EP1658372A2 - Nouvelles cetolases et procede de production de cetocarotenoides - Google Patents

Nouvelles cetolases et procede de production de cetocarotenoides

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Publication number
EP1658372A2
EP1658372A2 EP04763696A EP04763696A EP1658372A2 EP 1658372 A2 EP1658372 A2 EP 1658372A2 EP 04763696 A EP04763696 A EP 04763696A EP 04763696 A EP04763696 A EP 04763696A EP 1658372 A2 EP1658372 A2 EP 1658372A2
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EP
European Patent Office
Prior art keywords
sequence
sequence seq
amino acid
ketolase
nucleic acids
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Withdrawn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
EP04763696A
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German (de)
English (en)
Inventor
Matt Sauer
Christel Renate Schopfer
Ralf Flachmann
Karin Herbers
Irene Kunze
Martin Klebsattel
Thomas Luck
Dirk Voeste
Angelika-Maria Pfeiffer
Hendrik Tschoep
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SunGene GmbH
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SunGene GmbH
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Publication date
Priority claimed from PCT/EP2003/009102 external-priority patent/WO2004018693A2/fr
Priority claimed from PCT/EP2003/009101 external-priority patent/WO2004018688A1/fr
Priority claimed from DE102004007624A external-priority patent/DE102004007624A1/de
Application filed by SunGene GmbH filed Critical SunGene GmbH
Priority to EP04763696A priority Critical patent/EP1658372A2/fr
Publication of EP1658372A2 publication Critical patent/EP1658372A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates to a process for the production of ketocarotenoids by cultivating genetically modified, non-human organisms which have a modified ketolase activity compared to the wild type, the genetically modified organisms, their use as food and feed and for the production of ketocarotenoid extracts and new ketolases and nucleic acids encoding these ketolases ..
  • Ketocarotenoids are synthesized de novo in bacteria, algae, fungi and plants.
  • Ketocarotenoids i.e. carotenoids, which 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 in particular astaxanthin are used as pigmenting 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.
  • ketolases and processes for the preparation of ketocarotenoids described in the prior art and in particular the processes described for the preparation of astaxanthin have the disadvantage that on the one hand the yield is not yet satisfactory and on the other hand the transgenic organisms have a large amount of hydroxylated by-products , such as zeaxanthin and adonixanthin.
  • the object of the invention was therefore to provide a process for the production of ketocarotenoids by cultivating genetically modified, non-human organisms, or further genetically modified, non-human organisms which produce ketocarotenoids and new, advantageous ketolases to provide, which have the disadvantages of the prior art described above to a lesser extent or no longer or which provide the desired ketocarotenoids, in particular astaxanthin, in higher yields. Accordingly, a method for producing ketocarotenoids has been found by cultivating genetically modified non-human organisms which have an altered ketolase activity compared to the wild type and which altered ketolase activity is caused by a ketolase selected from the group
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • an altered ketolase activity compared to the wild type is preferably understood to mean a “ketolase activity caused compared to the wild type”.
  • an altered ketolase activity compared to the wild type is preferably understood to mean an “increased ketolase activity compared to the wild type”.
  • non-human 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 changed by genetic modification, such as re-regulating metabolic pathways or complementing them Be able to produce carotenoids such as ß-carotene or zeaxanthin.
  • Some organisms, as starting or wild type organisms, are already able to produce ketocarotenoids such as astaxanthin or canthaxanthin.
  • wild type is understood to mean the corresponding starting organism.
  • organism can be understood to mean the non-human starting organism (wild type) or an inventive, genetically modified, non-human organism or both.
  • wild type is used to increase or cause the ketolase activity, for the increase or cause described below, or to cause the hydroxylase activity for which Increase or cause of ⁇ -cyclase activity described below, for the increase in HMG-CoA reductase activity described below, for the increase in (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate described below Reductase activity, for the increase in 1-deoxy-D-xylose-5-phosphate synthase activity described below, for the increase in 1-deoxy-D-xylose-5-phosphate described below.
  • Reductoisomerase activity for the increase in isopenyl diphosphate- ⁇ -isomerase activity described below, for the increase in geranyl diphosphate synthase activity described below, for the increase in famesyl diphosphate synthase activity described below, for the increase in geranyl-geranyl diphosphate synthase activity described below, for the increase in phytoene synthase activity described below, for the increase in phytoene desaturase activity described below, for the increase in zeta-carotene described below Desaturase activity, for the increase in crtlSO activity described below, for the increase in FtsZ activity described below, for the increase in MinD activity described below, for the reduction in ⁇ -cyclase activity described below and for that described below Reduction of endogenous ß-hydroxylase activity u nd the increase in the content of ketocarotenoids was understood as a reference organism. This reference organism is preferably Haematococcus pluvialis for microorganisms which already have
  • This reference organism is preferably Blakeslea for microorganisms which, as a wild type, have no ketolase activity.
  • this reference organism is preferably Adonis aestivalis, Adonis flammeus or Adonis a ⁇ nuus, particularly preferably Adonis aestivalis.
  • This reference organism is particularly preferred for plants which, as wild type, have no ketolase activity in petals, preferably Tagetes erecta, Tagetes patula, Tagetes lucida, Tagetes pringlei, Tagetes palmeri, Tagetes minuta or Tagetes campanulata.
  • 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.
  • non-human organisms are used as starting organisms which already have a ketolase activity as wild type or starting organism, such as, for example, Haematococcus pluvialis, Paracoccus marcusii, Xanthophyllomyces dendrorhous, Bacillus circulans, Chlorococcum, Phaffia rhodozyma, Adonis florets (Adonis aestivalis), Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Scenedesmus vacuolatus, Chlorela zoofingiensis, Ankistrodesmus braunii, Euglena sanguinea or Bacillus atrophaeus.
  • the genetic modification causes an increase in ketolase activity compared to the wild type or parent organism.
  • the protein ketolase will convert the amount of ß- Carotene or the amount of canthaxanthin formed increases.
  • This increase in ketolase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, further preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the ketolase Wild type activity.
  • 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 Fraser 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 Wiid 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, according to the invention, in this embodiment also the manipulation of the expression of the organism's own endogenous ketolases. This can be achieved, for example, by changing the promoter DNA sequence for genes encoding 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 one that is not found in the wild-type organism or modified regulatory protein 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 selected from the group
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • the gene expression of a nucleic acid encoding a ketolase is increased by introducing nucleic acids encoding ketolases selected from the group
  • 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 80% on amino acids level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • non-human organisms are used as starting organisms which, as a wild type, have no ketolase activity, such as, for example, Blakeslea, Marigold, Tagetes erecta, Tagetes lucida, Tagetes minuta, Tagetes pringlei, Tagetes palmeri and 7a getes campanulata.
  • ketolase activity such as, for example, Blakeslea, Marigold, Tagetes erecta, Tagetes lucida, Tagetes minuta, Tagetes pringlei, Tagetes palmeri and 7a getes campanulata.
  • the genetic modification causes ketolase activity in the 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 preferably able to transgenically express a ketolase, the ketolases being selected from the group
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • the gene expression of a nucleic acid encoding a ketolase is caused analogously to that described above Increasing the gene expression of a nucleic acid encoding a ketolase, preferably by introducing nucleic acids encoding ketolases into the starting organism, the ketolases being selected from group 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2, B ketolase containing the amino acid sequence SEQ. ID. NO.
  • any ketolase gene that is to say any, can do this in both embodiments
  • ketolases are used, the ketolases being selected from the group
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • nucleic acids mentioned in the description can be, for example, an RNA, DNA or cDNA sequence.
  • nucleic acid sequences such as that which have already been processed to use corresponding cDNAs.
  • nucleic acids encoding a ketolase and the corresponding ketolases from group A that can be used in the method according to the invention are, for example, the ketolase sequences according to the invention
  • Nodularia spumigena strain NSOR10 Nodularia spumigena strain NSOR10
  • Nucleic acid SEQ ID NO: 1
  • protein SEQ ID NO: 2 (Acc. -No.AY210783, wrong sequence annotated as putative keto analysis),
  • Nodularia spumigena (Culture Collection of Algae at the University of Vienna, (CCAUV) 01-037), nucleic acid: SEQ ID NO: 3, protein: SEQ ID NO: 4),
  • Nodularia spumigena (Culture Collection of Algae at the University of Vienna (CCAUV) 01-053), nucleic acid: SEQ ID NO: 5, protein: SEQ ID NO: 6) and
  • Nodularia spumigena (Culture Collection of Algae at the University of Vienna (CCAUV) 01-061), nucleic acid: SEQ ID NO: 7, protein: SEQ ID NO: 8)
  • nucleic acids encoding a ketolase and the corresponding ketolases from group B which can be used in the method according to the invention are, for example, the ketolase sequences according to the invention Nostoc puntiforme (collection of algal cultures Göttingen (SAG) 60.79 nucleic acid: SEQ ID NO: 9, protein: SEQ ID NO: 10.
  • nucleic acids encoding a ketolase and the corresponding ketolases from group C that can be used in the method according to the invention are, for example, the ketolase sequences according to the invention
  • nucleic acids encoding a ketolase and the corresponding ketolases from group D that can be used in the method according to the invention are, for example, the ketolase sequences according to the invention
  • 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 described above and easy to find especially with the sequences SEQ ID NO: 2 and / or 10 and / or 12 and / or 14.
  • ketolases and ketolase genes can furthermore be derived from the nucleic acid sequences described above, in particular from the sequences SEQ ID NO: 1 and / or 9 and / or, 11 and / or 13 from different organisms, the genomic sequence of which is not is known to be easily found by hybridization techniques in a manner known per se.
  • hybridization and this condition applies to all nucleic acid sequences in the description, can take place under moderate (low stringency) or preferably under stringent (high stringency) conditions.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with 2X SSC at 50 ° C) and those with high stringency (with 0.2X SSC at 50 ° C, preferably at 65 ° C) (20X SSC: 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).
  • the temperature during the washing step can be raised from moderate conditions at room temperature, 22 ° C, to stringent conditions at 65 ° C.
  • Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can 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.
  • Group A ketolases contain 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 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • Group B ketolases contain the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90%, more preferably at least 95%, more preferably at least 97%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 10 has.
  • Group C ketolases contain the SEQ amino acid sequence. 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 90%, more preferably at least 95%, more preferably at least 97%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 12 has.
  • the group D ketolases contain the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • substitution is to be understood as the exchange 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 means the identity of the amino acids over the respective total protein length, in particular the identity that 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:
  • Pairwise alignment parameters FAST algorithm on K-tuplesize 1 Gap penalty 3 Window size 5 Number of best diagonals 5
  • a protein which has an identity of at least 80% at the amino acid level with a specific sequence is accordingly understood to be a protein which has an identity of at least 80% when comparing its sequence with the specific sequence, in particular according to the above-mentioned program logarithm with the above parameter set.
  • a protein which has, for example, an identity of at least 80% at the amino acid level with the sequence SEQ ID NO: 2 is accordingly understood to be a protein which, when its sequence is compared 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 80%.
  • a protein which has, for example, an identity of at least 90% at the amino acid level with the sequence SEQ ID NO: 10 is accordingly understood to be a protein which, when comparing its sequence with the sequence SEQ ID NO: 10, in particular according to the above program logarithm
  • the above parameter set has an identity of at least 90%.
  • a protein which has, for example, an identity of at least 90% at the amino acid level with the sequence SEQ ID NO: 12 is accordingly understood to be a protein which, when comparing its sequence with the sequence SEQ ID NO: 12, in particular according to the above program logarithm
  • the above parameter set has an identity of at least 90%.
  • a protein which has, for example, an identity of at least 50% at the amino acid level with the sequence SEQ ID NO: 14 is accordingly understood to be a protein which, when its sequence is compared with the sequence SEQ ID NO: 14, in particular according to the program logic above. with the above parameter set has an identity of at least 50%.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code. Those codons that are frequently used in accordance with the organism-specific codon usage are preferably used for this. The codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ ID NO: 2 is introduced into the organism.
  • a nucleic acid containing the sequence SEQ ID NO: 10 is introduced into the organism.
  • a nucleic acid containing the sequence SEQ ID NO: 12 is introduced into the organism.
  • a nucleic acid containing the sequence SEQ ID NO: 14 is introduced into the organism.
  • 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 attachment of synthetic oligonucleotides and the filling of gaps using the Klenow 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.
  • plants are cultivated which, in addition to the wild type, have an increased or caused hydroxylase activity and / or ⁇ -cyclase activity.
  • ⁇ -cyclase activity changed compared to the wild type is preferably understood to mean “ ⁇ -cyclase activity caused compared to the wild type”.
  • a .beta.-cyclase activity changed in comparison to the wild type preferably understood to mean an “increased ⁇ -cyclase activity compared to the Wiid type”.
  • hydroxylase activity that is changed in comparison with the wild type is preferably understood to mean “hydroxylase activity caused in comparison with the wild type”.
  • a “hydroxylase activity changed in comparison to the wild type” is preferably understood to mean “an increased hydroxylase activity in comparison 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 to convert ⁇ -carotene into zeaxanthin or canthaxanthin into astaxanthin.
  • hydroxyase 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 in the hydroxylase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the hydroxylase activity of the wild type.
  • ⁇ -cyclase activity means the enzyme activity of a ⁇ -cyclase.
  • a ß-cyclase is understood to mean a protein which has the enzymatic activity to convert a terminal, linear residue of lycopene into a ß-ionone ring. to lead.
  • 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 ⁇ -carotene converted or the amount of ⁇ -carotene formed is increased by the protein ß-cyclase in a certain time compared to the wild type.
  • This increase in the ⁇ -cyclase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the ⁇ - Wild-type cyclase activity.
  • 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 beta-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 characterization of carotenoid hydroxylases from 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 volume.
  • 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 a mixture of mono - and Digalactosylglyceriden (1: 1), 1 unit catalysis, 0.2 mg bovine serum albumin and organism extract in different volumes.
  • the reaction mixture is incubated at 30 ° C for 2 hours.
  • reaction products are extracted with organic solvent such as acetone or chloroform / methanol (2: 1) and determined by means of HPLC.
  • organic solvent such as acetone or chloroform / methanol (2: 1)
  • determination of the ⁇ -cyclase activity in genetically modified organisms according to the invention and in wiid-type or reference organisms is preferably carried out 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 is used as a buffer for a certain amount of organism extract (pH 7.6) , Lycopene as substrate, Stro- maprotein from paprika, NADP +, NADPH and ATP added.
