contains the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the nos toc punctiform NPl96 ketolase, fragment OCS terminator (192 bp) the octopine synthase polyadenylation signal.
  • AP3P PROM contains the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the nos toc punctiform NPl96 ketolase, fragment OCS terminator (192 bp) the octopine synthase polyadenylation signal.
  • Example 11 Example 11:
  • the DNA coding for the NOST ketolase from Nostoc punctiform PCC 7120 was amplified by means of PCR from Nostoc PCC 7120 (strain of the "Pasteur Culture Collection of Cyanobacterium”).
  • the bacterial cells were removed from a 10 ml liquid culture by 10
  • the cell suspension was incubated for 3 hours at 37 ° C. in 30 100 ⁇ l proteinase K (concentration: 20 mg / ml). The suspension was then extracted with 500 ⁇ l of phenol. After centrifugation at 13,000 rpm for 5 minutes, the upper, aqueous phase was transferred to a new 2 ml Eppendorf reaction vessel. The extraction
  • DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating to 65 ° C.
  • the nucleic acid encoding a ketolase from Nostoc PCC 7120 was determined by means of a "polymerase chain reaction” (PCR) from Nostoc PCC 7120 using a sense-specific primer (NOST-1, SEQ ID No. 66) and an antisense-specific primer NOST-2
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 66 and SEQ ID No. 67 resulted in an 809 bp fragment that codes for a protein consisting of the entire primary sequence (SEQ ID No. 68).
  • the amplificate was cloned into the PCR cloning vector pGEM-T (Promega) and the clone pNOST was obtained.
  • This clone pNOST was therefore used for the cloning into the expression vector pJOAP (described in Example 9).
  • the cloning was carried out by isolating the 799 bp Sphl fragment from pNOST and ligation into the SphI-cut vector pJOAP.
  • the clone that contains the NOSToc PCC7120 NOST ketolase in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJOAP: NOST Example 12:
  • NOST ketolase from Nostoc spp. PCC 7120 in L. Esculentum occurred with the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715). Expression was carried out under the control of the AP3P promoter from Arabidopsis thaliana (described in Example 5).
  • fragment AP3P PROM contains the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NOST KETO CDS (774 bp), coding for the Nostoc spp. PCC 7120 NOST-Ketolase, fragment OCS terminator (192 bp) the polyadenylation signal of octopine synthase.
  • the DNA which codes for the NPl95 ketolase from Nostoc punctiform ATCC 29133 was amplified by means of PCR from Nostoc punctiform ATCC 29133 (strain of the "American Type Culture Collection"). The preparation of genomic DNA from a suspension culture of Nostoc punctiforme ATCC 29133 was described in Example 9.
  • PCR polymerase chain reaction
  • the PCR conditions were as follows: The PCR for the amplification of the DNA, which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • This clone pNPl95 was therefore used for the cloning into the expression vector pJOAP (described in Example 9).
  • the cloning was carried out by isolating the 709 bp Sphl fragment from pNPl95 and ligating into the SphI-cut vector pJOAP.
  • the clone which contains the NPl95 ketolase from Nostoc punctiforme ATCC 29133 in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJOAP: NPl95.
  • fragment AP3P PROM contains the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), coding for the nostoc punctiform ATCC 29133 NP195 ketolase, Fragment OCS terminator (192 bp) the polyadenylation signal from octopine synthase.
  • the DNA encoding the ketolase from Nodularia spumignea NSOR10 was amplified by PCR from Nodularia spumignea NSOR10.
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml 10mM Tris_HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (2ml volume). After adding 100 ⁇ l Proteinase K (concentration: 20 mg / ml), the cell suspension was incubated for 3 hours at 37 ° C. The suspension was then extracted with 500 ⁇ l of phenol. After centrifugation at 13,000 rpm for 5 minutes, the upper, aqueous phase was transferred to a new 2 ml Eppendorf reaction vessel.
  • the extraction with phenol was repeated 3 times.
  • the DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating to 65 ° C. x
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions;
  • PCR amplification with SEQ ID No. 74 and SEQ ID No. 75 resulted in a 720 bp fragment coding for a protein consisting of the entire primary sequence (NODK, SEQ ID No. 76).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNODK was obtained.
  • the cloning was carried out by isolating the 710 bp Sphl fragment from pNODK and ligation into the SphI-cut vector pJOAP.
  • the clone that contains the NODK ketolase from Nodularia spumignea NSORIO in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJOAP: NODK.
