US20070094749A1 - Method for producing ketocarotinoids in plant fruit - Google Patents

Method for producing ketocarotinoids in plant fruit Download PDF

Info

Publication number
US20070094749A1
US20070094749A1 US10/524,829 US52482903A US2007094749A1 US 20070094749 A1 US20070094749 A1 US 20070094749A1 US 52482903 A US52482903 A US 52482903A US 2007094749 A1 US2007094749 A1 US 2007094749A1
Authority
US
United States
Prior art keywords
ketolase
plant
seq
genetically modified
sequence
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.)
Abandoned
Application number
US10/524,829
Other languages
English (en)
Inventor
Christel 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 DE10238979A external-priority patent/DE10238979A1/de
Priority claimed from DE10238978A external-priority patent/DE10238978A1/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
Assigned to SUNGENE GMBH & CO. KGAA reassignment SUNGENE GMBH & CO. KGAA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FLACHMANN, RALF, HERBERS, KARIN, KLEBSATTEL, MARTIN, KUNZE, IRENE, SAUER, MATT, SCHOPFER, CHRISTEL RENATE
Publication of US20070094749A1 publication Critical patent/US20070094749A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—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 method for the production of ketocarotenoids by culturing genetically modified plants which show a ketolase activity in fruits, to the genetically modified plants, and to their use as foods and feeds and for the production of ketocarotenoid extracts.
  • Ketocarotenoids are synthesized de novo in bacteria, algae, fungi and plants.
  • Ketocarotenoids i.e. carotenoids comprising at least one keto group, such as, for example, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin are natural antioxidants and pigments which are produced by some algae and microorganisms as secondary metabolites.
  • ketocarotenoids Owing to their color-imparting properties, the ketocarotenoids, and in particular astaxanthin, are used as pigmenting auxiliaries in animal nutrition, in particular in trout, salmon and shrimp farming.
  • Natural ketocarotenoids such as, for example, natural astaxanthin
  • WO 98/18910 describes the synthesis of ketocarotenoids in nectar glands 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 the seeds of oilseed plants such as oilseed rape, sunflower, soybean and mustard, using a seed-specific promoter and a ketolase from Haematococcus.
  • the invention was therefore based on the object of providing an alternative method for the production of ketocarotenoids by culturing plants, or of providing further transgenic plants which produce ketocarotenoids, which have the optimized characteristics, such as, for example, a higher ketocarotenoid content, and which do not suffer from the above-described disadvantage of the prior art.
  • Ketolase activity is understood as meaning the enzyme activity of a ketolase.
  • a ketolase is understood as meaning a protein with the enzymatic activity of introducing a keto group at the optionally substituted ⁇ -ionone ring of carotenoids.
  • ketolase is understood as meaning a protein with the enzymatic activity of converting ⁇ -carotene into canthaxanthin.
  • ketolase activity is understood as meaning the amount of ⁇ -carotene converted, or the amount of canthaxanthin formed, by the protein ketolase within a certain period of time.
  • a preferred embodiment involves the use of genetically modified plants which express a ketolase in fruits.
  • Genetically modified plants which comprise, in fruits, at least one nucleic acid encoding a ketolase are therefore preferably used in the method according to the invention.
  • No plants which show a ketolase activity in fruits as the wild type.
  • the preferred plants described hereinbelow show no ketolase activity in fruits as the wild type.
  • the ketolase activity in fruits of the genetically modified plants is caused by the genetic modification of the starting plant.
  • the genetically modified plant according to the invention shows a ketolase activity in fruits in comparison with the genetically non-modified starting plant and is thus preferably capable of expressing a ketolase in fruits.
  • starting plant or wild type is understood as meaning the corresponding non-genetically-modified starting plant.
  • genetically modified plant is preferably understood as meaning a plant which is genetically modified in comparison with the starting plant.
  • plant can be understood as meaning the starting plant (wild type), or a genetically modified plant according to the invention or both.
  • the invention therefore relates in particular to the above-described method, wherein genetically modified plants into which at least one nucleic acid encoding a ketolase has been introduced, starting from a starting plant.
  • ketolase gene that is to say any nucleic acid which encodes a ketolase
  • nucleic acids mentioned in the description can be for example an RNA, DNA or cDNA sequence.
  • nucleic acid sequences which are preferably to be used are, in the event that the host plant is not capable, or cannot be made capable, of expressing the ketolase in question, ready-processed nucleic acids 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 in particular 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: SEQ ID No. 9, protein SEQ ID No. 10).
  • Synechocystis sp. strain PC6803 (Accession No. 576617, NP442491; nucleic acid: SEQ ID No. 11, protein SEQ ID No. 12).
  • Bradyrhizobium sp. (Accession No. AF218415, BAB 74888; nucleic acid: SEQ ID No. 13, protein SEQ ID No. 14).
  • Nostoc sp. strain PCC7120 (Accession No. AP003592; nucleic acid: SEQ ID No. 15, protein SEQ ID No. 16).
  • 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 spumigena NSOR10 (Accession NO: AY210783, AAO64399; 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 accesion NO: NZ_AABC01000196; nucleic acid: SEQ ID NO: 47, protein: SEQ ID NO: 48)
  • Deinococcus radiodurans R1 (Accession NO: E75561, AE001872; nucleic acid: SEQ ID NO: 49, protein: SEQ ID NO: 50)
  • ketolases and ketolase genes which can be used in the method according to the invention can be found readily for example from various organisms whose genomic sequence is known by carrying out alignments of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with the above-described sequences, and in particular with the sequences SEQ ID NO. 2 and/or SEQ ID NO. 16.
  • ketolases and ketolase genes can furthermore be found readily from different organisms whose genomic sequence is not known by using hybridization techniques in the manner known per se, starting from the above-described nucleic acid sequences, in particular starting from the sequences SEQ ID NO. 1 and/or SEQ ID NO. 15.
  • the hybridization can be carried out 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 delimited by those with less stringency (with 2 ⁇ SSC at 50° C.) and those with high stringency (with 0.2 ⁇ SSC at 50° C., preferably at 65° C.) (20 ⁇ SSC: 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).
  • the temperature during the washing step can be increased from moderate conditions at room temperature, 22° C., to stringent conditions at 65° C.
  • Both parameters, salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant, while only the other one is varied.
  • denaturing agents such as, for example, formamide or SDS can be employed during the hybridization step. In the presence of 50% formamide, the hybridization is preferably carried out at 42° C.
  • nucleic acids are introduced which encode a protein comprising the amino acid sequence SEQ ID NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20%, by preference 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%, especially preferably at least 90% identity at the amino acid level with the sequence SEQ ID NO. 2 and which has the enzymatic characteristic of a ketolase.
  • This may take the form of a natural ketolase sequence which can be found from other organisms as described above by alignment of the sequences, or else an artificial ketolase sequence which has been modified starting from the sequence SEQ ID NO. 2 by artificial variation, for example by substitution, insertion or deletion of amino acids.
  • nucleic acids are introduced which encode a protein comprising 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 at least 20%, by preference 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%, especially preferably at least 90% identity at the amino acid level with the sequence SEQ ID NO. 16 and which has the enzymatic characteristic of a ketolase.
  • This may take the form of a natural ketolase sequence which can be found from other organisms as described above by alignment of the sequences, or else an artificial ketolase sequence which has been modified starting from the sequence SEQ ID NO. 16 by artificial variation, for example by substitution, insertion or deletion of amino acids.
  • substitution is understood as meaning the replacement of one or more amino acids by one or more amino acids. Substitutions which are preferably carried out are what are known as conservative substitutions, where the replaced amino acid has a similar property to the original amino acid, for example substitution of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, Ser by Thr.
  • Deletion is the replacement of an amino acid by a direct bond.
  • Preferred positions for deletion are the termini of the polypeptide and the linkages between the individual protein domains.
  • Insertions are introductions of amino acids into the polypeptide chain, where a direct bond is formally replaced by one or more amino acids.
  • a protein which has at least 20% identity at the amino acid level with the sequence SEQ ID NO. 2 or 16 is, accordingly, understood as meaning a protein which, upon comparison of its sequence with the sequence SEQ ID NO. 2 or 16, in particular by the above program algorithm with the above parameter set, has at least 20% identity.
  • Suitable nucleic acid sequences are obtainable, for example, by backtranslation of the polypeptide sequence according to the genetic code.
  • Codons which are preferably used for this purpose are those which are used frequently in accordance with the plant-specific codon usage.
  • the codon usage can be determined readily with the aid of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid comprising the sequence SEQ ID NO. 1 is introduced into the plant.
  • nucleic acid comprising the sequence SEQ ID NO. 15 is introduced into the plant.
  • ketolase genes can furthermore be generated in the known manner by chemical synthesis, starting with the nucleotide units, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix.
  • Oligonucleotides can be synthesized chemically in the known manner for example by the phosphoamidite method (Voet, Voet, 2 nd Edition, Wiley Press New York, pp. 896-897). The annealing of synthetic oligonucleotides and filling in of gaps by means of the Klenow fragment of the DNA polymerase and ligation reactions, and general cloning methods, are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
  • genetically modified plants which show the highest expression rate of a ketolase in fruits are used.
  • the above-described nucleic acids as described hereinbelow in detail are introduced into the plant in a nucleic acid construct in functional linkage with a fruit-specific promoter.
  • plants are preferably understood as meaning plants which, as the wild type, have chromoplasts in fruits.
  • Further preferred plants additionally have, as the wild type, carotenoids, in particular ⁇ -carotene, zeaxanthin, neoxanthin, violaxanthin or lutein, in the fruits.
  • carotenoids in particular ⁇ -carotene, zeaxanthin, neoxanthin, violaxanthin or lutein, in the fruits.
  • Further preferred plants have, as the wild type, additionally a hydroxylase activity in the fruits.
  • Hydroxylase activity is understood as meaning the enzyme activity of a hydroxylase.
  • a hydroxylase is understood as meaning a protein with the enzymatic activity of introducing a hydroxyl group at the optionally substituted ⁇ -ionone ring of carotenoids.
  • a hydroxylase is understood as meaning a protein with the enzymatic activity of converting ⁇ -carotene into zeaxanthin or cantaxanthin into astaxanthin.