  • the ⁇ -cyclase activity is particularly preferably determined under the following conditions according to Bouvier, d'Hariingue 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 batch contains 50 mM potassium phosphate (pH 7.6), different amounts of plant extract, 20 nM lycopene, 250 ⁇ g of chromoplastidic stromal protein from paprika, 0.2 mM NADP +, 0.2 mM NADPH and 1 mM ATP.
  • NADP / NADPH and ATP are dissolved in 10 ⁇ l ethanol with 1 mg Tween 80 immediately before the addition to the incubation medium.
  • 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 one ⁇ -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, as 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 ß-cyclase, in the organism.
  • Increasing the gene expression of a nucleic acid, encoding a hydroxylase and / or ⁇ -cyclase also means, according to the invention, the manipulation of the expression of the organisms' own endogenous hydroxylase and / or ⁇ -cyclase.
  • an altered or increased expression of an endogenous hydroxylase and / or ⁇ -cyclase gene can be achieved in that a regulator protein which does not occur in the non-transformed 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 described, for example, in WO 96/06166.
  • the gene expression of a nucleic acid encoding a hydroxylase is increased and / or the gene expression of a nucleic acid encoding a ⁇ -cyclase is increased by introducing at least one nucleic acid encoding a hydroxylase and / or by introducing at least one 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 for the case that the host organism is unable or cannot be able to express the corresponding hydroxylase or ⁇ -cyclase , preferably already processed nucleotides To use acid sequences like the corresponding cDNAs.
  • hydroxylase genes are nucleic acids encoding a hydroxylase from Haematococcus pluvialis, accession AX038729, WO 0061764); (Nucleic acid: SEQ ID NO: 15, protein: SEQ ID NO: 16), and coding hydroxylases of the following accession numbers:
  • Another particularly preferred hydroxylase is the hydroxylase from tomato (nucleic acid: SEQ. ID. No. 47; protein: SEQ. ID. No. 48).
  • ⁇ -cyclase genes are nucleic acids encoding a ⁇ -cyclase from tomato (Accession X86452). (Nucleic acid: SEQ ID NO: 17, protein: SEQ ID NO: 18), and ⁇ -cyclases of the following access numbers:
  • AAK07430 lycopene beta-cyclase [Adonis palaestina]
  • AAG 10429 beta cyclase [Tagetes erecta]
  • CAA67331 lycopene cyclase [Narcissus pseudonarcissus]
  • AAM45381 beta cyclase [Tagetes erecta]
  • AAG21133 chromoplast-specific lycopene beta-cyclase [Lycopersicon esculentum] AAF18989 lycopene beta-cyclase [Daucus carota]
  • ZP_001140 hypothetical protein [Prochlorococcus marinus str. MIT9313]
  • ZP_001050 hypothetical protein [Prochlorococcus marinus subsp. pastoris str. CCMP1378]
  • ZP_001046 hypothetical protein [Prochlorococcus marinus subsp. pastoris str. CCMP1378]
  • ZP_001134 hypothetical protein [Prochlorococcus marinus str. MIT9313] ZP_001150 hypothetical protein [Synechococcus sp. WH 8102] AAF10377 lycopene cyclase [Deinococcus radiodurans] BAA29250 393aa long hypothetical protein [Pyrococcus horikoshii] BAC77673 lycopene beta-monocyclase [marine bacterium P99-3] AAL01999 lycopene cyclase [Xanthobacter sp.
  • ZP_000190 hypothetical protein [Chloroflexus aurantiacus]
  • ZP_000941 hypothetical protein [Novosphingobium aromaticivorans]
  • AAF78200 lycopene cyclase [Bradyrhizobium sp. ORS278]
  • BAB79602 crtY [Pantoea agglomerans pv.
  • lycopene beta-cyclase [Paracoccus marcusii] BAA20275 lycopene cyclase [Erythrobacter longus] ZP_000570 hypothetical protein [Thermobifida fusca] ZP_000190 hypothetical protein [Chloroflexus aurantiacus] AAK07430 lycopene beta-cyclase [Adonis palaestina] CAA67331 lycopene cyclase [Narcissus pseudonarcissus] AAB53337 Lycopene beta cyclase BAC77673 lycopene beta monocyclase. [marine bacterium P99-3]
  • a particularly preferred ⁇ -cyclase is also the chromoplast-specific ⁇ -cyclase from tomato (AAG21133) (nucleic acid: SEQ. ID. No. 49; protein: SEQ. ID. No. 50)
  • 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 a ⁇ -Cyclase on.
  • the preferred hydroxylase genes used are nucleic acids which encode proteins, containing the amino acid sequence SEQ ID NO: 16 or 48 or a sequence derived from these sequences by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequences SEQ. ID. NO: 16 or 48, and which have the enzymatic property of a hydroxylase.
  • hydroxylases and hydroxylase genes can be obtained, for example, from various organisms whose genomic sequence is known, as described above, by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the sequences SEQ. ID. NO: Easily find 16 or 48.
  • hydroxylases and hydroxylase genes can also be found, for example, starting from the sequences SEQ ID NO: 15 or 47 from different organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques in a manner known per se Easy to find.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the hydroxylase of the sequence SEQ ID NO: 16 or 48.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • Those codons are preferably used for this which are frequently used in accordance with the organism-specific codon usage.
  • the codon usage can be. easily determined using computer evaluations of other known genes of the organisms concerned.
  • a nucleic acid containing the sequence SEQ is brought. ID. NO: 15 or 47, in the organism.
  • the ⁇ -cyclase genes used are preferably nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 18 or 50 or a sequence derived from these sequences by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the respective sequences SEQ ID NO: 18 or 50, and which have the enzymatic property of a ⁇ -cyclase exhibit.
  • ⁇ -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 or 50 ,
  • ⁇ -cyclases and ⁇ -cyclase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 17 or 49 from various organisms, the genomic sequence of which is not known, by hybridization and PCR techniques find oneself in a known manner.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the ⁇ -cyclase of the sequence SEQ. ID. NO: 18 or 50.
  • 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 concerned.
  • a nucleic acid containing the sequence SEQ is brought. ID. NO: 17 or 49 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).
  • genetically modified, non-human organisms are cultivated which, in addition to the wild type, have an increased activity of at least one of the activities selected from the group HMG-CoA reductase activity, (E) -4-hydroxy-3-methylbutyl 2-enyl-diphosphate reductase activity, 1-deoxy-D-xylose-5-phosphate synthase activity, 1-deoxy-D-xylose-5-phosphate reductoisomerase activity, isopentenyl-diphosphate- ⁇ -isomerase- Activity, geranyl diphosphate synthase activity, famesyl diphosphate synthase activity, geranyl geranyl diphosphate synthase activity, phytoene synthase activity, phytoene desaturase activity, zeta carotene desaturase activity, crtlSO- Have activity, FtsZ activity and MinD activity. •
  • HMG-CoA reductase activity is understood to mean the enzyme activity of an HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase).
  • An HMG-CoA reductase means a protein which has the enzymatic activity to convert 3-hydroxy-3-methyl-glutaryl-coenzyme-A to mevalonate.
  • HMG-CoA reductase activity is understood to mean the amount of 3-hydroxy-3-methyl-glutaryl-coenzyme A converted or amount of mevalonate formed in a certain time by the protein HMG-CoA reductase.
  • the HMG-CoA reductase activity is increased compared to the wild type, the amount of 3-hydroxy-3-methyl-glutaryl-coenzyme-A or the formed amount of mevalonate increased.
  • This increase in the HMG-CoA reductase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50% preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the HMG-CoA reductase activity of the wild type.
  • the determination of the HMG-CoA reductase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably carried out under the following conditions:
  • Frozen organism material is homogenized by intensive Mosern in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCI2, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • Organism tissue can be homogenized and extracted in cold buffer (100 mM potassium phosphate (pH 7.0), 4 mM MgCl 2 , 5 mM DTT). The homogenate is centrifuged at 10,000 g at 4C for 15 minutes. The supernatant is then centrifuged again at 100,000 g for 45-60 minutes.
  • the activity of the HMG-CoA reductase is determined in the supernatant and in the pellet of the microsomal fraction (after resuspending in 100 mM potassium phosphate (pH 7.0) and 50 mM DTT). Aliquots of the solution and the suspension (the protein content of the suspension corresponds to approximately 1-10 ⁇ g) are in 100 mM potassium phosphate buffer (pH 7.0 with 3 mM NADPH and 20 ⁇ M ( 14 C) HMG-CoA (58 ⁇ Ci / ⁇ M) ideally incubated in a volume of 26 ⁇ l for 15-60 minutes at 30 C.
  • the reaction is terminated by adding 5 ⁇ l mevalonate lactone (1 mg / ml) and 6 N HCl, after which the mixture is incubated at room temperature for 15 minutes ( 14 C) - Mevalonate formed in the reaction is quantified by adding 125 ⁇ l of a saturated potassium phosphate solution (pH 6.0) and 300 ⁇ l of ethyl acetate, the mixture is mixed well and centrifuged, and the radioactivity can be determined by scintillation measurement.
  • a saturated potassium phosphate solution pH 6.0
  • the (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase activity also called lytB or IspH, describes the enzyme activity of a (E) -4-hydroxy-3-methylbut-2-enyl- Diphosphate reductase understood.
  • An (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase means a protein which has the enzymatic activity, (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate in Convert isopentenyl diphosphate and dimethylallyldiphosphate.
  • This increase in the (E) -4-hydroxy-3-methylbut-2-enyldiphosphate reductase 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%, more preferably at least 500%, especially at least 600% of the (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase activity of the wild type.
  • the (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase activity is preferably determined in genetically modified, non-human organisms according to the invention and in wild-type or reference organisms under the following conditions:
  • Frozen organism material is homogenized by intensive Mosern in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCI2, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3.
  • LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis; FEBS Letters 532 (2002,) 437-440) an in vitro system which reduces the reduction of (E) -4-hydroxy-3-methyl-but-2-enyl diphosphate into the isopentenyl diphosphate and dimethyl allyl diphosphate.
  • 1-Deoxy-D-xylose-5-phosphate synthase activity means the enzyme activity of a 1-deoxy-D-xylose-5-phosphate synthase.
  • a 1-deoxy-D-xylose-5-phosphate synthase is understood to mean a protein which has the enzymatic activity to convert hydroxyethyl-ThPP and glyceraldehyde-3-phosphate into 1-deoxy-D-xylose-5-phosphate.
  • 1-deoxy-D-xylose-5-phosphate synthase activity means the amount of hydroxyethyl-ThPP and / or glyceraldehyde-3 converted by the protein 1-deoxy-D-xylose-5-phosphate synthase in a certain time Phosphate or the amount of 1-deoxy-D-xylose-5-phosphate formed.
  • the protein 1-deoxy-D-xylose-5-phosphate synthase With an increased 1-deoxy-D-xylose-5-phosphate synthase activity compared to the wild type, the protein 1-deoxy-D-xylose-5-phosphate synthase thus converts the amount converted in a certain time compared to the wild type Hydroxyethyl-ThPP and / or glyceraldehyde-3-phosphate or the amount -deoxy-D-xylose-5-phosphate formed increased.
  • This increase in 1-deoxy-D-xylose-5-phosphate synthase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600%, of the wild-type 1-deoxy-D-xylose-5-phosphate synthase activity.
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCI2, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ - Aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • the reaction solution (50-200 ⁇ l) for the determination of the D-1-deoxyxylulose-5-phosphate synthase activity consists of 100 mM Tris-HCl (pH 8.0), 3 mM MgCl 2 , 3 mM MnCl 2 , 3 mM ATP, 1 mM thiamine diphosphate, 0.1% Tween-60, 1 mM Ka liumfluorid, 30 uM (2- 14 C) pyruvate (0.5 uCi), 0.6 mM DL-Glyerinaldehyd-3-phosphate.
  • the organism extract is 1 to 2 hours in the reaction solution at 37C. incubated.
  • the quantification is carried out using a scintillation counter.
  • the method was described in Harker and Bramley (FEBS Letters 448 (1999) 115-119).
  • FEBS Letters 448 (1999) 115-119 Alternatively, a fluorometric assay to determine the DXS synthase activity of Queol and colleagues has been described (Analytical Biochemistry 296 (2001) 101-105).
  • 1-deoxy-D-xylose-5-phosphate reductoisomerase activity the enzyme activity of a 1-deoxy-D-xylose-5-phosphate reductoisomerase, also 1-deoxy-D-xylulose-5-phosphate reductoisomerase called, understood.
  • a 1-deoxy-D-xylose-5-phosphate reductoisomerase means a protein which has the enzymatic activity, 1-deoxy-D-xylose-5-phosphate in 2-C-methyl-D-erythritol 4-phosphate convert.
  • 1-deoxy-D-xylose-5-phosphate reductoisomerase activity becomes the amount of 1-deoxy-D-xylose-5 converted by the protein 1-deoxy-D-xylose-5-phosphate reductoisomerase in a certain time -Phosphate or formed Amount of 2-C-methyl-D-erythritol 4-phosphate understood.
  • the protein 1-deoxy-D-xylose-5-phosphate reductoisomerase With an increased 1-deoxy-D-xylose-5-phosphate reductoisomerase activity compared to the wild type, the protein 1-deoxy-D-xylose-5-phosphate reductoisomerase thus converts the amount converted in a certain time compared to the wild type 1-Deoxy-D-xylose-5-phosphate or the amount formed 2-C-methyl-D-erythritol 4-phosphate increased.
  • This increase in 1-deoxy-D-xylose-5-phosphate reductoisomerase 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%, 1-deoxy-D-xylose-5-phosphate reductoisomerase activity of the wild type.
  • the determination of the 1-deoxy-D-xylose-5-phosphate reductoisomerase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably carried out under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • D-1-deoxyxylulose-5-phosphate reductoisomerase (DXR) is measured in a buffer of 100 mM Tris-HCl (pH 7.5), 1 mM MnCl 2 , 0.3 mM NADPH and 0, 3 mM 1-deoxy-D-xylulose-4-phosphate, which can be synthesized, for example, enzymatically (Kuzuyama, Takahashi, Watanabe and Seto: Tetrahedon letters 39 (1998) 4509-4512).
  • the reaction is started by adding the organism extract.
