EP1532265A2 - Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies - Google Patents

Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies

Info

Publication number
EP1532265A2
EP1532265A2 EP03792348A EP03792348A EP1532265A2 EP 1532265 A2 EP1532265 A2 EP 1532265A2 EP 03792348 A EP03792348 A EP 03792348A EP 03792348 A EP03792348 A EP 03792348A EP 1532265 A2 EP1532265 A2 EP 1532265A2
Authority
EP
European Patent Office
Prior art keywords
sequence
ketolase
amino acid
seq
sequence seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03792348A
Other languages
German (de)
English (en)
Inventor
Matt Sauer
Ralf Flachmann
Martin Klebsattel
Christel Renate Schopfer
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 DE2002138980 external-priority patent/DE10238980A1/de
Priority claimed from DE10238978A external-priority patent/DE10238978A1/de
Priority claimed from DE2002153112 external-priority patent/DE10253112A1/de
Priority claimed from DE2002158971 external-priority patent/DE10258971A1/de
Application filed by SunGene GmbH filed Critical SunGene GmbH
Publication of EP1532265A2 publication Critical patent/EP1532265A2/fr
Withdrawn legal-status Critical Current

Links

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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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 process for the preparation of ketocarotenoids by cultivating genetically modified organisms which have a modified ketolase activity compared to the wild type, the genetically modified organisms, and their use as food and feed and for the production of ketocarotenoid extracts.
  • Ketocarotenoids are synthesized de novo in bacteria, algae, fungi and plants.
  • Ketocarotenoids i.e. carotenoids that contain at least one keto group, such as astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin and adonixanthin are natural antioxidants and pigments that are produced by some algae and microorganisms as secondary metabolites ,
  • ketocarotenoids and especially astaxanthin are used as pigmentation aids in animal nutrition, especially in trout, salmon and shrimp farming.
  • Natural ketocarotenoids such as natural astaxanthin
  • Nucleic acids encoding a ketolase and the corresponding protein sequences have been isolated and annotated from various organisms, such as nucleic acids encoding a ketolase from Agrobacterium aurantiacum (EP 735 137, Accession NO: D58420), from Alcaligenes sp. PC-1 (EP 735137, Accession NO: D58422), Haematococcus pluvialis Flotow em.
  • EP 735 137 describes the production of xanthophylls in microorganisms, such as, for example, E. coli by introducing ketolase genes (crtW) from Agrobacterium aurantiacum or Alcaligenes sp. PC-1 in microorganisms.
  • ketolase genes crtW
  • WO 98/18910 and Hirschberg et al. describe the synthesis of ketocarotenoids in nectaries of tobacco flowers by introducing the ketolase gene from Haematococcus pluvialis (crtO) into tobacco.
  • WO 01/20011 describes a DNA construct for the production of ketocarotenoids, in particular astaxanthin, in seeds of oilseed plants such as oilseed rape, sunflower, soybean and mustard using a seed-specific promoter and a ketolase from Haematococcus pluvialis.
  • ketocarotenoids All processes described in the prior art for the production of ketocarotenoids and in particular the processes described for the production of astaxanthin have the disadvantage that the transgenic organisms provide a large amount of hydroxylated by-products, such as zeaxanthin and adonixanthin.
  • the invention was therefore based on the object of providing a process for the preparation of ketocarotenoids by cultivating genetically modified organisms, or of providing further genetically modified organisms which produce ketocarotenoids which have the disadvantages of the prior art described above to a lesser extent or no longer.
  • a method for producing ketocarotenoids has been found by cultivating genetically modified organisms which have an altered ketolase activity compared to the wild type and the altered ketolase activity is caused by a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the organisms according to the invention are preferably naturally able, as starting organisms, to produce carotenoids such as, for example, ⁇ -carotene or zeaxanthin, or can be put into a position by carotinoids such as, for example, reorganization of metabolic pathways or complementation for example, to produce ⁇ -carotene or zeaxanthin.
  • ketocarotenoids such as astaxanthin or canthaxanthin.
  • These organisms such as Haematococcus pluvialis, Paracoccus marcusii, Xan- thophyllomyces dendrorhous, Bacillus circulans, Chlorococcum, Phaffia rhodozyma, Adonis, Neochloris wimmeri, vacuolatus Protosiphon botryoides, Scotiellopsis oocystifor- mis, Scenedesmus, Chlorela zofingiensis, braunii Ankistrodesmus, Euglena sanguinea, Bacillus atrophaeus, Blakeslea already have ketolase activity as a starting or wild-type organism.
  • organisms are therefore used as starting organisms which already have ketolase activity as a wild type or starting organism.
  • the genetic modification causes an increase in ketolase activity compared to the wild type or parent organism.
  • Ketolase activity means the enzyme activity of a ketolase.
  • a ketolase is understood to mean a protein which has the enzymatic activity of introducing a keto group on the optionally substituted ⁇ -ionone ring of carotenoids.
  • a ketolase is understood to be a protein which has the enzymatic activity to convert ⁇ -carotene into canthaxanthin.
  • ketolase activity is understood to mean the amount of ⁇ -carotene or amount of canthaxanthin formed by the protein ketolase in a certain time.
  • the amount of ⁇ -carotene converted or the amount of canthaxanthin formed is increased in a certain time by the protein ketolase compared to the wild type.
  • This increase in ketolase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the ketolase activity of the wild type.
  • wild type is understood to mean the corresponding starting organism.
  • organism can be understood to mean the starting organism (wild type) or a genetically modified organism according to the invention, or both.
  • wild type is used to increase or cause the ketolase activity, for the increase in hydroxylase activity described below, for the increase described below ⁇ -cyclase activity and the increase in the content of ketocarotenoids each understood a reference organism.
  • This reference organism is preferably Haematococcus pluvialis for microorganisms which already have ketolase activity as a wild type.
  • This reference organism is preferably Blakeslea for microorganisms which, as a wild type, have no ketolase activity.
  • This reference organism is for plants which already have a ketolase activity as a wild type, preferably Adonis aestivalis, Adonis flammeus or Adonis annuus, particularly preferably Adonis aestivalis.
  • This reference organism is particularly preferred for plants which have no ketolase activity in petals as a wild type, preferably Tagetes erecta, Tagetes patula, Tagetes lucida, Tagetes pringlei, Tagetes palmeri, Tagetes minuta or Tagetes campanulata, particularly preferably Tagetes erecta.
  • ketolase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • the ketolase activity in plant or microorganism material is determined in accordance with the method of Frazer et al., (J. Biol. Chem. 272 (10): 6128-6135, 1997).
  • the ketolase activity in plant or microorganism extracts is determined with the substrates ⁇ -carotene and canthaxanthin in the presence of lipid (soy lecithin) and detergent (sodium cholate).
  • Substrate / product ratios from the ketolase assays are determined by means of HPLC.
  • the ketolase activity can be increased in various ways, for example by switching off inhibitory regulatory mechanisms at the translation and protein levels or by increasing the gene expression of a nucleic acid encoding a ketolase compared to the wild type, for example by inducing the ketolase gene by activators or by introducing nucleic acids encoding a ketolase into the organism.
  • Increasing the gene expression of a nucleic acid encoding a ketolase means, in this embodiment, the manipulation of the expression of the organisms' own endogenous ketolases. For example can be achieved by changing the promoter DNA sequence for genes coding for ketolase. Such a change, which results in a changed or preferably increased expression rate of at least one endogenous ketolase gene, can be carried out by deleting or inserting DNA sequences.
  • an increased expression of at least one endogenous ketolase gene can be achieved in that a regulatory protein which is not found or modified in the wild-type organism interacts with the promoter of these genes.
  • Such a regulator can represent a chimeric protein which consists of a DNA binding domain and a transcription activator domain, as described, for example, in WO 96/06166.
  • the ketolase activity is increased compared to the wild type by increasing the gene expression of a nucleic acid encoding a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the gene expression of a nucleic acid encoding a ketolase is increased by introducing nucleic acids encoding ketolases into the organisms, the ketolases having 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • At least one further ketolase gene is present in the transgenic organisms according to the invention in comparison to the wild type, encoding a ketolase containing the amino acid sequence SEQ. ID. NO. 2 or one of these This sequence is derived by substitution, insertion or deletion of amino acids and has an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • organisms are used as the starting organisms which, as a wild type, have no ketase activity.
  • the genetic modification causes it. Ketolase activity in organisms.
  • the genetically modified organism according to the invention thus has a ketolase activity in comparison to the genetically unmodified wild type and is therefore preferred. It is able to transgenically express a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the gene expression of a nucleic acid encoding a ketolase is caused analogously to the above-described increase in gene expression of a nucleic acid.
  • Encoding a ketolase preferably by introducing nucleic acids encoding ketolases containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has, in the starting organism.
  • any nucleic acids encoding a ketolase containing the amino acid sequence SEQ can be used in both embodiments. ID. NO. 2 or one of these Sequence derived by substitution, insertion or deletion of amino acids, which has an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has to be used.
  • ketocarotenoids with a smaller amount of hydroxylated by-products in the method according to the invention than when using the ketolase genes used in the prior art.
  • nucleic acids mentioned in the description can be, for example, an RNA, DNA or cDNA sequence.
  • nucleic acid sequences which have already been processed such as the corresponding cDNAs, are preferred use.
  • Nostoc punctiforme ATTC 29133 nucleic acid: Acc.-No. NZ_AABC01000195, base pair 55.604 to 55.392 (SEQ ID NO: 3); Protein: Acc.-No. ZP_00111258 (SEQ ID NO: 4) (annotated as putative protein) or
  • Nostoc punctiforme ATTC 29133 nucleic acid: Acc.-No. NZ_AABC01000196, base pair 140.571 to 139.810 (SEQ ID NO: 5), protein: (SEQ ID NO: 6) (not annotated), Synechococcus sp. WH 8102, nucleic acid: Acc.-No. NZ_AABD01000001, base pair 1, 354.725-1, 355.528 (SEQ ID NO: 46), protein: Acc.-No. ZP_00115639 (SEQ ID NO: 47) (annotated as putative protein),
  • Nodularia spumigena NSOR10 (Accession NO: AY210783, AAO64399; Nucleic acid: SEQ ID NO: 52, Protein: SEQ ID NO: 53)
  • ketolases and ketolase genes that can be used in the method according to the invention can be obtained, for example, from different organisms whose genomic sequence is known by comparing the identity of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the sequences SEQ ID NO described above : 2 easy to find.
  • ketolases and ketolase genes can also be easily found using hybridization techniques in a manner known per se, starting from the nucleic acid sequences described above, in particular starting from the sequences SEQ ID NO: 1 from various organisms, the genomic sequence of which is not known.
  • the hybridization can take place under moderate (low stringency) or preferably under stringent (high stringency) conditions.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with 2X SSC at 50_C) and those with high stringency (with 0.2X SSC at 50_C, preferably at 65_C) (20X SSC: 0, 3 M sodium citrate, 3 M sodium chloride, pH 7.0).
  • the temperature during the washing step can be raised from moderate conditions at room temperature, 22 ° C, to stringent conditions at 65 ° C. s
  • Both parameters, salt concentration and temperature, can be varied at the same time, one of the two parameters can also be kept constant and only the other can be varied.
  • Denaturing agents such as formamide or SDS can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C.
  • 6X SSC 0.5% SDS, 100 mg / ml denatured, fragmented salmon sperm DNA at 68 ° C, or (v) 6XSSC, 0.5% SDS, 100 mg / ml denatured, fragmented salmon sperm DNA, 50% formamide at 42 ° C, or
  • nucleic acids encoding a ketolase are introduced, containing the amino acid sequence SEQ ID NO: 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which preferably has an identity of at least 50% at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, particularly preferably at least 98% at the amino acid level with the Sequence SEQ ID NO: 2 has.
  • This can be a natural ketolase sequence that can be found as described above by comparing the identity of the sequences from other organisms or an artificial ketolase sequence that can be started from the sequence SEQ ID NO: 2 by artificial variation, for example by Substitution, insertion or deletion of amino acids has been modified.
  • substitution is to be understood as meaning the replacement of one or more amino acids by one or more amino acids. So-called conservative exchanges are preferably carried out, in which the replaced amino acid has a similar property to the original amino acid, for example replacement of Glu by Asp, Gin by Asn, Val by Ile, Leu by Ile, Ser by Thr.
  • Deletion is the replacement of an amino acid with a direct link.
  • Preferred positions for deletions are the termini of the polypeptide and the links between the individual protein domains.
  • Inserts are insertions of amino acids into the polypeptide chain, with a direct bond being formally replaced by one or more amino acids.
  • Identity between two proteins is understood to mean the identity of the amino acids over the respective total protein length, in particular the identity which is obtained by comparison using the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp PM.Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5 (2): 151-1) is calculated using the following parameters:
  • Gap opening penalty 10 Gap extension penalty 10
  • a ketolase which has an identity of at least 42% at the amino acid level with the sequence SEQ ID NO: 2 is accordingly understood to be a ketolase which, when comparing its sequence with the sequence SEQ ID NO: 2, in particular according to the above program logarithm with the above parameter set has an identity of at least 42%.
  • the sequence of the ketolase from Nostoc punctiform ATTC 29133 (SEQ ID NO: 4) with the sequence of the ketolase from Nostoc sp.