  • NODK ketolase from Nodularia spumignea NSORIO was expressed in L. esculentum with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240: 709-715). The expression was carried out under the control of the AP3P promoter from Arabidopsis thaliana (described in Example 5).
  • fragment AP3P PROM contains the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NODK KETO CDS (690 bp), coding for the Nodularia spumignea NSORIO NODK ketolase, fragment OCS terminator (192 bp) the octopine synthase polyadenylation signal.
  • Example 17 Production of an expression cassette for fruit-specific overexpression of the chromoplast-specific b-hydroxylase from Lycopersicon esculentum.
  • chromoplast-specific ⁇ -hydroxylase from Lycopersicon esculentum in tomato takes place under the control of the fruit-specific promoter AP3P from Arabidopsis (Example 2).
  • LB3 database entry AX696005
  • Vicia faba is used as the terminator element.
  • the sequence of the chromoplast-specific ⁇ -hydroxylase was produced by RNA isolation, reverse transcription and PCR.
  • the DNA fragment containing the LB3 terminator region was isolated by PCR.
  • Genomic DNA from Vicia faJba tissue is isolated and used by genomic PCR using the primers PR206 (SEQ ID No. 78) and PR207 (SEQ ID No. 79).
  • the PCR for the amplification of this LB3 DNA fragment is carried out in a 50 ⁇ l reaction mixture which contains:
  • genomic DNA prepared as described above 0.25 mM dNTPs
  • PCR amplification with SEQ ID No. 78 and SEQ ID No. 79 results in a 307 bp fragment (SEQ ID No. 80) which contains the LB terminator.
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pLB3 was obtained. Sequencing of the clone pLB3 with the M13F and the M13R primer confirmed a sequence which corresponds to the DNA sequence from 3-298 of the database entry AX696005 is identical. This clone is called pLB3 and is therefore used for cloning into the vector pJAP3P (see example 5).
  • the expression cassette pJAP3P was modified by replacing the 35S- 5 terminator with the Legumin LB3 terminator from Vicia faba (database entry AX696005; WO03 / 008596) (see below).
  • Total RNA is used to produce the ß-hydr -xylase sequence
  • RNA is precipitated with a volume of isopropanol, washed with 75% ethanol and the pellet is dissolved in DEPC water (overnight incubation of water with 1/1000 volume of diethyl pyrocarbonate at room temperature, then autoclaved).
  • DEPC water overnight incubation of water with 1/1000 volume of diethyl pyrocarbonate at room temperature, then autoclaved.
  • RNA concentration 20 concentration is determined photometrically.
  • cDNA synthesis 2.5 ⁇ g of total RNA are denatured for 10 min at 60 a C, cooled on ice for 2 min and using a cDNA kit (Ready-to-go-you-prime beads, Pharmacia Biotech) according to the manufacturer's instructions using an antisense specific primer (PR215 SEQ ID No.
  • the nucleic acid encoding the ⁇ -hydroxylase was extracted from tomato by means of a "polymerase chain reaction” (PCR) using a sense-specific primer (VPR204, SEQ ID No. 81) and an antisense-specific primer (PR215 SEQ ID No . 82) amplified.
  • PCR polymerase chain reaction
  • the PCR for the amplification of the DNA which codes for a ⁇ -hydroxylase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR amplification with VPR204 and PR215 results in a 1,040 bp fragment (SEQ ID No. 83) which codes for the b-hydroxylase.
  • the amplificate is cloned into the PCR cloning vector pCR 2.1 (Invitrogen). This clone is called pCrtR-b2.
  • Sequencing of the clone pCrtR-b2 with the primers M13-R and M13-R confirmed a sequence which is identical to the DNA sequence from 33-558 of the database entry BE354440 and with the DNA sequence from 1-1009 of the database entry Y14810 is identical.
  • the clone pCrtR-b2 is therefore used for the cloning into the vector pCSP02 (see below).
  • the first cloning step is carried out by isolating the 1,034 bp HindIII-EcoRI fragment from pCrtR-b2, derived from the cloning vector pCR-2.1 (Invitrogen), and ligation with the HindIII-EcoRI cut vector pJAP3P (see Example 5).
  • the clone that contains the b-hydroxylase fragment CrtR-b2 is called pCSP02.
  • the second cloning step is carried out by isolating the 301 bp EcoRI-XhoI fragment from pLB3, derived from the cloning vector pCR-2.1 (Invitrogen), and ligation with the EcoRI-XhoI cut vector pCSP02.
  • the clone that contains the 296 bp terminator L ⁇ 3 is called pCSP03.
  • the ligation creates a transcriptional fusion between the terminator LB3 and the b-hydroxylase fragment CrtR-b2.
  • a transcriptional fusion occurs between the AP3P promoter and the b-hydroxylase fragment.
  • Example 18 Production of an expression cassette for fruit-specific overexpression of the B gene from Lycopersicon esculenurn.
  • the expression of the B gene from Lycopersicon esculentum in Tomat takes place under the control of the fruit-specific promoter PDS (phytoene desaturase; database entry U46919) from Lycopersicon esculentum. 35S from CaMV is used as the terminator element.
  • the sequence of the B gene was generated by PCR from genomic DNA from Lycopersicon esculentum.
  • the oligonucleotide primers BGEN-1 (SEQ ID No. 85) and BGEN-2 (SEQ ID No. 86) were used to isolate the B gene by means of PCR with genomic DNA from Lycopersicon esculentum.
  • the genomic DNA was isolated from Lycopersicon esculentum as described (Galbiati M et al. Funct. Integr. Genomics 2000, 20 1: 25-34).
  • the PCR amplification was carried out as follows:
  • the amplificate is in the PCR cloning vector pCR-2.1
  • genomic DNA from Lycopersicon esculentum tissue is isolated according to standard methods and used by genomic PCR using the primers PDS-1 and PDS-2.
  • the PCR for the amplification of this PDS-PRomotor fragment is carried out in a 50 ul. Reaction batch which contains: 35
  • 0.2 uM PDS-1 (SEQ ID No. 89) 0.2 uM PDS-2 (SEQ ID No. 90) 40 - 5 ul 10X Pfu-Turbo Polymerase (Stratagene) - 1 ul Pfu-Turbo Polymerase (Stratagene) 28.8 ul Aq. Dest.
  • the PCR amplification with PDS-1 and PDS-2 results in a fragment that contains the sequence for the PDS promoter.
  • the amplificate is cloned into the pCR4-BLUNT (Invitrogen). This clone is called pPDS.
  • the first cloning step is carried out by isolating the 1,499 bp Ncol / EcoRI fragment from pBGEN, derived from the cloning vector pCR2.1 (Invitrogen).
  • pBGEN is cut with BamHI, the 3 'ends are filled in according to standard methods (30 min at 30 ° C.) (Klenow-fill-in) and then a partial digestion is carried out with Ncol, in which the resulting 1,499 kb fragment is isolated.
  • This fragment was then cloned into pCSP02, which had previously been cut with EcoRI, the 3 'ends filled in according to standard methods (30 min at 30 ° C.) (Klenow fill-in) and then cut with Ncol.
  • the clone that contains the 1,497 bp B gene fragment BGEN is called pJAP: BGEN.
  • the ligation creates a transcriptional fusion between the 35S terminator and the B gene.
  • the second cloning step is carried out by isolating the 2,078 bp PDS PROM fragment from pPDS.
  • pPDS is cut with Smal and then a partial digestion with Sacl is carried out, in which the resulting 2,088 bp fragment is isolated.
  • Sacl is carried out, in which the resulting 2,088 bp fragment is isolated.
  • This fragment was then cloned into the pJAP: BGEN, which had previously been cut with BamHI, the 3 'ends filled in using standard methods (30 min at 30 ° C.) (Klenow fill-in) and then cut with SacI.
  • the ligation creates a transcriptional fusion between the promoter PDS and the B gene.
  • the clone that contains the 2,078 bp PDS promoter BGEN is called pJPDS: BGEN.
  • Example 19 Production of a triple expression vector for the overexpression of the B gene, the expression of the Nostoc punctiform ketolase NP196, and the overexpression of the chromoplast-specific B-hydroxylase from Lycopersicon esculentum fruit-specifically in Lycopersicon esculentum.
  • a double construct which contains expression cassettes for the overexpression of the Nostoc punctiform ATCC 29133 NP196 ketolase and for the overexpression of the B-hydroxylase.
  • fragment AP3P b-hydroxy lase: LB3, which contains the B-hydroxylase expression cassette, as a 2104 bp Ecll36lI-XhoI fragment isolated from pCSP03 (described in Example 18).
  • the 3 'ends (30 min at 30 a C) are filled using standard methods (Klenow fill-in).
  • this fragment was cut in the vector MSP120 (described in Example 10) with Ecll36ll and EcoRI, the 3 'ends were filled in according to standard methods (30 min at 30 ° C.) (Klenow fill-in).
  • the ligation results in a T-DNA which contains two expression cassettes: firstly a cassette for the chromoplast-specific overexpression of the B-hydroxylase from Lycopersicon esculentum, and secondly a cassette for the overexpression of the ketolase NP196 from Nostoc punctiforme.