  • the reaction volume can typically be 0.2 to 0.5 mL; incubation takes place at 37C for 30-60 minutes. During this time, the oxidation of NADPH is monitored photometrically at 340 nm.
  • Isopentenyl diphosphate ⁇ isomerase activity is understood to mean the enzyme activity of an isopentenyl diphosphate ⁇ isomerase.
  • An isopentenyl diphosphate ⁇ isomerase is understood to mean a protein which has the enzymatic activity to convert isopentenyl diphosphate to dimethylallyl phosphate.
  • isopentenyl diphosphate ⁇ -isomerase activity is understood to mean the amount of isopentenyl diphosphate or dimethylallyl phosphate formed in a certain time by the protein isopentenyl diphosphate D- ⁇ isomerase.
  • the type isopentenyl diphosphate- ⁇ -isomerase increases the amount of isopentenyl diphosphate converted or the amount of dimethylallyl phosphate formed in a certain time compared to the wild type.
  • This increase in isopentenyl diphosphate is preferably
  • ⁇ -isomerase activity 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 isopentenyl diphosphate ⁇ isomerase Wild type activity.
  • the determination of the isopentenyl diphosphate ⁇ isomerase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably carried out under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • IPP isomerase activity determinations of isopentenyl diphosphate isomerase (IPP isomerase) can be carried out using the method presented by Fräser and colleagues (Fräser, Römer, Shipton, Mills, Kiano, Misawa, Drake, Schuch and Bramley: Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner; Proc. Natl. Acad. Sci. USA 99 (2002), 1092-1097, based on Fraser, Pinto, Holloway and Bramley, Plant Journal 24 (2000), 551-558).
  • Geranyl diphosphate synthase activity means the enzyme activity of a geranyl diphosphate synthase.
  • a geranyl diphosphate synthase is understood to mean a protein which has the enzymatic activity of converting isopentenyl diphosphate and dimethylallyl phosphate into geranyl diphosphate.
  • geranyl diphosphate synthase activity means the amount of isopentenyl diphosphate and / or dimethylallyl phosphate or amount of geranyl diphosphate formed in a certain time by the protein geranyl diphosphate synthase.
  • the amount of isopentenyl diphosphate and / or dimethylallyl phosphate or the amount of geranyl Diphosphate increased.
  • This increase in geranyl diphosphate synthase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the Wild-type geranyl diphosphate synthase activity.
  • the geranyl diphosphate synthase activity is determined in genetically modified organisms according to the invention and in wild-type or reference organisms preferably under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCI2, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ - Aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • geranyl diphosphate synthase can be found in 50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 5 mM MnCl 2 , 2 mM DTT, 1 mM ATP, 0.2% Tween-20.5 ⁇ M ( 14C ) IPP and 50 ⁇ M DMAPP (dimethylallyl pyrophosphate) can be determined after adding organism extract (according to Bouvier, Suire, d'Harlingue, Backhaus and Camara: Meolcular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells, plant Journal 24 (2000) 241-252). After incubation of, for example, 2 hours at 37 ° C., the reaction products are dephosphyrylated (according to Koyama, Fuji and Ogura: Enzymatic hydrolysis of polyprenyl pyrophosphates,
  • Famesyl diphosphate synthase activity means the enzyme activity of a farnesyl diphosphate synthase.
  • a farnesyl diphosphate synthase is understood to mean a protein which has the enzymatic activity of sequentially converting 2 molecular sopentenyl diphosphate with dimethyl allyl diphosphate and the resulting geranyl diphosphate into famesyl diphosphate.
  • farnesyl diphosphate synthase activity is understood to mean the amount of dimethylallyl diphosphate and / or isopentenyl diphosphate or amount of famesyl diphosphate formed in a certain time by the protein farnesyl diphosphate synthase.
  • the protein ferns syl diphosphate synthase increases the amount of dimethylallyl diphosphate and / or isopentenyl diphosphate or the amount of farnesyl diphosphate formed.
  • This increase in the farnesyl diphosphate synthase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the Wild-type famesyl diphosphate synthase activity.
  • Farnesyl diphosphate synthase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCI2, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ - Aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • FPP synthase farnesyl pyrophosphate snthase
  • the enzyme activity in a buffer of 10 mM HEPES (pH 7.2), 1 mM MgCl 2, 1 mM dithiothreitol, 20 uM and 40 uM will geranyl pyrophosphate (1- 14 C) isopentenyl pyrophosphate (4 Ci / mmol) measured.
  • the reaction mixture is incubated at 37 ° C; the reaction is stopped by adding 2.5 N HCl (in 70% ethanol with 19 ⁇ g / ml farnesol).
  • the reaction products are thus hydrolyzed within 30 minutes by acid hydrolysis at 37C.
  • the mixture is neutralized by adding 10% NaOH and extracted with hexane. An aliquot of the hexane phase can be measured using a scintillation counter to determine the built-in radioactivity.
  • reaction products can be separated into benzene / methanol (9: 1) by means of thin layer chromatography (silica gel SE60, Merck). Products labeled with radioactivity are eluted and radioactivity determined (according to Gaffe, Bru, Causse, Vidal, Stamitti-Bert, Carde and Gallusci: LEFPS1, a tomato farnesyl pyrophosphate gene highly ex- pressed during early fruit development; Plant Physiology 123 (2000) 1351-1362).
  • Geranyl-geranyl diphosphate synthase activity is understood to mean the enzyme activity of a geranyl-geranyl diphosphate synthase.
  • a geranyl-geranyl diphosphate synthase is understood to be a protein which has the enzymatic activity to convert famesyl diphosphate and isopentenyl diphosphate into geranyl-geranyl diphosphate.
  • geranyl-geranyl diphosphate synthase activity is understood to mean the amount of farnesyl diphosphate and / or isopentenyl diphosphate or the amount of geranyl-geranyl diphosphate formed in a certain time by the protein geranyl-geranyl diphosphate synthase.
  • the amount of farnesyl diphosphate and / or isopentenyl diphosphate or the formed amount of geranyl-geranyl diphosphate increased.
  • This increase in geranyl-geranyl diphosphate synthase 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 wild-type geranyl-geranyl-piphosphate synthase activity.
  • the geranyl-geranyl-diphosphate synthase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3.
  • GGPP synthase geranylgeranyl pyrophosphate synthase
  • Activity measurements of geranylgeranyl pyrophosphate synthase can be carried out by the method described by Dogbo and Camara (in Biochim. Biophys. Acta 920 (1987), 140-148: Purification of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicochrome chromoplasts by affinity ) can be determined.
  • a buffer 50 mM Tris-HCl (pH 7.6), 2 mM MgCl 2, 1 mM MnCl 2, 2 mM dithiothreitol, (1- 14 C) IPP (0.1 uCi is, 10 uM), 15 uM DMAPP, GPP or FPP) with a total volume of about 200 ul organism extract.
  • Incubation can be for 1-2 hours (or longer) at 30C.
  • the reaction is carried out by adding 0.5 ml of ethanol and 0.1 ml of 6N HCl. After incubation at 37 ° C.
  • reaction mixture is neutralized with 6N NaOH, mixed with 1 ml of water and extracted with 4 ml of diethyl ether.
  • amount of radioactivity is determined in an aliquot (for example 0.2 ml) of the ether phase by means of scintillation counting.
  • the radioactively labeled prenyl alcohols can be shaken out in ether and HPLC (25 cm column Spherisorb ODS-1, 5um; elution with methanol / water (90:10; v / v) at a flow rate of 1 ml / min) are separated and quantified using a radioactivity monitor (according to Wiedemann, Misawa and Sandmann: Purification and enzymatic characterization of the geranylgeranyl pyrophosphate synthase from Erwinia uredovora after expression in Escherichia coli; Archives Biochemistry and Biophysics 306 (1993), 152-157 ).
  • Phytoene synthase activity means the enzyme activity of a phytoene synthase.
  • a phytoene synthase is understood to be a protein which has the enzymatic activity to convert geranyl-geranyl diphosphate into phytoene.
  • phytoene synthase activity is understood to mean the amount of geranyl-geranyl diphosphate or amount of phytoene formed in a certain time by the protein phytoene synthase.
  • the amount of geranyl-geranyl diphosphate or the amount of phytoene formed is increased in a certain time by the protein phytoene synthase compared to the wild type.
  • This increase in phytoene synthase 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 phytoene Wild-type synthase activity.
  • the phytoene synthase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • PY phytoene synthase
  • Fräser and colleagues Fräser, Romer, Shipton, Mills, Kiano, Misawa, Drake, Schuch and Bramley: Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit- specific manner; Proc. Natl. Acad. Sci. USA 99 (2002), 1092-1097, based on Fraser, Pinto, Holloway and Bramley, Plant Journal 24 (2000) 551-558).
  • GeranyIgeranyl pyrophosphate 15 mCi / mM, American Radiolabeled Chemicals, St.
  • Organism extracts are mixed with buffer, eg 295 ul buffer with extract in a total volume of 500 ul. Incubate for at least 5 hours at 28C. Then, phytoene is in each case extracted by shaking twice '500 ul) with chloroform. The radioactively labeled phytoene formed during the reaction is separated by thin layer chromatography on silica plates in methane / water (95: 5; v / v).
  • Phytoenes can be identified on the silica plates in an iodine-enriched atmosphere (by heating fewer iodine crystals).
  • a phytoene standard serves as a reference.
  • the amount of radioactively labeled product is determined by measurement in a scintillation counter.
  • phytoenes can also be quantified using HPLC, which is equipped with a radioactivity detector (Fräser, Albrecht and Sandmann: Development of high Performance liquid Chromatographie Systems for the Separation of radiolabeled carotenes and precursors formed in specific enzymatic reactions; J. Chromatogr. 645 ( 1993) 265-272).
  • Phytoene desaturase activity means the enzyme activity of a phytoene desaturase.
  • a phytoene desaturase is understood to mean a protein which has the enzymatic activity to convert phytoene into phytofluene and / or phytofluene into ⁇ -carotene (zeta-carotene).
  • phytoene desaturase activity is understood to mean the amount of phytoene or phytofluene or amount of phytofluene or ⁇ -carotene formed in a certain time by the protein phytoene desaturase.
  • the amount of phytoene or phytofluene or the amount of phytofluen or inclin.-carotene formed is increased in a certain time by the protein phytoen desaturase compared to the wild type.
  • This increase in phytoene desaturase 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 phytoene Wild-type desaturase activity.
  • Phytoene desaturase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions:
  • Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20.
  • the respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -
  • Aminocaproic acid 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • the activity of phytoene desaturase can be measured by incorporating radioactively labeled ( 14 C) phytoene in unsaturated carotenes (according to Römer, Fraser, Kiano, Shipton, Misawa, Schuch and Bramley: Elevation of the provitamin A content of transgenic tomato plants; Nature Biotechnology 18 (2000) 666-669).
  • Radioactively labeled phytoenes can be synthesized according to Fräser (Fräser, De la Rivas, Mackenzie, Bramley: Phycomyces blakesleanus CarB mutants: their use in assays of phytoene desaturase; Phytochemistry 30 (1991), 3971-3976).
  • Membranes from plastic The target tissue can be mixed with 100 M MES buffer (pH 6.0) with 10 mM MgCl 2 and
  • pigments are extracted three times with about 5 mL petroleum ether (mixed with 10% diethyl ether) and separated and quantified by HPLC.
  • Zeta-carotene desaturase activity means the enzyme activity of a zeta-carotene desaturase.
  • a zeta-carotene desaturase is understood to mean a protein which has the enzymatic activity to convert ot-carotene into neurosporin and / or neurosporin into lycopene.
  • zeta-carotene desaturase activity means the amount of ⁇ -carotene or neurosporin or the amount of neurosporin or lycopene formed in a certain time by the protein zeta-carotene desaturase.
  • the amount of ⁇ -carotene or neurosporin or the amount of neurosporin or lycopene formed is increased in a certain time by the protein zeta-carotene desaturase compared to the wild type.
  • This increase in zeta-carotene desaturase 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 Zeta-carotene desaturase - wild-type activity.
  • the zeta-carotene desaturase activity in genetically modified organisms according to the invention and in wild-type or reference organisms is preferably determined under the following conditions: Frozen organism material is homogenized by intensive mortar in liquid nitrogen and extracted with extraction buffer in a ratio of 1: 1 to 1:20. The respective ratio depends on the enzyme activities in the available organism material, so that a determination and quantification of the enzyme activities within the linear measuring range is possible.
  • the extraction buffer can consist of 50 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% (v / v) Triton X-100, 2 mM ⁇ -aminocaproic acid, 10% glycerin, 5 mM KHCO3. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • ZDS desaturase can be carried out in 0.2 M potassium phosphate (pH 7.8, buffer volume of about 1 ml).
  • the analysis method was developed by Schunbach and colleagues (Breitenbach, Kuntz, Takaichi and Sandmann: Catalytic properties of an expressed and purified higher plant type ⁇ -carotene desaturase from Capsicum annuum; European Journal of Biochemistry. 265 (1): 376-383 , 1999 Oct).
  • Each analysis batch contains 3 mg phosphytidylcholine suspended in 0.4 M potassium phosphate buffer (pH 7.8), 5 ocg ⁇ -carotene or neurosporin, 0.02% butylated hydroxytoluene, 10 ⁇ l decyl plastoquinone (1 mM methanolic stock solution) and organism extract.
  • the volume of the organism extract must be adjusted to the amount of ZDS desaturase activity present in order to enable quantifications in a linear measuring range.
  • Incubations typically take place for about 17 hours with vigorous shaking (200 revolutions / minute) at about 28 ° C in the dark.
  • Carotenoids are extracted by adding 4 ml acetone at 50 ° C for 10 minutes with shaking.
  • the carotenoids are transferred from this mixture to a petroleum ether phase (with 10% diethyl ether).
  • the ethyl ether / petroleum ether phase is evaporated under nitrogen, the carotenoids redissolved in 20 ⁇ l and separated and quantified by HPLC.
  • CrtlSO activity means the enzyme activity of a crtlSO protein.
  • a crtlSO protein is understood to mean a protein which has the enzymatic activity of converting 7,9,7 ', 9'-tetra-cis-lycopene into all-trans-lycopene.
  • crtlSO activity is understood to mean the amount of 7,9,7 ', 9'-tetra-cis-lycopene or amount of all-trans-lycopene formed in a certain time by the crtlSO protein.