  • Strain PCC7120 (SEQ ID NO: 2) has an identity of 65%.
  • the sequence of the second ketolase from Nostoc punctiform ATTC 29133 (SEQ ID NO: 6) with the sequence of the ketolase from Nostoc sp.
  • Strain PCC7120 (SEQ ID NO: 2), for example, has an identity of 58%.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons are preferably used for this which are frequently used in accordance with the organism-specific "codon usage".
  • the "codon usage” can easily be determined on the basis of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ ID NO: 1 is introduced into the organism.
  • All of the above-mentioned ketolase genes can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pp. 896-897).
  • the sequence of the ketolase from Nostoc sp. Strain PCC7120 (SEQ ID NO: 2) has an identity of 39% (Agrobacterium aurantiacum (EP 735 137, Accession NO: D58420), 40% (Alcaligenes) with the sequences of the ketolases used in the methods of the prior art sp. PC-1 (EP 735137, Accession NO: D58422) and 20 to 21% (Haematococcus pluvialis Flotow em.
  • organisms are cultivated which, in addition to the increased ketolase activity, have an increased hydroxylase activity and / or ⁇ -cyclase activity compared to the wild type.
  • Hydroxylase activity means the enzyme activity of a hydroxylase.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity of introducing a hydroxyl group on the optionally substituted ⁇ -ionone ring of carotenoids.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity, ⁇ -carotene in zeaxanthin or canthaxanthin in
  • hydroxylase activity is understood to mean the amount of ⁇ -carotene or canthaxanthin converted or the amount of zeaxanthin or astaxanthin formed in a certain time by the protein hydroxylase.
  • the amount of ⁇ -carotene or canthaxantine or the amount of zeaxanthin or astaxanthin formed is increased by the protein hydroxylase in a certain time compared to the wild type.
  • This increase is preferably the hydroxylase activity of at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, more preferably at least • 500%, especially at least 600% of the hydroxylase activity of the wild type.
  • ⁇ -cyclase activity means the enzyme activity of a ⁇ -cyclase.
  • a ⁇ -cyclase is understood to mean a protein which has the enzymatic activity to convert a terminal, linear residue of lycopene into a ⁇ -ionone ring.
  • a ⁇ -cyclase is understood to be a protein which has the enzymatic activity to convert ⁇ -carotene into ⁇ -carotene.
  • ß-cyclase activity is understood to mean the amount of ⁇ -carotene converted or the amount of ß-carotene formed in a certain time by the protein ß-cyclase.
  • the amount of lycopene or ⁇ -carotene converted or the amount of ⁇ -carotene formed from lycopene or the formed amount of ß-carotene from ⁇ -carotene increased.
  • This increase in the ⁇ -cyclase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably min. 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.
  • hydroxylase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • the activity of the hydroxylase is according to Bouvier et al. (Biochim. Biophys. Acta 1391 (1998), 320-328) in vitro. Ferredoxin, ferredoxin-NADP oxidoreductase, catalase, NADPH and ⁇ -carotene with mono- and digalactosylglycerides are added to a certain amount of organism extract.
  • the hydroxylase activity is particularly preferably determined under the following conditions according to Bouvier, Keller, d'Harlingue and Camara (Xanthophyll bio-synthesis: molecular and functional identification of carotenoid hydroxylases for rom pepper fruits (Capsicum annuum L; Biochim. Biophys. Acta 1391 (1998), 320-328):
  • the in vitro assay is carried out in a volume of 0.250 ml.
  • the mixture contains 50 mM potassium phosphate (pH 7.6), 0.025 mg ferredoxin from spinach, 0.5 units ferredoxin-NADP + oxidoreductase from spinach, 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 of catalysis, 200 mono- and digalactosylglycerides (1: 1), 0.2 mg bovine serum albumin and organism extract in different volumes.
  • the reaction mixture is incubated at 30 ° C for 2 hours.
  • the reaction products are extracted with organic solvent such as acetone or chloroform / methanol (2: 1) and determined by HPLC.
  • ⁇ -cyclase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • the activity of the ⁇ -cyclase is determined according to Fräser and Sandmann (Biochem. Biophys. Res. Comm. 185 (1) (1992) 9-15) / n vitro. Potassium phosphate as a buffer (pH 7.6), lycopene as a substrate, stomaprotein from paprika, NADP +, NADPH and ATP are added to a certain amount of organism extract.
  • the ⁇ -cyclase activity is particularly preferably determined under the following conditions according to Bouvier, d'Harlingue and Camara (Molecular Analysis of carotenoid cyclae inhibition; Arch. Biochem. Biophys. 346 (1) (1997) 53-64):
  • the in vitro assay is carried out in a volume of 250 ⁇ l volume.
  • the mixture contains 50 mM potassium phosphate (pH 7.6), different amounts of organism extract, 20 nM lycopene, 250 ⁇ g of chromoplastid stromal protein from paprika, 0.2 mM NADP +, 0.2 mM NADPH and 1 mM ATP.
  • NADP / NADPH and ATP are dissolved in 10 ml ethanol with 1 mg Tween 80 immediately before adding to the incubation medium. After a reaction time of 60 minutes at 30 ° C., the reaction is terminated by adding chloroform / methanol (2: 1). The reaction products extracted in chloroform are analyzed by HPLC.
  • the hydroxylase activity and / or ⁇ -cyclase activity can be increased in various ways, for example by switching off inhibitory regulatory mechanisms at the expression and protein level or by increasing the gene expression of nucleic acids encoding a hydroxylase and / or of nucleic acids , encoding a ⁇ -cyclase, compared to the wild type.
  • the increase in the gene expression of the nucleic acids encoding a hydroxylase and / or the increase in the gene expression of the nucleic acid encoding a ⁇ -cyclase compared to the wild type can also be achieved in various ways, for example by inducing the hydroxylase gene and / or ⁇ -cyclase Gene by activators or by introducing one or more hydroxylase gene copies and / or ⁇ -cyclase gene copies, ie by introducing at least one nucleic acid encoding a hydroxylase and / or at least one nucleic acid encoding a ⁇ -cyclase into the organism ,
  • Increasing the gene expression of a nucleic acid encoding a hydroxylase and / or ⁇ -cyclase means according to the invention the manipulation of the expression of the organism's own endogenous hydroxylase and / or ⁇ -cyclase. This can be achieved, for example, by changing the promoter DNA sequence for genes encoding hydroxylases and / or ⁇ -cyclases. Such a change, which results in an increased expression rate of the gene, can take place, for example, by deleting or inserting DNA sequences.
  • an altered or increased expression of an endogenous hydroxylase and / or ⁇ -cyclase gene can be achieved in that a regulator protein which does not occur in the non-transformed organism interacts with the promoter of this gene.
  • Such a regulator can represent a chimeric protein which consists of a DNA binding domain and a transcription activator domain, as for example in
  • the gene expression of a nucleic acid encoding a hydroxylase is increased and / or the gene expression of a nucleic acid encoding a ⁇ -cyclase is increased by introducing at least one nucleic acid encoding a hydroxylase and / or by introducing at least one a nucleic acid encoding a ⁇ -cyclase in the organism.
  • any hydroxylase gene or each ⁇ -cyclase gene that is to say any nucleic acid which codes for a hydroxylase and any nucleic acid which codes for a ⁇ -cyclase, can be used for this purpose.
  • genomic hydroxylase or. ⁇ -cyclase nucleic acid sequences from eukaryotic sources which contain introns are, in the event that the host organism is unable or cannot be able to express the corresponding hydroxylase or ⁇ -cyclase, preferably to use already processed nucleic acid sequences, such as the corresponding cDNAs.
  • An example of a hydroxylase gene is a nucleic acid encoding a hydroxylase from Haematococcus pluvialis, accession AX038729, WO 0061764); (Nucleic acid: SEQ ID NO: 15, protein: SEQ ID NO: 16).
  • ⁇ -cyclase gene is a nucleic acid encoding a ⁇ -cyclase from tomato (Accession X86452) (nucleic acid: SEQ ID NO: 17, protein: SEQ ID NO: 18).
  • the preferred transgenic organisms according to the invention therefore have at least one further hydroxylase gene and / or ⁇ -cyclase gene compared to the wild type.
  • the genetically modified organism 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 one ⁇ -cyclase.
  • nucleic acids encoding proteins are preferably used as the hydroxylase genes, containing the amino acid sequence SEQ ID NO: 16 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the
  • Sequence SEQ ID NO: 16 which have the enzymatic property of a hydroxylase.
  • hydroxylases and hydroxylase genes can be obtained, for example, from various organisms whose genomic sequence is known, as described above, by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SEQ ID. NO: 16 easy to find.
  • hydroxylases and hydroxylase genes can also be found, for example, starting from the sequence SEQ ID NO: 15 from various organisms. whose genomic sequence is not known, as described above, can easily be found by hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the hydroxylase of the sequence SEQ ID NO: 16.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons which are frequently used in accordance with the organism-specific "codon usage” are preferably used for this. This "codon usage” can easily be determined on the basis of computer evaluations of other known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ is brought. ID. NO: 15, in the organism.
  • nucleic acids which encode proteins are preferably used as the ⁇ -cyclase genes, comprising the amino acid sequence SEQ ID NO: 18 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 30 %, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 18, and which have the enzymatic property of a ⁇ -cyclase.
  • ⁇ -cyclases and ⁇ -cyclase genes can easily be found, for example, from various organisms whose genomic sequence is known, as described above, by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with SEQ ID NO: 18.
  • ⁇ -cyclases and ⁇ -cyclase genes can also be derived, for example, from the sequence SEQ ID NO: 17 from different organisms, whose genomic sequence is not known can easily be found by hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the ⁇ -cyclase of the sequence SEQ. ID. NO: 18.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons which are frequently used in accordance with the organism-specific "codon usage” are preferably used for this. This "codon usage” can easily be determined on the basis of computer evaluations of other known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ is brought. ID. NO: 17 in the organism.
  • All of the above-mentioned hydroxylase genes or ⁇ -cyclase genes can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the attachment of synthetic oligonucleotides and the filling of gaps using the Kleenow fragment of DNA polymerase and ligation reactions as well as general cloning methods are described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
  • these genetically modified organisms can be produced, for example, by introducing individual nucleic acid constructs (expression cassettes) or by introducing multiple constructs which contain up to two or three of the activities described.
  • organisms are preferably understood to mean organisms which, as wild-type or starting organisms, naturally or by genetic complementation and / or reorganization of the metabolic pathways, are capable of producing carotenoids, in particular ⁇ -carotene and / or zeaxanthin and / or neoxanthine and / or violaxanthin and / or to produce lutein.
  • Further preferred organisms already have hydroxylase activity as wild-type or starting organisms and are therefore capable of producing zeaxanthin as wild-type or starting organisms.
  • Preferred organisms are plants or microorganisms, such as bacteria, yeasts, algae or fungi.
  • Both bacteria can be used as bacteria that are able to synthesize xanthophylls due to the introduction of genes of the carotenoid biosynthesis of a carotenoid-producing organism, such as bacteria of the genus Escherichia, which contain, for example, crt genes from Erwinia, as well as bacteria. which are capable of synthesizing xanthophylls, such as, for example, bacteria of the genus Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc or cyanobacteria of the genus Synechocystis.
  • Preferred bacteria are Escherichia coli, Erwinia herbicola, Erwinia uredovora, Agrobacterium aurantiacum, Alcaligenes sp. PC-1, Flavobacterium sp. strain R1534, the Cyanobacterium Synechocystis sp. PCC6803, Paracoccus marcusii or Paracoccus carotinifaciens.
  • yeasts are Candida, Saccharomyces, Hansenula, Pichia or Phaffia. Particularly preferred yeasts are Xanthophyllomyces dendrorhous or Phaffia rhodozyma.
  • Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Blakeslea, Phycomyces, Fusarium or others in Indian Chem. Engr. Section B. Vol. 37, No. 1, 2 (1995) on page 15, table 6 described mushrooms.
  • Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Particularly preferred algae are Haematococcus puvialis or Dunaliella bardawil.
  • Plants selected from the Ranuncu families are particularly preferred plants. iaceae, Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae, Fabaceae, Lina- ceae, Vitaceae, Brassicaceae, Cucurbitaceae, Primulaceae, Caryophyl Iaceae, Amaranthaceae, Gentianaceae, Geraniaceae, Caprifoliaceae, Oleaceae, Tropaeolaceae, Solanaceae, Scrophulariaceae, Asteraceae, Liliaceae, Amaryllidaceae , Poaceae, Orchidaceae, Malvaceae, liliaceae or Lamiaceae.
  • Very particularly preferred plants are selected from the group of the plant genera Marigold, Tagetes errecta, Tagetes patula, Acacia, Aconitum, Adonis, Arnica, Aquilegia, Aster, Astragalus, Bignonia, Calendula, Caltha, Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum , Citrus, Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus, Dimorphotheca, Doronicum, Eschscholtzia, Forsythia, Fremontia, Gazania, Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillaea, Helenium, Helianthus, Hepatica , Heracleum, Hisbiscus, Heliopsis, Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kenya, Labumum, Lathyrus, Leontodon, Lili
  • the cultivation step of the genetically modified organisms is preferably followed by harvesting the organisms and, more preferably, additionally isolating ketocarotenoids from the organisms.
  • the organisms are harvested in a manner known per se in accordance with the respective organism.
  • Microorganisms such as bacteria, yeast, algae or fungi or plant cells, which are cultivated by fermentation in liquid nutrient media, can be separated off, for example, by centrifuging, decanting or filtering. Plants are grown in a conventional manner on nutrient media and corresponds' speaking harvested.
  • the cultivation of the genetically modified microorganisms is preferably carried out in the presence of oxygen at a cultivation temperature of at least about 20 ° C, e.g. 20 ° C to 40 ° C, and a pH of about 6 to 9.
  • the microorganisms are preferably first cultivated in the presence of oxygen and in a complex medium, such as e.g. TB or LB medium at a cultivation temperature of about 20 ° C or more, and a pH of about 6 to 9 until a sufficient cell density is reached.