  • hydroxylase activity is understood as meaning the amount of ⁇ -carotene or cantaxanthin converted, or the amount of zeaxanthin or astaxanthin formed, by the protein hydroxylase within a certain period of time.
  • plants are cultured which additionally show an increased hydroxylase activity and/or ⁇ -cyclase activity in comparison with the wild type.
  • Hydroxylase activity is understood as meaning the enzyme activity of a hydroxylase.
  • a hydroxylase is understood as meaning a protein with the enzymatic activity of introducing a hydroxyl group at the optionally substituted ⁇ -ionone ring of carotenoids.
  • a hydroxylase is understood as meaning a protein with the enzymatic activity of converting ⁇ -carotene into zeaxanthin or cantaxanthin into astaxanthin.
  • hydroxylase activity is understood as meaning the amount of ⁇ -carotene or cantaxanthin converted, or the amount of zeaxanthin or astaxanthin formed, by the protein hydroxylase within a certain period of time.
  • the converted amount of ⁇ -carotene or cantaxanthin, or the amount of zeaxanthin or astaxanthin formed, by the protein hydroxylase is increased within a certain period of time in comparison with the wild type.
  • This increase of the hydroxylase activity amounts by preference to at least 5%, furthermore preferably at least 20%, furthermore preferably at least 50%, furthermore preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the hydroxylase activity of the wild type.
  • ⁇ -Cyclase activity is understood as meaning the enzyme activity of a ⁇ -cyclase.
  • a ⁇ -cyclase is understood as meaning a protein with the enzymatic activity of converting a terminal, linear residue of lycopene into a ⁇ -ionone ring.
  • a ⁇ -cyclase is understood as meaning a protein with the enzymatic activity of converting ⁇ -carotene into ⁇ -carotene.
  • ⁇ -cyclase activity is understood as meaning the amount of ⁇ -carotene converted, or the amount of ⁇ -carotene formed, by the protein ⁇ -cyclase within a certain period of time.
  • the amount of ⁇ -carotene converted, or the amount of ⁇ -carotene formed, by the protein ⁇ -cyclase within a certain period of time is increased in comparison with the wild type.
  • This increase of the ⁇ -cyclase activity amounts by preference to at least 5%, furthermore preferably at least 20%, furthermore preferably at least 50%, furthermore preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the ⁇ -cyclase activity of the wild type.
  • wild type is understood as meaning the corresponding non-genetically-modified starting plant.
  • wild type for increasing the hydroxylase activity, for increasing the ⁇ -cyclase activity and for increasing the ketocarotenoid content is understood as meaning in each case one 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 hydroxylase activity is determined in vitro by the method of Bouvier et al. (Biochim. Biophys. Acta 1391 (1998), 320-328). Ferredoxin, ferredoxin-NADP oxidoreductase, catalase, NADPH and beta-carotene together with mono- and digalactosylglycerides are added to a certain amount of plant extract.
  • the hydroxylase activity is especially preferably determined by the method of Bouvier, Keller, d'Harlingue and Camara (Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits ( Capsicum annuum L.; Biochim. Biophys. Acta 1391 (1998), 320-328) under the following conditions:
  • the in-vitro assay is carried out in a volume of 0.250 ml.
  • the mixture comprises 50 mM potassium phosphate (pH 7.6), 0.025 mg spinach ferredoxin, 0.5 units spinach ferredoxin-NADP+ oxidoreductase, 0.25 mM NADPH, 0.010 mg beta-carotene (emulsified in 0.1 mg Tween 80), 0.05 mM of a mixture of mono- and digalactosylglycerides (1:1), 1 unit catalase, 200 mono- and digalactosylglycerides (1:1), 0.2 mg bovine serum albumin and plant extract in different volumes.
  • the reaction mixture is incubated for 2 hours at 30° C.
  • the reaction products are extracted with organic solvents 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 determined under the following conditions:
  • the ⁇ -cyclase activity is determined in vitro by the method of Fraser and Sandmann (Biochem. Biophys. Res. Comm. 185(1) (1992) 9-15). The following are added to a certain amount of plant extract: potassium phosphate to act as buffer (pH 7.6), lycopene to act as substrate, stromaprotein from Capsicum, NADP+, NADPH and ATP.
  • the hydroxylase activity is especially preferably carried out by the method of Bouvier, d'Harlingue and Camara (Molecular Analysis of carotenoid cyclae inhibition; Arch. Biochem. Biophys. 346(1) (1997) 53-64) under the following conditions:
  • the in-vitro assay is carried out in a volume of 250 ⁇ l.
  • the mixture comprises 50 mM potassium phosphate (pH 7.6), different amounts of plant extract, 20 nM lycopene, 250 ⁇ g of chromoplastidial stromaprotein from Capsicum, 0.2 mM NADP+, 0.2 mM NADPH and 1 mM ATP.
  • NADP/NADPH and ATP are dissolved in 10 ml of ethanol together with 1 mg of Tween 80 immediately prior to addition to the incubation medium. After a reaction time of 60 minutes at 30° C., the reaction is quenched by addition of chloroform/methanol (2:1). The reaction products which are extracted in chloroform are analyzed by means of HPLC.
  • Increasing the hydroxylase activity and/or ⁇ -cyclase activity can be effected in various ways, for example by eliminating inhibiting regulatory mechanisms at the expression and protein level, or by increasing the gene expression of nucleic acids encoding a hydroxylase and/or nucleic acids encoding a ⁇ -cyclase in comparison with the wild type.
  • Increasing the gene expression of the nucleic acids encoding a hydroxylase and/or increasing the gene expression of the nucleic acid encoding a ⁇ -cyclase in comparison with the wild type can likewise be effected 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, i.e. by introducing, into the plant, at least one nucleic acid encoding a hydroxylase and/or at least one nucleic acid encoding an ⁇ -cyclase.
  • Increasing the gene expression of a nucleic acid encoding a hydroxylase and/or ⁇ -cyclase is also understood as meaning, in accordance with the invention, the manipulation of the expression of the plants' homologous, endogenous hydroxylase and/or ⁇ -cyclase.
  • a modified, or increased, expression of an endogenous hydroxylase and/or ⁇ -cyclase gene can be achieved by a regulator protein which does not occur in the untransformed plant interacting with the promoter of this gene.
  • Such a regulator can be 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 and/or increasing the gene expression of a nucleic acid encoding a ⁇ -cyclase is effected by introducing, into the plant, at least one nucleic acid encoding a hydroxylase and/or by introducing, into the plant, at least one nucleic acid encoding a ⁇ -cyclase.
  • any hydroxylase gene, or any ⁇ -cyclase gene i.e. any nucleic acid which encodes a hydroxylase and any nucleic acid which encodes a ⁇ -cyclase can be used for this purpose.
  • nucleic acid sequences from eukaryotic sources, which comprise introns
  • ready-processed nucleic acid sequences such as the corresponding cDNAs
  • hydroxylase genes are nucleic acids encoding a hydroxylase from Haematococcus pluvialis, Accession AX038729, WO 0061764); (nucleic acid: SEQ ID NO: 51, protein: SEQ ID NO: 52),
  • an especially preferred 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: S66350 lycopene beta-cyclase (EC 5.5.1.—) - tomato CAA60119 lycopene synthase [ Capsicum annuum ] S66349 lycopene beta-cyclase (EC 5.5.1.—) - common tobacco CAA57386 lycopene cyclase [ Nicotiana tabacum ] AAM21152 lycopene beta-cyclase [ Citrus sinensis ] AAD38049 lycopene cyclase [ Citrus ⁇ paradisi ] AAN86060 lycopene cyclase [ Citrus unshiu ] AAF44700 lycopene beta-
  • WH 8102 AAF10377 lycopene cyclase [ Deinococcus radiodurans ] BAA29250 393aa long hypothetical protein [ Pyrococcus horikoshii ] BAC77673 lycopene beta-monocyclase [marine bacterium P99-3] AAL01999 lycopene cyclase [ Xanthobacter sp. Py2] ZP_000190 hypothetical protein [ Chloroflexus aurantiacus ] ZP_000941 hypothetical protein [ Novosphingobium aromaticivorans ] AAF78200 lycopene cyclase [ Bradyrhizobium sp.
  • BAB79602 crtY [ Pantoea agglomerans pv. milletiae ] CAA64855 lycopene cyclase [ Streptomyces griseus ] AAA21262 dycopene cyclase [ Pantoea agglomerans ] C37802 crtY protein - Erwinia uredovora
  • BAB79602 crtY [ Pantoea agglomerans pv.
  • ZP_000941 hypothetical protein [ Novosphingobium aromaticivorans ] CAB56061 lycopene beta-cyclase [ Paracoccus marcusii ] BAA20275 lycopene cyclase [ Erythrobacter longus ] ZP_000570 hypothetical protein [ Thermobifida fusca ] ZP_000190 hypothetical protein [ Chloroflexus aurantiacus ] AAK07430 lycopene beta-cyclase [ Adonis palaestina ] CAA67331 lycopene cyclase [ Narcissus pseudonarcissus ] AAB53337 Lycopene beta cyclase BAC77673 lycopene beta-monocyclase [marine bacterium P99-3]
  • an especially preferred ⁇ -cyclase is the chromoplast-specific ⁇ -cyclase from tomato (AAG21133) (nucleic acid: SEQ ID No. 57; protein: SEQ ID No. 58)
  • At least one further hydroxylase gene and/or ⁇ -cyclase gene is present in this preferred embodiment in comparison with the wild type.
  • the genetically modified plant has, for example, at least one exogenous nucleic acid encoding a hydroxylase or at least two endogenous nucleic acids encoding a hydroxylase and/or at least one exogenous nucleic acid encoding a ⁇ -cyclase or at least two endogenous nucleic acids encoding a ⁇ -cyclase.
  • Hydroxylase genes which are preferably used in the above-described preferred embodiment are nucleic acids which encode proteins comprising the amino acid sequence SEQ ID NO: 52 or a sequence which is derived from this sequence by substitution, insertion or deletion of amino acids and which have at least 30%, by preference at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% identity 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 found readily, as described above, for example from various organisms whose genomic sequence is known, by homology comparisons of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with SEQ ID NO: 52.
  • hydroxylases and hydroxylase genes can furthermore be found readily in the manner known per se from various organisms whose genomic sequence is not known, by hybridization and PCR techniques as described above, for example starting from the sequence SEQ ID NO: 51.
  • nucleic acids which encode proteins comprising the amino acid sequence of the hydroxylase of the sequence SEQ ID NO: 52 are introduced into organisms in order to increase the hydroxylase activity.
  • suitable nucleic acid sequences can be obtained by backtranslation of the polypeptide sequence in accordance with the genetic code.
  • Codons which are preferably used for this purpose are codons which are used frequently in accordance with the plant-specific codon usage.
  • the codon usage can be determined readily with the aid of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid comprising the sequence SEQ ID NO: 51 is introduced into the organism.
  • ⁇ -Cyclase genes which are preferably used in the above-described preferred embodiment are nucleic acids which encode proteins comprising the amino acid sequence SEQ ID NO: 54 or a sequence which is derived from this sequence by substitution, insertion or deletion of amino acids and which have at least 30%, by preference at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% identity 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 found readily, as described above, for example from various organisms whose genomic sequence is known, by homology comparisons of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with SEQ ID NO: 54.
  • ⁇ -cyclases and ⁇ -cyclase genes can furthermore be found readily in the manner known per se from various organisms whose genomic sequence is not known, by hybridization and PCR techniques, for example starting from the sequence SEQ ID NO: 53.
  • nucleic acids which encode proteins comprising the amino acid sequence of the ⁇ -cyclase of the sequence SEQ ID NO: 54 are introduced into organisms in order to increase the ⁇ -cyclase activity.
  • nucleic acid sequences can be obtained by backtranslation of the polypeptide sequence in accordance with the genetic code.