  • the amount converted by the crtlSO protein in a certain time compared to the wild type 7,9,7 ', 9'-tetra-cis-lycopene or the amount of all-trans-lycopene formed increased.
  • This increase in crtlSO activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, further preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the crtlSO- Wild type activity.
  • FtsZ activity is understood to mean the physiological activity of an FtsZ protein.
  • FtsZ protein is understood to be a protein which has a cell division and plastid division promoting effect and has homologies to tubulin proteins.
  • MinD activity is understood to mean the physiological activity of a MinD protein.
  • a MinD protein is understood to be a protein that has a multifunctional role in cell division. It is a membrane-associated ATPase and can show an oscillating movement from pole to pole within the cell.
  • enzymes in the non-mevalonate pathway can lead to a further increase in the desired ketocarotenoid end product.
  • examples of this are the 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase and the 2-C-methyl-D-erythritol kinase 2,4-cyclodiphoshate synthase.
  • the activity of the enzymes mentioned can be increased by changing the gene expression of the corresponding genes.
  • the changed concentrations of the relevant proteins can be detected as standard by means of antibodies and corresponding blotting techniques.
  • the increase in HMG-CoA reductase activity and / or (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase activity and / or 1-deoxy-D-xylose-5-phosphate synthase -Activity and / or 1-deoxy-D-xylose-5-phosphate reductoisomerase activity and / or isopentenyl diphosphate- ⁇ -isomerase activity and / or geranyl diphosphate synthase activity and / or farnesyl diphosphate synthase -Activity and / or geranyl-geranyl-diphosphate synthase activity and / or phytoene synthase activity and / or phytoene desaturase activity and / or zeta-carotene desaturase activity and / or crtlSO activity and / or FtsZ Activity and / or MinD activity can take place in various ways, for example by switching off inhibitory regulatory mechanisms at the
  • Diphosphate- ⁇ -isomerase and / or nucleic acids encoding a geranyl diphosphate synthase and / or nucleic acids encoding a farnesyl diphosphate synthase and / or nucleic acids encoding a geranyl geranyl diphosphate synthase and / or nucleic acids encoding a phytoene synthase and / or nucleic acids encoding a phytoene desaturase and / or nucleic acids encoding a zeta-carotene desaturase and / or nucleic acids encoding a crtlSO protein and / or nucleic acids encoding an FtsZ protein and / or nucleic acids encoding a MinD protein compared to the wild type also take place in various ways, for example by inducing the HMG-CoA reductase gene and / or (E) -4-hydroxy-3-methylbut-2-enyl-
  • Such a change, which results in an increased expression rate of the gene can take place, for example, by deleting or inserting DNA sequences.
  • the gene expression of a nucleic acid encoding an HMG-CoA reductase is increased and / or the gene expression is increased.
  • Synthase gene or phytoene desaturase gene or zeta-carotene desaturase gene or crtlSO gene or FtsZ gene or MinD gene can be used.
  • the preferred transgenic organisms according to the invention therefore have at least one further HMG-CoA reductase gene and / or (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase gene and compared to the wild type / or 1-deoxy-D-xylose-5-phosphate synthase gene and / or 1-deoxy-D-xylose-5-phosphate reductoisomerase gene and / or isopentenyl-diphosphate- ⁇ -isomerase gene and / or Geranyl diphosphate synthase gene and / or farnesyl diphosphate synthase gene and / or geranyl geranyl diphosphate synthase gene and / or phytoene synthase gene and / or phytoene desaturase gene and / or zeta Carotene desaturase gene and / or crtlSO gene and / or FtsZ gene and / or MinD gene.
  • the genetically modified organism has, for example, at least one exogenous nucleic acid, coding for an HMG-CoA reductase or at least two endogenous nucleic acids, coding for an HMG-CoA reductase and / or at least one exogenous nucleic acid, coding for an (E) -4 - Hydroxy-3-methylbut-2-enyl diphosphate reductase or at least two endogenous nucleic acids encoding an (E) -4-hydroxy-3-methylbut-2-enyl diphosphate reductase and / or at least one exogenous nucleic acid, coding a 1-deoxy-D-xylose-5-phosphate synthase or at least two endogenous nucleic acids encoding a 1-deoxy-D-xyiose-5-phosphate synthase and / or at least one exogenous nucleic acid encoding a 1-deoxy-D -Xylose-5-phosphate
  • HMG-CoA reductase genes are:
  • HMG-CoA reductase genes as well as other HMG-CoA reductase genes from other organisms with the following accession numbers:
  • AF270978 NP_485028.1, NP_442089.1, NP_681832.1, ZP_00110421.1, ZP_00071594.1, ZP_00114706.1, ISPH_SYNY3, ZP_00114087.1, ZP_00104269.1, AF398145_71, AF3985156A5, AF39814567, AF398145_1, AF398145141A, AF3981456_1, AF398145A5.
  • Examples of 1-deoxy-D-xylose-5-phosphate synthase genes are:
  • nucleic acid encoding a 1-deoxy-D-xylose-5-phosphate synthase from Lycopersicon esculentum, ACCESSION # AF143812 (nucleic acid: SEQ ID NO: 23, protein: SEQ ID NO: 24),
  • NP_540415.1 NP_196699.1, NP_384986.1, ZP_00096461.1, ZP_00013656.1
  • NP_790545.1 ZP_00125266.1, CAC17468.1, NP_252733.1, ZP_00092466.1,
  • Examples of 1-deoxy-D-xylose-5-phosphate reductoisomerase genes are:
  • nucleic acid encoding a 1-deoxy-D-xylose-5-phosphate reductoisomerase from Arabidopsis thaliana, ACCESSION # AF148852, (nucleic acid: SEQ ID NO: 25, protein: SEQ ID NO: 26),
  • NP_224545.1 ZP_00038451.1, DXR_KITGR, NP_778563.1.
  • isopentenyl diphosphate ⁇ isomerase genes are:
  • geranyl diphosphate synthase genes are:
  • Examples of farnesyl diphosphate synthase genes are:
  • Arabidopsis thaliana contains two differentially expressed famesyl-diphosphate synthase genes, J. Biol. Chem. 271 (13), 7774-7780 (1996), (nucleic acid: SEQ ID NO: 31, protein: SEQ ID NO: 32),
  • geranyl-geranyl diphosphate synthase genes are:
  • phytoene synthase genes are: a nucleic acid encoding a phytoene synthase from Erwinia uredovora, ACCESSION # D90087; published by Misawa.N., Nakagawa, M., Kobayashi.K., Yamama, S., lzawa, Y., Nakamura, K. and Harashima.K .: Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli; J. Bacteriol.
  • phytoene desaturase genes are:
  • zeta-carotene desaturase genes are:
  • a nucleic acid encoding a Narcissus pseudonarcissus zeta-carotene desaturase ACCESSION # AJ224683, published by AI-Babili, S., Oelschlegel.J. and Beyer.P .: A cDNA encoding for beta carotene desaturase (Accession No.AJ224683) from Narcissus pseudonarcissus L. (PGR98-103), Plant Physiol. 117, 719-719 (1998), (nucleic acid: SEQ ID NO: 39, protein: SEQ ID NO: 40),
  • crtlSO genes are:
  • nucleic acid encoding a crtlSO from Lycopersicon esculentum; ACCESSION # AF416727, published by IsaacsonT., Ronen, G., Zamir.D. and Hirschberg, J .: Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants; Plant Cell 14 (2), 333-342 (2002), (nucleic acid: SEQ ID NO: 41, protein: SEQ ID NO: 42),
  • FtsZ genes are:
  • MinD genes are:
  • nucleic acids encoding proteins are preferably used as HMG-CoA reductase genes, containing the amino acid sequence SEQ ID NO: 20 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which have 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: 20, and which have the enzymatic property of an HMG-CoA reductase ,
  • HMG-CoA reductases and HMG-CoA reductase 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: 20 easy to find.
  • HMG-CoA reductases and HMG-CoA reductase genes can also be found, for example, starting from the sequence SEQ ID NO: 19 from various organisms, the genomic sequence of which is not known, as described above, can easily be found by hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the HMG-CoA reductase of the sequence SEQ ID NO: 20.
  • 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.
  • 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: 19 is introduced into the organism.
  • nucleic acids encoding proteins are preferably used as (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase genes, containing the amino acid sequence SEQ ID NO: 22 or one of these sequences Substitution, insertion or deletion of a sequence derived from amino acids, which has an identity of at least 30%, preferably at least 50%, more preferably at least 70%, more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 22, and which have the enzymatic property of an (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase.
  • (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductases and (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase genes can also be obtained, for example, from the sequence SEQ ID NO: 21 from different orga- nisms 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 (E) - 4- Hydroxy-3-methylbut-2-enyl diphosphate reductase of sequence SEQ ID NO: 22.
  • 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.
  • 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: 21 is introduced into the organism.
  • (1-deoxy-D-xylose-5-phosphate synthase and (1-deoxy-D-xylose-5-phosphate synthase genes can be obtained, for example, from various organisms whose genomic sequence is known, as described above , easy to find by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SeQ ID NO: 24.
  • (1-qeoxy-D-xylose-5-phosphate synthases and (1-deoxy-D-xylose-5-phosphate synthase genes can also be obtained from different organisms, for example, starting from the sequence SEQ ID NO: 23 whose genomic sequence is not known, as described above, by hybridization and Easily find 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 (1-deoxy-D-xylose-5 Phosphate synthase of sequence SEQ ID NO: 24.
  • 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.
  • 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: 23 is introduced into the organism.
  • nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 26 or one of these sequences by substitution, insertion or deletion of are used as 1-deoxy-D-xylose-5-phosphate reductoisomerase genes
  • Amino acid-derived sequence which has an identity of at least 30%, preferably at least 50%, more preferably at least 70%, more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 26, and which is the enzymatic Have property of a 1-deoxy-D-xylose-5-phosphate reductoisomerase.
  • 1-deoxy-D-xylose-5-phosphate reductoisomerase and 1-deoxy-D-xylose-5-phosphate reductoisomerase genes can be obtained, for example, from various organisms, the genomic sequence of which is known, as described above easily find homology comparisons of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with SeQ ID NO: 26.
  • 1-deoxy-D-xylose-5-phosphate reductoisomerases and 1-deoxy-D-xylose-5-phosphate reductoisomerase genes can also be found, for example, starting from the sequence SEQ ID NO: 25 from different organisms and their genomic Sequence is not known, as described above, by hybridization tion and PCR techniques can be easily found in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the 1-deoxy-D-xylose-5-phosphate Reductoisomerase of sequence SEQ ID NO: 26.
  • 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.
  • 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: 25 is introduced into the organism.
  • nucleic acids which encode proteins are preferably used as isopentenyl-D-isomerase genes, containing the amino acid sequence SEQ ID NO: 28 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: 28, and which have the enzymatic property of an isopentenyl-D-isornerase ,
  • isopentenyl-D-isomerases and isopentenyl-D-isomerase 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: 28 easy to find.
  • isopentenyl-D-isomerases and isopentenyl-D-isomerase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 27 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques easy to find in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the isopentenyl-D-isomerase of the sequence SEQ ID NO: 28.
  • 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: 27 is introduced into the organism.
  • the geranyl diphosphate synthase genes used are nucleic acids which encode proteins, containing the amino acid sequence SEQ ID NO: 30 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 30 and which have the enzymatic property of a geranyl diphosphate synthase.
  • geranyl diphosphate synthases and geranyl diphosphate synthase 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: 30 easy to find.
  • geranyl diphosphate synthases and geranyl diphosphate synthase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 29 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR - easily find techniques in a manner known per se.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the geranyl diphosphate Synthase of sequence SEQ ID NO: 30.
  • 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.
  • 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: 29 is introduced into the organism.
  • the famesyl diphosphate synthase genes used are preferably nucleic acids which encode proteins, containing the amino acid sequence SEQ ID NO: 32 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 32, and which have the enzymatic property of a farnesyl diphosphate synthase.
  • famesyl diphosphate synthases and farnesyl diphosphate synthase 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: 32 easy to find.
  • famesyl diphosphate synthases and famesyl diphosphate synthase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 31 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques easy to find in a manner known per se.
  • 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: 31 is introduced into the organism.
  • the geranyl-geranyl-diphosphate synthase genes used are preferably nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 34 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is a Identity of at least 30%, preferably at least 50%, more preferably at least 70%, more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 34, and which the enzymatic property of a geranyl-geranyl-diphosphate Have synthase.
  • geranyl-geranyl-diphosphate synthases and geranyl-geranyl-diphosphate synthase 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 easy to find with SeQ ID NO: 22.
  • geranyl-geranyl-diphosphate synthases and geranyl-geranyl-diphosphate synthase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 33 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques can be easily found in a manner known per se.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the geranyl-geranyl-diphosphate synthase of the sequence SEQ ID NO: 34.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code. Those codons which are frequently used in accordance with the organism-specific codon usage are preferably used for this. The codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ ID NO: 33 is introduced into the organism.
  • the phytoene synthase genes used are preferably nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 36 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: 36, and which have the enzymatic property of a phytoene synthase.
  • phytoene synthases and phytoene synthase 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 the SeQ ID NO: 36.
  • phytoene synthases and phytoene synthase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 35 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques in a manner known per se Easy to find.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the phytoene synthase of the sequence SEQ ID NO: 36.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codon usage can be determined on the basis of computer evaluations of other known genes of the relevant organ easily identify nisms.
  • a nucleic acid containing the sequence SEQ ID NO: 35 is introduced into the organism.
  • the phytoene desaturase genes used are preferably nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 38 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: 38, and which have the enzymatic property of a phytoene desaturase.
  • phytoene desaturases and phytoene desaturase 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 the SeQ ID NO: 38.
  • phytoene desaturases and phytoene desaturase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 37 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques in a manner known per se Easy to find.
  • nucleic acids which encode proteins containing the amino acid sequence of the phytoene desaturase of the sequence SEQ ID NO: 38 are introduced into organisms to increase the phytoene desaturase activity.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • Those codons which are frequently used in accordance with the organism-specific codon usage are preferably used for this.
  • the codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organisms in question.
  • a nucleic acid containing the sequence SEQ ID NO: 37 is introduced into the organism.
  • the zeta-carotene desaturase genes used are preferably nucleic acids which encode proteins, containing the amino acid sequence SEQ ID NO: 40 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 40, and which have the enzymatic property of a zeta-carotene desaturase ,
  • zeta-carotene desaturases and zeta-carotene desaturase 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: 40 easy to find.