  • a complex medium such as e.g. TB or LB medium
  • the cultivation is carried out after induction of ketolase expression in the presence of oxygen, e.g. 12 hours to 3 days continued.
  • ketocarotenoids are isolated from the harvested biomass in a manner known per se, for example by extraction and, if appropriate, further chemical or physical purification processes, such as, for example, precipitation methods, crystallography, thermal separation processes, such as rectification processes or physical separation processes, such as, for example, chromatography.
  • the ketocarotenoids in the genetically modified plants according to the invention can preferably be produced specifically in various plant tissues, such as, for example, seeds, leaves, fruits, flowers, in particular in petals.
  • Ketocarotenoids are isolated from the harvested petals in a manner known per se, for example by drying and subsequent extraction and, if appropriate, further chemical or physical purification processes, such as, for example, precipitation methods, crystallography, thermal separation processes, such as rectification processes or physical separation processes, such as chromatography. Ketocarotenoids are isolated from the petals, for example, preferably using organic solvents such as acetone, hexane, ether or tert-methylbutyl ether.
  • ketocarotenoids in particular from petals, are described, for example, in Egger and Kleinig (Phytochemistry (1967) 6, 437-440) and Egger (Phytochemistry (1965) 4, 609-618).
  • ketocarotenoids are preferably selected from the group consisting of astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin and adonixanthin.
  • ketocarotenoid is astaxanthin.
  • ketocarotenoids are obtained in free form or as fatty acid esters.
  • the ketocarotenoids are obtained in the process according to the invention in the form of their mono- or diesters with fatty acids.
  • Some proven fatty acids are e.g. Myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, and lauric acid (Kamata and Simpson (1987) Comp. Biochem. Physiol. Vol. 86B (3), 587-591).
  • the ketocarotenoids can be produced in the whole plant or, in a preferred embodiment, specifically in plant tissues which contain chromoplasts.
  • Preferred plant tissues are, for example, roots, seeds, leaves, Fruits, flowers and especially nectaries and petals, which are also called petals.
  • genetically modified plants are used which have the highest expression rate of a ketolase in flowers.
  • the gene expression of the ketolase takes place under the control of a flower-specific promoter.
  • the nucleic acids described above, as described in detail below are introduced into the plant in a nucleic acid construct functionally linked with a flower-specific promoter.
  • genetically modified plants are used which have the highest expression rate of a ketolase in fruits.
  • the gene expression of the ketolase takes place under the control of a fruit-specific promoter.
  • the nucleic acids described above, as described in detail below are introduced into the plant in a nucleic acid construct functionally linked with a fruit-specific promoter.
  • genetically modified plants are used which have the highest expression rate of a ketolase in seeds.
  • the gene expression of the ketolase takes place under the control of a seed-specific promoter.
  • the nucleic acids described above, as described in detail below are introduced into the plant in a nucleic acid construct functionally linked with a seed-specific promoter.
  • the targeting in the chrome peaks is carried out by a functionally linked plastid transit peptide.
  • a functionally linked plastid transit peptide The production of genetically modified plants with increased or caused ketolase activity is described as an example.
  • Other activities such as the hydroxylase activity and / or the ⁇ -cyclase activity, can be increased analogously using nucleic acid sequences encoding a hydroxylase or ⁇ -cyclase instead of nucleic acid sequences encoding a ketolase.
  • the transformation can take place individually or through multiple constructs.
  • the transgenic plants are preferably produced by transforming the starting plants, using a nucleic acid construct which contains the nucleic acids described above, encoding a ketolase, which are functionally linked to one or more regulation signals which ensure transcription and translation in plants.
  • nucleic acid constructs in which the coding nucleic acid sequence is functionally linked to one or more regulatory signals which ensure transcription and translation in plants, are also called expression cassettes below.
  • the regulation signals preferably contain one or more promoters which ensure transcription and translation in plants.
  • the expression cassettes contain regulation signals, that is to say regulative nucleic acid sequences, which control the expression of the coding sequence in the host cell.
  • an expression cassette upstream, ie at the 5 'end of the coding sequence, a promoter and downstream, ie at the 3' end, a polyadenylation signal and, if appropriate, further regulatory elements which match the coding sequence for at least one of the above genes described are operatively linked.
  • An operative link is understood to mean 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 fulfill its function as intended when expressing the coding sequence.
  • the preferred nucleic acid constructs, expression cassettes and vectors for plants and methods for producing transgenic plants and the transgenic plants themselves are described below by way of example.
  • sequences which are preferred, but not limited to, for operative linking are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil bodies or other compartments and Translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).
  • any promoter which can control the expression of foreign genes in plants is suitable as the promoter of the expression cassette.
  • Constant promoter means those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development.
  • a plant promoter or a plant virus-derived promoter is preferably used.
  • Particularly preferred is the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228), the 19S CaMV promoter (US 5,352,605; WO 84/02913; Benfey et al.
  • TPT triose phosphate translocator
  • Another suitable constitutive promoter is the pds promoter (Pecker et al. (1992) Proc. Natl. Acad. Be USA 89: 4962-4966) or the "Rubisco small subunit (SSU)" promoter (US 4,962,028), the LeguminB Promoter (GenBank Acc. No. X03677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), the ubiquitin 1 promoter (Christensen et al.
  • the expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the ketolase gene in the plant is controlled at a specific point in time can.
  • a chemically inducible promoter e.g. the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0388 186), a tetracycline-inducible promoter Promoter (Gatz et al.
  • promoters that are induced by biotic or abiotic stress such as 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 (US 5,187,267), the cold-inducible alpha-amylase promoter from the potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinII promoter (EP375091).
  • 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 US 5,187,267
  • the cold-inducible alpha-amylase promoter from the potato
  • the light-inducible PPDK promoter or the wound-induced pinII promoter EP375091.
  • Pathogen-inducible promoters include those of genes that are induced as a result of a pathogen attack, such as, for example, genes from PR proteins, SAR proteins, b-1, 3-glucanase, chitinase etc. (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.
  • suitable promoters are, for example, fruit ripening-specific promoters, such as the fruit ripening-specific promoter from tomato (WO
  • Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.
  • promoters are particularly preferred which ensure expression in tissues or parts of plants in which, for example, the biosynthesis of ketocarotenoids or their precursors takes place.
  • promoters with specificities for the anthers, ovaries, petals, sepals, flowers, leaves, stems, seeds and roots and combinations thereof.
  • Tuber, storage root or root-specific promoters are, for example, the patatin class I (B33) promoter or the potato cathepsin D inhibitor promoter.
  • Leaf-specific promoters are, for example, the cytosolic promoter
  • FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al. (1989) EMBO J 8: 2445- 2451).
  • Flower-specific promoters are, for example, the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593), the AP3 promoter from Arabidopsis thaliana (see Example 5), the CHRC promoter (chromoplast-specific carotenoid-associated protein (CHRC) gene promoter from Cucumis sativus Acc.-No. AF099501, base pair 1 to 1532), the EPSP_Synthase promoter (5-enol-pyruvylshikimate-3-phosphate synthase gene promoter from Petunia hybrida, Acc.-No.
  • CHRC chromoplast-specific carotenoid-associated protein
  • the PDS promoter (Phytoene desaturase gene pro- moter from Solanum lycopersicum, Acc.-No. U46919, base pair 1 to 2078), the DFR-A promoter (dihydroflavonol 4-reductase gene A promoter from Petunia hybrida, Acc.- No. X79723, base pair 32 to 1902) or the FBP1 promoter (Floral Binding Protein 1 gene promoter from Petunia hybrida, Acc.-No. L10115, base pair 52 to 1069).
  • the DFR-A promoter dihydroflavonol 4-reductase gene A promoter from Petunia hybrida, Acc.- No. X79723, base pair 32 to 1902
  • the FBP1 promoter Floral Binding Protein 1 gene promoter from Petunia hybrida, Acc.-No. L10115, base pair 52 to 1069.
  • Anther-specific promoters are, for example, the 5126 promoter (US 5,689,049, US 5,689,051), the glob-1 promoter or the g-zein promoter.
  • Seed-specific promoters are, for example, the ACP05 promoter (acyl carrier protein gene, WO9218634), the promoters AtS1 and AtS3 from Arabidopsis
  • the present invention therefore relates in particular to a nucleic acid construct containing functionally linked a flower-specific or in particular a petal-specific promoter and a nucleic acid encoding a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • An expression cassette is preferably produced by fusing a suitable promoter with a nucleic acid described above, encoding a ketolase, and preferably a nucleic acid inserted between promoter and nucleic acid sequence, which codes for a plastid-specific transit peptide, and a polyadenylation signal according to common recombination and cloning technology. techniques such as those described in T. Maniatis, EF Fritsch and J.
  • nucleic acids encoding a plastid transit peptide ensure localization in plastids and in particular in chromoplasts.
  • Expression cassettes can also be used, the nucleic acid sequence of which codes for a ketolase fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
  • Preferred transit peptides are preferred for the chromoplasts, which are cleaved enzymatically from the ketolase part after translocation of the ketolase into the chromoplasts.
  • the transit peptide which is derived from the plastid Nicotiana tabacum transketolase or another transit peptide (for example the transit peptide of the small subunit of the Rubisco (rbcS) or the ferredoxin NADP oxidoreductase as well as the isopentenyl pyrophosphate isomerase-2) or its functional equivalent is particularly preferred ,
  • Nucleic acid sequences of three cassettes of the plastid transit peptide of plastid transketolase from tobacco in three reading frames are particularly preferred as Kpnl / BamHI fragments with an ATG codon in the Ncol interface:
  • plastid transit peptide examples include the transit peptide of the plastid
  • IPP-2 X ren isopentenyl pyrophosphate isomerase-2
  • rbcS ribulose bisphosphate carboxylase
  • nucleic acids according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural nucleic acid constituents, and can consist of different heterologous gene segments from different organisms.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter can be native or homologous as well as foreign or heterologous to the host plant.
  • the expression cassette preferably contains the promoter, a coding nucleic acid sequence or a nucleic acid construct and a region for the transcriptional termination in the 5'-3 'transcription direction. Different termination areas are interchangeable.
  • Examples of a terminator are the 35S terminator (Guerineau et al. (1988) Nucl Acids Res. 16: 11380), the nos terminator (Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1982; 1 (6): 561-73) or the ocs terminator (Gielen, J, de Beuckeleer, M, Seurinck, J, Debroek, H, de Greve, H , Lemmers, M, van Montagu, M,
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as, for example, transitions and transversions come into question, w ' tro mutagenesis, "primer repair", restriction or ligation can be used.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially contain T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Ti Plasmids correspond to pTiACH ⁇ (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • Suitable methods for the transformation of plants are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - the so-called "particle bombardment” method, electroporation, the incubation of dry embryos in DNA-containing solution, the Microinjection and the Agrobacterium-mediated gene transfer described above.
  • the methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published 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 preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711) or particularly preferably pSUN2, pSUN3, pSUN4 or pSUN5 (WO 02/00900).
  • Agrobacteria transformed with an expression plasmid can be used in a known manner to transform plants, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the fused expression cassette which expresses a ketolase is cloned into a vector, for example pBin19 or in particular pSUN5 and pSUN3, which is suitable for being transformed into Agrobacterium tumefaciens.
  • Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular cultivated plants. are used, for example, by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • transgenic plants can be regenerated in a known manner which contain a gene integrated into the expression cassette for the expression of a nucleic acid encoding a ketolase.
  • an expression cassette is inserted as an insert in a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
  • additional functional regulation signals for example sequences for replication or integration.
  • Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119 (1993).
  • the expression cassettes can be cloned into suitable vectors that allow their proliferation, for example in E. coli.
  • suitable cloning vectors include PJIT117 (Guerineau et al. (1988) Nucl. Acids Res. 16: 11380), pBR332, pUC series, M13mp series and pACYC184.
  • Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.
  • Hydroxylase or ⁇ -cyclase are preferably incorporated into expression constructs containing, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for an enzyme according to the invention; and vectors comprising at least one of these expression constructs.
  • Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence.
  • An “operative linkage” is understood to mean 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 perform its function as intended when expressing the coding sequence.
  • sequences which can be linked operatively are targeting sequences as well