  • Codons which are preferably used for this purpose are codons which are frequently used in accordance with the plant-specific codon usage.
  • the codon usage can be determined readily with the aid of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid comprising the sequence SEQ ID NO: 53 is introduced into the organism.
  • All of the abovementioned hydroxylase genes or ⁇ -cyclase genes can furthermore be generated in a known manner by chemical synthesis, starting with the nucleotide units, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix.
  • Oligonucleotides can be synthesized chemically in a known manner for example by the phosphoamidite method (Voet, Voet, 2 nd Edition, Wiley Press New York, pp. 896-897). The annealing of synthetic oligonucleotides and filling in of gaps by means of the Klenow fragment of the DNA polymerase and ligation reactions, and general cloning methods, are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
  • Especially preferred plants are plants selected from the plant genera Actinophloeus, Aglaeonema, Ananas, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Cliptocalix, 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, Hibiscus, Hippophaea, Iris, Lathyrus, Lonicera, Luffa, Lycium, Lycopersicum, Malpighia, Man
  • the ketolase activity in genetically modified plants according to the invention is determined by a method similar to that of Frazer et al., (J. Biol. Chem. 272(10): 6128-6135, 1997).
  • the ketolase activity in plant extracts is determined using the substrates beta-carotene and canthaxanthin in the presence of lipid (soya lecithin) and detergent (sodium cholate). Substrate/product ratios from the ketolase assays are determined by HPLC.
  • the cultivation step of the genetically modified plants, hereinbelow also referred to as transgenic plants is preferably followed by harvesting of the plants and isolating ketocarotenoids from the fruits of the plants.
  • the transgenic plants are grown in a manner known per se on substrates and harvested in a suitable manner.
  • Ketocarotenoids are isolated from the harvested fruits in a manner known per se, for example by drying followed by extraction and, if appropriate, further chemical or physical purification processes such as, for example, precipitation methods, crystallography, thermal separation methods such as rectification methods or physical separation methods such as, for example, chromatography.
  • ketocarotenoids are isolated from the fruits with 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 selected from the group astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin.
  • ketocarotenoid is astaxanthin.
  • the transgenic plants are preferably generated by transforming the starting plants with a nucleic acid construct which comprises at least one, preferably also a plurality of the above-described nucleic acids which are functionally linked to one or more regulatory signals which ensure the 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 the transcription and translation in plants are hereinbelow also referred to as expression cassettes.
  • the regulatory signals comprise one or more promoters which ensure the transcription and translation in plants.
  • the expression cassettes comprise regulatory signals, i.e. regulatory nucleic acid sequences which regulate the expression of the coding sequence in the host cell.
  • an expression cassette comprises upstream, i.e. at the 5′terminus of the coding sequence, a promoter and downstream, i.e. at the 3′terminus, a polyadenylation signal and, if appropriate, further regulatory elements which are linked operably with the interjacent coding sequence for at least one of the above-described genes.
  • Operable linkage is understood as meaning the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfil its intended function when the coding sequence is expressed.
  • sequences which are preferred for the operable linkage are targeting sequences for ensuring the subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrium, in the endoplasmic reticulum (ER), in the nucleus, in oil bodies or other compartments, and translation enhancers such as the tobacco mosaic virus 5′-leader sequence (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).
  • any promoter which is capable of controlling the expression of foreign genes in plants is suitable as promoter of the expression cassette.
  • Constant promoter means those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of the plant's development, preferably at all points in time of the plant's development.
  • a promoter which is used by preference is, in particular, a plant promoter or a promoter derived from a plant virus.
  • a promoter of the CaMV cauliflower mosaic virus 35S transcript (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 6:221-228) or the 19S CaMV promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8:2195-2202).
  • a further suitable constitutive promoter is the pds promoter (Pecker et al. (1992) Proc. Natl. Acad. Sci USA 89: 4962-4966) or the “Rubisco small subunit (SSU)” promoter (U.S. Pat. No. 4,962,028), the legumin B promoter (GenBank Acc. No. X03677), the promoter of the Agrobacterium nopaline synthase, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al.
  • the expression cassettes may also comprise a chemically inducible promoter (review paper: 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 can be controlled at a particular point in time.
  • a chemically inducible promoter such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), salicylic-acid-inducible promoter (WO 95/19443), a benzene-sulfonamide-inducible promoter (EP 0 388 186), a tetracyclin-inducible promoter (Gatz et al.
  • promoters are those which are induced by biotic or abiotic stress such as, for example, the pathogen-inducible promoter of the PRP1 gene (Ward et al. (1993) Plant Mol Biol 22:361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (U.S. Pat. No. 5,187,267), the cold-inducible alpha-amylase promoter from potato (WO 96/12814), the light-inducible PPDK promoter or the wounding-induced pinII promoter (EP375091).
  • the pathogen-inducible promoter of the PRP1 gene Ward et al. (1993) Plant Mol Biol 22:361-366
  • the heat-inducible hsp70 or hsp80 promoter from tomato U.S. Pat. No. 5,187,267
  • the cold-inducible alpha-amylase promoter from potato
  • Pathogen-inducible promoters comprise the promoters of genes which are induced as the result of a pathogen attack such as, for example, genes of PR proteins, SAR proteins, ⁇ -1,3-glucanase, chitinase and the like (for example Redolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al. (1992) The Plant Cell 4:645-656; Van Loon (1985) Plant Mol Viral 4:111-116; Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2:325-342; Somssich et al.
  • genes of PR proteins, SAR proteins, ⁇ -1,3-glucanase, chitinase and the like for example Redolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes
  • wounding-inducible promoters such as that of the promoter of the pinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) Nat Biotech 14:494-498), of the wun1 and wun2 gene (U.S. Pat. No. 5,428,148), of the win1 and win2 gene (Stanford et al. (1989) Mol Gen Genet 215:200-208), of the systemin (McGurl et al. (1992) Science 225:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol 22:783-792; Ekelkamp et al. (1993) FEBS Letters 323:73-76), of the MPI gene (Corderok et al. (1994) The Plant J 6(2):141-150) and the like.
  • wounding-inducible promoters such as that of the promoter of the pinII
  • suitable promoters are, for example, fruit-maturation-specific promoters such as, for example, the fruit-maturation-specific promoter from tomato (WO 94/21794, EP 409 625).
  • Some of the promoters which the development-promoters comprise are the tissue-specific promoters since, naturally, the individual tissues are formed as a function of the development.
  • promoters which ensure the expression in tissues or plant parts 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 hereof.
  • Tuber-specific, storage-root-specific or root-specific promoters are, for example, the patatin promoter class I (B33) or the promoter of the cathepsin D inhibitor from potato.
  • 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 the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
  • Examples of anther-specific promoters are the 5126 promoter (U.S. Pat. No. 5,689,049, U.S. Pat. No. 5,689,051), the glob-1 promoter or the g-zein promoter.
  • Fruit-specific promoters are, for example,
  • Especially preferred in the method according to the invention are constitutive and, in particular, fruit-specific promoters.
  • An expression cassette is prefereably prepared by fusing a suitable promoter with an above-described nucleic acid encoding a ketolase and preferably a nucleic acid which is inserted between promoter and nucleic acid sequence and which encodes a plastid-specific transit peptide, and with a polyadenylation signal, using customary recombination and cloning techniques as are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman und L. W.
  • nucleic acids which encode a plastidic transit peptide and which are preferably inserted ensure the localization in plastids and in particular in chromoplasts.
  • expression cassettes whose nucleic acid sequence encodes a ketolase fusion protein, where part of the fusion protein is a transit peptide which governs the translocation of the polypeptide.
  • Preferred are chromoplast-specific transit peptides which are cleaved enzymatically from the ketolase moiety after translocation of the ketolase into the chromoplasts.
  • the transit peptide which is derived from the plastidic Nicotiana tabacum transketolase or from another transit peptide (for example the transit peptide of the Rubisco small subunit (rbcS) or the transit peptide of the ferredoxin-NADP oxidoreductase and of the isopentenyl-pyrophosphate isomerase-22) or its functional equivalent.
  • a plastidic transit peptide examples include the transit peptide of the plastidic isopentenyl-pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana and the transit peptide of the ribulose-bisphosphate carboxylase small subunit (rbcS) from pea (Guerineau, F, Woolston, S, Brooks, L, Mullineaux, P (1988) An expression cassette for targeting foreign proteins into the chloroplasts. Nucl. Acids Res. 16: 11380).
  • IPP-2 plastidic isopentenyl-pyrophosphate isomerase-2
  • rbcS ribulose-bisphosphate carboxylase small subunit
  • nucleic acids according to the invention can be generated synthetically or obtained naturally or comprise a mixture of synthetic and natural nucleic acid constituents, and consist of various heterologous gene segments from a variety of organisms.
  • Preferrred are, as described above, synthetic nucleotide sequences with codons which are preferred by plants. These codons which are preferred by plants can be determined from codons with the highest protein frequency which are expressed in most of the plant species of interest.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame.
  • adaptors or linkers may be added to the fragments.
  • the promoter and the terminator regions can be provided, in the direction of transcription, with a linker or polylinker comprising one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, in most cases 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, frequently less than 60 bp, but at least 5 bp, within the regulatory regions.
  • the promoter can either be native, or homologous, or else foreign, or heterologous, to the host plant.
  • the expression cassette comprises, in the 5′-3′ direction of transcription, the promoter, a coding nucleic acid sequence or a nucleic acid construct and a region for transcriptional termination. Various termination regions can be exchanged for one another as desired.
  • 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 H M. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular the gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 et seq.), or functional equivalents.
  • transformation The transfer of foreign genes in the genome of a plant is referred to as transformation.