  • zeta-carotene desaturases and zeta-carotene desaturase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 39 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques easy to find in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the zeta-carotene desaturase of the sequence SEQ ID NO: 40.
  • 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.
  • 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: 39 is introduced into the organism.
  • nucleic acids encoding proteins are preferably used as CrtlSO genes, containing the amino acid sequence SEQ ID NO: 42 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: 42, and which have the enzymatic property of a Crtlso.
  • CrtlSO and CrtlSO 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 the SeQ ID NO: 42.
  • CrtlSO and CrtlSO genes can also be easily found, for example, starting from the sequence SEQ ID NO: 41 from various organisms whose genomic sequence is not known, as described above, 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 CrtlSO of the sequence SEQ ID NO: 42.
  • 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.
  • 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: 41 is introduced into the organism.
  • the FtsZ genes used are preferably nucleic acids which encode proteins, comprising the amino acid sequence SEQ ID NO: 44 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and which have an identity of at least 30%, preferably at least 50%, more preferably at least 70%, even more preferred at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 44 and which have the enzymatic property of an FtsZ.
  • FtsZn and FtsZ 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 the SeQ ID NO: 44.
  • FtsZn and FtsZ genes can also be easily found, for example, starting from the sequence SEQ ID NO: 43 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques in a manner known per se.
  • FtsZ activity Nucleic acids introduced into organisms which encode proteins, containing the amino acid sequence of the FtsZ of the sequence SEQ ID NO: 44
  • 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.
  • the codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organisms concerned.
  • a nucleic acid containing the sequence SEQ ID NO: 43 is introduced into the organism.
  • nucleic acids which encode proteins are preferably used as MinD genes, containing the amino acid sequence SEQ ID NO: 46 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: 46, and which have the enzymatic property of a MinD.
  • MinDn and MinD genes can be obtained, for example, from various organisms whose genomic sequence is known, as described above, by comparing the amino acid sequences or the corresponding ones with homology easily locate DO back-translated nucleic acid sequences from databases with SeQ ID NO: 46.
  • MinDn and MinD genes can also easily be obtained, for example, starting from the sequence SEQ ID NO: 45 from various organisms whose genomic sequence is not known, as described above, by hybridization and PCR techniques in a manner known per se find.
  • nucleic acids which encode proteins containing the amino acid sequence of the MinD of the sequence SEQ ID NO: 46 are introduced into organisms to increase the MinD activity.
  • 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.
  • 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: 45 is introduced into the organism.
  • 5-phosphate synthase genes 1-deoxy-D-xylose-5-phosphate reductoisomerase genes, isopentenyl diphosphate ⁇ isomerase genes, geranyl diphosphate synthase genes, fernesyl diphosphate synthase genes Genes, geranyl-geranyl diphosphate synthase genes, phytoene synthase genes, phytoene desaturase genes, zeta-carotene desaturase genes, crtl-SO genes, FtsZ genes or MinD genes are still in themselves can be prepared in a known manner 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.
  • 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 Klenow 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.
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • the genetically modified, non-human organisms can be produced as described below, for example by introducing individual nucleic acid constructs (expression cassettes) containing an effect gene, or by introducing multiple constructs which contain up to two or three or more of the effect genes included.
  • 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.
  • a carotenoid-producing organism such as bacteria of the genus Escherichia, which contain, for example, crt genes from Erwinia, as well as bacteria.
  • teries that are capable of synthesizing xanthophylls such as 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 mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Blakeslea, in particular Blakeslea trispora, 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. Further useful microorganisms and their preparation for carrying out the method according to the invention are known, for example, from DE-A-199 16 140, to which reference is hereby made.
  • plants are used as non-human organisms.
  • genetically modified plants are used which have the highest expression rate of a ketolase according to the invention in flowers.
  • the gene expression of the ketolase according to the invention 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.
  • the genetically modified plants additionally have a reduced ⁇ -cyclase activity compared to the wild type.
  • ⁇ -Cyclase activity means the enzyme activity of an ⁇ -cyclase.
  • An ⁇ -cyclase is understood to mean a protein which has the enzymatic activity to convert a terminal, linear residue of lycopene into an ⁇ -ionone ring.
  • ⁇ -cyclase is therefore understood to mean in particular a protein which has the enzymatic activity to convert lycopene to ⁇ -carotene.
  • ⁇ -cyclase activity is understood to mean the amount of lycopene converted or amount of ⁇ -carotene formed by the protein ⁇ -cyclase in a certain time.
  • the amount of lycopene converted or the amount of ⁇ -carotene formed is reduced in a certain time by the protein ⁇ -cyclase compared to the wild type.
  • the partially or essentially complete, based on different cell biological mechanisms is preferably de Understanding or blocking the functionality of an ⁇ -cyclase in a plant cell, plant or a part, tissue, organ, cells or seeds derived therefrom.
  • the ⁇ -cyclase activity in plants can be reduced compared to the wild type, for example by reducing the amount of ⁇ -cyclase protein or the amount of ⁇ -cyclase mRNA in the plant. Accordingly, ⁇ -cyclase activity which is reduced compared to the wild type can be determined directly or by determining the amount of ⁇ -cyclase protein or the amount of ⁇ -cyclase mRNA of the plant according to the invention in comparison to the wild type.
  • a reduction in ⁇ -cyclase activity includes a quantitative reduction in ⁇ -cyclase up to an essentially complete absence of ⁇ -cyclase (i.e. lack of detectability of ⁇ -cyclase activity or lack of immunological detectability of ⁇ -cyclase).
  • the ⁇ -cyclase activity (or the ⁇ -cyclase protein amount or the ⁇ -cyclase mRNA amount) in the plant, particularly preferably in flowers compared to the wild type is preferably increased by at least 5%, more preferably by at least 20% , more preferably reduced by at least 50%, more preferably by 100%.
  • “reduction” also means the complete absence of the ⁇ -cyclase activity (or the ⁇ -cyclase protein or the ⁇ -cyclase mRNA).
  • ⁇ -cyclase activity in genetically modified plants according to the invention and in wild-type or reference plants is preferably carried out under the following conditions:
  • the ⁇ -cyclase activity can be determined according to Fräser and Sandmann (Biochem. Biophys. Res. Comm. 185 (1) (1992) 9-15) / n vitro if potassium phosphate is used as a buffer for a certain amount of plant extract (pH 7.6 ), Lycopene as substrate, stro- maprotein from paprika, NADP +, NADPH and ATP are added.
  • ⁇ -cyclase activity in genetically modified plants according to the invention and in wild-type or reference plants is carried out particularly preferably according to Bouvier, Darlingue and Camara (Molecular Analysis of carotenoid cyclase inhibition; Arch. Biochem. Biophys. 346 (1) (1997) 53-64):
  • the in vitro assay is carried out in a volume of 0.25 ml.
  • the mixture contains 50 mM potassium phosphate (pH 7.6), different amounts of plant extract, 20 nM lycopene, 0.25 mg 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 0.01 ml ethanol with 1 mg Tween 80 immediately before adding to the incubation medium.
  • the reaction is ended by adding chloroform / methanol (2: 1).
  • the reaction products extracted in chloroform are analyzed by HPLC.
  • the ⁇ -cyclase activity in plants is preferably reduced by at least one of the following methods:
  • ⁇ -cyclase dsRNA a double-stranded ⁇ -cyclase ribonucleic acid sequence
  • ⁇ -cyclase dsRNA an expression cassette or cassettes ensuring expression thereof.
  • ⁇ -cyclase-ds.RNA is directed against an ⁇ -cyclase gene (that is to say genomic DNA sequences such as the promoter sequence) or an ⁇ -cyclase transcript (that is to say mRNA sequences),
  • ⁇ -cyclase antisenseRNA introduction of at least one ⁇ -cyclase antisense ribonucleic acid sequence, hereinafter also called ⁇ -cyclase antisenseRNA, or an expression cassette ensuring its expression.
  • ⁇ -cyclase antisenseRNA introduction of at least one ⁇ -cyclase antisense ribonucleic acid sequence, hereinafter also called ⁇ -cyclase antisenseRNA, or an expression cassette ensuring its expression.
  • ⁇ -cyclase senseRNA introduction of at least one ⁇ -cyclase sense ribonucleic acid sequence, hereinafter also referred to as ⁇ -cyclase senseRNA, for inducing a co-suppression or an expression cassette ensuring its expression
  • Knockout mutants can preferably be generated by means of targeted insertion into said ⁇ -cyclase gene by homologous recombination or introduction of sequence-specific nucleases against ⁇ -cyclase gene sequences.
  • ⁇ -cyclase-dsRNA a double-stranded ⁇ -cyclase-ribonucleic acid sequence
  • double-stranded RNA interference double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • Matzke MA et al. (2000) Plant Mol Biol 43: 401-415; Fire A. et al (1998) Nature 391: 806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035 or WO 00/63364.
  • dsRNAi double-stranded RNA interference
  • double-stranded ribonucleic acid sequence means one or more ribonucleic acid sequences which are theoretically based on complementary sequences, for example in accordance with the base pair rules of Waston and Crick and / or factually, for example based on hybridization experiments, in vitro and / or in vivo are able to form double-stranded RNA structures.
  • the person skilled in the art is aware that the formation of double-stranded RNA structures represents an equilibrium state.
  • the ratio of double-stranded molecules to corresponding dissociated forms is preferably at least 1 to 10, preferably 1: 1, particularly preferably 5: 1, most preferably 10: 1.
  • a double-stranded ⁇ -cyclase-ribonucleic acid sequence or ⁇ -cyclase-dsRNA is preferably understood to mean an RNA molecule which has a region with a double-strand structure and which contains a nucleic acid sequence in this region which
  • a) is identical to at least part of the plant's own ⁇ -cyclase transcript and / or
  • b) is identical to at least part of the plant's own ⁇ -cyclase promoter sequence.
  • an RNA which has a region with a double-strand structure and which contains a nucleic acid sequence in this region is therefore preferably introduced into the plant in order to reduce the ⁇ -cyclase activity
  • a) is identical to at least part of the plant's own ⁇ -cyclase transcript and / or
  • b) is identical to at least part of the plant's own ⁇ -cyclase promoter sequence.
  • ⁇ -cyclase transcript is understood to mean the transcribed part of an ⁇ -cyclase gene which, in addition to the ⁇ -cyclase coding sequence, also contains, for example, non-coding sequences, such as UTRs.
  • "A part" of the plant's own ⁇ -cyclase transcript or the plant's own ⁇ -cyclase promoter sequence is understood to mean partial sequences which can range from a few base pairs to complete sequences of the transcript or the promoter sequence. The person skilled in the art can easily determine the optimal length of the partial sequences by routine experiments.
  • the length of the partial sequences is at least 10 bases and at most 2 kb, preferably at least 25 bases and at most 1.5 kb, particularly preferably at least 50 bases and at most 600 bases, very particularly preferably at least 100 bases and at most 500 am most preferably at least 200 bases or at least 300 bases and at most 400 bases.
  • the partial sequences are preferably selected in such a way that the highest possible specificity is achieved and activities of other enzymes, the reduction of which is not desired, are not reduced. It is therefore advantageous to select parts of the ⁇ -cyclase transcript and / or partial sequences of the ⁇ -cyclase promoter sequences for the partial sequences of the ⁇ -cyclase dsRNA that do not occur in other activities.
  • the ⁇ -cyclase dsRNA therefore contains a sequence which is identical to a part of the plant's own ⁇ -cyclase transcripts and the 5 'end or the 3' end of the plant's own nucleic acid, coding for an ⁇ -Cyclase contains.
  • non-translated regions in the 5 'or 3' of the transcript are suitable for producing selective double-strand structures.
  • Another object of the invention relates to double-stranded RNA molecules (dsRNA molecules) which, when introduced into a plant organism (or a cell, tissue, organ or propagation material derived therefrom), reduce ⁇ -cyclase.
  • dsRNA molecules double-stranded RNA molecules
  • a double-stranded RNA molecule for reducing the expression of an ⁇ -cyclase preferably comprises
  • RNA strand comprising at least one ribonucleotide sequence which is essentially identical to at least part of a “sense” RNA ⁇ -Cy ase transcript, and
  • RNA strand which is essentially, preferably completely, complementary to the RNA “sense” strand under a).
  • a nucleic acid construct is preferably used which is introduced into the plant and which is transcribed into the ⁇ -cyclase dsRNA in the plant.
  • the present invention therefore also relates to a nucleic acid construct that can be transcribed into
  • RNA strand comprising at least one ribonucleotide sequence which is essentially identical to at least part of the “sense” RNA ⁇ -cyclase transcript
  • RNA strand which is essentially — preferably completely — complementary to the RNA sense strand under a).
  • nucleic acid constructs are also called expression cassettes or expression vectors below.
  • ⁇ -cyclase nucleic acid sequence is preferably understood to be the sequence according to SEQ ID NO: 38 or a Tel thereof.
  • dsRNA sequence can also have insertions, deletions and individual point mutations in comparison to the ⁇ -cyclase target sequence and nevertheless brings about an efficient reduction in expression.
  • the homology is preferably at least 75%, preferably at least 80%, very particularly preferably at least 90%, most preferably 100% between the “sense” strand of an inhibitory dsRNA and at least part of the “sense” RNA transcript of an ⁇ -Cyclase Ge ⁇ s, or between the "antisense” strand the complementary strand of an ⁇ -cyclase gene.
  • dsRNA preferably comprises sequence regions of ⁇ -cyclase gene transcripts which correspond to conserved regions. said conserved areas can easily be derived from sequence comparisons.
  • an "essentially identical" dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with part of an ⁇ -cyclase gene transcript (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50 ° C or 70 ° C for 12 to 16 h).
  • “Essentially complementary” means that the “antisense” RNA strand can also have inserts, deletions and individual point mutations in comparison to the complement of the “sense” RNA strand.
  • the homology is preferably at least 80%, preferably at least 90%, very particularly preferably at least 95%, most preferably 100% between the "antisense” RNA strand and the complement of the "sense” RNA strand.
  • the ⁇ -cyclase dsRNA comprises
  • RNA strand comprising at least one ribonucleotide sequence which is essentially identical to at least part of the “sense” RNA transcript of the promoter region of an ⁇ -cyclase gene
  • RNA strand which is essentially — preferably completely — complementary to the RNA “sense” strand under a).