  • Translation enhancers include selectable markers, amplification signals, origins of replication and the like.
  • the natural regulatory sequence can still be present before the actual structural gene. This natural regulation can possibly be switched off by genetic modification and the
  • genes can be increased or decreased.
  • the gene construct can also have a simpler structure, ie no additional regulation signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased or decreased.
  • the nucleic acid sequences can be contained in one or more copies in the gene construct.
  • Examples of useful promoters in microorganisms are: cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, laclq-, T7-, T5-, T3-, gal- , trc, ara, SP6, lambda PR or in the lambda PL promoter, which are advantageously used in gram-negative bacteria; as well as the gram-positive promoters amy and SPO2 or the yeast promoters ADC1, MFa, AC, P-60, CYC1, GAPDH.
  • inducible promoters such as, for example, light and in particular temperature-inducible promoters, such as the P r P r promoter
  • inducible promoters such as, for example, light and in particular temperature-inducible promoters, such as the P r P r promoter
  • all natural prorriotors with their regulatory sequences can be used.
  • synthetic promoters can also be used advantageously.
  • the regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences and the protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • An expression cassette is produced by fusing a suitable promoter with the nucleic acid sequences described above, encoding a ketolase, ⁇ -hydroxylase or ⁇ -cyclase and a terminator or polyadenylation signal.
  • Common recombination and cloning techniques are used, such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausu, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
  • the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host.
  • Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
  • vectors also include all other vectors known to the person skilled in the art, such as, for example, phages, viruses such as SV40, CMV, baculovirus and adevirus, transposons, IS elements, phasmids, cosmids, and linear or circular Understand DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.
  • fusion expression vectors such as pGEX (Pharmacia Biotech ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ) which glutathione-S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST glutathione-S-transferase
  • Non-fusion protein expression vectors such as pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (19g ⁇ ) 60-89) or pBluescript and pUC vectors.
  • yeast expression vector for expression in the yeast S. cerevisiae such as pYepSed (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943) , pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Vectors and methods of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol .. 3: 2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • recombinant constructs according to the invention described above are advantageously introduced and expressed in a suitable host system.
  • Common cloning and transfection methods known to the person skilled in the art such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used to bring the nucleic acids mentioned into expression in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997.
  • marker genes which are also contained in the vector or in the expression cassette.
  • marker genes are genes for antibiotic resistance and for enzymes which catalyze a coloring reaction which stains the transformed cell. These can then be selected using automatic cell sorting.
  • Microorganisms successfully transformed with a vector and carrying an appropriate antibiotic resistance gene can be selected using appropriate antibiotic-containing media or nutrient media.
  • Marker proteins that are presented on the cell surface can be used for selection by means of affinity chromatography.
  • the invention further relates to a method for producing genetically modified organisms, characterized in that a nucleic acid construct comprising functionally linked a promoter and nucleic acids encoding a ketolase containing the amino acid sequence SEQ. ID. NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which a Identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2, and optionally introduces a terminator into the genome of the starting organism or extrachromosomally into the starting organism.
  • the invention further relates to the genetically modified organisms, the genetic modification being the activity of a ketolase
  • ketolase activity increased after A or caused after B is caused by a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the ketolase activity is increased or caused compared to the wild type, preferably by increasing or causing the gene expression of a nucleic acid encoding a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the gene expression of a nucleic acid, coding for a ketolase is increased or caused by introducing nucleic acids, coding for a ketolase, into the plants and thus preferably for overexpression or transgenic expression of nucleic acids, coding a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the invention further relates to a genetically modified organism containing at least one transgenic nucleic acid encoding a ketolase containing the amino acid sequence SEQ. ID. NO.
  • the invention further relates to a genetically modified organism containing at least two endogenous nucleic acids encoding a ketolase containing 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 an identity of at least 42% at the amino acid level with the sequence SEQ. ID. NO. 2 has. This is the case if the starting organism has an endogenous ketolase and the endogenous ketolase is overexpressed.
  • genetically modified organisms additionally have an increased hydroxlase activity and / or ⁇ -cyclase activity compared to a wild-type organism. Further preferred embodiments are described above in the method according to the invention.
  • organisms are preferably understood to mean organisms which, as wild-type or starting organisms, naturally or by genetic complementation and / or reorganization of the metabolic pathways, are capable of producing carotenoids, in particular ⁇ -carotene and / or zeaxanthin and / or neoxanthine and / or violaxanthin and / or to produce lutein.
  • Further preferred organisms already have hydroxylase activity as wild-type or starting organisms and are therefore capable of producing zeaxanthin as wild-type or starting organisms.
  • Preferred organisms are plants or microorganisms, such as bacteria, yeasts, algae or fungi.
  • Both bacteria can be used as bacteria which, due to the introduction of genes of the carotenoid biosynthesis of a carotenoid-producing organism, must be able to synthesize xanthophylls, such as, for example, bacteria of the genus Escherichia, which, for example, contain crt genes from Erwinia, and also bacteria which are capable of synthesizing xanthophylls, such as, for example, bacteria of the genus Erwinia, Agrobacterium , Flavobacterium, Alcaligenes, Paracoccus, Nostoc or cyanobacteria of the genus Synechocystis.
  • xanthophylls such as, for example, bacteria of the genus Escherichia, which, for example, contain crt genes from Erwinia
  • bacteria which are capable of synthesizing xanthophylls such as, for example, bacteria of the genus Erwinia, Agrobacterium , Flavobacter
  • Preferred bacteria are Escherichia coli, .Erwinia herbicola, Erwinia uredovora, Agrobacterium aurantiacum, Alcaligenes sp. PC-1, Flavobacterium sp. strain R1534, the Cyanobacterium Synechocystis sp. PCC6803, Paracoccus marcusii or Paracoccus carotinifaciens.
  • yeasts are Candida, Saccharomyces, Hansenula, Pichia or Phaffia. Particularly preferred yeasts are Xanthophyllomyces dendrorhous or Phaffia rhodozyma.
  • Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Blakeslea, Phycomyces, Fusarium or others in Indian Chem. Engr. Section B. Vol. 37, No. 1, 2
  • Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Particularly preferred algae are Haematococcus puvialis or Dunaliella bardawil.
  • Particularly preferred plants are plants selected from the Ranuncu-laceae, Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae, Fabaceae, Lina- ceae, Vitaceae, Brassicaceae, Cucurbitaceae, Primulaceae, Caryophyllaceae, Amaranthaceae, Geraniaceaeaceae, Gentianaceaeaea, Gentianaceae , • Solanaceae, Scrophulariaceae, Asteraceae, Liliaceae, Amaryllidaceae, Poaceae, Orchidaceae, Malvaceae, liliaceae or Lamiaceae.
  • Very particularly preferred plants are selected from the group of the plant species chard, tagetes errectta, tagetes patula, acacia, aconite, adonis, ami ca, Aquilegia, Aster, Astragalus, Bignonia, Calenduia, Caltha, Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum, Citrus, Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus, Dimorphotheca, Doronicum, Eschscholtzia, Foria, Forsy Gazania, Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillea, Helenium, Helianthus, Hepatica, Heracleum, Hisbiscus, Heliopsis, Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kerria, Laburnum, Lathyrus, Leonto
  • Very particularly preferred genetically modified plants are selected from the plant genera Marigold, Tagetes erecta, Tagetes patula, Adonis, Lycopersicon; Rosa, Calenduia, Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium or Tropaeolum, the genetically modified plant containing at least one transgenic nucleic acid, coding for a ketolase.
  • the present invention further relates to the transgenic plants, their reproductive material and their plant cells, tissue or parts, in particular their fruits, seeds, flowers and petals.
  • the genetically modified plants can be used to produce ketocarotenoids, in particular astaxanthin.
  • Genetically modified organisms according to the invention which can be consumed by humans and animals, in particular plants or parts of plants, such as in particular petals with an increased content of ketocarotenoids, in particular astaxanthin, can also be used, for example, directly or after processing known per se as foods or feeds or as feed and food supplements become. Furthermore, the genetically modified organisms can be used for the production of ketocarotenoid-containing extracts of the organisms and / or for the production of feed and food supplements.
  • the genetically modified organisms have an increased ketocarotenoid content compared to the wild type.
  • An increased ketocarotenoid content is generally understood to mean an increased total ketocarotenoid content.
  • ketocarotenoids is also understood to mean, in particular, a changed content of the preferred ketocarotenoids, without the total carotenoid content necessarily having to be increased.
  • the genetically modified plants according to the invention have an increased astaxanthin content compared to the wild type.
  • an increased content is also understood to mean a caused content of ketocarotenoids or astaxanthin.
  • the invention further relates to the new ketolases and the new nucleic acids encoding them.
  • the invention relates to ketolases containing the amino acid sequence SEQ. ID. NO. 8 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 70%, preferably at least 75%, particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% at the amino acid level with the sequence SEQ. ID. NO. 8, with the proviso that the amino acid sequences SEQ ID NO: 4 is not included.
  • the sequence SEQ ID NO: 4, as mentioned above, is annotated as a putative protein in databases.
  • the invention further relates to ketolases containing the amino acid sequence SEQ. ID. NO. 6 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 70% on amino acid level with the sequence SEQ. ID. NO. 6 has.
  • the sequence SEQ ID NO: 6, as mentioned above, is not annotated in databases.
  • the invention relates to ketolases containing the amino acid sequence SEQ. ID. NO. 12 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 70%, preferably at least 75%, particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% at the amino acid level with the sequence SEQ. ID. NO. 12, with the proviso that the amino acid sequences SEQ ID NO: 6 is not included.
  • the invention further relates to ketolases containing the amino acid sequence SEQ. ID. NO. 49 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50%, preferably at least 60%, particularly preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% at the amino acid level with the sequence SEQ. ID. NO. 49, with the proviso that the amino acid sequences SEQ ID NO: 47 is not included.
  • the sequence SEQ ID NO: 47 is, as mentioned above, annotated as a putative protein in databases.
  • the invention further relates to nucleic acids encoding a protein described above, with the proviso that the nucleic acid does not contain the sequence SEQ ID NO: 5.
  • a protein containing the amino acid sequence SEQ. ID. NO. 4 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 70%, preferably at least 75%, particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% at the amino acid level with the sequence SEQ. ID. NO. 4 and has the property of a ketolase has a property as a ketolase.
  • the invention therefore also relates to the use of a protein containing the amino acid sequence SEQ. ID. NO. 4 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 70%, preferably at least 75%, particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% at the amino acid level with the SEQ sequence. ID. NO. 4 and has the property of a ketolase as a ketolase.
  • a protein containing the amino acid sequence SEQ. ID. NO. 6 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has an identity of at least 65%, preferably at least 70%, preferably at least 75%, particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% at the amino acid level with the SEQ sequence. ID. NO. 6 and has the property of a ketolase, has a property as a ketolase.
  • the invention therefore also relates to the use of a protein containing the amino acid sequence SEQ. ID. NO. 6 or one of this sequence by substitution,
  • Insertion or deletion of amino acid-derived sequence which has an identity of at least 65%, preferably at least 70%, preferably at least 75%, x particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% at the amino acid level the sequence SEQ. ID. NO. 6 and has the property of a ketolase as a ketolase.
  • a protein containing the amino acid sequence SEQ. ID. NO. 47 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has an identity of at least 50%, preferably at least 60%, preferably at least 70%, particularly preferably at least 80%, more preferably at least 85%, more preferably at least 90% , more preferably at least 95% at the amino acid level with the sequence SEQ. ID. NO. 47 and has the property of a ketolase, has a property as a ketolase.
  • the invention therefore also relates to the use of a protein containing the amino acid sequence SEQ. ID. NO. 47 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50%, preferably at least 60%, preferably at least 70%, particularly preferably at least 80%, more preferably at least 85% at least 90%, more preferably at least 95% at the amino acid level with the SEQ sequence. ID. NO. 47 and has the property of a ketolase as ketolase.
  • the process according to the invention provides a higher amount of ketocarotenoids, in particular astaxanthin with a lower amount of hydroxylated by-products.
  • the sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from Licor (distributed by MWG Biotech, Ebersbach) according to the