  • Suitable methods for the transformation of plants are the transformation of protoplasts by means of polyethylene-glycol-induced DNA uptake, the biolistic method using the gene gun—what is known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-comprising solution, microinjection, and the above-described Agrobacterium -mediated gene transfer.
  • the above methods are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. 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 cloned into a vector which is suitable for the transformation of 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 which have been transformed with such a vector can then be used in the known manner for the transformation of plants, in particular crop plants, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently growing them in suitable media.
  • an expression cassette is incorporated, as insertion, into a recombinant vector whose vector DNA comprises additional functional regulatory signals, for example sequences for replication or integration.
  • additional functional regulatory signals for example sequences for replication or integration.
  • Suitable vectors are described, inter alia, in “Methods in Plant Molecular Biology and Biotechnology” (CRC Press), chapter 6/7, pp. 71-119 (1993).
  • expression can take place constitutively or, preferably, specifically in the fruits, depending on the choice of the promoter.
  • the invention furthermore relates to a method for the production of genetically modified plants, wherein a nucleic acid construct comprising, in functional linkage, a fruit-specific promoter and nucleic acids encoding a ketolase is introduced into the genome of the starting plant.
  • the invention furthermore relates to the genetically modified plants with a ketolase activity in fruits in comparison with the starting plant.
  • the preferred genetically modified plants therefore comprise, in fruits, at least one nucleic acid encoding a ketolase.
  • the invention therefore especially preferably relates to an above-described genetically modified plant, wherein, starting from a starting plant, at least one nucleic acid encoding a ketolase has been introduced into the plant.
  • the invention relates to genetically modified plants selected from the plant genera Actinophloeus, Aglaeonema, Ananas, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Cliptocalix, 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, Hibiscus, Hippophaea, Iris, Lathyrus, Lonicera, Luffa, Lycium, Lycopersicum, Malp
  • Very especially preferred plant genera are Ananas, Asparagus, Capsicum, Citrus, Cucumis, Cucurbita, Citrullus, Lycopersicum, Passiflora, Prunus, Physalis, Solanum, Vaccinium and Vitis, comprising at least one transgenic nucleic acid encoding a ketolase.
  • the ketolase is expressed in the fruits; especially preferably, the expression of the ketolase is highest in the fruits.
  • Especially preferred genetically modified plants have, as mentioned hereinabove, additionally an increased hydroxylase activity and/or ⁇ -cyclase activity in comparison with a wild plant. Further preferred embodiments are described above in the method according to the invention.
  • the present invention furthermore relates to the transgenic plants, their propagation material and their plant cells, tissues or parts, in particular their fruits.
  • the genetically modified plants can be used for the production of ketocarotenoids, in particular astaxanthin.
  • Genetically modified plants according to the invention which can be consumed by humans and animals and which have an increased ketocarotenoid content can also be used for example directly or after processing known per se as foodstuff or feedstuff, or else as food or feed supplement. Furthermore, the genetically modified plants can be used for the production of ketocarotenoid-comprising extracts of the plants and/or for the production of feed and food supplements.
  • the genetically modified plants have an increased ketocarotenoid content in comparison with the wild type.
  • An increased ketocarotenoid content is, as a rule, understood as meaning an increased total ketocarotenoid content.
  • an increased ketocarotenoid content is also understood as meaning, in particular, a modified 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 in comparison with the wild type.
  • an increased content is in particular understood as meaning a generated content of ketocarotenoids or astaxanthin.
  • Recombinant DNA molecules were sequenced using a laser fluorescence DNA sequencer from Licor (available from MWG Biotech, Ebersbach) following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
  • the cDNA which encodes the ketolase from Haematococcus pluvialis was amplified from Haematococcus pluvialis (strain 192.80 of the “Sammlung von Algenkulturen der Vietnamese Göttingen” [Collection of algal cultures of the university of Göttingen]) suspension culture by means of PCR.
  • RNA from a suspension culture of Haematococcus pluvialis (strain 192.80) which had grown for 2 weeks with indirect daylight at room temperature in Haematococcus medium (1.2 g/l sodium acetate, 2 g/l yeast extract, 0.2 g/l MgCl 2 ⁇ 6H 2 O, 0.02 CaCl 2 ⁇ 2H 2 O; pH 6.8; after autoclaving addition of 400 mg/l L-asparagine, 10 mg/l FeSO 4 ⁇ H 2 O), the cells were harvested, frozen in liquid nitrogen and ground to a powder in a mortar.
  • Haematococcus medium 1.2 g/l sodium acetate, 2 g/l yeast extract, 0.2 g/l MgCl 2 ⁇ 6H 2 O, 0.02 CaCl 2 ⁇ 2H 2 O; pH 6.8; after autoclaving addition of 400 mg/l L-asparagine, 10 mg/l FeSO 4 ⁇ H 2 O
  • RNA For the cDNA synthesis, 2.5 ug of total RNA were denatured for 10 min at 60° C., cooled on ice for 2 minutes and transcribed into cDNA by means of a cDNA kit (Ready-to-go-you-prime-beads, Pharmacia Biotech) following the manufacturer's instructions and using an antisense-specific primer (PRI SEQ ID No. 29).
  • a cDNA kit Ready-to-go-you-prime-beads, Pharmacia Biotech
  • PRI SEQ ID No. 29 an antisense-specific primer
  • the PCR conditions were as follows:
  • the PCR for the amplification of the cDNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 53° C. 2 minutes 72° C. 3 minutes 1X 72° C. 10 minutes
  • the PCR amplification with SEQ ID No. 29 and SEQ ID No. 30 results in a 1155 bp fragment which encodes a protein consisting of the entire primary sequence (SEQ ID No. 22).
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega), giving rise to the clone pGKETO2.
  • the cDNA which encodes the ketolase from Haematococcus pluvialis (strain 192.80) which is truncated at the N terminus by 14 amino acids was amplified by means of PCR from Haematococcus pluvialis suspension culture (strain 192.80 of the “Sammlung von Algenkulturen der Vietnamese Göttingen”).
  • the preparation of total RNA from a suspension culture of Haematococcus pluvialis was carried out as described in Example 1.
  • the nucleic acid encoding a ketolase from Haematococcus pluvialis (strain 192.80) which is truncated at the N-terminus by 14 amino acids was amplified by means of polymerase chain reaction (PCR) from Haematococcus pluvialis using a sense-specific primer (PR3 SEQ ID No. 31) and an antisense-specific primer (PR1 SEQ ID No. 29).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 53° C. 2 minutes 72° C. 3 minutes 1X 72° C. 10 minutes
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) using standard methods and the clone pGKETO3 was obtained. Sequencing reactions with the primers T7 and SP6 confirmed a sequence which is identical to the sequence SEQ ID No. 22, the 5′ region (positions 1-53) of SEQ ID No. 22 in the amplificate SEQ ID No. 24 having been replaced by a nonamer sequence whose sequence deviates. This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • the cDNA which encodes the ketolase from Haematococcus pluvialis (strain 192.80) consisting of the entire primary sequence and fused C-terminal myc tag was prepared by means of PCR using the plasmid pGKETO2 (described in Example 1) and the primers PR15 (SEQ ID No. 32).
  • the primer PR15 is compsed of an antisense-specific 3′ region (nucleotides 40-59) and a myc-tag encoding 5′ region (nucleotides 1-39).
  • reaction mixture comprising:
  • the nucleic acid encoding a ketolase from Haematococcus pluvialis (strain 192.80) consisting of the entire primary sequence and fused C-terminal myc tag was amplified from Haematococcus pluvialis by means of polymerase chain reaction (PCR) 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 encodes a ketolase protein with fused C-terminal myc tag was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 53° C. 1 minute 72° C. 1 minute 1X 72° C. 10 minutes
  • the PCR amplification with SEQ ID No. 32 and SEQ ID No. 30 results in a 1032 bp fragment which encodes a protein consisting of the entire primary sequence of the ketolase from Haematococcus pluvialis as double translational fusion with the rbcs transit peptide at the N terminus und 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 pGKETO4 was obtained. Sequencing reactions with the primers T7 and SP6 confirmed a sequence which was identical to the sequence SEQ ID No. 22, the 3′ region (positions 993-1155) of SEQ ID No. 22 in the amplificate SEQ ID No. 26 having been replaced by a 39 bp sequence which deviated. This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • Cloning was effected by isolating the 1038 bp EcoRI/SpHI fragment from pGKETO4 and ligation with the EcoRI-SpHI-cut vector pJIT117. The ligation gives rise to a translational fusion between the C terminus of the rbcS transit peptide sequence and the N terminus of the ketolase sequence.
  • the clone which comprises the Haematococcus pluvialis ketolase with fused C-terminal myc tag in correct orientation as translational N-terminal fusion with the rbcs transit peptide is named pJKET4.
  • ketolase from Haematococcus pluvialis in L. esculentum and in Tagetes erecta was under the control of the constitutive promoter d35S from CaMV (Franck et al. 1980, Cell 21: 285-294). The expression was carried out with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715).
  • the ketolase from Haematococcus pluvialis was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was under the control of a modified version AP3P of the promoter AP3 of Arabidopsis thaliana (AL132971: nucleotide region 9298-10200; Hill et al. (1998) Development 125: 1711-1721).
  • the DNA fragment which comprises the AP3 promoter region ⁇ 902 to +15 from Arabidopsis thaliana was prepared by means of PCR using genomic DNA (isolated from Arabidopsis thaliana by standard methods) and the primers PR7 (SEQ ID No. 33) and PR10 (SEQ ID No. 36).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which comprises the AP3 promoter fragment was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 50° C. 1 minute 72° C. 1 minute 1X 72° C. 10 minutes
  • the 922 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods, giving rise to the plasmid pTAP3.
  • Sequencing the clone pTAP3 confirms a sequence which differs from the published AP3 sequence (AL132971, nucleotide region 9298-10200) only by one insertion (one G in position 9765 of the sequence AL132971) and one base substitution (one G instead of one A in position 9726 of the sequence AL132971). These nucleotide differences were reproduced in an independent amplification experiment and thus represent the actual nucleotide sequence in the Arabidopsis thaliana plants used.
  • the PCR conditions were as follows:
  • PCR reactions for the amplification of the DNA fragments which comprise the region 10200-9771 and the region 9526-9285 of the AP3 promoter were carried out in 50- ⁇ l batches of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 50° C. 1 minute 72° C. 1 minute 1X 72° C. 10 minutes
  • the recombinant PCR comprises annealing of the amplificates A7/9 and A8/10, which overlap over a sequence of 25 nucleotides, complementation to give a double strand, and subsequent amplification.
  • Denaturation (5 minutes at 95° C.) and annealing (slow cooling at room temperature to 40° C.) of the two amplificates A7/9 and A8/10 were carried out in 17.6 ⁇ l of reaction mixture comprising:
  • the nucleic acid encoding 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 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 50° C. 1 minute 72° C. 1 minute 1X 72° C. 10 minutes
  • the PCR amplification with SEQ ID No. 33 and SEQ ID No. 36 resulted in a 778 bp fragment which encodes the modified promoter version AP3P.