  • the B-hydroxylase downregulation cassette can ligate into the vector in two orientations.
  • the version in which both expression cassettes match in their orientation is preferred (see Figure 14). This version can be identified by PCR as described:
  • the PCR for the amplification of the PR206-PR010 plasmid fragment which contains the compound of LB3 terminator of the B-hydroxylase cassette and the AP3P promoter of the ketolase cassette, is carried out in a 50 ⁇ l reaction mixture which contains:
  • PCR amplification with PR010 and PR206 results in a 1,080 bp fragment, which indicates the presence of the above-described connection of LB3 terminator and AP3P promoter, and thus the preferred orientation of both expression cassettes.
  • This clone is called pBHYX: NPl96.
  • this B gene overexpression cassette into expression vectors for the Agrobacterium -mediated transformation of tomato, isolation of the 4,362 bp EcoRV-XhoI fragment from pJPDS-.BGEN (see Example 19) and ligation in the Smal-Xhol-cut vector pBHYX: NPl96 (described above).
  • the ligation results in a T-DNA which contains three expression cassettes: first a cassette for overexpressing the B gene, secondly a cassette for overexpressing the ketolase NP196-1 from Nostoc punctiforme, and thirdly a cassette for chromoplast-specific overexpression of the B-hydroxylase Lycopersicon esculentum ( Figure 14, construct map).
  • This clone is called MSP124.
  • fragment AP3P PROM (765 bp) contains the AP3P promoter, fragment BHYX b2 CDS (2 bp) the B-hydroxylase CrtRb2, fragment LB3 TERM (296 bp) the LB3 terminator.
  • Fragment AP3P PROM (765 bp) also contains the AP3P promoter, fragment rbcS TP FRAGMENT (194 bp) the transit peptide of the rbcS gene from pea, NP196 KETO CDS (761 bp) the ketolase from Noctoc punctiform ATCC29133, and OCS TERM (192 bp) the polyadenylation signal of the octopine synthase gene.
  • fragment PDS PROM (2078 bp) contains the PDS promoter, fragment BGEN CDS (1,497 bp) the B gene sequence, and fragment 35S TERM (746 bp) the 35S terminator.
  • MSP121 MSP121-1, MSP121-2, MSP121-3
  • MSP123 MSP123-1, MSP123-2, MSP123-3

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EP03792349A 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des fruits de plantes Withdrawn EP1532266A2 (fr)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
DE10238978 2002-08-20
DE10238978A DE10238978A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Ketocarotinoiden in Früchten von Pflanzen
DE10238979A DE10238979A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Zeaxanthin und/oder dessen biosynthetischen Zwischen- und/oder Folgeprodukten
DE10238979 2002-08-20
DE10238980 2002-08-20
DE2002138980 DE10238980A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Ketocarotinoiden in Blütenblättern von Pflanzen
DE10253112 2002-11-13
DE2002153112 DE10253112A1 (de) 2002-11-13 2002-11-13 Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten Organismen
DE10258971 2002-12-16
DE2002158971 DE10258971A1 (de) 2002-12-16 2002-12-16 Verwendung von astaxanthinhaltigen Pflanzen oder Pflanzenteilen der Gattung Tagetes als Futtermittel
PCT/EP2003/009107 WO2004018695A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des fruits de plantes

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EP03792345A Withdrawn EP1532264A2 (fr) 2002-08-20 2003-08-18 Procede de production de cetocarotenoides dans les petales de plantes
EP03792348A Withdrawn EP1532265A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies
EP03792350A Expired - Lifetime EP1531683B1 (fr) 2002-08-20 2003-08-18 Utilisation de plantes ou de parties de plantes contenant de l' astaxanthine du genre tagetes comme produit de fourrage
EP03792349A Withdrawn EP1532266A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des fruits de plantes
EP03792347A Withdrawn EP1542945A2 (fr) 2002-08-20 2003-08-18 Procede de fabrication de zeaxanthine et/ou de ses produits intermediaires et/ou produits ses produits secondaires biosynthetiques

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EP03792345A Withdrawn EP1532264A2 (fr) 2002-08-20 2003-08-18 Procede de production de cetocarotenoides dans les petales de plantes
EP03792348A Withdrawn EP1532265A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies
EP03792350A Expired - Lifetime EP1531683B1 (fr) 2002-08-20 2003-08-18 Utilisation de plantes ou de parties de plantes contenant de l' astaxanthine du genre tagetes comme produit de fourrage

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