  • the DNA required for the NOST ketolase from Nostoc sp. PCC 7120 coded was by means of PCR from Nostoc sp. PCC 7120 (strain of the "Pasteur Culture Collection of Cyanobacterium”) amplified.
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8,000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (2 ml volume). After adding 100 ⁇ l Proteinase K (concentration: 20 mg / ml), the cell suspension was incubated for 3 hours at 37 ° C. The suspension was then extracted with 500 ⁇ l of phenol. After centrifugation at 13,000 rpm for ⁇ minutes, the upper, aqueous phase was transferred to a new 2 ml Eppendorf reaction vessel.
  • the extraction with phenol was repeated 3 times.
  • the DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating to 65 ° C.
  • the nucleic acid encoding a ketolase from Nostoc PCC 7120 was determined by means of a "polymerase chain reaction” (PCR) from Nostoc sp.
  • PCC 7120 was amplified using a sense-specific primer (NOSTF, SEQ ID No. 19) and an antisense-specific primer (NOSTG SEQ ID No. 20).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions: 1X 94 ° C 2 minutes 35X 94 ° C 1 minute 55 ° C 1 minutes 72 ° C 3 minutes
  • PCR amplification with SEQ ID No. 19 and SEQ ID No. 20 resulted in an 805 bp fragment that codes for a protein consisting of the entire primary sequence (SEQ ID No. 21).
  • the amplificate was cloned into the PCR cloning vector pGEM-T (Promega) and the clone pNOSTF-G was obtained.
  • This clone pNOSTF-G was therefore used for the cloning into the expression vector pJIT117 (Guerineau et al. I g88, Nucl. Acids Res. 16: 11380). Cloning was carried out by isolating the 799 bp Sphl fragment from pNOSTF-G and ligation into the Sphl cut vector pJIT117.
  • the clone that is the ketolase from Nostoc sp. PCC 7120, in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide, is called pJNOST.
  • PMCL-CrtYlBZ / idi / gps was constructed in three steps using the intermediate stages pMCL-CrtYlBZ and pMCL-CrtYlBZ / idi.
  • the plasmid pMCL200 compatible with high-copy-number vectors was used as the vector (Nakano, Y., Yoshida, Y., Yamashita, Y. and Koga, T .; Construction of a series of pACYC-derived plasmid vectors; Gene 162 ( 1995), 157-158).
  • Example 2.1. Construction of pMCL-CrtYlBZ
  • the biosynthetic genes crtY, crtB, crtl and crtZ come from the bacterium Erwinia ure- dovora and were amplified by PCR.
  • Erwinia uredovora genomic DNA (DSM 30080) was prepared by the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig) as part of a service.
  • the PCR reaction was carried out according to the manufacturer's instructions (Röche, Long Template PCR: Procedure for amplification of 5-20 kb targets with the expand long template PCR system).
  • the PCR conditions for the amplification of the Erwinia uredovora biosynthesis cluster were as follows:
  • the plasmid pCR2.1-CrtYIBZ was cut Sall and Hindill, the resulting Sall / Hindlll fragment isolated and transferred by ligation into the Sall / Hindlll cut vector pMCL200.
  • the Sall / Hindlll fragment from pCR2.1-CrtYIBZ cloned in pMCL 200 is 4624 bp long, codes for the genes CrtY, CrtI, crtB and CrtZ and corresponds to the sequence from positions 2295 to 6918 in D90087 (SEQ ID No. 24).
  • the resulting clone is called pMCL-CrtYlBZ.
  • Example 2.2 Construction of pMCL-CrtYlBZ / idi
  • the gene / d / ' (isopentenyl diphosphate isomerase; IPP isomerase) was amplified from E coli by means of PCR.
  • the nucleic acid encoding the entire / cf gene with idi promoter and ribosome binding site was extracted from E. coli by means of "polymerase chain reaction” (PCR) using a sense-specific primer (5'-idi SEQ ID No. 28) and an antisense-specific primer (3'-idi SEQ ID No.29).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions: 1X 94 ° C 2 minutes 20X 94 ° C 1 minute
  • PCR amplification with SEQ ID No. 28 and SEQ ID No. 29 resulted in a 679 bp fragment coding for a protein consisting of the entire primary sequence (SEQ ID No. 30).
  • the amplificate was cloned into the PCR cloning vector pCR2.1 (Invitrogen) and the clone pCR2.1-idi was obtained.
  • Sequencing of the clone pCR2.1-idi confirmed a sequence that does not differ from the published sequence AE000372 in position 8774 to position 9440.
  • This region comprises the promoter region, the potential ribosome binding site and the entire "open reading frame" for the IPP isomerase.
  • the fragment cloned into pCR2.1-idi has a total length of 679 bp by inserting an Xhol site at the 5 'end and a SalI site at the 3' end of the gene.
  • This clone was therefore used for the cloning of the / oY gene in the vector pMCL-CrtYlBZ.
  • the cloning was carried out by isolating the Xhol / Sall fragment from pCR2.1-idi and ligating into the Xhol / Sall cut vector pMCL-CrtYlBZ.
  • the resulting clone is called pMCL-CrtYlBZ / idi.
  • Example 2.3 Construction of pMCL-CrtYlBZ / idi / gps
  • the gene gps (geranylgeranyl pyrophosphate synthase; GGPP synthase) was amplified from Archaeoglobus fulgidus by means of PCR.
  • the DNA of Archaeoglobus fulgidus was prepared by the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig) as part of a service.
  • the PCR conditions were as follows: The PCR for the amplification of the DNA, which codes for a GGPP synthase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • SEQ ID No. 32 and SEQ ID No. 33 amplified DNA fragments were eluted from the agarose gel using methods known per se and cut with the restriction enzymes Ncol and Hindill. This results in a 962 bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 34).
  • Ncol / HindIII cut amplificate was cloned into the vector pCB97-30 and the clone pCB-gps was obtained.
  • Sequencing of the clone pCB-gps confirmed a sequence for the GGPP synthase from A. fulgidus, which differs from the published sequence AF120272 in one nucleotide.
  • the second codon of the GGPP synthase was changed by inserting an Ncol site in the gps gene.
  • CTG position 4-6
  • this second codon was changed to GTG, which codes for valine.
  • the clone pCB-gps was therefore used for the cloning of the gps gene into the vector pMCL-CrtYlBZ / idi.
  • the cloning was carried out by isolating the Kpnl / Xhol fragment from pCB-gps and ligation into the Kpnl and Xhol cut vector pMCL-CrtYlBZ / idi.
  • the cloned Kpnl / Xhol fragment (SEQ ID No.
  • GGPP synthase 34 carries the Prm16 promoter together with a minimal 5 'UTR sequence of rbcL, the first 6 codons of rbcL, which extend the GGPP synthase N-terminally, and 3 'from the gps gene the psbA sequence.
  • the N-terminus of the GGPP synthase thus has the changed amino acid sequence Met-Thr-Pro-Gln-Thr-Ala-Met instead of the natural amino acid sequence with Met-Leu-Lys-Glu (amino acid 1 to 4 from AF120272) -Val-Lys- GIu.
  • the recombinant GGPP synthase starting with Lys in position 3 (in AF120272), is identical and has no further changes in the amino acid sequence.
  • the rbcL and psbA sequences were based on a reference according to Eibl et al. (Plant J. 1. (I 99), 1-13).
  • the resulting clone is called pMCL-CrtYlBZ / idi / gps.
  • E. co // strains were produced which are capable of producing zeaxanthin by heterologous complementation.
  • Strains of E. coli TOP10 were used as host cells for the complementation experiments with the plasmids pNOSTF-G and pMCL-CrtYlBZ / idi / gps.
  • the plasmid pMCL-CrtYlBZ / idi / gps was constructed in order to produce E. co // strains which enable the synthesis of zeaxanthin in high concentrations.
  • the plasmid carries the genes crtY, crtB, crtl and crtY from Erwinia uredovora, the gene gps (for geranylgeranyl pyrophoshate synthastase) from Archaeoglobus fulgidus and the gene / ⁇ / (isopentenyl diphosphate isomerase) from E. coli. Limiting steps for a high accumulation of carotenoids and their biosynthetic precursors were eliminated with this construct.
  • the cells were extracted with acetone, the organic solvent was evaporated to dryness and the carotenoids were separated by means of HPLC on a C30 column. The following procedural conditions were set.
  • the spectra were determined directly from the elution peaks using a photodiode array detector.
  • the isolated substances were identified by their absorption spectra and their retention times in comparison to standard samples.
  • Figure 1 shows the chromatographic analysis of a sample obtained from an E. co // strain transformed with pNOSTF-G and pMCL-CrtYlBZ / idi / gps. It appears, that this strain can synthesize various ketocarotenoids due to the heterologous complementation. With increasing retention time, astaxanthin (peak 1), adonirubin (peak 2) and canthaxanthin (peak 3) are eluted.
  • a E. coli strain was produced as a comparative example, which contains a ketolase from Haematococcus pluvialis Flotow em. Will expressed.
  • the cDNA was used for the entire primary sequence of the ketolase from Haematococcus pluvialis Flotow em. Wille coded amplified and cloned into the same expression vector according to Example 1.
  • the cDNA coding for the ketolase from Haematococcus pluvialis was amplified by means of PCR from Haematococcus pluvialis (strain 192.80 from the "Collection of algal cultures of the University of Göttingen") suspension culture.
  • RNA For the preparation of total RNA from a suspension culture of Haematococcus pluvialis (strain 192.80), which with indirect daylight at room temperature for 2 weeks in Haematococcus medium (1.2 g / l sodium acetate, 2 g / l yeast extract, 0.2 g / l MgCI2x6H2O , 0.02 CaCI2x2H2O; pH 6.8; after autoclaving 400 mg / l L-Asparagine, 10 mg / l FeSO4xH2O) had been grown, the cells were harvested, frozen in liquid nitrogen and pulverized in a mortar.
  • Haematococcus pluvialis strain 192.80
  • RNA For the cDNA synthesis, 2.5 ⁇ g of total RNA were denatured for 10 min at 60_C, cooled on ice for 2 min and used using a cDNA kit (ready-to-go-you-prime-beads, Pharmacia Biotech) according to the manufacturer's instructions of an antisense-specific primer PR1 (gcaagctcga cagctacaaa cc) was rewritten in cDNA.
  • a cDNA kit ready-to-go-you-prime-beads, Pharmacia Biotech
  • the nucleic acid encoding a kematolase from Haematococcus pluvialis was amplified by means of a polymerase chain reaction (PCR) from Haematococcus pluvialis using a sense-specific primer PR2 (gaagcatgca gctagcagcg acag) and an antisense-specific primer PR1.
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the cDNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ml reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the PCR amplification with PR1 and PR2 resulted in a 1155 bp fragment consisting encodes a protein consisting of the entire primary sequence: gaagcatgca gctagcagcg acagtaatgt tggagcagct taccggaagc gctgaggcac 60 tcaaggagaa ggagaaggag gttgcaggca gctctgacgt gtgtaca tgggcgaccc 120 agtactcgct tccgtcagag gagtcagacg cggccccc gggactgaag aatgcctaca 180 agccaccaccacc ttccgacaca aagggcatca caatggcgct agctcatc ggctctggg 240 ccgcagtgttt c
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) and the clone pGKETO2 was obtained.
  • This clone was used for the cloning in the expression vector described in Example 1.
  • the cloning was carried out analogously to that described in Example 1.
  • the transformation of the E. coli strains, their cultivation and the analysis of the carotenoid profile were carried out as described in Example 3.
  • Figure 2 shows the chromatographic analysis of a sample obtained from an E. co // strain transformed with this expression vector and pMCL-CrtYlBZ / idi / gps.
  • a ketolase from Haematococcus pluvialis as described for example in EP 725137, elute with increasing retention time astaxanthin (peak 1), adonixanthin (peak 2) and unreacted zeaxanthin (peak 3).
  • This carotenoid profile has already been described in EP 0725137.
  • Table 1 shows a comparison of the bacterially produced amounts of carotenoids:
  • Table 1 Comparison of the bacterial ketocarotenoid synthesis using two different ketolases, the NOST ketolase from Nostoc sp. PCC7120 (Example 3) and the ketolase from Haematococcus pluvialis as a comparative example (Example 3.1). Amounts of carotenoids are given in ng / ml culture fluid.
  • ketolase from Nostoc sp. Strain PCC7120 leads to a carotenoid pattern, which differs significantly from the carotenoid pattern after expression of a ketolase from Haematococcus pluvialis. While the keto lens from the prior art provides the desired ketocarotenoid astaxanthin only incompletely, astaxanthin is the main product when using the ketolase according to the invention. In the process according to the invention, hydroxylated by-products occur in a significantly smaller amount.
  • the expression of the NOST ketolase from Nostoc sp. PCC7120 in Lesculentum and in Tagetes erecta was carried out under control of the constitutive promoter FNR (ferredoxin-NADPH-oxidoreductase, database entry AB011474 position 70127 to 69493; WO03 / 006660) from Arabidopsis thallana.
  • FNR constitutive promoter
  • the FNR gene begins at base pair 69492 and is annotated with "ferredoxin-NADP + reductase”. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the DNA fragment containing the FNR promoter region from Arabidopsis thallana was PCR-analyzed using genomic DNA (isolated from Arabidopsis thallana according to standard methods) as well as the primers FNR-A (SEQ ID No.38) and FNR-B (SEQ ID No. 39).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the FNR promoter fragment FNR # 1), was carried out in a 50 ⁇ l reaction mixture which contained: 100 ng genomic DNA from A.thaliana 0.25 mM dNTPs 0.2 mM FNR-A (SEQ ID No. 38) 0.2 mM FNR-B (SEQ ID No. 39) - 5 ⁇ l IOX PCR buffer (Stratagene)
  • the PCR was carried out under the following cycle conditions:
  • the 647 bp amplificate was determined using standard methods in the PCR
  • Cloning vector pCR 2.1 (Invitrogen) cloned and the plasmid pFNR # 1 obtained.
  • Sequencing of clone pFNR # 1 confirmed a sequence which corresponds to a sequence section on chromosome 5 of Arabidopsis thaliana (database entry AB011474; WO03 / 006660) from position 70127 to 6g4g3.
  • the FNR gene begins - at base pair 6g4 2 and is annotated with "ferredoxin-NADP + reductase".
  • pFNR was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • the cloning was carried out by isolating the 637 bp Sacl-Hindlll fragment from pFNR # 1 (partial Sacl hydrolysis) and ligating into the Sacl-Hindlll cut vector pJIT117.
  • the clone that contains the promoter FNR # 1 instead of the original promoter d35S is called pJITFNR.
  • the 799 bp SpHI fragment NOSTF-G (described in Example 1) was cut into the SpHI vector pJITFNR cloned.
  • the clone that contains the fragment NOSTF-G in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJFNRNOST.
  • An expression cassette for the Agrobacterium -mediated transformation of the ketolase from Nostoc into lesculentum was produced using the binary vector pSUN3 (WO0200900).
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS TP fragment the rbcS transit peptide from pea (194 bp), fragment Nost Ketolase CDS (777 bp) the entire primary sequence coding for the Nostoc Ketolase, fragment 35S Term (746 bp) the polyadenylation signal of CaMV.
  • An expression cassette for the Agrobacterium-vet rMeWe transformation of the expression vector with the ketolase from Nostoc in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00900).
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS transit peptide the rbcS transit peptide from pea (1 g4 bp), fragment Nost ketolase (777 bp) the entire primary sequence, coding for the nostoc ketolase, fragment 35S Terminator (746 bp) the CaMV polyadenylation signal.
  • the expression of the ketolase from Nostoc in L. esculentum and Tagetes erecta was carried out with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the expression was carried out under the control of a modified version AP3P of the flower-specific promoter AP3 from Arabidopsis thaliana (AL132971: nucleotide region 9298-10200; Hill et al. (1998) Development 125: 1711-1721).
  • the DNA fragment which contains the AP3 promoter region -902 to +15 from Arabidopsis thalia- na, was PCR-analyzed using genomic DNA (isolated from Arabidopsis thaliana according to standard methods) and the primers AP3-1 (SEQ ID No.41) and AP3 -2 (SEQ ID No. 42).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the AP3 promoter fragment (-902 to +15), was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 2 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods and the plasmid pAP3 was obtained.
  • Sequencing of the clone pAP3 confirmed a sequence consisting only of an insertion (a G in position g765 of the sequence AL132g71) and a base exchange (a G instead of an A in position g726 of the sequence AL132971) of the published AP3 sequence (AL132g71, nucleotide region g2g8-10200) differs. These nucleotide differences were reproduced in an independent amplification experiment and thus represent the actual nucleotide sequence in the Arabidopsis thaliana plants used.
  • the modified version AP3P was produced by means of recombinant PCR using the plasmid pAP3.
  • the region 10200 - g771 was amplified with the primers AP3-1 (SEQ ID No. 41) and primers AP3-4 (SEQ ID No. 44) (amplificate A1 / 4), the region g526-g285 with the AP3-3 (SEQ ID No. 43) and AP3-2 (SEQ ID No. 42) amplified (amplificate A2 / 3).
  • the PCR conditions were as follows:
  • the PCR was carried out under the following cycle conditions:
  • the recombinant PCR includes annealing of the amplificates A1 / 4 and A2 / 3, which overlap over a sequence of 25 nucleotides, completion into a double strand and subsequent amplification.
  • the denaturation (5 min at 95 ° C.) and annealing (slow cooling at room temperature to 40 ° C.) of both amplicons A1 / 4 and A2 / 3 was carried out in a 17.6 ⁇ l reaction mixture which contained:
  • the nucleic acid coding for the modified promoter version AP3P was amplified by means of PCR using a sense-specific primer (AP3-1 SEQ ID No. 41) and an antisense-specific primer (AP3-2 SEQ ID No. 42).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the AP3P fragment was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • the amplificate was cloned into the cloning vector pCR2.1 (Invitrogen) and the plasmid pAP3P was obtained. Sequencing with the primers T7 and M13 confirmed a sequence identical to the sequence AL132971, region 10200-92g8, the internal region g285-g526 being deleted. This clone was therefore used for the cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • the cloning was carried out by isolating the 767 bp SacI-HindIII fragment from pAP3P and ligating into the SacI-HindIII cut vector pJIT117.
  • the clone that contains the promoter AP3P instead of the original promoter d35S is called pJITAP3P.
  • the 79 g bp SpHI fragment NOSTF-G (described in Example 1) was cloned into the SpHI-cut vector pJITAP3P.
  • the clone that contains the fragment NOSTF-G in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJAP3PNOST.
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled ketolase from Nostoc to Lesculentum was produced using the binary vector pSUN3 (WO02 / 00900).
  • fragment AP3P PROMOTER contains the modified AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NOST KETOLASE CDS (777bp) the entire primary sequence coding for the nostoc ketolase, fragment 35S TERM (746 bp) the polyadenylation signal of CaMV.
  • An expression vector for the Agrobacterium -mediated transformation of the AP3P-controlled ketolase from Nostoc into Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00900).
  • fragment AP3P PROMOTER contains the modified AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (207 bp), fragment NOST KETOLASE CDS (777 bp) the entire primary sequence coding for the nostoc ketolase, fragment 35S TERM (746 bp) the polyadenylation signal of CaMV.
  • X was amplified by PCR from Nostoc punctiform ATCC 29133 (strain of the "American Type Culture Collection").
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris_HCI (pH 7.5) and placed in an Eppendorf reaction vessel (2 ml of lumen) transferred. After adding 100 ⁇ l Proteinase K (concentration: 20 mg / ml), the cell suspension was incubated for 3 hours at 37 ° C. The suspension was then extracted with 500 ⁇ l of phenol.
  • the upper, aqueous phase was transferred to a new 2 ml Eppendorf reaction vessel i.
  • the extraction with phenol was repeated 3 times.
  • the DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating to 65 ° C.
  • the nucleic acid encoding a ketolase from Nostoc punctiform ATCC 29133 was determined by means of a "polymerase chain reaction” (PCR) from Nostoc punctiform ATCC 29133 using a sense-specific primer (NP196-1, SEQ ID No. 54) and an antisense-specific Primers (NP196-2 SEQ ID No. 55) amplified.
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 54 and SEQ ID No. 55 resulted in a 792 bp fragment that codes for a protein consisting of the entire primary sequence (NPig ⁇ , SEQ ID No. 56).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNP196 was obtained.
  • pJIT117 was modified by using the 35S terminator through the OCS terminator (octopine synthase) of the Ti plasmid pTi15g55 from Agrobacterium tumefaciens (database entry X004g3 from position 12.541-12.350, Gielen et al. (ig84) EMBO J. 3835-846 ) was replaced.
  • the DNA fragment which contains the OCS terminator region was PCR-isolated using the plasmid pHELLSGATE (database entry AJ311874, Wesley et al. (2001) Plant J. 27581-590, isolated from E. coli by standard methods) and the primer OCS -1 (SEQ ID No. 58) and OCS-2 (SEQ ID No. 59).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the octopine synthase (OCS) terminator region (SEQ ID No. 60), was carried out in a 50 ⁇ l reaction mixture, which contained: 100 ng pHELLSGATE plasmid DNA 0.25 mM dNTPs 0.2 mM OCS-1 (SEQ ID No. 58) 0.2 mM OCS-2 (SEQ ID No. 5) 5 ul IOX PCR buffer (Stratagene) 0.25 ul Pfu polymerase (Stratagene) 28.8 ul aq. Least.
  • OCS octopine synthase
  • the PCR was carried out under the following cycle conditions:
  • the 210 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods and the plasmid pOCS was obtained.
  • Sequencing of the clone pOCS confirmed a sequence which corresponds to a sequence section on the Ti plasmid pTi15955 from Agrobacterium tumefaciens (database entry X00493) from positions 12,541 to 12,350.
  • the cloning was carried out by isolating the 210 bp Sall-Xhol fragment from pOCS and ligation into the Sall-Xhol cut vector pJIT117.
  • This clone is called pJO and was therefore used for the cloning into the expression vector pJONP196.
  • the cloning was carried out by isolating the 782 bp Sphl fragment from pNP196 and ligating into the SphI cut vector pJO.
  • the clone that contains the Nostoc punctiforme NPig6 ketolase in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJONP196.
  • the expression of the NP196 ketolase from Nostoc punctiforme in L. esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase, database entry AB011474 position 70127 to 694g3; WO03 / 006660), from Arabidopsis thallana.
  • FNR constitutive promoter
  • the FNR gene begins at base pair 69492 and is annotated with "ferredoxin-NADP + reductase”. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the DNA fragment containing the FNR promoter region from Arabidopsis thallana was PCR-analyzed using genomic DNA (isolated from Arabidopsis thaliana according to standard methods) and the primers FNR-1 (SEQ ID No. 61) and FNR-2 (SEQ ID No. 62).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the FNR promoter fragment FNR (SEQ ID No. 63), was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 652 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Jnvitrogen) using standard methods and the plasmid pFNR was obtained.
  • Sequencing of the clone pFNR confirmed a sequence which corresponds to a sequence section on chromosome 5 of Arabidopsis thaliana (database entry AB011474) from position 70127 to 6g493.
  • This clone is called pFNR and was therefore used for the cloning into the expression vector pJONP196 (described in Example 6).
  • the cloning was carried out by isolating the 644 bp Smal-Hindlll fragment from pFNR and ligating into the Ecl136ll-Hindlll cut vector pJONP196.
  • the clone which contains the promoter FNR instead of the original promoter d35S and the fragment NP196 in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJOFNR: NP196.
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (ig4 bp), fragment NP196 KETO CDS (761 bp), coding for the nostoc punctiform NPig ⁇ ketolase, fragment OCS W2 bp) the polyadenylation signal from the octopine synthase.
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the nostoc punctiform NP196 ketolase, Fragment OCS terminator (192 bp) the octopine synthase polyadenylation signal.
  • Example 8 Production of expression vectors for the flower-specific expression of the NP196 ketolase from Nostoc punctiforme ATCC 29133 in Lycopersicon esculentum and Tagetes erecta
  • NPig ⁇ -ketolase from Nostoc punctiforme in Lesculentum and Unes erecta was carried out with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240: 709-715). Expression was carried out under the control of the flower-specific promoter EPSPS from Petunia hybrida (database entry M37029: nucleotide region 7-1787; Benfey et al. (1990) Plant Cell 2: 849-856).
  • the DNA fragment which contains the EPSPS promoter region (SEQ ID No. 66) from Petunia hybrida, was PCR-analyzed using genomic DNA (isolated from Petunia hybrida according to standard methods) and the primers EPSPS-1 (SEQ ID No. 64) and EPSPS -2 (SEQ ID No. 65).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the EPSPS promoter fragment (database entry M37029: nucleotide region 7-1787), was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 1773 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods and the plasmid pEPSPS was obtained.
  • Sequencing of the clone pEPSPS confirmed a sequence consisting only of two deletions (bases ctaagtttcagga in position 46-58 of sequence M3702g; bases aaaaatat in positions 1422-142g of sequence M3702g) and the base exchanges (T instead of G in position 1447 of sequence M3702g ; A instead of C in position 1525 of sequence M3702g; A instead of G in position 1627 of sequence M3702g) differs from the published EPSPS sequence (database entry M3702g: nucleotide region 7-1787).
  • the two deletions and the two base changes at positions 1447 and 1627 of the sequence M3702g were reproduced in an independent amplification experiment and thus represent the actual nucleotide sequence in the Petunia hybrida plants used.
  • the clone pEPSPS was therefore used for the cloning into the expression vector pJONPI g6 (described in Example 6).
  • the cloning was carried out by isolating the 1763 bp SacI-HindIII fragment from pEPSPS and ligation into the SacI-HindIII cut vector pJONPI 6.
  • the clone which contains the promoter EPSPS instead of the original promoter d35S is called pJOESP: NP196.
  • This expression cassette contains the fragment NP196 in the correct orientation as an N-terminal fusion with the rbcS transit peptide.
  • fragment EPSPS contains the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (1 g4 bp), fragment NP196 KETO CDS (761 bp), coding for the Nostoc punctiform NPig ⁇ -ketolase, fragment OCS terminator (ig2 bp) the polyadenylation signal of octopine synthase.
  • An expression vector for the Agrobacterium -mediated transformation of the EPSPS-controlled NP196 ketolase from Nostoc punctiforme in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00g00).
  • fragment EPSPS contains the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (1 4 bp), fragment NP196 KETO CDS (761 bp), coding for the Nostoc punctiform NPig ⁇ ketolase, fragment OCS terminator (192 bp) the polyadenylation signal of octopine synthase.
  • Example g Amplification of a DNA encoding the entire primary sequence of the NP1 g5 ketolase from Nostoc punctiforme ATCC 29133
  • the nucleic acid encoding a Nostoc punctiform ATCC 29133 ketolase was synthesized by means of a "polymerase chain reaction” (PCR) from Nostoc punctiform ATCC 29133 using a sense-specific primer (NP195-1, SEQ ID No. 67) and an antisense-specific one Primers (NPig5-2 SEQ ID No. 68) amplified.
  • PCR polymerase chain reaction
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 67 and SEQ ID No. 68 resulted in an 819 bp fragment coding for a protein consisting of the entire primary sequence (NP195, SEQ ID No. 69).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNP195 was obtained.
  • This clone pNP105 was therefore used for the cloning into the expression vector pJO (described in Example 6).
  • the cloning was carried out by isolating the 800 bp Sphl fragment from pNPigs and ligation into the SphI cut vector pJO.
  • the clone which contains the NPig ⁇ ketolase from Nostoc punctiforme in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJONPI 95.
  • the expression of the NP195 ketolase from Nostoc punctiforme in L. esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase, database entry AB011474 position 70127 to 69493; WO03 / 006660), from Arabidopsis thallana.
  • FNR constitutive promoter
  • the FNR gene begins at base pair 694g2 and is annotated with "ferredoxin-NADP + reductase”. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1086, Biochem J. 240: 700-715).
  • the clone pFNR (described in Example 7) was therefore used for the cloning into the expression vector pJONPI 05 (described in Example 10).
  • the cloning was carried out by isolating the 644 bp Sma-Hindlll fragment from pFNR and ligating into the Ecl136ll-Hindlll cut vector pJONPI 05.
  • the clone which contained the promoter FNR instead of the original promoter d35S and the fragment NPi ⁇ in the correct orientation as contains terminal fusion with the rbcS transit peptide is called pJOFNR: NP195.
  • An expression cassette for the Agrobacterium-mediated transformation of the NP195 ketolase from Nostoc punctiforme into lesculentum was produced using the binary vector pSUN3 (WO02 / 00000).
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (78g bp), coding for the nostoc punctiform NPi g ⁇ -ketolase, fragment OCS terminator (ig2 bp) the polyadenylation signal of octopine synthase.
  • the expression of the NP1 g5 ketolase from Nostoc punctiforme in L. esculentum and Tagetes erecta was carried out with the transit peptide rbcS from pea (Anderson et al. Ig86, Biochem J. 240: 700-715). The expression was carried out under the control of the flower-specific promoter EPSPS from Petunia hybrida (database entry M37020: nucleotide region 7-1787; Benfey et al. (1990) Plant Cell 2: 840-856).
  • the clone pEPSPS (described in Example 8) was therefore used for the cloning into the expression vector pJONPI 95 (described in Example 10).
  • the cloning was carried out by isolating the 1763 bp SacI-HindIII fragment from pEPSPS and ligation into the SacI-HindIII cut vector pJONPI 05.
  • the clone which contains the promoter EPSPS instead of the original promoter d35S, is called pJOESP: NPig5.
  • This expression cassette contains the fragment NPi ⁇ in the correct orientation as an N-terminal fusion with the rbcS transit peptide.
  • fragment EPSPS contains the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), coding for the Nostoc punctiform NPig ⁇ -ketolase, fragment OCS terminator (192 bp) the polyadenylation signal of octopine synthase.
  • An expression vector for the Agrobacterium -mediated transformation of the EPSPS-controlled NPig ⁇ -ketolase from Nostoc punctiforme in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00 ⁇ 00).
  • fragment EPSPS contains the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (1 g4 bp), fragment NP195 KETO CDS (78g bp), coding for the Nostoc punctiform NPig ⁇ -ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.
  • Example 12 Amplification of a DNA encoding the entire primary sequence of the NODK ketolase from Noularia spumignea ⁇ / SOft 70.
  • the DNA encoding the ketolase from Nodularia spumignea NSOR 10 was amplified by PCR from Nodularia spumignea NSOR10.
  • genomic DNA from a suspension culture of Nodularia spumignea NSOR10, d ⁇ e 1 week with continuous light and constant shaking (150 rpm) at 25 ° C in BG 77 medium (1.5 g / l NaN0 3 , 0.04 g / l K 2 P0 4 x3H 2 0, 0.075 g / l MgS0 4 xH 2 O, 0.036 g / l CaCI 2 x2H 2 O, 0.006 g / l citric acid, 0.006 g / l Ferric ammonium citrate, 0.001 g / l EDTA disodium magnesium, 0.04 g / l Na 2 CO 3 , 1 ml trace metal mix "A5 + Co" (2.86 g / l H 3 BO 3 ,
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (2 ml volume). After adding 100 ⁇ l Proteinase K (concentration: 20 mg / ml), the cell suspension was incubated for 3 hours at 37 ° C. Then the
  • the nucleic acid encoding a ketolase from Nodularia spumignea NSOR10 was determined by means of a "polymerase chain reaction” (PCR) from Nodularia spumignea NSOR10 using a sense-specific primer (NODK-1, SEQ ID No. 71) and an antisense-specific primer ( NODK-2 SEQ ID No. 72).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture, which contained: 1 ul of a Nodularia spumignea NSOR10 DNA (prepared as described above)
  • 0.2 mM NODK-1 (SEQ ID No. 71) - 0.2 mM NODK-2 (SEQ ID No. 72) 5 ul 10X PCR buffer (TAKARA) 0.25 ul R Taq polymerase (TAKARA) 25.8 ul Aq. Least.
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 71 and SEQ ID No. 72 resulted in a 720 bp fragment coding for a protein consisting of the entire primary sequence (NODK, SEQ ID No. 73).
  • 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 the cloning into the expression vector pJO (described in Example 6).
  • the cloning was carried out by isolating the 710 bp Sphl fragment from pNODK and ligation into the SphI-cut vector pJO.
  • the clone that shows the NODK ketolase from Nodularia spumignea in the correct orientation contains as an N-terminal translational fusion with the rbcS transit peptide is called pJONODK.
  • Example 13 Production of expression vectors for the constitutive expression of the NODK ketolase from Nodularia spumignea NSOR10 in Lycopersicon esculentum and Tagetes erecta.
  • the expression of the NODK ketolase from Nodularia spumignea NSOR10 in L. esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin-NADPH-oxidoreductase, database entry AB011474 position 70127 to 60403; WO03 / 006660) from Arabidopsis thall.
  • FNR constitutive promoter
  • the FNR gene begins at base pair 604g2 and is annotated with "ferredoxin-NADP + reductase”. Expression was carried out using the pea transit peptide rbcS (Anderson et al. Ig86, Biochem J. 240: 700-715).
  • the clone pFNR (described in Example 7) was therefore used for the cloning into the expression vector pJONODK (described in Example 12).
  • the cloning was carried out by isolating the 644 bp Sma-HindIII fragment from pFNR and ligating into the Ecl136II-HindIII cut vector pJONODK.
  • the clone which contains the promoter FNR instead of the original promoter d35S and the fragment NODK in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJOFNR: NODK.
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (104 bp), fragment NODK KETO CDS (600 bp), coding for the Nodularia spumignea NSOR10 NODK ketolase, fragment OCS terminator (102 bp) the polyadenylation signal from the octopine synthase.
  • An expression cassette for the / 4grobactet / um-mediated transformation of the expression vector with the NODK ketolase from Nodularia spumignea NSOR10 punctiforme in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00900).
  • fragment FNR promoter contains the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NODK KETO CDS (6g ⁇ bp), coding for the Nodularia spumignea NSOR10 NODK ketolase , Fragment OCS terminator (102 bp) the polyadenylation signal of octopine synthase.
  • Example 14 Production of expression vectors for the flower-specific expression of the NODK ketolase from Nodularia spumignea NSORW in Lycopersicon esculentum and Tagetes erecta.
  • the NODK ketolase from Nodularia spumignea NSOR10 ⁇ n L. esculenum and Tagetes erecta was expressed with the transit peptide rbcS from pea (Anderson et al: 1086, Biochem J. 240: 709-715). The expression was carried out under the control of the flower-specific promoter EPSPS from Petunia hybrida (database entry M37029: nucleotide region 7-1787; Benfey et al. (1990) Plant Cell 2: 840-856).
  • the clone pEPSPS (described in Example 8) was therefore used for the cloning into the expression vector pJONODK (described in Example 12).
  • the cloning was carried out by isolating the 1763 bp SacI-HindIII fragment from pEPSPS and ligation into the SacI-HindIII cut vector pJONODK.
  • the clone that contains the promoter EPSPS instead of the original promoter d35S is called pJOESP: NODK.
  • This expression cassette contains the fragment NODK in the correct orientation as an N-terminal fusion with the rbcS transit peptide.
  • fragment EPSPS contains the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NODK KETO CDS (690 bp), coding for the Nodularia spumignea NSOR 10 NODK ketolase, fragment OCS terminator (102 bp) the polyadenylation signal of octopine synthase.
  • An expression vector for the Agrobacterium -mediated transformation of the EPSPS-controlled NODK ketolase from Nodularia spumignea NSOR10 in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00 ⁇ 00).
  • the 2,880 KB bp Sacl-Xhol was used to produce the expression vector MSP116
  • fragment EPSPS contains the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT ' the rbcS transit peptide from pea (104 bp), fragment NODK KETO CDS (600 bp), coding for the Nodularia spumignea NSOR10 NODK-Ketolase, fragment OCS terminator (102 bp) the polyadenylation signal of octopine synthase.
  • Transformation and regeneration of tomato plants was carried out according to the published method by Ling and co-workers (Plant Cell Reports (1998), 17: 843-847).
  • Ling and co-workers Plant Cell Reports (1998), 17: 843-847).
  • kanamycin concentrations 100 mg / L were selected.
  • the starting explant for the transformation was cotyledons and hypocotyls, seven to ten day old seedlings of the Microtome line.
  • the culture medium according to Murashige and Skoog (1062: Murashige and Skoog, 1062, Physiol. Plant 15, 473-) with 2% sucrose, pH 6.1 was used for the germination. Germination took place at 21 ° C in low light (20 - 100 ⁇ E).
  • the cotyledons became divided transversely and the hypocotyls cut into sections about 5 to 10 mm long and placed on the medium MSBN (MS, pH 6.1, 3% sucrose + 1 mg / l BAP, 0.1 mg / l NAA), which on The previous day was loaded with suspension-cultivated tomato cells.
  • the tomato cells were covered with sterile filter paper without air bubbles.
  • the explants were precultured on the medium described for three to five days.
  • Cells from the Agrobacterium tumefaciens LBA4404 strain were individually transformed with the plasmids pS3FNR: NOST, pS3AP3: NOST, pS3FNR: NP106, pS3EPS: NP106, pS3FNR: NP105, pS3EPS: NP105, pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: NODK and pS3FNR: transform.
  • the bacterial pellet was resuspended with liquid MS medium (3% sucrose, pH 6.1) and adjusted to an optical density of 0.3 (at 600 nm).
  • the precultivated explants were transferred to the suspension and incubated for 30 minutes at room temperature with gentle shaking.
  • the explants were then dried with sterile filter paper and placed back on their preculture medium for the three-day co-culture (21 ° C.).
  • 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 for selective regeneration at 21 ° C stored under low light conditions (20 - 100 ⁇ E, light rhythm 16h / 8h).
  • MSZ2 medium MS pH 6.1 + 3% sucrose, 2 mg / l zeatin, 100 mg / l kanamycin, 160 mg / l timentin
  • the explants were transferred every two to three weeks until shoots formed. Small shoots could be separated from the explant and rooted on MS (pH 6.1 + 3% sucrose) 160 mg / l timentin, 30 mg / l kanamycin, 0.1 mg / l IAA. Rooted plants were transferred to the greenhouse.
  • NP196 was obtained: MSP107-1, MSP107-2, MSP107-3
  • NP105 was obtained: MSP100-1, MSP100-2, MSP100-3
  • germination medium MS medium; Murashige and Skoog, Physiol. Plant. 15 (1062), 473-407) pH 5.8, 2% sucrose). Germination takes place in a temperature / light / time interval of 18-28 ° C / 20-200 ⁇ E / 3-16 weeks, but preferably at 21 ° C, 20-70 ⁇ E, for 4-8 weeks.
  • pS5FNR NOST
  • pS5AP3 NOST
  • pS5FNR NP106
  • pS5EPS NP196
  • pS5FNR NP1
  • pS5EPS NP1 g5
  • pS5FNR NODK
  • pS5EPS NODK
  • the bacterial strain can be grown as follows: A single colony of the corresponding strain is in YEB (0.1% yeast extract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.5% magnesium sulfate x 7 H) 2 0) inoculated with 25 mg / l kanamycin and dressed at 28 ° C for 16 to 20 h. The bacterial suspension is then harvested by centrifugation at 6000 g for 10 min and resuspended in liquid MS medium such that an OD 600 of approximately 0.1 to 0.8 was formed. This suspension is used for C cultivation with the leaf matter! used.
  • the MS medium in which the leaves have been kept is replaced by the bacterial suspension.
  • the leaflets were incubated in the agrobacterial suspension for 30 min with gentle shaking at room temperature.
  • the infected explants are then placed on an MS medium solidified with agar (for example 0.8% plant agar (Duchefa, NL) with growth regulators, such as 3 mg / l benzylaminopurine (BAP) and 1 mg / l indolylacetic acid (IAA).
  • agar for example 0.8% plant agar (Duchefa, NL) with growth regulators, such as 3 mg / l benzylaminopurine (BAP) and 1 mg / l indolylacetic acid (IAA).
  • the orientation of the leaves on the medium is irrelevant: the explants are cultivated for 1 to 8 days, but preferably for 6 days, the following conditions can be used: light intensity: 30-80 ⁇ mol / m 2 x sec, temperature: 22-24 ° C., light / dark change of 16/8 hours, after which the co-cultivated explants are transferred to fresh MS medium, preferably with the same growth regulators, this second medium additionally containing an antibiotic to suppress bacterial growth
  • a concentration of 200 to 500 mg / l is very suitable for this purpose, and the second selective component is one used to select the success of the transformation.
  • Phosphinothricin in a concentration of 1 to 5 mg / l selects very efficiently, but other selective components according to the method to be used are also conceivable.
  • the explants are transferred to fresh medium until shoot buds and small shoots develop, which are then on the same basal medium including timentin and PPT or alternative components with growth regulators, namely, for example, 0.5 mg / l indolylbutyric acid (IBA) and 0.5 mg / l gibberillic acid GA 3 , are transferred for rooting. Rooted shoots can be transferred to the greenhouse.
  • IBA 0.5 mg / l indolylbutyric acid
  • GA 3 gibberillic acid
  • the explants Before the explants are infected with the bacteria, they can be pre-incubated for 1 to 12 days, preferably 3-4, on the medium described above for the co-culture become. The infection, co-culture and selective regeneration then take place as described above.
  • the pH value for regeneration (normally 5.8) can be lowered to pH 5.2. This improves the control of agrobacterial growth.
  • Liquid culture medium can also be used for the entire process.
  • the culture can also be incubated on commercially available carriers which are positioned on the liquid medium.
  • pS5FNR NOST, for example, the following was obtained: MSP102-1, MSP102-2, MSP102-3,
  • pS5AP3 NOST, for example, the following was obtained: MSP104-1, MSP104-2, MSP104-3
  • NP106 was obtained: MSP106-1, MSP106-2, MSP106-3
  • NP106 was obtained: MSP108-1, MSP108-2, MSP108-3
  • NP105 was obtained: MSP110-1, MSP110-2, MSP110-3
  • the petals of the transgenic plants are mortarized in liquid nitrogen and the petalen powder (about 40 mg) extracted with 100% acetone (three times 500 ul). The solvent is evaporated and the carotenoids are resuspended in 100-200 ul of petroleum ether / acetone (5: 1, v / v).
  • the carotenoids are separated in concentrated form by means of thin layer chromatography (TLC) on Silica ⁇ O F254 plates (Merck) in an organic solvent (petroleum ether / acetone; 5: 1) according to their phobicity. Yellow (xanthophyll esters), red (ketocarotenoid esters) and orange bands (mixture of xanthophyll and ketocarotenoid esters) are scraped out on the TLC.
  • TLC thin layer chromatography
  • the carotenoids bound to silica are eluted three times with 500 ⁇ l of acetone, the solvent is evaporated and the carotenoids are separated and identified by means of HPLC.
  • a C30 reverse phase column can be used to differentiate between mono- and diesters of carotenoids.
  • HPLC running conditions were almost identical to a published method (Frazer et al. (2000), Plant Journal 24 (4): 551-558). The following process conditions were set.
  • Solvent B 80% methanol, 0.2% ammonium acetate solvent
  • C 100% t-butyl methyl ether gradient profile:
  • the carotenoids can be identified on the basis of the UV-VIS spectra.
  • Petal material from the transgenic tomato plants is ground and extracted with acetone. Extracted carotenoids are separated by means of TLC. In the lines can
  • Mono and diesters of ketocarotenoids can be detected; the monoesters are present in a significantly lower concentration than the diesters.
  • Mortar petal material (30-100 mg fresh weight) is extracted with 100% acetone (three times 500 ul; shake for about 15 minutes each). The solvent is evaporated. Carotenoids are then taken up in 405 ⁇ l of acetone, 4.95 ml of potassium phosphate buffer (100 mM, pH 7.4) are added and mixed well. Then about 17 mg of Bile salts (Sigma) and 140 ⁇ l of a NaCI / CaCI2 solution (3M NaCI and 75 mM CaCI2) are added. The suspension is incubated for 30 minutes at 37C.
  • a lipase solution 50 mg / ml lipase type 7 from Candida rugosa (Sigma)
  • 505 ⁇ l of a lipase solution 50 mg / ml lipase type 7 from Candida rugosa (Sigma)
  • a lipase solution 50 mg / ml lipase type 7 from Candida rugosa (Sigma)
  • 595 ⁇ l of lipase was added again and incubation was continued for at least 5 hours at 37C.
  • be about 700 mg of Na2SO4x10H20 dissolved in the solution After adding 1800 ⁇ l of petroleum ether, the carotenoids are extracted into the organic phase by vigorous mixing. This shaking is repeated until the organic phase remains indolent.
  • the petroleum ether fractions are combined and the petroleum ether evaporated. Free carotenoids are taken up in 100-120 ul acetone. Free carotenoids can be identified on the basis of retention time and UV-VlS spectra by means of HPLC