  • the amplificate was cloned into the cloning vector pCR2.1 (Invitrogen) and the clone pTAP3P was obtained. Sequencing reactions with the primers T7 and M13 confirmed a sequence with identity to the sequence AL132971, region 10200-9298, with the internal region 9285-9526 having been deleted. This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • Cloning was carried out by isolating the 771 bp SacI/HindIII fragment from pTAP3P and ligation into the SacI/HindIII-cut vector pJIT117.
  • the clone which comprises the promoter AP3P instead of the original promoter d35S is named pJAP3P.
  • the 1027 bp SpHI fragment KETO2 (described in Example 1) was cloned into the SpHI-cut vector pJAP3P.
  • the clone which comprises the fragment KETO2 in the correct orientation as N-terminal fusion with the rbcS transit peptide is named pJAP3PKETO2.
  • Tomato plants were transformed and regenerated by the published method of Ling and coworkers (Plant Cell Reports (1998), 17:843-847). A higher kanamycine concentration (100 mg/l) was used for the selection for the variety Microtom.
  • the starting explants for the transformation were cotyledons and hypocotyls of seven- to ten-day old seedlings of the line Microtom.
  • the cotyledons were divided horizontally and the hypocotyls were cut into segments 5 to 10 mm in length and placed on the medium MSBN (MS, pH 6.1, 3% sucrose+1 mg/l BAP, 0.1 mg/l NAA) which had been charged on the day before with tobacco cells grown in suspension culture.
  • MSBN MS, pH 6.1, 3% sucrose+1 mg/l BAP, 0.1 mg/l NAA
  • the tobacco cells were covered with sterile paper filters in such a way that there were no air bubbles.
  • the explants were precultured on the above-described medium for three to five days.
  • Cells of the strain Agrobacterium tumefaciens LBA4404 were transformed individually with the plasmids pS3KETO2, pS3KETO3 and pS3AP3KETO2, respectively.
  • the explants were transferred to MSZ2 medium (MS pH 6.1+3% sucrose, 2 mg/l zeatin, 100 mg/l kanamycin, 160 mg/l Timentin) and stored under low light conditions (20 to 100 ⁇ E, ohotoperiod 16 h/8 h) at 21° C. for the selective regeneration.
  • MSZ2 medium MS pH 6.1+3% sucrose, 2 mg/l zeatin, 100 mg/l kanamycin, 160 mg/l Timentin
  • low light conditions (20 to 100 ⁇ E, ohotoperiod 16 h/8 h
  • the explants were transferred every two to three weeks until shoots formed. Small shoots were separated from the explants and rooted on MS (pH 6.1+3% sucrose), 160 mg/l Timentin, 30 mg/l kanamycine, 0.1 mg/l IAA. Rooted plants were transferred to the greenhouse.
  • the fruit material of the transgenic plants was crushed in liquid nitrogen and the powder (approximately 250 to 500 mg) was extracted with 100% acetone (three portions of 500 ⁇ l each). The solvent was evaporated and the carotenoids were resuspended in 100 ⁇ l of acetone.
  • the spectra were recorded using a photodiode array detector.
  • the carotenoids were identified via their absorption spectra and their retention times in comparison with standard samples.
  • Table 1 shows the carotenoid profile in tomato fruits of the transgenic tomatoes produced in accordance with the above-described examples and of control tomato plants.
  • the genetically modified plants show a ketocarotenoid content, in particular an astaxanthin content.
  • Table 2a shows the amount of carotenoid in mature fruits of transgenic tomatoes and control plants. The data are means of different lines and shown as a percentage of the total carotenoid content.
  • Table 2b shows the amount of carotenoid in mature fruits of transgenic tomatoes and control plants. The data are means of different lines and shown as a percentage of the total carotenoid content.
  • the DNA which encodes the NP196-ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 (strain of the “American Type Culture Collection”) by means of PCR.
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation for 10 minutes at 8000 rpm. Thereafter, the bacterial cells were comminuted and ground in liquid nitrogen using a pestle and mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (volume 2 ml). After addition of 100 ⁇ l of Proteinase K (concentration: 20 mg/ml), the cell suspension was incubated for 3 hours at 37° C. Thereafter, the suspension was extracted with 500 ⁇ l of phenol.
  • the aqueous top phase was transferred to a fresh 2 ml Eppendorf reaction vessel.
  • the phenol extraction was repeated 3 times.
  • the DNA was precipitated by addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 0.6 volume isopropanol and subsequently washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating at 65° C.
  • the nucleic acid encoding a ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 by means of polymerase chain reaction (PCR) using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NP196-2 SEQ ID No. 60).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cyclic conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 55° C. 1 minute 72° C. 3 minutes 1X 72° C. 10 minutes
  • the PCR amplification with SEQ ID No. 59 and SEQ ID No. 60 resulted in a 792 bp fragment which encodes a protein consisting of the entire primary sequence (NP196, SEQ ID No. 61).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods, giving rise to the clone pNP196.
  • This clone pNP196 was therefore used for cloning into the expression vector pJAP3P described in Example 5.
  • pJAP3P was modified by replacing the 35S terminator by the OCS terminator (octopine synthase) of the Ti plasmid pTi15955 of Agrobacterium tumefaciens (database entry X00493, position 12.541-12.350, Gielen et al. (1984) EMBO J. 3 835-846).
  • the DNA fragment which comprises the OCS terminator region was prepared by means of PCR using the plasmid pHELLSGATE (database entry AJ311874, Wesley et al. (2001) Plant J. 27 581-590, isolated from E. coli by standard methods) and the primers 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 comprises the octopine synthase (OCS) terminator region (SEQ ID 65) was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 50° C. 1 minute 72° C. 1 minute 1X 72° C. 10 minutes
  • the 210 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard conditions, giving rise to the plasmid pOCS.
  • Sequencing of the clone pOCS verified a sequence which agrees with a sequence segment on the Ti plasmid pTi15955 of Agrobacterium tumefaciens (database entry X00493) from position 12.541 to 12.350.
  • Cloning was carried out by isolating the 210 bp SalI/XhoI fragment from pOCS and ligation into the SalI/XhoI-cut vector pJAP3P.
  • This clone is named pJOAP and was therefore used for cloning into the expression vector pJOAP:NP196.
  • Cloning was effected by isolating the 782 bp SphI fragment from pNP196 and ligation into the SphI-cut vector pJOAP.
  • the clone which comprises the NP196 ketolase of Nostoc punctiforme in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NP196.
  • the NP196-ketolase from Nostoc punctiforme was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was effected under the control of the promoter AP3P from Arabidopsis thaliana described in Example 5.
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled NP196-ketolase from Nostoc punctiforme ATCC 29133 into L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).
  • fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), encoding the Nostoc punctiforme NP196-ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.
  • the DNA which encodes the NOST-ketolase from Nostoc punctiforme PCC 7120 was amplified by means of PCR from Nostoc PCC 7120 (strain of the Pasteur Culture Collection of Cyanobacterium).
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation for 10 minutes at 8000 rpm. Thereafter, the bacterial cells were crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (volume 2 ml). After addition of 100 ⁇ l of Proteinase K (concentration: 20 mg/ml), the cell suspension was incubated for 3 hours at 37° C. Thereafter, the suspension was extracted with 500 ⁇ l of phenol. After centrifugation for 5 minutes at 13 000 rpm, the aqueous top phase was transferred to a fresh 2 ml Eppendorf reaction vessel.
  • the phenol extraction was repeated 3 times.
  • the DNA was precipitated by addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 0.6 volume isopropanol and subsequently washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating at 65° C.
  • the nucleic acid encoding a ketolase from Nostoc PCC 7120 was amplified from Nostoc PCC 7120 by means of polymerase chain reaction (PCR) using a sense-specific primer (NOST-1, SEQ ID No. 66) and an antisense-specific primer (NOST-2, SEQ ID No. 67).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 ⁇ l of reaction mixture comprising:
  • This clone pNOST was therefore used for cloning into the expression vector pJOAP (described in Example 9).
  • Cloning was effected by isolating the 799 bp SphI fragment from pNOST and ligation into the SphI-cut vector pJOAP.
  • the clone which comprises the NOST-ketolase from Nostoc PCC 7120 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NOST.
  • the NOST-ketolase from Nostoc spp. PCC 7120 was expressed in L. esculentum with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was effected under the control of the promoter AP3P from Arabidopsis thaliana (described in Example 5).
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled NOST-ketolase from Nostoc spp. PCC 7120 into L. esculentum was generated using the binary vector pSUN3 (WO02/00900).
  • fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NOST KETO CDS (774 bp), encoding the Nostoc spp. PCC 7120 NOST-ketolase, fragment OCS terminator (192 bp) the polyadenylation signal of octopine synthase.
  • the DNA which encodes the NP195-ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 (strain of the American Type Culture Collection) by means of PCR.
  • the preparation of genomic DNA from a suspension culture of Nostoc punctiforme ATCC 29133 was described in Example 9.
  • the nucleic acid encoding a ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 by means of polymerase chain reaction (PCR) using a sense-specific primer (NP195-1, SEQ ID No. 70) and an antisense-specific primer (NP195-2, SEQ ID No. 71).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 55° C. 1 minute 72° C. 3 minutes 1X 72° C. 10 minutes
  • the PCR amplification with SEQ ID No. 70 and SEQ ID No. 71 resulted in an 819 bp fragment which encodes a protein consisting of the entire primary sequence (SEQ ID No. 72).
  • the amplificate was cloned into the PCR cloning vector pGEM-T (Promega) and the clone pNP195 was obtained.
  • This clone pNP195 was therefore used for cloning into the expression vector pJOAP (described in Example 9).
  • Cloning was effected by isolating the 709 bp SphI fragment from pNP195 and ligation into the SphI-cut vector pJOAP.
  • the clone which comprises the NP195-ketolase from Nostoc punctiforme ATCC 29133 in correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NP195.
  • the NP195-ketolase from Nostoc punctiforme ATCC 29133 was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was effected under the control of the promoter AP3P from Arabidopsis thaliana (described in Example 5).
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled NP195-ketolase from Nostoc punctiforme ATCC 29133 into L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).
  • fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), encoding the Nostoc punctiforme ATCC 29133 NP195-ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.
  • the DNA which encodes the ketolase from Nodularia spumignea NSOR10 was amplified from Nodularia spumignea NSOR10 by means of PCR.
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation for 10 minutes at 8000 rpm. Thereafter, the bacterial cells were comminuted and ground in liquid nitrogen using a pestle and mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (volume 2 ml). After addition of 100 ⁇ l of Proteinase K (concentration: 20 mg/ml), the cell suspension was incubated for 3 hours at 37° C. Thereafter, the suspension was extracted with 500 ⁇ l of phenol.
  • the aqueous top phase was transferred to a fresh 2 ml Eppendorf reaction vessel.
  • the phenol extraction was repeated 3 times.