  • nucleic acid construct comprises
  • a “sense” DNA strand which is essentially identical to at least part of the promoter region of an ⁇ -cyclase gene
  • an “antisense” DNA strand which is essentially — preferably completely — complementary to the DNA “sense” strand under a).
  • the promoter region of an ⁇ -cyclase is preferably understood to mean a sequence according to SEQ ID NO: 51 or a part thereof.
  • the following partial sequences are particularly preferably used to produce the ⁇ -cyclase dsRNA sequences for reducing the ⁇ -cyclase activity, in particular for Tagetes erecta: SEQ ID NO: 52: Sense fragment of the 5'-terminal region of the ⁇ -cyclase
  • SEQ ID NO: 53 Antisense fragment of the 5'-terminal region of the ⁇ -cyclase
  • SEQ ID NO: 54 Sense fragment of the 3'-terminal region of the ⁇ -cyclase
  • SEQ ID NO: 55 Antisense fragment of the 3'-terminal region of the ⁇ -cyclase
  • SEQ ID NO: 56 Sense fragment of the ⁇ -cyclase promoter
  • SEQ ID NO: 57 Antisense fragment of the ⁇ -cyclase promoter
  • the dsRNA can consist of one or more strands of polyribonucleotides.
  • several individual dsRNA molecules, each comprising one of the ribonucleotide sequence sections defined above, can also be introduced into the cell or the organism.
  • the double-stranded dsRNA structure can be formed from two complementary, separate RNA strands or - preferably - from a single, self-complementary RNA strand.
  • the “sense” RNA strand and the “antisense” RNA strand are preferably covalently linked to one another in the form of an inverted “repeat”.
  • the dsRNA can also comprise a hairpin structure, in that the “sense” and “antisense” strand are connected by a connecting sequence (“linker”; for example an intron).
  • linker for example an intron
  • the self-complementary dsRNA structures are preferred since they only require the expression of an RNA sequence and the complementary RNA strands always comprise an equimolar ratio.
  • the connecting sequence is an intron (e.g. an intron of the ST-LS1 gene from potato; Vancänneyt GF et al. (1990) Mol Gen Genet 220 (2): 245-250).
  • the nucleic acid sequence coding for a dsRNA can contain further elements. such as transcription termination signals or polyadenylation signals.
  • the dsRNA is directed against the promoter sequence of an ⁇ -cyclase, it preferably does not include any transcription termination signals or polyadenylation signals. This enables a retention of the dsRNA in the nucleus of the cell and prevents a distribution of the dsRNA in the whole plant "Spreadinng"). If the two strands of the dsRNA are to be brought together in a cell or plant, this can be done, for example, in the following way:
  • RNA duplex The formation of the RNA duplex can be initiated either outside the cell or inside it.
  • the dsRNA can be synthesized either in vivo or in vitro.
  • a DNA sequence coding for a dsRNA can be placed in an expression cassette under the control of at least one genetic control element (such as, for example, a promoter). Polyadenylation is not required, and there is no need for elements to initiate translation.
  • the expression cassette for the MP-dsRNA is preferably contained on the transformation construct or the transformation vector.
  • the expression of the dsRNA takes place starting from an expression construct under the functional control of a flower-specific promoter.
  • the expression cassettes coding for the “antisense” and / or the “sense” strand of an ⁇ -cyclase dsRNA or for the self-complementary strand of the dsRNA are preferably inserted into a transformation vector for this purpose and into the plant cell using the methods described below brought in.
  • a stable insertion into the genome is advantageous for the method according to the invention.
  • the dsRNA can be introduced in an amount that enables at least one copy per cell. Larger quantities (e.g. at least 5, 10, 100, 500 or 1000 copies per cell) can possibly result in an efficient reduction. b) introduction of an antisense ribonucleic acid sequence of an ⁇ -cyclase ( ⁇ -cyclase-antisenseRNA)
  • the antisense nucleic acid molecule hybridizes or binds with the cellular mRNA and / or genomic DNA coding for the ⁇ -cyclase to be reduced. As a result, the transcription and / or translation of the ⁇ -cyclase is suppressed.
  • Hybridization can occur in a conventional manner via the formation of a stable duplex or - in the case of genomic DNA - by binding of the antisense nucleic acid molecule with the duplex of the genomic DNA through specific interaction in the major groove of the DNA helix.
  • An ⁇ -cyclase antisenseRNA can be derived using the nucleic acid sequence coding for this ⁇ -cyclase, for example the nucleic acid sequence according to SEQ ID NO: 58, according to the base pair rules of Watson and Crick.
  • the ⁇ -cyclase antise ⁇ seRNA can be complementary to the entire transcribed mRNA of the ⁇ -cyclase, limited to the coding region or consist only of an oligonucleotide which is complementary to part of the coding or non-coding sequence of the mRNA.
  • the oligonucleotide can be complementary to the region that comprises the translation start for the ⁇ -cyclase.
  • the ⁇ -cyclase antisenseRNA can have a length of, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides, but can also be longer and at least 100, 200, 500, 1000, 2000 or comprise 5000 nucleotides.
  • ⁇ -Cyclase antisenseRNAs are preferably recombinantly expressed in the target cell in the context of the method according to the invention.
  • Another object of the invention relates to transgenic expression cassettes containing a nucleic acid sequence coding for at least part of an ⁇ -cyclase, said nucleic acid sequence being functionally linked to a promoter which is functional in plant organisms in an antisense orientation.
  • the expression of the antisenseRNA takes place starting from an expression construct under the functional control of a flower-specific promoter.
  • Said expression cassettes can be part of a transformation construct or transformation vector, or can also be introduced as part of a co-transformation.
  • the expression of an ⁇ -cyclase can be inhibited by nucleotide sequences which are complementary to the regulatory one
  • Region of an ⁇ -cyclase gene e.g. an ⁇ -cyclase promoter and / or enhancer
  • ⁇ -cyclase promoter and / or enhancer form triple-helical structures with the DNA double helix there, so that the transcription of the ⁇ -cyclase gene is reduced.
  • Appropriate methods have been described (Helene C (1991) Anticancer Drug Res 6 (6): 569-84; Helene C et al.
  • the ⁇ -cyclase antisenseRNA can be an ⁇ -anomeric nucleic acid.
  • ⁇ -anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA in which, in contrast to the conventional ⁇ -nucleic acids, the two strands run parallel to one another (Gautier C et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense strategy described above can advantageously be coupled with a ribozyme method.
  • Catalytic RNA molecules or ribozymes can be adapted to any target RNA and cleave the phosphodiester framework at specific positions, whereby the target RNA is functionally deactivated (Tanner NK (1999) FEMS Microbiol Rev 23 (3): 257-275 ). This does not modify the ribozyme itself, but is able to cleave further target RNA molecules analogously, which gives it the properties of an enzyme.
  • the incorporation of ribozyme sequences in "antisense" RNAs gives these "antisense” RNAs this enzyme-like, RNA-cleaving property and thus increases their efficiency in inactivating the target RNA.
  • RNA molecules The preparation and use of corresponding ribozyme "antisense” RNA molecules is described (inter alia in Haseloff et al. (1988) Nature 334: 585-591); Haselhoff and Gerlach (1988) Nature 334: 585-591; Steinecke P et al. (1992) EMBO J 11 (4): 1525-1530; de Feyter R et al. (1996) Mol Gen Genet. 250 (3): 329-338).
  • ribozymes for example "Hammerhead”ribozymes; Haselhoff and Gerlach (1988) Nature 334: 585-591
  • Ribozyme technology can increase the efficiency of an antisense strategy.
  • Methods for the expression of ribozymes for the reduction of certain proteins are described in (EP 0 291 533, EP 0 321 201, EP 0 360 257). Ribozyme expression is also described in plant cells (Steinecke P et al. (1992) EMBO J 11 (4): 1525-1530; de Feyter R et al.
  • Suitable target sequences and ribozymes can be described, for example, as in "Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. Eds, Academic Press, Inc. (1995), pp. 449-460". were determined by secondary structure calculations of ribozyme and target RNA and by their interaction (Bayley CC et al. (1992) Plant Mol Biol. 18 (2): 353-361; Lloyd AM and Davis RW et al. (1994) Mol Gen Genet. 242 (6): 653-657).
  • Tetrahymena L-19 IVS RNA can be constructed which have regions complementary to the mRNA of the ⁇ -cyclase to be suppressed (see also US 4,987,071 and US 5,116,742).
  • ribozymes can also be identified via a selection process from a library of diverse ribozymes (Bartel D and Szostak JW (1993) Science 261: 1411-1418).
  • ⁇ -cyclase ribonucleic acid sequence (or a part thereof) in sense orientation can lead to a co-suppression of the corresponding ⁇ -cyclase gene.
  • sense RNA with homology to an endogenous ⁇ -cyclase gene can reduce or switch off the expression of the same, similarly as has been described for antisense approaches (Jorgensen et al. (1996) Plant Mol Biol 31 (5): 957-973; Goring et al. (1991) Proc Natl Acad Sei USA 88: 1770-1774; Smith et al. (1990) Mol Gen Genet 224: 447-481; Napoli et al.
  • the cosuppression is preferably implemented using a sequence which is essentially identical to at least part of the nucleic acid sequence coding for an ⁇ -cyclase, for example the nucleic acid sequence according to SEQ ID NO: 38.
  • the ⁇ -cyclase-senseRNA is preferably selected such that translation of the ⁇ -cyclase or a part thereof cannot occur.
  • the ⁇ '-untranslated or 3'-untranslated region can be selected or the ATG start codon deleted or mutated.
  • a reduction in ⁇ -cyclase expression is also possible with specific DNA-binding factors, for example with factors of the type of zinc finger transcription factors. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory areas, and cause a reduction in expression. Appropriate processes for the production of such factors are described (Dreier B et al. (2001) J Biol Chem 276 (31): 29466-78; Dreier B et al. (2000) J Mol Biol 303 (4): 489-502; Beerli RR et al. (2000) Proc Natl Acad Sei USA 97 (4): 1495-1500; Beerli RR et al.
  • ⁇ -cyciase gene can be selected using any piece of an ⁇ -cyciase gene.
  • This section is preferably in the region of the promoter region. For gene suppression, however, it can also lie in the area of the coding exons or introns.
  • proteins can be introduced into a cell that inhibit the ⁇ -cyclase itself.
  • protein binding factors can e.g. Aptamers (Famulok M and Mayer G (1999) Curr Top Microbiol Immunol 243: 123-36) or antibodies or antibody fragments or single-chain antibodies. The extraction of these factors has been described (Owen M et al. (1992) Biotechnology (NY) 10 (7): 790-794; Franken E et al. (1997) Curr Opin Biotechnol 8 (4): 411-416; Whitelam ( 1996) Trend Plant Be 1: 286-272).
  • the ⁇ -cyclase expression can also be effectively achieved by induction of the specific ⁇ -cyclase RNA degradation by the plant using a viral expression system (Amplikon; Angell SM et al. (1999) Plant J 20 (3): 357-362) , These systems - also referred to as "VIGS” (viral induced gene silencing) - introduce nucleic acid sequences into the plant with homology to the transcript of an ⁇ -cyclase to be reduced by means of viral vectors. The transcription is then switched off - presumably mediated by plant defense mechanisms against viruses. Appropriate techniques and processes are described (Ratcliff F et al.
  • the VIGS-mediated reduction is preferably implemented using a sequence which is essentially identical to at least part of the nucleic acid sequence coding for an ⁇ -cyclase, for example the nucleic acid sequence according to SEQ ID NO: 1.
  • genomic sequences can be modified in a targeted manner. These include in particular methods such as the generation of knockout mutants by means of targeted homologous recombination e.g. by generating stop codons, shifts in the reading frame etc. (Hohn B and Puchta H (1999) Proc Natl Acad Sei USA 96: 8321-8323) or the targeted deletion or inversion of sequences using e.g. sequence-specific recombinases or nucleases (see below)
  • -Activity can also be realized by a targeted insertion of nucleic acid sequences (for example the nucleic acid sequence to be inserted in the process according to the invention) into the sequence coding for an ⁇ -cyclase (for example by means of intermolecular homologous recombination).
  • a DNA construct is preferably used which comprises at least a part of the sequence of an ⁇ -cyclase gene or neighboring sequences and can thus be recombined in a targeted manner in the target cell, so that at least by deletion, addition or substitution of a nucleotide the ⁇ -cyclase gene is changed in such a way that the functionality of the ⁇ -cyclase gene is reduced or completely eliminated.
  • the change can also affect the regulatory elements (eg the promoter) of the ⁇ -cyclase gene, so that the coding sequence remains unchanged, but expression (transcription and / or translation) is omitted and reduced.
  • the sequence to be inserted is flanked at its 5 'and / or 3' end by further nucleic acid sequences (A 1 or B ') which are of sufficient length and homology to corresponding sequences of the ⁇ -cyclase - Show genes (A or B) to enable homologous recombination.
  • the length is usually in the range from several hundred bases to several kilobases (Thomas KR and Capecchi MR (1987) Cell 51: 503; Strepp et al.
  • the plant cell with the recombination construct is transformed using the methods described below and successfully recombined clones are selected based on the ⁇ -cyclase which is inactivated as a result.
  • the efficiency of the recombination is increased by combination with methods which promote homologous recombination. Such methods are described and include, for example, the expression of proteins such as RecA or the treatment with PARP inhibitors. It could be shown that the intrachromosomal homologous recombination in tobacco plants can be increased by using PARP inhibitors (Puchta H et al.
  • Inhibitors such as 3-aminobenzamide, 8-hydroxy-2-methyiquinazolin-4-one (NU1025), 1.11 b-dihydro- [2H] benzopyrano- [4,3,2-de] isoquinolin-3-one are preferably included (GPI 6150), 5-aminoisoquinolinone, 3,4-dihydro-5- [4- (1-piperidinyl) butoxy] -1 (2H) -isoquinolinone or those described in WO 00/26192, WO 00/29384, WO 00 / 32579, WO 00/64878, WO 00/68206, WO 00/67734, WO 01/23386 and WO 01/23390.
  • RNA / DNA oligonucleotides into the plant
  • Knockout mutants with the help of e.g. T-DNA mutagenesis
  • Point mutations can also be generated using DNA-RNA hybrids, also known as "chimeraplasty” (Cole-Strauss et al. (1999) Nucl Acids Res 27 (5): 1323-1330; Kmiec (1999) Gene therapy American Scientist 87 (3): 240-247).