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)
  • Biophysics (AREA)
  • Animal Husbandry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Pregnancy & Childbirth (AREA)
  • Birds (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physiology (AREA)
  • Reproductive Health (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Insects & Arthropods (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Fodder In General (AREA)
EP03792348A 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies Withdrawn EP1532265A2 (fr)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
DE10238978 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
DE2002138980 DE10238980A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Ketocarotinoiden in Blütenblättern von Pflanzen
DE10238979 2002-08-20
DE10238980 2002-08-20
DE10238978A DE10238978A1 (de) 2002-08-20 2002-08-20 Verfahren zur Herstellung von Ketocarotinoiden in Früchten von Pflanzen
DE10253112 2002-11-13
DE2002153112 DE10253112A1 (de) 2002-11-13 2002-11-13 Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten Organismen
DE2002158971 DE10258971A1 (de) 2002-12-16 2002-12-16 Verwendung von astaxanthinhaltigen Pflanzen oder Pflanzenteilen der Gattung Tagetes als Futtermittel
DE10258971 2002-12-16
PCT/EP2003/009106 WO2004018694A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies

Publications (1)

Publication Number Publication Date
EP1532265A2 true EP1532265A2 (fr) 2005-05-25

Family

ID=31950810

Family Applications (5)

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

Family Applications After (4)

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

Country Status (12)

Country Link
US (5) US20060253927A1 (fr)
EP (5) EP1532265A2 (fr)
CN (1) CN1675367A (fr)
AT (1) ATE484198T1 (fr)
AU (5) AU2003258623A1 (fr)
CA (5) CA2495235A1 (fr)
DE (1) DE50313184D1 (fr)
IL (4) IL166507A0 (fr)
MX (5) MXPA05001948A (fr)
NO (5) NO20050598L (fr)
WO (5) WO2004018693A2 (fr)
ZA (1) ZA200602230B (fr)

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003258622A1 (en) * 2002-08-20 2004-03-11 Sungene Gmbh And Co. Kgaa Method for the production of $g(b)-carotinoids
US20060107352A1 (en) * 2002-08-20 2006-05-18 Sungene Gmbh & Co. Kgaa Transgenic expression cassettes for expression of nucleic acids in the plant blooms
EP1532265A2 (fr) * 2002-08-20 2005-05-25 Sungene GmbH & Co. KGaA Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies
EP1554388A1 (fr) * 2002-10-11 2005-07-20 Sungene GmbH & Co. KGaA Cassettes d'expression transgenique pour l'expression d'acides nucleiques dans une fleur vegetale
DE10300649A1 (de) * 2003-01-09 2004-07-22 Basf Ag Verfahren zur Herstellung von Ketocarotinoiden durch Kultivierung von genetisch veränderten Organismen
EP1780281B1 (fr) * 2004-06-04 2013-12-11 Kirin Holdings Kabushiki Kaisha Procédé de production de l'astaxanthine ou d'un produit métabolique de ce composé en utilisant les gènes de la caroténoïde cétolase et de la caroténoïde hydrolase
AU2006227165B2 (en) 2005-03-18 2011-11-10 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
CA2614659A1 (fr) * 2005-07-11 2007-01-18 Commonwealth Scientific And Industrial Research Organisation Pigment de ble
WO2008042338A2 (fr) 2006-09-28 2008-04-10 Microbia, Inc. Production de caroténoïdes dans des levures et des champignons oléagineux
EP2087096A4 (fr) * 2006-10-20 2009-11-25 Univ Arizona Cyanobacterie modifiee
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 (fr) * 2008-12-17 2010-06-23 BASF Plant Science GmbH Production de cétocaroténoïdes dans les plantes
WO2010079032A1 (fr) * 2008-12-17 2010-07-15 Basf Plant Science Gmbh Production de cétocaroténoïdes dans des plantes
HK1199286A1 (en) * 2011-08-08 2015-06-26 Evolva Sa Methods and materials for recombinant production of saffron compounds
US9493515B2 (en) 2011-11-29 2016-11-15 Proclara Biosciences, Inc. Bacteriophage gene 3 protein compositions and use as amyloid binding agents
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
WO2019222711A1 (fr) 2018-05-17 2019-11-21 Lumen Bioscience, Inc. Plate-forme d'administration de vaccin oral d'arthrospira platensis
US12252513B2 (en) 2018-07-16 2025-03-18 Lumen Bioscience, Inc. Thermostable phycobiliproteins produced from recombinant arthrospira
EP3994152A4 (fr) 2019-07-03 2023-08-02 Lumen Bioscience, Inc. Plate-forme d'administration non parentérale d'agent thérapeutique d'arthrospira platensis
WO2021085575A1 (fr) * 2019-10-30 2021-05-06 アスタリール株式会社 Suppresseur d'hémolyse induit par l'exercice et composition pour inhiber/améliorer l'anémie hémolytique induite par l'exercice
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
AU702584B2 (en) * 1993-12-27 1999-02-25 Kirin Holdings Kabushiki Kaisha DNA strands useful for the synthesis of xanthophylls and the process for producing the xanthophylls
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
BR9713462A (pt) * 1996-08-09 2000-03-28 Calgene Inc Métodos para produção de compostos carotenóides e óleos especiais em sementes de plantas.
US5876782A (en) * 1997-05-14 1999-03-02 Kemin Industries, Inc. Method for the conversion of xanthophylls in plant material
US6221417B1 (en) * 1997-05-14 2001-04-24 Kemin Industries, Inc. Conversion of xanthophylls in plant material for use as a food colorant
WO1999061652A1 (fr) * 1998-05-22 1999-12-02 University Of Maryland Genes et produits geniques de cetolase de carotenoide, production de carotenoides et procedes permettant de modifier des carotenoides au moyen de ces genes
CA2330167A1 (fr) * 1998-06-02 1999-12-09 University Of Maryland Genes de la biosynthese et du metabolisme du carotenoide et techniques d'utilisation
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
US7575766B2 (en) * 2001-06-29 2009-08-18 Ball Horticultural Company Tagetes erecta with altered carotenoid compositions and ratios
US6784351B2 (en) * 2001-06-29 2004-08-31 Ball Horticultural Company Targetes erecta marigolds 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 (fr) 2002-03-11 2003-09-25 Prodemex, S.A. De C.V. Nourrissage d'animaux aquatiques avec de l'astaxanthine, produit derive de la calendula, en vue d'ameliorer leurs taux d'engraissement et de croissance
US7223909B2 (en) * 2002-03-21 2007-05-29 Ball Horticultural 4-ketocarotenoids in flower petals
EP1532265A2 (fr) * 2002-08-20 2005-05-25 Sungene GmbH & Co. KGaA Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004018694A2 *

Also Published As

Publication number Publication date
IL166770A0 (en) 2006-01-15
NO20050755L (no) 2005-05-19
WO2004018385A3 (fr) 2004-10-21
NO20050705L (no) 2005-05-18
US20060253927A1 (en) 2006-11-09
WO2004018385A2 (fr) 2004-03-04
CA2495235A1 (fr) 2004-03-04
AU2003258623A1 (en) 2004-03-11
EP1542945A2 (fr) 2005-06-22
AU2003253416A1 (en) 2004-03-11
WO2004018695A3 (fr) 2004-10-14
AU2003264062B2 (en) 2008-01-03
NO20050703L (no) 2005-05-09
NO20050598L (no) 2005-04-07
MXPA05001944A (es) 2005-06-22
US20060031963A1 (en) 2006-02-09
IL166507A0 (en) 2006-01-15
ZA200602230B (en) 2007-05-30
WO2004018693A3 (fr) 2004-12-09
CA2496207A1 (fr) 2004-03-04
EP1532264A2 (fr) 2005-05-25
MXPA05001659A (es) 2005-07-22
EP1532266A2 (fr) 2005-05-25
AU2003264062A1 (en) 2004-03-11
CA2496133A1 (fr) 2004-03-04
DE50313184D1 (de) 2010-11-25
WO2004018693A2 (fr) 2004-03-04
CA2495878A1 (fr) 2004-03-04
IL166767A0 (en) 2006-01-15
EP1531683A2 (fr) 2005-05-25
US20060112451A1 (en) 2006-05-25
WO2004017749A3 (fr) 2004-10-14
IL166771A0 (en) 2006-01-15
WO2004018695A2 (fr) 2004-03-04
US20070094749A1 (en) 2007-04-26
MXPA05001948A (es) 2005-09-08
WO2004018694A3 (fr) 2004-09-10
WO2004018694A2 (fr) 2004-03-04
WO2004017749A2 (fr) 2004-03-04
EP1531683B1 (fr) 2010-10-13
AU2003260423A1 (en) 2004-03-11
US7381541B2 (en) 2008-06-03
CN1675367A (zh) 2005-09-28
NO20050704L (no) 2005-05-13
CA2495444A1 (fr) 2004-03-04
US7385123B2 (en) 2008-06-10
US20050281909A1 (en) 2005-12-22
ATE484198T1 (de) 2010-10-15
MXPA05001899A (es) 2005-08-29
MXPA05001811A (es) 2005-10-19
AU2003260424A1 (en) 2004-03-11

Similar Documents

Publication Publication Date Title
US7385123B2 (en) Process for preparing ketocarotenoids in genetically modified organisms
DE10300649A1 (de) Verfahren zur Herstellung von Ketocarotinoiden durch Kultivierung von genetisch veränderten Organismen
WO2005019467A1 (fr) Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies
JP2007502605A6 (ja) 遺伝子的に改変された非ヒト生物におけるケトカロテノイドの製造方法
DE10238980A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in Blütenblättern von Pflanzen
WO2005019461A2 (fr) Nouvelles cetolases et procede de production de cetocarotenoides
EP1532256A1 (fr) Procede de preparation de $g(b)-carotinoides
DE10253112A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten Organismen
DE10238978A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in Früchten von Pflanzen
EP1658372A2 (fr) Nouvelles cetolases et procede de production de cetocarotenoides
EP1658377A1 (fr) Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies
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
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050321

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20070426

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20070828