  • the DNA was precipitated by addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 0.6 volume isopropanol and subsequently washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating at 65° C.
  • the nucleic acid encoding a ketolase from Nodularia spumignea NSOR10 was amplified from Nodularia spumignea NSOR10 by means of polymerase chain reaction (PCR) using a sense-specific primer (NODK-1, SEQ ID No. 74) and an antisense-specific primer (NODK-2 SEQ ID No. 75).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR was carried out under the following cycling conditions: 1X 94° C. 2 minutes 35X 94° C. 1 minute 55° C. 1 minute 72° C. 3 minutes 1X 72° C. 10 minutes
  • the PCR amplification with SEQ ID No. 74 and SEQ ID No. 75 resulted in an 720 bp fragment which encodes 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.
  • This clone pNODK was therefore used for cloning into the expression vector pJOAP (described in Example 9).
  • Cloning was effected by isolating the 710 bp SphI fragment from pNODK and ligation into the SphI-cut vector pJOAP.
  • the clone which comprises the NODK-ketolase from Nodularia spumignea NSOR10 in correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NODK.
  • NODK-ketolase from Nodularia spumignea NSOR10 was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was under the control of the promoter AP3P from Arabidopsis thaliana (described in Example 5).
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled NP195-ketolase from Nostoc punctiforme ATCC 29133 into L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).
  • fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NODK KETO CDS (690 bp), encoding the Nodularia spumignea NSOR10 NODK-ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.
  • the chromoplast-specific ⁇ -hydroxylase from Lycopersicon esculentum is expressed in tomato under the control of the fruit-specific promoter AP3P from Arabidopsis (Example 2).
  • the terminator element used is LB3 (database entry AX696005) from Vicia faba.
  • the sequence of the chromoplast-specific ⁇ -hydroxylase was prepared by RNA isolation, reverse transcription and PCR.
  • the DNA fragment which comprises the LB3 terminator region was isolated by means of PCR.
  • Genomic DNA is isolated from Vicia faba tissue by standard methods 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 50 ⁇ l of reaction mixture comprising:
  • the PCR amplification with SEQ ID No. 78 and SEQ ID No. 79 results in a 307 bp fragment (SEQ ID No. 80) which comprises 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 is identical to the DNA sequence of 3-298 of the database entry AX696005. This clone is named pLB3 and is therefore used for cloning into the vector pJAP3P (see Example 5).
  • the expression cassette pJAP3P was modified by replacing the 35S terminator by the legumin LB3 terminator of Vicia faba (database entry AX696005; WO03/008596) (see below).
  • RNA is prepared from tomato.
  • 100 mg of the frozen pulverized flowers are transferred to a reaction vessel and taken up in 0.8 ml of Trizol buffer (LifeTechnologies).
  • the suspension is extracted with 0.2 ml of chloroform. After centrifugation for 15 minutes at 12 000 g, the aqueous supernatant is removed and transferred to a fresh reaction vessel and extracted with one volume of ethanol.
  • the RNA is precipitated with one volume of isopropanol, washed with 75% of ethanol, and the pellet is dissolved in DEPC water (overnight incubation of water with 1/1000 volume of diethyl pyrocarbonate at room temperature, followed by autoclaving).
  • RNA concentration is determined photometrically.
  • cDNA synthesis 2.5 ⁇ g of total RNA are denatured for 10 minutes at 60° C., cooled on ice for 2 minutes, and transcribed into cDNA using a cDNA kit (Ready-to-go-you-prime-beads, Pharmacia Biotech) following the manufacturer's instructions and using an antisense-specific primer (PR17 SEQ ID NO: 56).
  • a cDNA kit Ready-to-go-you-prime-beads, Pharmacia Biotech
  • the nucleic acid encoding the ⁇ -hydroxylase was amplified from tomato by means of polymerase chain reaction (PCR) using a sense-specific primer (VPR204, SEQ ID No. 81) and an antisense-specific primer (PR215 SEQ ID No. 82).
  • PCR polymerase chain reaction
  • the PCR for the amplification of the DNA which encodes a ⁇ -hydroxylase protein consisting of the entire primary sequence was carried out in 50 ⁇ l of a reaction mixture comprising:
  • the PCR amplification with VPR204 and PR215 results in a 1040 bp fragment (SEQ ID No. 83) which encodes the b-hydroxylase.
  • the amplificate is cloned into the PCR cloning vector pCR 2.1 (Invitrogen). This clone is named pCrtR-b2.
  • Sequencing reactions of the clone pCrtR-b2 with the primers M13-R and M13-R confirmed a sequence which is identical to the DNA sequence of 33-558 of the database entry BE354440 and which is identical to the DNA sequence of 1-1009 of the database entry Y14810.
  • the clone pCrtR-b2 is therefore used for cloning into the vector pCSP02 (see below).
  • the first cloning step was carried out by isolating the 1034 bp HindIII/EcoRI fragment from pCrtR-b2, derived from the cloning vector pCR-2.1 (Invitrogen), and ligation with the HindIII/EcoRI-cut vector pJPA3P (see Example 5).
  • the clone which comprises the b-hydroxylase fragment CrtR-b2 is named 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 which comprises the 296 bp terminator LB3 is named pCSP03.
  • Ligation gives rise to a transcriptional fusion between the terminator LB3 and the b-hydroxylase fragment CrtR-b2.
  • it gives rise to a transcriptional fusion between the AP3P promoter and the b-hydroxylase fragment.
  • the expression of the B gene from Lycopersicon esculentum in tomato takes place under the control of the fruit-specific promoter PDS (phytoene desaturase; database entry U46919) from Lycopersicon esculentum.
  • the terminator element used is 35S from CaMV.
  • the sequence of the B gene was prepared from genomic DNA from Lycopersicon esculentum by means of PCR.
  • the oligonucleotide primers BGEN-1 (SEQ ID No. 85) and BGEN-2 (SEQ ID No. 86) were used for isolating 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 PCR amplification with BGEN-1 and BGEN-2 results in a 1505 kb fragment (SEQ ID No. 87) which encodes the b-hydroxylase.
  • the amplificate is cloned into the PCR cloning vector pCR-2.1 (Invitrogen). This clone is named pBGEN.
  • genomic DNA is isolated from Lycopersicon esculentum tissue by standard methods and employed by genomic PCR using the primers PDS-1 and PDS-2.
  • the PCR for the amplification of this PDS promoter fragment is carried out in 50 ⁇ l of reaction mixture comprising:
  • the PCR amplification with PDS-1 and PDS-2 results in a fragment which comprises the sequence for the PDS promoter.
  • the amplificate is cloned into pCR4-BLUNT (Invitrogen). This clone is named pPDS.
  • the first cloning step is carried out by isolating the 1499 bp NcoI/EcoRI fragment from pBGEN, derived from the cloning vector pCR2.1 (Invitrogen).
  • pBGEN is cut with BamHI, and the 3′ ends are filled in by standard methods (30 minutes at 30° C.) (Klenew fill-in) and then a partial digest with NcoI is carried out in which the resulting 1499 kb fragment isolates.
  • This fragment was subsequently cloned into pCSP02 which, previously cut with EcoRI, the 3′ ends filled in by standard methods (30 minutes at 30° C.) (Klenow fill-in), and is then cut with NcoI.
  • the clone which comprises the 1497 bp B gene fragment BGEN is named pJAP:BGEN.
  • the ligation gives rise to a transcriptional fusion between the 35S terminator and the B gene.
  • the second cloning step is carried out by isolating the 2078 bp PDS PROM fragment from pPDS.
  • pPDS is cut with SmaI and then a partial digest with SacI is carried out in which the resulting 2088 bp fragment isolates.
  • SacI SacI
  • This fragment was subsequently cloned into pJAP:BGEN which previously cut with BamHI, the 3′ ends filled in by standard methods (30 minutes at 30° C.) (Klenow fill-in), and is then cut with SacI.
  • the ligation gives rise to transcriptional fusion between the promoter PDS and the B gene.
  • the clone which comprises the 2078 bp PDS promoters BGEN is named pJPDS:BGEN.
  • a double construct which comprises expression cassettes for overexpressing the Nostoc punctiforme ATCC 29133 NP196 ketolase and for overexpressing the B-hydroxylase is prepared.
  • the fragment AP3P:b-hydroxylase:LB3 which comprises the B-hydroxylase expression cassette, is isolated from pCSP03 as a 2104 bp Ecl136II/XhoI fragment (described in Example 18).
  • the 3′ ends are filled in by standard methods (Klenow fill-in; 30 minutes at 30° C.). Thereafter, this fragment was cut with Ecl136II and EcoRI in the vector MSP120 (described in Example 10), and the 3′ ends were filled in by standard methods (30 minutes at 30° C.; Klenow fill-in).
  • T-DNA which comprises two expression cassettes: firstly, a cassette for the chromoplast-specific overexpression of the B-hydroxylase from Lycopersicon esculentum and, secondly, a cassette for overexpressing the ketolase NP196 from Nostoc punctiforme.
  • the B-hydroxylase downregulation cassette can ligate into the vector in two orientations. It is preferred to use the version in which the two expression cassettes have the same orientation (see FIG. 14 ). This version can be identified by PCR as described:
  • the PCR for the amplification of the PR206-PR010 plasmid fragment which comprises the ligation of LB3 terminator of the B-hydroxylase cassette and the AP3P promoter of the ketolase cassette is carried out in 50 ⁇ l of reaction mixture comprising:
  • this B gene overexpression cassette into expression vectors for the Agrobacterium -mediated transformation of tomato is carried out by isolating the 4362 bp EcoRV/XhoI fragment from pJPDS:BGEN (see Example 19) and ligation into the SmaI/XhoI-cut vector pBHYX:NP196 (described above).
  • the ligation gives rise to a T-DNA which comprises three expression cassettes: firstly, a cassette for overexpressing the B gene, secondly, a cassette for overexpressing the NP196-1 ketolase from Nostoc punctiforme, and, thirdly, a cassette for the chromoplast-specific overexpression of the B-hydroxylase from Lycopersicon esculentum ( FIG.
  • fragment AP3P PROM (765 bp) comprises the AP3P promoter, the fragment BHYX b2 CDS (2 bp) the B-hydroxylate CrtRb2, fragment LB3 TERM (296 bp) the LB3 terminator.
  • fragment AP3P PROM (765 bp) comprises 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 Nostoc punctiforme ATCC29133, and OCS TERM (192 bp) the polyadenylation signal of the octopin synthase gene.
  • fragment PDS PROM (2078 bp) comprises the PDS promoter, fragment BGEN CDS (1497 bp) the B gene sequence, and fragment 35S TERM (746 bp) the 35 S terminator.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Nutrition Science (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Animal Husbandry (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Reproductive Health (AREA)
  • Insects & Arthropods (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Pregnancy & Childbirth (AREA)
  • Birds (AREA)
  • Botany (AREA)
  • Physiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Fodder In General (AREA)
US10/524,829 2002-08-20 2003-08-18 Method for producing ketocarotinoids in plant fruit Abandoned US20070094749A1 (en)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
DE10238980.2 2002-08-20
DE10238979A DE10238979A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Zeaxanthin und/oder dessen biosynthetischen Zwischen- und/oder Folgeprodukten
DE10238978.0 2002-08-20
DE10238978A DE10238978A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Ketocarotinoiden in Früchten von Pflanzen
DE2002138980 DE10238980A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Ketocarotinoiden in Blütenblättern von Pflanzen
DE10238979.9 2002-08-20
DE2002153112 DE10253112A1 (de) 2002-11-13 2002-11-13 Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten Organismen
DE10253112.9 2002-11-13
DE2002158971 DE10258971A1 (de) 2002-12-16 2002-12-16 Verwendung von astaxanthinhaltigen Pflanzen oder Pflanzenteilen der Gattung Tagetes als Futtermittel
DE10258971.2 2002-12-16
PCT/EP2003/009107 WO2004018695A2 (de) 2002-08-20 2003-08-18 Verfahren zur herstellung von ketocarotinoiden in früchten von pflanzen