  • PTGS post-transcriptional gene silencing
  • TGS transcriptional gene silencing
  • the ⁇ -cyclase activity is reduced compared to the wild type by:
  • the ⁇ -cyclase activity is reduced compared to the wild type by introducing at least one double-stranded ⁇ -cyclase ribonucleic acid sequence or an expression cassette or expression cassettes ensuring its expression in plants.
  • genetically modified plants are used which have the lowest expression rate of an ⁇ -cyclase in flowers.
  • this is achieved in that the transcription of the ⁇ -cyclase dsRNA sequences takes place under the control of a flower-specific promoter or, even more preferably, under the control of a flower-leaf-specific promoter.
  • Particularly preferred plants are plants selected from the families Amaranthaceae, Amaryllidaceae, Apocynaceae, Asteraceae, Balsaminaceae, Begonia- ceae, Berberidaceae, Brassicaceae, Cannabaceae, Caprifoliaceae, Caryophyllaceae, Chenopodiaceae, Compitaceae, Compositaceae, Compositaceae, Compositeaeae, Compositeae, Compositeae , Graminae, llliaceae, Labiatae, Lamiaceae, Leguminosae, Liliaceae, Linaceae, Lobeliaceae, Malvaceae, Oleaceae, Orchidaceae, Papaveraceae, Plumbaginaceae, Poaceae, Polemoniaceae, Primulacaceae, Roseaaceae, Rosunceae , Vitaceae and Violaceae.
  • Very particularly preferred plants are selected from the group of the plant genera Marigold, Tagetes erreeta, 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, Grevillea, Helenium, Helianthus, Hepatica, Heracleum, Hisbiscus, Heliopsis, Hypericum, Hypochoeris Impatiens, Iris, Jacaranda, Kenya, Laburnum, Lathyrus, Leontodon, Lilium
  • Calendula Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium, Tropaeolum or Adonis.
  • 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 on nutrient media in a manner known per se and harvested accordingly.
  • 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
  • an inducible promoter is preferred. 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 see separation methods such as chromatography.
  • 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 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 or as diglucosides.
  • the ketocarofinlides 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 in particular nectaries and petals, which also have petals. be drawn.
  • 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.
  • the modified ketolase activity being caused by a ketolase selected from group 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2, B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has.
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • Synthase activity, geranyl-geranyl diphosphate synthase activity, phytoene synthase activity, phytoene desaturase activity, zeta-carotene desaturase activity, crtlSO activity, FtsZ activity and / or MinD activity can be analogous The corresponding effect genes are used.
  • 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 at least one of the effect genes described above and which are functionally linked to one or more regulation signals which ensure transcription and translation in plants.
  • nucleic acid constructs in which the effect genes are functionally linked to one or more regulation 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.
  • an expression cassette contains regulatory signals, that is to say regulative nucleic acid sequences which control the expression of the effect genes in the host cell.
  • an expression cassette comprises a promoter upstream, ie at the 5 'end of the coding sequence, and downstream, ie at 3'-end, a polyadenylation signal and optionally further regulatory elements which are operatively linked to the intermediate coding sequence of the effect gene for at least one of the genes described above.
  • 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.
  • sequences which are preferred, but not limited to, for operative linking are targeting sequences for ensuring 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 promoter derived from a plant virus is preferably used in particular. 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 effect genes in the plant can be 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 0 388 186), a tetracycline inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), an abscisic acid inducible promoter (EP 0 335 528) or an ethanol or cyclohexanone inducible promoter (WO 93/21334) can also be used become.
  • 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
  • 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 are preferred.
  • 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-enolpyruvylshikimate-3-phosphate synthase gene promoter from Petunia hybrida, Acc.- No.
  • CHRC chromoplast-specific carotenoid-associated protein
  • the PDS promoter (Phytoene desaturase gene promoter 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).
  • Anther-specific promoters are, for example, the 5126 promoter (US Pat. No. 5,689,049, US Pat. No. 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 (WO 9920775), the LeB4 promoter from Vicia faba (WO 9729200 and US 06403371), the napin Promoter from Brassica napus (US 5608152; EP 255378; US 5420034), the
  • SBP promoter from Vicia faba (DE 9903432) or the maize promoters End1 and End2 (WO 0011177).
  • An expression cassette is preferably produced by fusing a suitable promoter with at least one of the effect genes described above, 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 techniques, such as those for example in T. Maniatis, EF 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 Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
  • nucleic acids encoding a plastid transit peptide ensure localization in plastids and in particular in chromoplasts.
  • Expression cassettes the nucleic acid sequence of which codes for an effect gene-product fusion protein, can also be used, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
  • Preferred transit peptides are preferred for the chromoplasts, which after translocation the effect genes are split off enzymatically from the effect gene product part 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:
  • a plastid transit peptide are the transit peptide of the plastid isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabisopsis thaliana and the transit peptide of the small subunit of ribulose bisphosphate carboxylase (rbcS) from pea (Guerineau, F, Woolston, S, Brook L, Mullineaux, P (1988) An expression casette fortargeting
  • 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, in the 5'-3 'transcription direction, the promoter, a coding nucleic acid sequence or a nucleic acid construct and a region for the transcriptional termination. Different termination areas are interchangeable.
  • 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.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • Suitable methods for the transformation of plants are the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene cannon - the so-called "particle bombardment” method, the 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, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the fused expression cassette 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 crop plants, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • Transgenic plants which contain one or more genes integrated into the expression cassette can be regenerated in a known manner from the transformed cells of the wounded leaves or leaf pieces.
  • an expression cassette is inserted as an insertion into a recombinant vector, the vector DNA of which contains additional functional regulatory signals, for example sequences for replication or integration.
  • additional functional regulatory signals for example sequences for replication or integration.
  • Suitable vectors are 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 which enable their multiplication, 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.
  • the production of genetically modified microorganisms according to the invention with increased or caused ketolase activity is described by way of example, the changed ketolase activity being caused by a ketolase selected from the group 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14. or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • Synthase activity, geranyl-geranyl diphosphate synthase activity, phytoene synthase activity, phytoene desaturase activity, zeta-carotene desaturase activity, crtlSO activity, FtsZ activity and / or MinD activity can be analogous The corresponding effect genes are used.
  • nucleic acids described above encoding a ketolase, ⁇ -hydroxylase or ⁇ -cyclase, and the nucleic acids encoding an HMG-CoA reductase, nucleic acids encoding an (E) -4-hydroxy-3-methylbut-2-enyl diphosphate reductase , Nucleic acids encoding a 1-deoxy-D-xylose-5-phosphate synthase, nucleic acids encoding a 1-deoxy-D-xylose-5-phosphate reductoisomerase, nucleic acids encoding an isopentenyl-diphosphate- ⁇ -isomerase, nucleic acids encoding a geranyl -Diphosphate synthase, nucleic acids encoding a farnesyl diphosphate synthase, nucleic acids encoding a geranyl-geranyl diphosphate synthase, nucleic acids encoding a phytoene synthase, nucleic acids
  • 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 effect gene.
  • An “operative linkage” is understood to mean the sequential arrangement of promoter, coding sequence (effect gene), terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can perform its function as intended in the expression of the coding sequence.
  • sequences which can be linked operatively are targeting sequences as well.
  • Further regulatory elements include selectable markers, amplification signals, origins of replication and the like.
  • the natural regulation sequence can still be present before the actual effect gene. This natural regulation can possibly be switched off by genetic modification and the expression of the genes 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 promoters 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, ⁇ -cyclase, HMG-CoA reductase, (E) -4-hydroxy-3-methylbut-2-enyl- Diphosphate reductase, 1-deoxy-D-xylose-5-phosphate synthase, 1-deoxy-D-xylose-5-phosphate reductoisomerase, isopentenyl-diphosphate- ⁇ -isomerase, geranyl-diphosphate-synthase, farnesyl-diphosphate- Synthase, geranyl-geranyl-diphosphate synthase, phytoene synthase, phytoene desaturase, zeta-carotene desaturase, crtlSO protein, FtsZ protein and / or a MinD protein and a terminator or polyadenylation signal.
  • 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 adeovirus, 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 (1990) 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).
  • genetically modified microorganisms can be produced, for example with at least one vector according to the invention are transformed.
  • 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 which have been successfully transformed with a vector and carry 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 the genetically modified, non-human organisms, the genetic modification being the activity of a ketolase
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • the increase (according to E) or causation (according to F) of the ketolase activity compared to the wild type is preferably carried out by the
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or one of this sequence by substitution, insertion or deletion of amino acids derived sequence that is at least 90% identical at the amino acid level to the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% at the amino acid level with the sequence SEQ. ID. NO. 14 has.
  • the gene expression of a nucleic acid encoding a ketolase is increased by introducing into the organism nucleic acids encoding ketolases selected from the group
  • 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 80% at the amino acid level with the sequence SEQ. ID. NO. 2 has
  • B ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ. ID. NO. 10 has
  • C ketolase 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 90% at the amino acid level with the sequence SEQ. ID. NO. 12 or
  • D ketolase containing the amino acid sequence SEQ. ID. NO. 14 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 50% on amino acids. level with the sequence SEQ. ID. NO. 14 has.
  • the transgenic organisms according to the invention therefore have at least one further ketolase gene according to the invention compared to the wild type.
  • genetically modified organisms additionally have an increased or induced hydroxlase activity and / or ⁇ -cyclase activity compared to the wild type. Further preferred embodiments are described above in the method according to the invention.
  • genetically modified non-human organisms additionally have at least one further increased activity compared to the wild type, selected from the group HMG-CoA reductase activity, (E) - 4-hydroxy-3 -Methylbut-2-enyl-diphosphate reductase activity, 1 -deoxy-D-xylose-5-phosphate synthase activity, 1-deoxy-D-xylose-5-phosphate reductoisomerase activity, isopentenyl-diphosphate- ⁇ -Isomerase activity, geranyl diphosphate synthase activity, farnesyl diphosphate synthase activity, geranyl geranyl diphosphate synthase activity, phytoene synthase activity, phytoene desaturase activity, zeta-carotene desaturase activity , crtlSO activity, FtsZ activity and MinD activity. Further preferred embodiments are described above in the method according to the invention.
  • genetically modified plants as mentioned above, additionally have a reduced ⁇ -cyclase activity compared to a Wiid type plant. 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 able to produce 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 Bacteria that are able to synthesize xanthophylls, such as, for example, bacteria of the genus Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc or cyanobacteria of the genus Synechocystis.
  • bacteria of the genus Escherichia which contain, for example, crt genes from Erwinia
  • Bacteria that are able to synthesize xanthophylls such as, for example, bacteria of the genus Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc or cyan
  • 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 mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Blakeslea, in particular Blakeslea trispora, 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.
  • Particularly preferred plants are selected from the Amaranthaceae.Amaryllidaceae, Apocynaceae, Asteraceae, Balsaminaceae, Begoniaceae, Berberidaceae, Brassicaceae, Cannabaceae, Caprifoliaceae, Caryophyllaceae, Chenopaceae, Compateaceae Gentianaceae, Geraniaceae, Graminae, llliaceae, Labiatae, Lamiaceae, Leguminosae, Liliaceae, Linaceae, Lobeliaceae, Malvaceae, Oleaceae, Orchidaceae, Papaveraceae, Plumbaginaceae, Poaceae, Polemoniaceae Verbana- ceae, Vitaceae and Violaceae.
  • 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, Laburnum, Lathyrus, Leontodon, Lili
  • Calendula Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium, Tropaeolum or Adonis.
  • Very particularly preferred genetically modified plants are selected from the plant genera Marigold, Tagetes erecta, Tagetes patula, Adonis, Lycopersicon, Rosa, Calendula, Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium or Tropaeolum, the genetic modified plant contains at least one transgenic nucleic acid encoding 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 in particular plants or parts of plants, such as in particular petals with an increased content of ketocarotenoids, in particular astaxanthin, which can be consumed by humans and animals can also be used, for example, directly or after processing known per se as food or Feed or used as feed and food supplements.
  • 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, an altered 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 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 and having an identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • ketolases contain the sequence SEQ. ID. NO. 2, 4, 6 or 8. Particularly preferred ketolases are ketolases with the sequences SEQ. ID. NO. 2, 4, 6 or 8.
  • the invention further relates to nucleic acids encoding ketolases described above.
  • Preferred nucleic acids contain the sequence SEQ. ID. NO. 1, 3, 5 or 7. Particularly preferred nucleic acids are nucleic acids with the sequence SEQ. ID. NO. 1, 3, 5 or 7.
  • the invention further relates to a ketolase containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 10 has.
  • ketolases contain the sequence SEQ. ID. NO. 10. Particularly preferred ketolases are ketolases of the sequence SEQ. ID. NO. 10th
  • the invention further relates to nucleic acids encoding a ketolase described above.
  • Preferred nucleic acids contain the sequence SEQ. ID. NO. 9. Particularly preferred nucleic acids are nucleic acids of the sequence SEQ. ID. NO. 9th
  • the invention further 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 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, particularly preferably at least 99% at the amino acid level with the sequence SEQ. ID. NO. 12 has.
  • ketolases contain the sequence SEQ. ID. NO. 12. Particularly preferred ketolases are ketolases of the sequence SEQ. ID. NO. 12th
  • the invention further relates to nucleic acids encoding a ketolase described above.
  • Preferred nucleic acids contain the sequence SEQ. ID. NO. 11. Particularly preferred nucleic acids are nucleic acids of the sequence SEQ. ID. NO. 11th
  • ketolase NP60.79 BKT from Nostoc punctiforme SAG 60.79
  • the DNA coding for the ketolase NP60.79: BKT was amplified by means of PCR from Nostoc punctiforme SAG 60.79 (SAG: Collection of algal cultures in Göttingen).
  • 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 coding for the ketolase NP60.79 was determined by means of a "polymerase chain reaction” (PCR) from Nostoc punctiforme SAG 60.79 using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NP196-2 SEQ ID No. 60).
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which 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. 59 and SEQ ID No. 60 resulted in a 792 bp fragment coding for a protein consisting of the entire primary sequence (SEQ ID No. 61).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1-TOPO (Invitrogen) and the clone pNP60.79 was obtained.
  • ketolase NP60.79 BKT from Nostoc punctiforme SAG 60.79 in Lycopersicon esculentum and Tagetes erecta
  • the expression of the ketolase from Nostoc punctiform SAG 60.79 in Lycopersicon esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreduetase) from Arabidopsis thaliana. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the DNA fragment which contains the FNR promoter region -635 to -1 from Arabidopsis thaliana (SEQ ID No. 65) was PCR-analyzed using genomic DNA (isolated from Arabidopsis thaliana according to standard methods) and the primer FNR-1 (SEQ ID No .63) and FNR-2 (SEQ ID No. 64).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the FNR promoter fragment (-635 to -1), was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 653 bp amplificate (SEQ ID No. 65) was cloned into the PCR cloning vector pCR 2.1-TOPO (Invitrogen) 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 69493.
  • the gene begins at base pair 69492 and is annotated with "ferredoxin-NADP + reductase".
  • the clone pFNR 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 637 bp Kpnl-Hindlll fragment from pFNR and ligating into the Kpnl-Hindlll cut vector pJIT117.
  • the clone that uses the FNR promoter instead of the original d35S promoter is called pJFNR.
  • the clone pNP60.79 was used for the cloning into the expression vector pJFNR (example 2). The cloning was carried out by isolating the 790 bp Sphl fragment from pNP60.79 and ligating into the Sphl cut vector pJFNR.
  • the clone that contains the Nostoc punctiforme SAG 60.79 ketolase in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJFNRNP60.79.
  • the 2.4 Kb Kpnl fragment from pJFNRNP60.79 was ligated with the Kpnl-cut vector pSUN3. This clone is called MSP1.
  • ketolase NP60.79 BKT from Nostoc punctiforme SAG 71.79
  • the DNA which codes for the ketolase NP71.79: BKT was amplified by means of PCR from Nostoc punctiforme SAG 71.79 (SAG: Collection of algal cultures in Göttingen).
  • 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.
  • 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. 59 and SEQ ID No. 60 resulted in a 792 bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 66).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1-TOPO (Invitrogen) and the clone pNP71, 79 was obtained.
  • ketolase NP71.79 BKT from Nostoc punctiforme SAG 71.79 in Lycopersicon esculentum and Tagetes erecta
  • the expression of the ketolase from Nostoc punctiform SAG 71.79 in Lycopersicon esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreduetase) from Arabidopsis thaliana. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the clone pNP71.79 was used for the cloning into the expression vector pJFNR (example 2). The cloning was carried out by isolating the 790 bp Sphl fragment. vector pJFNR cut from pNP71.79 and ligation into the coil. The clone that contains the Nostoc punctiforme SAG 71.79 ketolase in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJFNRNP71.79.
  • An expression cassette for the Agrobacterium -mediated transformation of the ketolase NP71.79: BKT from Nostoc punctiforme SAG 71.79 in Lycopersicon esculentum was produced using the binary vector pSUN3 (WO02 / 00900).
  • pS3FNRNP71.79 the 2.4 Kb Kpnl fragment from pJFNRNP71.79 was ligated with the Kpnl-cut vector pSUN3. This clone is called MSP3.
  • Example 5 Amplification of a DNA encoding the entire primary sequence of the ketolase NS037: BKT from Nodularia spumigena CCAUV 01-037
  • the DNA coding for the ketolase NS037: BKT was amplified by PCR from Nodularia spumigena CCAUV 01-037 (CCAlN.Culture Collection ofAlgae at the University of Vienna).
  • 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 ⁇ minute centrifugation at 13,000 rpm, 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 coding for the ketolase NS037 BKT from Nodularia spumigena CCAUV 01-037 was synthesized by means of a "polymerase chain reaction” (PCR) from Nodularia spumigena CCAUV 01-037 using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NSK-2 SEQ ID No. 68).
  • 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:
  • NSK-2 SEQ ID No. 68
  • TAKARA 5 ul 10X PCR buffer
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 59 and SEQ ID No. 68 resulted in an 807 bp fragment that codes for a protein consisting of the entire primary sequence (SEQ ID No. 69).
  • the amplificate in the PCR cloning vector pCR 2.1-TOPO was cloned and the clone pNS037 obtained.
  • NS037 BKT from Nodularia spumigena CCAUV 01-037 in Lycopersicon esculentum and Tagetes erecta
  • the expression of the ketolase from Nodularia spumigena CCAUV 01-037 in Lycopersicon esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreduetase) from Arabidopsis thaliana. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the clone pNS037 was used for the cloning into the expression vector pJFNR (example 2).
  • the cloning was carried out by isolating the 797 bp Sphl fragment from pNS037 and ligation into the Sphl cut vector pJFNR.
  • the clone which contains the ketolase from Nodularia spumigena CCAUV 01-037 in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJFNRNS037.
  • the 2.4 Kb Kpnl fragment from pJFNRNS037 was ligated with the Kpnl-cut vector pSUN5. This clone is called MSP6.
  • the DNA coding for the ketolase NS053: BKT was amplified by means of PCR from Nodularia spumigena CCAUV 01-053 (CCAUV: Culture Collection of Algae at the University of Vienna).
  • 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 phenol extraction 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 coding for the ketolase NS053 BKT from Nodularia spumigena CCAUV 01-053 was synthesized by means of a "polymerase chain reaction” (PCR) from Nodularia spumigena CCAUV 01-053 using a sense-specific primer (NP196-1, SEQ ID No . 59) and an antisense-specific primer (NSK-2 SEQ ID No. 68).
  • 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. 59 and SEQ ID No. 68 resulted in an 807 bp fragment coding for a protein consisting of the entire primary sequence (SEQ ID No. 71).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1-TOPO (Invitrogen) and the clone pNS053 was obtained.
  • ketolase NS053 BKT from Nodularia spumigena CCAUV 01-053 in Lycopersicon esculentum and Tagetes erecta
  • the expression of the ketolase from Nodularia spumigena CCAUV 01-053 in Lycopersicon esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreduetase) from Arabidopsis thaliana. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715). The clone pNS053 was used for the cloning into the expression vector pJFNR (example
  • the cloning was carried out by isolating the 797 bp Sphl fragment from pNS053 and ligating into the SphI cut vector pJFNR. The clone that the
  • pJFNRNS053 ketolase from Nodularia spumigena CCAUV 01-053 in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide.
  • the 2.4 Kb Kpnl fragment from pJFNRNS053 was ligated with the Kpnl-cut vector pSUN3. This clone is called MSP7.
  • the 2.4 Kb Kpnl fragment from pJFNRNS053 was ligated with the Kpnl-cut vector pSUN5. This clone is called MSP8.
  • ketolase GV35.87 BKT from Gloeobacter violaceus SAG 35.87
  • the DNA coding for the ketolase GV35.87.BKT was amplified by means of PCR from Gloeobacter violaceus SAG 35.87 (SAG: Collection of algal cultures in Göttingen).
  • 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 washed in liquid nitrogen with a. Crush and ground the mortar. The cell material was resuspended in 1 ml 10mM Tris HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (2ml volume). After adding 100 ⁇ l Proteinase K (concentration: 20 mg / ml), the cell suspension was incubated for 3 hours at 37 ° C. The suspension was then extracted with 500 ⁇ l of phenol. After centrifugation at 13,000 rpm for ⁇ 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 coding for the ketolase GV35.87.BKT from Gloeobacter violaceus SAG 35.87 was determined by means of "polymerase chain reaction” (PCR) from Gloeobacter violaceus SAG 35.87 using a sense-specific primer (GVK-F1, SEQ ID No. 73 ) and an antisense-specific primer (GVK-R1 SEQ ID No. 74).
  • 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:
  • PCR amplification with SEQ ID No. 73 and SEQ ID No. 74 resulted in a 785 bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 75).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1-TOPO (Invitrogen) and the clone pGV35.87 was obtained.
  • ketolase GV35.87 BKT from Gloeobacter violaceus SAG 35.87 in Lycopersicon esculentum and Tagetes erecta
  • the expression of the ketolase from Gloeobacter violaceus SAG 35.87 m Lycopersicon esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreduetase) from Arabidopsis thaliana. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the clone pGV35.87 was used for the cloning into the expression vector pJFNR (example 2). The cloning was carried out by isolating the 797 bp Sphl fragment from pGV35.87 and ligation into the SphI-cut vector pJFNR.
  • the clone which contains the ketolase from Gloeobacter violaceus SAG 35.87 in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJFNRGV35.87.
  • the expression vector pS3FNRGV35.87 To produce the expression vector pS3FNRGV35.87, the 2.4 Kb Kpnl fragment (partial Kpnl hydrolysis) from pJFNRGV35.87 was ligated with the Kpnl-cut vector pSUN3. This clone is called MSP9.
  • An expression cassette for the> 4gro ⁇ acter / - / m -mediated transformation of the expression vector with the ketolase GV35.87; ßKT from Gloeobacter violaceus SAG 35.87 in Tagetes erecta was carried out using the binary vector pSUN ⁇ (WO02 / 00900).
  • 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), 157-168).
  • Example 11.1 Construction of pMCL-CrtYlBZ
  • the biosynthetic genes crtY, crtB, crtl and crtZ come from the bacterium Erwinia uredovora and were amplified by PCR. Genomic DNA from Erwinia uredovora (DSM 30080), prepared by the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig) as part of a service.
  • the 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:
  • PCR amplification with SEQ ID No. 77 and SEQ ID No. 78 resulted in a fragment (SEQ ID NO. 79) which is responsible for the genes CrtY (protein: SEQ ID NO. 80), Crtl (protein: SEQ ID NO. 81), crtB (protein: SEQ ID NO. 82) and CrtZ (iDNA) encoded.
  • the amplificate was cloned into the PCR cloning vector pCR2.1 (Invitrogen) and the clone pCR2.1-CrtYIBZ was obtained.
  • the plasmid pCR2.1-CrtYIBZ was cut Sall and Hindlll, 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, Crtl, crtB and CrtZ and corresponds to the sequence from positions 229 ⁇ to 6918 in D90087 (SEQ ID No. 79).
  • the gene CrtZ is transcribed against the reading direction of the genes CrtY, Crtl and CrtB by means of its endogenous promoter.
  • the resulting clone is called pMCL-CrtYlBZ.0
  • Example 11.2 Construction of pMCL-CrtYlBZ / idi
  • the gene idi isopentenyl diphosphate isomerase; IPP isomerase
  • the nucleic acid encoding the entire idi gene with idi-5 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. 81) and an antisense-specific primer (3'-idi SEQ ID No. 82) was amplified.
  • PCR polymerase chain reaction
  • the PCR conditions were as follows: 0
  • 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:
  • PCR amplification with SEQ ID No. 81 and SEQ ID No. 82 resulted in a 679 bp fragment coding for a protein consisting of the entire primary sequence (SEQ ID No. 83).
  • 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 includes the promoter region, the potential ribosome binding site and the entire "open reading frame" for the IPP isomerase.
  • the fragment cloned in 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 / ' oY gene.
  • This clone was therefore used for the cloning of the / / gene into the vector pMCL-CrtYIBZ.
  • the cloning was carried out by isolating the Xhol / Sall fragment from pCR2.1-idi and ligation into the Xhol / Sall cut vector pMCL-CrtYIBZ.
  • the resulting clone is called pMCL-CrtYlBZ / idi.
  • Example 11.3 Construction of pMCL-CrtYlBZ idi / gps
  • the gene gps (geranylgeranyl pyrophosphate synthase; GGPP synthase) was amplified from Archaeoglobus fulgidus by means of PCR.
  • the nucleic acid encoding gps Archaeoglobus fulgidus was determined by means of "polymerase chain reaction” (PCR) using a sense-specific primer (5'-gps SEQ ID No. 85) and an anti-sense-specific primer (3'-gps SEQ ID No. 86) amplified.
  • 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 ⁇ O ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • SEQ ID No. 86 and SEQ ID No. 86 amplified DNA fragments were eluted from the agarose gel using methods known per se and cut with the restriction enzymes Ncol and HindIII. This results in a 962 bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 87).
  • Ncol / HindIII cut amplificate was cloned into the vector pCB97-30 and the clone pCB-gps was obtained.

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Abstract

La présente invention concerne un procédé de production de cétocaroténoïdes par culture d'organismes non humains génétiquement modifiés qui présentent une activité de cétolase modifiée par rapport au type sauvage. L'invention concerne également lesdits organismes génétiquement modifiés, leur utilisation comme produits alimentaires et produits fourragers et pour la production d'extraits de cétocaroténoïdes, ainsi que de nouvelles cétolases et des acides nucléiques codant pour ces cétolases.
EP04763696A 2003-08-18 2004-07-31 Nouvelles cetolases et procede de production de cetocarotenoides Withdrawn EP1658372A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP04763696A EP1658372A2 (fr) 2003-08-18 2004-07-31 Nouvelles cetolases et procede de production de cetocarotenoides

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
PCT/EP2003/009102 WO2004018693A2 (fr) 2002-08-20 2003-08-18 Procede de production de cetocarotenoides dans les petales de plantes
PCT/EP2003/009105 WO2004018385A2 (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
PCT/EP2003/009107 WO2004018695A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des fruits de plantes
PCT/EP2003/009106 WO2004018694A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies
PCT/EP2003/009109 WO2004017749A2 (fr) 2002-08-20 2003-08-18 Utilisation de plantes ou de parties de plante scontenant de l'astaxanthine du genre tagetes comme produit de fourrage
PCT/EP2003/009101 WO2004018688A1 (fr) 2002-08-20 2003-08-18 Procede de preparation de $g(b)-carotinoides
DE102004007624A DE102004007624A1 (de) 2004-02-17 2004-02-17 Neue Ketolasen und Verfahren zur Herstellung von Ketocarotinoiden
PCT/EP2004/008625 WO2005019461A2 (fr) 2003-08-18 2004-07-31 Nouvelles cetolases et procede de production de cetocarotenoides
EP04763696A EP1658372A2 (fr) 2003-08-18 2004-07-31 Nouvelles cetolases et procede de production de cetocarotenoides

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102888425A (zh) * 2012-07-02 2013-01-23 中国科学院昆明植物研究所 利用转基因植物生产虾青素的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005019461A2 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102888425A (zh) * 2012-07-02 2013-01-23 中国科学院昆明植物研究所 利用转基因植物生产虾青素的方法
CN102888425B (zh) * 2012-07-02 2015-02-25 中国科学院昆明植物研究所 利用转基因植物生产虾青素的方法

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