Publications (1)

Publication Number Publication Date
US20070094749A1 true US20070094749A1 (en) 2007-04-26

Family

ID=31950810

Family Applications (5)

Application Number Title Priority Date Filing Date
US10/524,972 Abandoned US20060031963A1 (en) 2002-08-20 2003-08-18 Method for the production of ketocarotinoids in flower petals on plants
US10/524,827 Expired - Fee Related US7385123B2 (en) 2002-08-20 2003-08-18 Process for preparing ketocarotenoids in genetically modified organisms
US10/524,652 Abandoned US20060253927A1 (en) 2002-08-20 2003-08-18 Method for the production of zeaxanthin and/or biosynthetic intermediates and/or subsequent products thereof
US10/524,647 Expired - Fee Related US7381541B2 (en) 2002-08-20 2003-08-18 Methods for producing animal feed preparations with astaxanthin-containing plants or parts of plants of the genus Tagetes
US10/524,829 Abandoned US20070094749A1 (en) 2002-08-20 2003-08-18 Method for producing ketocarotinoids in plant fruit

Family Applications Before (4)

Application Number Title Priority Date Filing Date
US10/524,972 Abandoned US20060031963A1 (en) 2002-08-20 2003-08-18 Method for the production of ketocarotinoids in flower petals on plants
US10/524,827 Expired - Fee Related US7385123B2 (en) 2002-08-20 2003-08-18 Process for preparing ketocarotenoids in genetically modified organisms
US10/524,652 Abandoned US20060253927A1 (en) 2002-08-20 2003-08-18 Method for the production of zeaxanthin and/or biosynthetic intermediates and/or subsequent products thereof
US10/524,647 Expired - Fee Related US7381541B2 (en) 2002-08-20 2003-08-18 Methods for producing animal feed preparations with astaxanthin-containing plants or parts of plants of the genus Tagetes

Country Status (12)

Country Link
US (5) US20060031963A1 (de)
EP (5) EP1531683B1 (de)
CN (1) CN1675367A (de)
AT (1) ATE484198T1 (de)
AU (5) AU2003258623A1 (de)
CA (5) CA2496133A1 (de)
DE (1) DE50313184D1 (de)
IL (4) IL166507A0 (de)
MX (5) MXPA05001659A (de)
NO (5) NO20050598L (de)
WO (5) WO2004018695A2 (de)
ZA (1) ZA200602230B (de)

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060107352A1 (en) * 2002-08-20 2006-05-18 Sungene Gmbh & Co. Kgaa Transgenic expression cassettes for expression of nucleic acids in the plant blooms
CA2496133A1 (en) * 2002-08-20 2004-03-04 Sungene Gmbh & Co. Kgaa Method for the production of ketocarotinoids in flower petals on plants
EP1532256A1 (de) * 2002-08-20 2005-05-25 Sungene GmbH & Co. KGaA VERFAHREN ZUR HERSTELLUNG VON &bgr;-CAROTINOIDEN
EP1554388A1 (de) * 2002-10-11 2005-07-20 Sungene GmbH & Co. KGaA Transgene expressionskassetten zur expression von nukleinsäuren in der pflanzlichen blüte
DE10300649A1 (de) * 2003-01-09 2004-07-22 Basf Ag Verfahren zur Herstellung von Ketocarotinoiden durch Kultivierung von genetisch veränderten Organismen
EP1780281B1 (de) * 2004-06-04 2013-12-11 Kirin Holdings Kabushiki Kaisha Verfahren zur herstellung von astaxanthin oder einem stoffwechselprodukt davon unter verwendung von carotinketolase und carotinketolase-genen
EP2371967B1 (de) 2005-03-18 2015-06-03 DSM IP Assets B.V. Produktion von Carotenoiden in Ölhaltigen Hefen und Pilzen
AU2006269823B2 (en) * 2005-07-11 2012-08-02 Commonwealth Scientific And Industrial Research Organisation Wheat pigment
EP2078092A2 (de) 2006-09-28 2009-07-15 Microbia, Inc. Herstellung von carotenoiden in öliger hefe und pilzen
US8753840B2 (en) * 2006-10-20 2014-06-17 Arizona Board Of Regents On Behalf Of Arizona State University Modified cyanobacteria
BRPI0806029A2 (pt) * 2007-07-19 2009-11-10 Biosigma Sa plasmìdeos para transformação de bactérias do gênero aciditiobacillus spp. e método de transformação
US20090093015A1 (en) * 2007-10-09 2009-04-09 Kemin Foods, L.C. Beta-cryptoxanthin production using a novel lycopene beta-monocyclase gene
EP2199399A1 (de) 2008-12-17 2010-06-23 BASF Plant Science GmbH Ketokarotinoidproduktion in Pflanzen
WO2010079032A1 (en) * 2008-12-17 2010-07-15 Basf Plant Science Gmbh Production of ketocarotenoids in plants
KR101983115B1 (ko) * 2011-08-08 2019-05-29 에볼바 에스아 사프란 화합물의 재조합 생성을 위한 방법 및 물질
CN114748605A (zh) 2011-11-29 2022-07-15 普罗克拉拉生物科学股份有限公司 噬菌体基因3蛋白组合物及作为淀粉样蛋白结合剂的用途
ES2558953B1 (es) * 2015-11-23 2016-11-18 Universitat De Lleida Maíz enriquecido en antioxidantes para mejorar la calidad nutricional del huevo
JP2019165635A (ja) * 2016-08-10 2019-10-03 味の素株式会社 Ｌ−アミノ酸の製造法
US10004253B1 (en) * 2017-09-05 2018-06-26 Jose-Odon Torres-Quiroga Method for increasing the health condition of crustaceans in aquaculture, survival rate and pigmentation
CN112458103B (zh) * 2021-01-28 2022-09-30 青岛农业大学 一种调控辣椒红素积累的基因CaBBX20及其应用

Family Cites Families (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9016012D0 (en) 1990-07-20 1990-09-05 Unilever Plc Pigments
EP0735137B1 (de) * 1993-12-27 2004-08-25 Kirin Beer Kabushiki Kaisha Dna - kette zur synthese von xanthophyll und prozess zur herstellung von xanthophyll
US5916791A (en) * 1995-11-24 1999-06-29 Hirschberg; Joseph Polynucleotide molecule from Haematococcus pluvialis encoding a polypeptide having a β--C--4--oxygenase activity for biotechnological production of (3S,3S)astaxanthin
US6429356B1 (en) * 1996-08-09 2002-08-06 Calgene Llc Methods for producing carotenoid compounds, and specialty oils in plant seeds
JP2001505409A (ja) * 1996-08-09 2001-04-24 カルジーン エルエルシー 植物種子中のカロチノイド化合物と特殊油の製造方法
US6221417B1 (en) * 1997-05-14 2001-04-24 Kemin Industries, Inc. Conversion of xanthophylls in plant material for use as a food colorant
US5876782A (en) * 1997-05-14 1999-03-02 Kemin Industries, Inc. Method for the conversion of xanthophylls in plant material
JP2002516117A (ja) * 1998-05-22 2002-06-04 ユニバーシティ オブ メリーランド カロチノイド・ケトラーゼ遺伝子及び遺伝子生成物、ケトカロチノイドの生成及び遺伝子を用いてカロチノイドを変成する方法
BR0007128A (pt) * 1998-06-02 2001-07-17 Univ Maryland Genes para a biossìntese e metabolismo de carotenoides e método de aplicação dos mesmos
US6232530B1 (en) 1998-11-30 2001-05-15 University Of Nevada Marigold DNA encoding beta-cyclase
DE19916140A1 (de) * 1999-04-09 2000-10-12 Basf Ag Carotinhydroxylase und Verfahren zur Herstellung von Xanthophyllderivaten
SE9903336D0 (sv) 1999-09-17 1999-09-17 Astacarotene Ab DNA construct and its use
US20050003474A1 (en) * 2001-01-26 2005-01-06 Desouza Mervyn L. Carotenoid biosynthesis
DE10201458A1 (de) * 2001-04-11 2002-10-17 Adelbert Bacher Intermediate und Enzyme des Mevalonat-unabhängigen Isoprenoidbiosyntheseweg
US6784351B2 (en) * 2001-06-29 2004-08-31 Ball Horticultural Company Targetes erecta marigolds with altered carotenoid compositions and ratios
US7575766B2 (en) * 2001-06-29 2009-08-18 Ball Horticultural Company Tagetes erecta with altered carotenoid compositions and ratios
US6372946B1 (en) * 2001-09-13 2002-04-16 Prodemex, S.A. De C.V. Preparation of 4,4′-diketo-β-carotene derivatives
WO2003077950A1 (en) 2002-03-11 2003-09-25 Prodemex, S.A. De C.V. Enhanced feeding and growth rates of aquatic animals fed an astaxanthin product derived from marigold extract
US7223909B2 (en) 2002-03-21 2007-05-29 Ball Horticultural 4-ketocarotenoids in flower petals
CA2496133A1 (en) * 2002-08-20 2004-03-04 Sungene Gmbh & Co. Kgaa Method for the production of ketocarotinoids in flower petals on plants
DE10300649A1 (de) * 2003-01-09 2004-07-22 Basf Ag Verfahren zur Herstellung von Ketocarotinoiden durch Kultivierung von genetisch veränderten Organismen
DE102004007623A1 (de) * 2004-02-17 2005-08-25 Sungene Gmbh & Co. Kgaa Promotoren zur Expression von Genen in Tagetes

Also Published As

Publication number Publication date
MXPA05001948A (es) 2005-09-08
MXPA05001899A (es) 2005-08-29
WO2004018693A3 (de) 2004-12-09
WO2004018695A3 (de) 2004-10-14
ATE484198T1 (de) 2010-10-15
US20060112451A1 (en) 2006-05-25
WO2004017749A3 (de) 2004-10-14
ZA200602230B (en) 2007-05-30
AU2003264062A1 (en) 2004-03-11
WO2004018693A2 (de) 2004-03-04
EP1531683B1 (de) 2010-10-13
MXPA05001659A (es) 2005-07-22
US7385123B2 (en) 2008-06-10
US7381541B2 (en) 2008-06-03
AU2003260424A1 (en) 2004-03-11
NO20050703L (no) 2005-05-09
CA2495235A1 (en) 2004-03-04
EP1532266A2 (de) 2005-05-25
CN1675367A (zh) 2005-09-28
DE50313184D1 (de) 2010-11-25
EP1531683A2 (de) 2005-05-25
WO2004018695A2 (de) 2004-03-04
MXPA05001944A (es) 2005-06-22
IL166771A0 (en) 2006-01-15
EP1542945A2 (de) 2005-06-22
AU2003260423A1 (en) 2004-03-11
WO2004018694A2 (de) 2004-03-04
WO2004017749A2 (de) 2004-03-04
MXPA05001811A (es) 2005-10-19
EP1532264A2 (de) 2005-05-25
WO2004018694A3 (de) 2004-09-10
NO20050598L (no) 2005-04-07
NO20050705L (no) 2005-05-18
CA2495444A1 (en) 2004-03-04
WO2004018385A3 (de) 2004-10-21
NO20050755L (no) 2005-05-19
NO20050704L (no) 2005-05-13
US20050281909A1 (en) 2005-12-22
AU2003253416A1 (en) 2004-03-11
CA2496133A1 (en) 2004-03-04
AU2003258623A1 (en) 2004-03-11
IL166507A0 (en) 2006-01-15
AU2003264062B2 (en) 2008-01-03
CA2496207A1 (en) 2004-03-04
CA2495878A1 (en) 2004-03-04
IL166770A0 (en) 2006-01-15
IL166767A0 (en) 2006-01-15
US20060031963A1 (en) 2006-02-09
US20060253927A1 (en) 2006-11-09
WO2004018385A2 (de) 2004-03-04
EP1532265A2 (de) 2005-05-25

Similar Documents

Publication Publication Date Title
US20070094749A1 (en) Method for producing ketocarotinoids in plant fruit
US20060053513A1 (en) Method for producing ketocarotenoids by cultivating genetically modified organisms
US20060194274A1 (en) Method for producing ketocarotinoids in genetically modified, non-human organisms
DE10238980A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in Blütenblättern von Pflanzen
US20060059584A1 (en) Method for the production of $g(b)-carotinoids
US20080060096A1 (en) Novel Ketolases and Method for Producing Ketocarotinoids
DE10238978A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in Früchten von Pflanzen
DE10253112A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten Organismen
DE10238979A1 (de) Verfahren zur Herstellung von Zeaxanthin und/oder dessen biosynthetischen Zwischen- und/oder Folgeprodukten
AU2004267196A1 (en) Method for producing ketocarotinoids in genetically modified, non-human organisms

Legal Events

Date Code Title Description
AS Assignment

Owner name: SUNGENE GMBH & CO. KGAA, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHOPFER, CHRISTEL RENATE;FLACHMANN, RALF;HERBERS, KARIN;AND OTHERS;REEL/FRAME:015978/0917

Effective date: 20030911

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION