EP1658377A1 - Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies - Google Patents

Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies

Info

Publication number
EP1658377A1
EP1658377A1 EP04741347A EP04741347A EP1658377A1 EP 1658377 A1 EP1658377 A1 EP 1658377A1 EP 04741347 A EP04741347 A EP 04741347A EP 04741347 A EP04741347 A EP 04741347A EP 1658377 A1 EP1658377 A1 EP 1658377A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acids
activity
sequence
sequence seq
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04741347A
Other languages
German (de)
English (en)
Inventor
Ralf Flachmann
Christel Renate Schopfer
Karin Herbers
Irene Kunze
Matt Sauer
Martin Klebsattel
Thomas Luck
Dirk Voeste
Angelika-Maria Pfeiffer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SunGene GmbH
Original Assignee
SunGene GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/EP2003/009105 external-priority patent/WO2004018385A2/fr
Priority claimed from PCT/EP2003/009101 external-priority patent/WO2004018688A1/fr
Priority claimed from DE102004007622A external-priority patent/DE102004007622A1/de
Application filed by SunGene GmbH filed Critical SunGene GmbH
Priority to EP04741347A priority Critical patent/EP1658377A1/fr
Publication of EP1658377A1 publication Critical patent/EP1658377A1/fr
Withdrawn legal-status Critical Current

Links

Definitions

  • the present invention relates to a process for the preparation of ketocarotenoids by cultivating genetically modified organisms which, compared to the wild type, have an altered ketolase activity and an altered ⁇ -cyclase activity, the genetically modified organisms, and their use as food and feed and for the production of ketocarotenoid extracts.
  • Carotenoids are synthesized de novo in bacteria, algae, fungi and plants. .. ketocarotenoids, ie carotenoids, which contain at least one keto group, such as, "for example astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin and adonixanthin are natural antioxidants and pigments, which are considered by some algae and microorganisms Secondary metabolites are produced.
  • ketocarotenoids ie carotenoids, which contain at least one keto group, such as, "for example astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin and adonixanthin are natural antioxidants and pigments, which are considered by some algae and microorganisms Secondary metabolites are produced.
  • ketocarotenoids and in particular astaxanthin are used as pigmenting aids in animal nutrition, especially in trout, salmon and shrimp farming.
  • ketocarotenoids such as natural astaxanthin
  • biotechnological processes by cultivating algae, for example Haematococcus pluvialis or by fermentation of genetically optimized microorganisms and subsequent isolation.
  • An economical biotechnological process for the production of natural ketocarotenoids is therefore of great importance.
  • 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.
  • the invention was therefore based on the object of providing a process for the preparation of ketocarotenoids by cultivating genetically modified, non-human organisms, or of providing further genetically modified, non-human organisms which produce ketocarotenoids which have the disadvantages of the prior art described above to a lesser extent or no longer or which provide the desired ketocarotenoids 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 and an altered ⁇ -cyclase activity compared to the wild type, and the altered ⁇ -cyclase activity by a ⁇ -cyclase is caused, 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 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”.
  • ⁇ -cyclase activity changed compared to the wild type is preferably understood to mean “ ⁇ -cyclase activity caused compared to the wild type”.
  • ⁇ -cyclase activity changed compared to the wild type is preferably understood to mean “ ⁇ -cyclase activity increased 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 enabled by genetic modification, such as re-regulation of metabolic pathways or complementation are to produce carotenoids such as ß-carotene or zeaxanthin.
  • ketocarotenoids such as astaxanthin or canthaxanthin.
  • These organisms such as Haematococcus pluvialis, Paracoccus marcusii, Xan- thophyllomyces dendrorhous, Bacillus circulans, Chlorococcum, Phaffia rhodozyma, Adonis, Neochlohs wimmeri, Protosiphon vacuolätus botryoides, Scotiellopsis oocystifor- mis, Scenedesmus, Chlorela zofingiensis, Ankistrodesmus braunii, Euglena sanguinea and Bacillus atrophaeus, already as starting or wild-type organisms a ketolase activity and a ⁇ -cyclase activity.
  • 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 for increasing or causing ketolase activity, for increasing or causing hydroxylase activity described below, for that described below
  • Increasing or causing the ⁇ -cyclase activity for the increase in the HMG-CoA reductase activity described below, for the increase in the (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase described below
  • Increase in isopentenyl diphosphate ⁇ isomerase activity for the increase in geranyl diphosphate synthase activity described below, for the E described below increase in farnesyl diphosphate synthase activity, for the increase in geranyl-geranyl diphosphate synthase activity described below, for the increase in phytoen
  • This reference organism is preferably Haematococcus pluvialis for microorganisms which already have ketolase activity as a wild type.
  • This reference organism is preferably Blakeslea for microorganisms which, as a wild type, have no ketolase activity.
  • This reference organism is preferably Adonis aestivalis, Adonis flammeus or Adonis for plants which already have a ketolase activity as a wild type annuus, 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 palms, 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 Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Scenedesmus vacuolatus, Cholrela zofingiensis, 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 ketolase activity is higher than that of the wild type, the amount of ⁇ -carotene or the amount of canthaxanthin formed is increased by the protein ketolase in a certain time compared to the wild type.
  • ketolase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the ketolase activity of the wild type.
  • the 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 wild type, for example by inducing the ketolase gene by activators or by Introduction of nucleic acids encoding a ketolase into the organism.
  • Increasing the gene expression of a nucleic acid encoding a ketolase is understood according to the invention in this embodiment as 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 a regulator protein which is not found or modified in the wild-type organism interacts with the promoter of these genes.
  • Such a regulator can represent a chimeric protein which consists of a DNA binding domain and a transcription activator domain, as described, for example, in WO 96/06166.
  • the ketolase activity is increased compared to the wild type by increasing the gene expression of a nucleic acid encoding a ketolase.
  • the gene expression of a nucleic acid encoding a ketolase is increased by introducing nucleic acids encoding ketolases into the organism.
  • At least one further ketolase gene is thus present in the transgenic organisms according to the invention compared to the wild type.
  • 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 Tagetes 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 with the genetically unmodified wild type and is therefore preferably capable of transgenically expressing a ketolase.
  • the gene expression of a nucleic acid encoding a ketolase is caused analogously to the above-described increase in gene expression of a nucleic acid encoding a ketolase, preferably by introducing nucleic acids which encode ketolases into the starting organism.
  • any ketolase gene that is to say any nucleic acids encoding a ketolase, can be used in both embodiments.
  • nucleic acids mentioned in the description can be, for example, an RNA, DNA or cDNA sequence.
  • genomic ketolase sequences from eukaryotic sources which contain introns in the event that the host organism is unable or not able to the position can be shifted to express the corresponding ketolase, preferably to use already processed nucleic acid sequences, such as the corresponding cDNAs.
  • nucleic acids encoding a ketolase and the corresponding ketolases that can be used in the method according to the invention are, for example, sequences from
  • Haematoccus pluvialis especially from Haematoccus pluvialis Flotow em. Wille (Accession NO: X86782; nucleic acid: SEQ ID NO: 3, protein SEQ ID NO: 4),
  • Agrobacterium aurantiacum (Accession NO: D58420; nucleic acid: SEQ ID NO: 37, protein SEQ ID NO: 38),
  • Alicaligenes spec. (Accession NO: D58422; nucleic acid: SEQ ID NO: 39, protein SEQ ID NO: 40),
  • Paracoccus marcusii (Accession NO: Y15112; nucleic acid: SEQ ID NO: 41, protein SEQ ID NO: 42).
  • Synechocystis sp. Strain PC6803 (Accession NO: NP442491; nucleic acid: SEQ ID NO: 43, protein SEQ ID NO: 44).
  • Bradyrhizobium sp. (Accession NO: AF218415; nucleic acid: SEQ ID NO: 45, protein SEQ ID NO: 46).
  • Nostoc punctiforme ATCC 29133 (Accession NO: NZ_AABC01000195, ZP_00111258; nucleic acid: SEQ ID NO: 57, protein: SEQ ID NO: 58)
  • Nucleic acid Acc.-No. NZ_ AABD01000001, base pair 1, 354.725-1, 355.528 (SEQ ID NO: 75), protein: Acc.-No. ZP_00115639 (SEQ ID NO: 76) (annotated as putative protein),
  • sequences derived from these sequences such as, for example
  • ketolases of the sequence SEQ ID NO: 64 or 66 and the corresponding coding nucleic acid sequences SEQ ID NO: 63 or SEQ ID NO: 65 which result, for example, from the sequence SEQ ID NO: 58 or SEQ ID NO: 57 by variation / mutation .
  • ketolases of the sequence SEQ ID NO: 68 or 70 and the corresponding coding nucleic acid sequences SEQ ID NO: 67 or SEQ ID NO: 69 which result, for example, from the sequence SEQ ID NO: 60 or SEQ ID NO: 59 by variation / mutation , or
  • 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: 4 and / or 48 and / or 58 and / or 60.
  • ketolases and ketolase genes can furthermore be derived from the nucleic acid sequences described above, in particular from the sequences SEQ ID NO: 3 and / or 47 and / or 57 and / or 59 from different organisms, the genomic sequence of which is not known is easy to find by hybridization techniques in a manner known per se.
  • the hybridization can take place under moderate (low stringency) or preferably under stringent (high stringency) conditions.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with 2X SSC at 50 ° C) and those with high stringency (with 0.2X SSC at 50 ° C, preferably at 65 ° C) (20X SSC: 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).
  • the temperature during the washing step can be raised from moderate conditions at room temperature, 22 ° C, to stringent conditions at 65 ° C.
  • Both parameters, salt concentration and temperature, can be varied 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.
  • nucleic acids are encoded which encode a protein containing the amino acid sequence SEQ ID NO: 4 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which have an identity of at least 70% , preferably at least 80%, more preferably at least 85%, more preferably at least 90%, 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: 4 and the enzymatic property has a ketolase.
  • This can be a natural ketolase sequence, which can be found as described above, by comparing the identity of the sequences from other organisms, or an artificial ketolase sequence which, starting from the sequence SEQ ID NO: 4, can be found by artificial variation, for example by Substitution, insertion or deletion of amino acids has been modified.
  • nucleic acids are encoded which encode a protein containing the amino acid sequence SEQ ID NO: 48 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and which have an identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, 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: 48 and exhibits enzymatic property of a ketolase.
  • This can be a natural ketolase sequence which, as described above, can be found by comparing the identity of the sequences from other organisms or an artificial ketolase sequence which can be derived from the sequence SEQ ID NO: 48 by artificial variation , for example by substitution, insertion or deletion of amino acids.
  • nucleic acids which encode a protein are introduced, containing the amino acid sequence SEQ ID NO: 58 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, 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 of the sequence SEQ ID NO: 58 and has the enzymatic property of a ketolase.
  • This can be a natural ketolase sequence which, as described above, can be found by comparing the identity of the sequences from other organisms or an artificial ketolase sequence which, starting from the sequence SEQ ID NO: 58, can be found by artificial variation, for example was modified by substitution, insertion or deletion of amino acids.
  • nucleic acids which encode a protein are introduced, comprising the amino acid sequence SEQ ID NO: 60 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, 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: 60 and the enzymatic Has property of a ketolase.
  • This can be a natural ketolase sequence which, as described above, can be found by comparing the identity of the sequences from other organisms or an artificial ketolase sequence which, starting from the sequence SEQ ID NO: 60, can be found by artificial variation, for example was modified by substitution, insertion or deletion of amino acids.
  • substitution is understood to mean the replacement of one or more amino acids by one or more amino acids for all proteins. So-called conservative exchanges are preferably carried out, in which the replaced amino acid has a similar property to the original amino acid, for example replacement of Glu by Asp, Gin by Asn, Val by Ile, Leu by Ile, Ser by Thr.
  • Deletion is the replacement of an amino acid with a direct link.
  • Preferred positions for deletions are the termini of the polypeptide and the links between the individual protein domains.
  • Inserts are insertions of amino acids into the polypeptide chain, with a direct bond being formally replaced by one or more amino acids.
  • Identity between two proteins is understood to mean the identity of the amino acids over the respective total protein length, in particular the identity which is obtained by comparison using the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp PM.Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5 (2): 151-1) is calculated using the following parameters:
  • Gap opening penalty 10 Gap extension penalty 10
  • a protein which has an identity of at least 70% at the amino acid level with a specific sequence is accordingly understood to be a protein which, when comparing its sequence with the specific sequence, in particular according to the above program logarithm with the above parameter set, has an identity of at least 70%.
  • a protein which has, for example, an identity of at least 70% at the amino acid level with the sequence SEQ ID NO: 4 or 48 or 58 or 60 is accordingly understood to be a protein which, when its sequence is compared with the sequence SEQ ID NO: 4 or 48 or 58 or 60, in particular according to the above program logarithm with the above parameter set, has an identity of at least 70%.
  • 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: 3 is introduced into the plant.
  • a nucleic acid containing the sequence SEQ ID NO: 48 is introduced into the plant.
  • nucleic acid containing the sequence SEQ ID NO: 58 is introduced into the plant.
  • a nucleic acid containing the sequence SEQ ID NO: 60 is introduced into the plant.
  • ketolase genes can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can 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
  • non-human organisms used in the process according to the invention have an altered ketolase activity and an altered ⁇ -cyclase activity compared to the wild type, the altered ⁇ -cyclase activity being caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • non-human organisms are used as starting organisms which already have a ⁇ -cyclase activity as wild type or starting organism.
  • the genetic modification brings about an increase in the ⁇ -cyclase activity in comparison to the wild type or starting organism, the increased ⁇ -cyclase activity being caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • ⁇ -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.
  • a ⁇ -cyclase is understood to be a protein which has the enzymatic activity to convert ⁇ -carotene into ⁇ -carotene.
  • ß-cyclase activity is understood to mean the amount of ⁇ -carotene converted or the amount of ß-carotene formed in a certain time by the protein ß-cyclase.
  • the amount of lycopene or ⁇ -carotene converted or the amount of ⁇ -carotene formed from lycopene or the formed amount of ß-carotene from ⁇ -carotene increased.
  • This increase in the ⁇ -cyclase activity is preferably at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably 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.
  • ⁇ -cyclase activity in genetically modified organisms according to the invention and in wild-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) / ⁇ vitro. It becomes a certain amount Potassium phosphate as buffer (pH 7.6), lycopene as substrate, paprika stromal protein, NADP +, NADPH and ATP added to organism extract.
  • the ⁇ -cyclase activity is particularly preferably determined under the following conditions according to Bouvier, d'Harlingue and Camara (Molecular Analysis of carotenoid cyclae inhibition; Arch. Biochem. Biophys. 346 (1) (1997) 53-64):
  • the in vitro assay is carried out in a volume of 250 ⁇ l volume.
  • the mixture contains 50 mM potassium phosphate (pH 7.6), different amounts of organism extract, 20 nM lycopene, 250 ⁇ g of chromoplastid stromal protein from paprika, 0.2 M NADP +, 0.2 M NADPH and 1 mM ATP.
  • NADP / NADPH and ATP are dissolved in 10 ml ethanol with 1 mg Tween 80 immediately before adding to the incubation medium. After a reaction time of 60 minutes at 30 ° C., the reaction is terminated by adding chloroform / methanol (2: 1). The reaction products extracted in chloroform are analyzed by HPLC.
  • the ß-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 gene expression compared to the wild type of nucleic acids encoding a ß-cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the increase in the gene expression of the nucleic acids encoding a ⁇ -cyclase compared to the wild type can also be achieved in various ways, for example by inducing the ⁇ -cyclase gene by activators or by introducing one or more ⁇ -cyclase gene copies, ie by introducing at least one nucleic acid encoding a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has in the organism.
  • nucleic acid encoding a ⁇ -cyclase By increasing the gene expression of a nucleic acid encoding a ⁇ -cyclase, the manipulation of the expression of the organism's own endogenous ⁇ -cyclase containing the amino acid sequence SEQ is also carried out according to the invention.
  • an altered or increased expression of an endogenous ⁇ -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 coding for a ⁇ -cyclase is increased by introducing into the organism at least one nucleic acid coding for a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • non-human organisms are used as starting organisms which, as a wild type, have no ⁇ -cyclase activity.
  • the genetic modification causes the ⁇ -cyclase activity in the organisms.
  • the genetically modified organism according to the invention thus has in this, Embodiment compared to the genetically unmodified wild type on a ß-cyclase activity and is therefore preferably able to transgenically express a ß-cyclase.
  • the gene expression of a nucleic acid encoding a ⁇ -cyclase is caused analogously to the above-described increase in gene expression of a nucleic acid encoding a ⁇ -cyclase, preferably by introducing nucleic acids which encode ⁇ -cyclase into the starting organism.
  • each ⁇ -cyclase gene that is to say any nucleic acid which encodes a ⁇ -cyclase, contains 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has to be used.
  • a particularly preferred ⁇ -cyclase is the chromoplast-specific ⁇ -cyclase from tomato (AAG21133) (nucleic acid: SEQ ID No. 1; protein: SEQ ID No. 2).
  • ⁇ -cyclase genes which can be used according to the invention are nucleic acids which encode proteins, 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 which have an identity of at least 70%, preferably at least 80 %, preferably at least 85%, more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 2, and which have the enzymatic property of a ⁇ -cyclase.
  • ⁇ -cyclases and ⁇ -cyclase genes can easily be found, for example, from various organisms whose genomic sequence is known, as described above, by comparing the homology of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SEQ ID NO: 2. Further examples of ⁇ -cyclases and ⁇ -cyclase genes can also be easily found, for example, starting from the sequence SEQ ID NO: 1 from various organisms whose genomic sequence is not known, using hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the ⁇ -cyclase of the sequence SEQ ID NO: 2.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons that are frequently used in accordance with the 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: 1 is introduced into the organism.
  • All of the ⁇ -cyclase genes mentioned above can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the attachment of synthetic oligonucleotides and the filling of gaps using the 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.
  • non-human organisms are cultivated which, compared to the wild type, have an altered hydroxylase activity in addition to the altered ketolase activity and altered ⁇ -cyclase activity.
  • an “hydroxylase activity changed compared to the wild type” is preferably understood to mean “hydroxylase activity caused compared to the wild type”.
  • an “hydroxylase activity changed in comparison to the wild type” is preferably understood to mean an “increased hydroxylase activity in comparison to the wild type”.
  • non-human organisms are cultivated which, compared to the wild type, have a caused or increased hydroxylase activity in addition to the changed ketolase activity and changed ⁇ -cyclase activity.
  • Hydroxylase activity means the enzyme activity of a hydroxylase.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity of introducing a hydroxyl group on the optionally substituted ⁇ -ionone ring of carotenoids.
  • a hydroxylase is understood to mean a protein which has the enzymatic activity to convert ⁇ -carotene into zeaxanthin or canthaxanthin into astaxanthin.
  • hydroxyase activity is understood to mean the amount of ⁇ -carotene or canthaxanthin or the amount of zeaxanthin or astaxanthin formed in a certain time by the protein hydroxylase.
  • the amount of ⁇ -carotene or cantaxantin or the amount of zeaxanthin or astaxanthin formed is increased in a certain time by the protein hydroxylase compared to the wild type.
  • This increase in 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.
  • 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. There is a certain amount of organizing Mus extract, ferredoxin, ferredoxin-NADP oxidoreductase, catalase, NADPH and beta-carotene with mono- and digalactosylglycerides added.
  • 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.
  • the reaction products are extracted with organic solvent such as acetone or chloroform / methanol (2: 1) and determined by means of HPLC.
  • the hydroxylase activity can be increased or caused in various ways, for example by switching off inhibitory regulatory mechanisms at the expression and protein level or by increasing or causing the gene expression of nucleic acids encoding a hydroxylase compared to the wild type.
  • the increase or causation of the gene expression of the nucleic acids encoding a hydroxylase compared to the wild type can also take place in different ways, for example by inducing the hydroxylase gene, by activators or by introducing one or more hydroxylase gene copies, i.e. by introducing at least one nucleic acid encoding one Hydroxylase in the organism.
  • Increasing the gene expression of a nucleic acid encoding a hydroxylase also means manipulating the expression of the organism's own endogenous hydroxylase.
  • a caused or increased expression of an endogenous hydroxylase 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 or caused by introducing at least one nucleic acid encoding a hydroxylase into the organism.
  • any hydroxylase gene that is to say any nucleic acid which codes for a hydroxylase, can be used for this purpose.
  • nucleic acid sequences which have already been processed such as the corresponding cDNAs, are preferred use.
  • hydroxylase gene examples include:
  • nucleic acid encoding a hydroxylase from Haematococcus pluvialis, Accession AX038729, WO 0061764); (Nucleic acid: SEQ ID NO: 77, protein: SEQ ID NO: 78),
  • a particularly preferred hydroxylase is also the hydroxylase from tomato (Accession Y14810) (nucleic acid: SEQ ID NO: 5; protein: SEQ ID NO. 6).
  • the preferred transgenic organisms according to the invention therefore have at least one further hydroxylase 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.
  • the preferred hydroxylase genes used are nucleic acids encoding proteins containing the amino acid sequence SEQ ID NO: 6 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 6, and which have the enzymatic property of a hydroxylase.
  • hydroxylases and hydroxylase 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: 6.
  • hydroxylases and hydroxylase genes can also be easily found, for example, starting from the sequence SEQ ID NO: 5 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 hydroxylase of the sequence SEQ ID NO: 6.
  • 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: 5 is introduced into the organism.
  • All of the above-mentioned hydroxylase genes can also be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the attachment of synthetic oligonucleotides and the filling of gaps using the 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.
  • Genetically modified non-human organisms which have as starting organisms a .beta.-cyclase activity and no ketolase activity are particularly preferably used, the genetically modified organisms having an increased .beta.-cyclase activity, caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 and have caused ketolase activity.
  • Genetically modified non-human organisms which have no ⁇ -cyclase activity and no ketolase activity as starting organisms are particularly preferably used in the process according to the invention, the genetically modified organisms compared to the wild type having a ⁇ -cyclase activity, caused by a ß-cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 and has caused ketolase Have activity.
  • Genetically modified non-human organisms which have a .beta.-cyclase activity and a ketolase activity as starting organisms are particularly preferably used in the process according to the invention, the genetically modified organisms having an increased .beta.-cyclase activity, caused by, in comparison to the wild type a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 and have an increased ketolase activity.
  • Genetically modified non-human organisms are particularly preferably used in the process according to the invention which have as starting organisms a ⁇ -cyclase activity, no ketolase activity and no hydroxylase activity, the genetically modified organisms having an increased ⁇ -cyclase activity compared to the wiid type.
  • Activity caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 have a caused ketolase activity and a caused hydroxylase activity.
  • Genetically modified non-human organisms are particularly preferably used in the process according to the invention which have as starting organisms a ⁇ -cyclase activity, a hydroxylase activity and no ketolase activity, the genetically modified organisms having an increased ⁇ -cyclase activity compared to the wild type.
  • Activity caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has an increased hydroxylase activity and a caused ketolase activity.
  • Genetically modified, non-human organisms which have no ⁇ -cyclase activity, no hydroxylase activity and no ketolase activity as starting organisms are particularly preferably used in the process according to the invention, the genetically modified organisms causing a ⁇ - Cyclase activity caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has a caused hydroxylase activity and a caused ketolase activity te hydroxylase activity and have caused ketolase activity.
  • Genetically modified non-human organisms which have a .beta.-cyclase activity, a hydroxylase activity and a ketolase activity as starting organisms are particularly preferably used in the process according to the invention, the genetically modified organisms having an increased .beta.-cyclase compared to the wild type Activity, caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has an increased ⁇ -cyclase activity, an increased hydroxylase activity and an increased ketolase activity.
  • 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- 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, have crtlSO 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).
  • HMG-CoA reductase is understood to be a protein which has the enzymatic activity to convert 3-hydroxy-3-methyl-glutaryl-coenzyme-A into 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%, 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 HMG-CoA reductase activity.
  • 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, 5mM KHC03. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • HMG-CoA reductase The activity of HMG-CoA reductase can be measured according to published descriptions (e.g. Schaller, Grausem, Benveniste, Chye, Tan, Song and Chua, Plant Physiol. 109 (1995), 761-770; Chappell, Wolf, Proulx, Cuellar and Saunders, Plant Physiol. 109 (1995) 1337-1343).
  • Organism tissue can be homogenized and extracted in cold buffer (100 M 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 dissolved in 100 mM potassium phosphate buffer (pH 7.0 with 3 mM NADPH and 20 ⁇ M ( 4 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
  • the ( 4 C) -evalonate 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.
  • 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.
  • the protein (E) -4-hydroxy-3- Methylbut-2-enyl diphosphate reductase increases the amount of (E) -4-hydroxy-3-methylbut-2-enyl diphosphate converted or the amount of isopentenyl diphosphate and / or dimethylallyldiphosphate formed.
  • 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 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 KHC03. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF added.
  • the (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase activity can be determined by immunological detection.
  • the production of specific antibodies is by Rohdich and colleagues (Rohdich, Hecht, Gärtner, A-dam, Krieger, Amslinger, Arigoni, Bacher and Eisenreich: Studies on the nonmevalonat terpene biosynthetic pathway: metabolic role of IspH (LytB) protein, Natl Acad. Natl. USA 99 (2002), 1158-1163).
  • 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 tracked dimethyl allyl diphosphate.
  • 1-Deoxy-D-xylose-5-phosphate synthase activity means the enzyme activity of a 1-deoxy-D-xylose-5-phosphate synthase activity.
  • 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 converted by the protein 1-deoxy-D-xylose-5-phosphate synthase in a certain time -3-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 formed -deoxy-D-xylose-5-phosphate increased.
  • This increase in the 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.
  • the determination of the 1-deoxy-D-xylose-5-phosphate synthase 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 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 KHC03. 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 ⁇ M (2- 14 C) pyruvate (0.5 ⁇ Ci), 0.6 mM DL-Glyerinaldehyd-3-phosphate.
  • the organism extract is incubated for 1 to 2 hours in the reaction solution at 37C.
  • 1-Deoxy-D-xylose-5-phosphate reductoisomerase activity describes the enzyme activity of a 1-deoxy-D-xylose-5-phosphate reductoisomerase, also called 1-deoxy-D-xylulose-5-phosphate reductoisomerase. Roger that.
  • 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-xyiose-5-phosphate reductoisomerase - activity which is determined in a certain time by the protein 1-deoxy-D-xylose-5-phosphate - Reductoisomerase understood amount of 1-deoxy-D-xylose-5-phosphate or amount of 2-C-methyl-D-erythritol 4-phosphate formed.
  • the protein 1-deoxy-D-xylose-5-phosphate-reductoisomerase in a certain time compared to the wild type converted amount of 1-deoxy-D-xylose-5-phosphate or the amount of 2-C-methyl-D-erythritol 4-phosphate formed increased.
  • This increase in 1-deoxy-D-xylose-5-phosphate is preferably
  • Reductoisomerase 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% 1-deoxy-D-xylose-5- Wild-type phosphate reductoisomerase activity.
  • 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 KHC03. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • D-1-deoxyxylulose-5-phosphate reductoisomerase 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 amount of dimethylallylphosphate formed in a certain time by the protein isopentenyl-diphosphate-D- ⁇ -isomerase.
  • the protein isopentenyl-diphosphate- ⁇ -isomerase increases the amount of isopentenyl-diphosphate or the amount of dimethylallylphosphate formed in a certain time compared to the wild type.
  • This increase in the isopentenyl diphosphate ⁇ -isomerase 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 isopentenyl diphosphate ⁇ isomerase 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 mortaring 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 KHC03. 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. Be. USA 99 (2002), 1092-1097, based on milling cutters, 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 to convert isopentenyl diphosphate and dimethylallyl phosphate to 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 formed is increased by the protein geranyl diphosphate synthase in a certain time compared to the wild type ,
  • 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% the geranyl Wild-type diphosphate synthase activity.
  • the 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 M 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 KHC03. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • the activity of 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).
  • reaction products are dephosphyrylated (according to Koyama, Fuji and Ogura: Enzymatic hydrolysis of polyprenyl pyrophosphats, Methods Enzymol. 110 (1985), 153-155) and analyzed by means of thin layer chromatography and measurement of the incorporated radioactivity (Dogbo, Bardat, Quennemet and Camara: Metabolism of plastid terpenoids: In vitrp inhibition of phytoene synthesis by phenethyl pyrophosphate derivates, FEBS Letters 219 (1987) 211-215).
  • Farnesyl diphosphate synthase activity means the enzyme activity of a farnesyl diphosphate synthase.
  • a famesyl diphosphate synthase is understood to mean a protein which has the enzymatic activity to sequentially convert 2 molecular sopentenyl diphosphate with dimethyl allyl diphosphate and the resulting geranyl diphosphate into farnesyl diphosphate.
  • the amount of dimethylallyl diphosphate and / or isopentenyl diphosphate or the amount formed in a certain time by famesyl diphosphate synthase activity is converted by the protein famesyl diphosphate synthase Farnesyl diphosphate understood.
  • the converted amount of dimethylallyl diphosphate and / or isopentenyl diphosphate or the amount formed is thus in a certain time compared to the wild type by the protein farnesyl diphosphate synthase Farnesyl diphosphate increased.
  • This increase in the famesyl 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 farnesyl diphosphate synthase activity.
  • the determination of the farnesyl diphosphate synthase 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 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 KHC03. Shortly before the extraction, 2 mM DTT and 0.5 M PMSF are added.
  • the activity of franesyl pyrophosphate snthase can be determined according to a protocol by Joly and Edwards (Journal of Biological Chemistry 268 (1993), 26983-26989). The enzyme activity is then measured in a buffer of 10 mM HEPES (pH 7.2), 1 mM MgCl 2 , 1 mM dithiothreitol, 20 ⁇ M geranyl pyrophosphate and 40 ⁇ M (1- 4 C) isopentenyl pyrophosphate (4 Ci / mmol). 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 Famesol).
  • 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.
  • the reaction products can be separated into benzene / methanol (9: 1) by means of thin layer chromatography (silica gel SE60, Merck).
  • Radioactively labeled products are eluted and the radioactivity determined (according to Gaffe, Bru, Causse, Vidal, Stamitti-Bert, Carde and Gallusci: LEFPS1, a tomato farnesyl pyrophosphate gene highly expressed 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 mean a protein which has the enzymatic activity to convert farnesyl diphosphate and isopentenyl diphosphate into geranyl-geranyl diphosphate. Accordingly, 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 buffers 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 KHC03. Shortly before the extraction, 2 mM DTT and 0.5 mM PMSF are added.
  • GGPP synthase Activity measurements of geranylgeranyl pyrophosphate synthase (GGPP 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 is ⁇ Ci, 10 "M), 15 ": M DMAPP, GPP or FPP) with a total volume of about 200 ul of 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, 5 ⁇ m; 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 mean a protein which has the enzymatic activity of converting 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 KHC03. 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
  • the radioactively labeled phytoene formed during the reaction is separated by thin layer chromatography on silica plates in methanol / water (95: 5; v / v).
  • Phytoene 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.
  • phytoene can also be quantified using HPLC, which is equipped with a radioactivity detector (Fräser, Albrecht and Sandmann: Development of high performance liquid chromatography 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 ⁇ -caotin converted 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.
  • the 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 KHC03.
  • PDS phytoene desaturase
  • Radioactive 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 of plastids of the target tissue can be incubated with 100 mM MES buffer (pH 6.0) with 10 mM MgCl 2 and 1 mM dithiothreitol in a total volume of 1 mL.
  • 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 ⁇ -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 00%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the Zeta-carotene desaturase - Wild type activity.
  • 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 KHC03. 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).
  • Each analysis batch contains 3 mg phosphytidylcholine, which is suspended in 0.4 M potassium phosphate buffer (pH 7.8), 5 ⁇ g ⁇ -carotene or neurosporin, 0.02% butylhydroxytoluene, 10 ⁇ l decyl plastoquinone (1 mM methanolic see 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 while 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 ul 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. Accordingly, 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.
  • crtlSO activity is higher than that of the wild type, the amount of 7,9,7 ', 9'-tetra-cis-lycopene converted or the amount of all-trans formed by the crtlSO protein is reduced in a certain time compared to the wild type - Lycopene increased.
  • This increase in crtlSO 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 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 using antibodies and corresponding blotting techniques as standard.
  • 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 famesyl diphosphate Synthase activity and / or geranylgeranyl 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 expression and protein level or by increasing the gene expression of nucleic acids encoding an HMG-CoA reductase and / or nucleic acids encoding an (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate reductase and /
  • a change which is an increased expression rate of the gene can result, for example, by deletion or insertion of DNA sequences.
  • the gene expression of a nucleic acid encoding an HMG-CoA reductase is increased and / or the gene expression of a nucleic acid encoding an (E) -4-hydroxy-3-methylbut-2-enyldiphosphate reductase and / or is increased.
  • 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 famesyl 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 plant has, for example, at least one exogenous nucleic acid encoding an HMG-CoA reductase or at least two endogenous nucleic acids encoding an HMG-CoA reductase and / or at least one exogenous nucleic acid encoding 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 encoding a 1-deoxy-D-xylose-5-phosphate synthase or at least two endogenous Nucleic acids encoding a 1-deoxy-D-xylose-5-phosphate synthase and / or at least one exogenous nucleic acid encoding a 1-deoxy-D-xylose-5-phosphate reductoi
  • 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:
  • 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: 103, protein: SEQ ID NO: 12), as well as further 1-deoxy-D-xylose-5-phosphate synthase genes from other organisms with the following accession numbers:
  • 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: 13, protein: SEQ ID NO: 14),
  • isopentenyl diphosphate ⁇ isomerase genes are:
  • geranyl 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: 19, protein: SEQ ID NO: 112),
  • geranyl-geranyl diphosphate synthase genes are:
  • phytoene synthase genes examples include:
  • phytoene desaturase genes are:
  • phytoene desaturase genes from other organisms with the following accession numbers: AAL15300, A39597, CAA42573, AAK51545, BAB08179, CAA48195, BAB82461, AAK92625, CAA55392, AAG10426, AAD02489, AA024235, AAC12846, AAA99519, AAL38046, CAA60479, CAA75094, ZP_001041, ZP_001163, CAA39004, CAA44452, ZP_001142, ZP_000718, BAB82462, AAM45380, CAB56040, ZP_001091, BAC091 13, AAP79175, AAL80005, AAM72642, AAM72043, ZP_000745, ZP_001141, BAC07889, CAD55814, ZP_001041, CAD27442, CAE00192, ZP_001163, ZP_000197, BAA18400, AAG10425, ZP
  • 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: 119, protein: SEQ ID NO: 28),
  • crtlSO genes are:
  • FtsZ genes are:
  • MinD genes are: A nucleic acid encoding a MinD from Tagetes erecta, ACCESSION # AF251019, published by Moehs.CP, Tian.L, Osteryoung.KW and Dellapena, D .: Analysis of carotenoid biosynthetic gene expression during marigold petal development; Plant Mol. Biol. 45 (3), 281-293 (2001), (nucleic acid: SEQ ID NO: 33, protein: SEQ ID NO: 34),
  • nucleic acids which encode proteins are preferably used as HMG-CoA reductase genes, comprising the amino acid sequence SEQ ID NO: 8 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: 8, and which have the enzymatic property of an HMG-CoA reductase.
  • HMG-CoA reductases and HMG-CoA reductase genes can be found, for example, from different organisms, their genomic sequence is known, as described above, by homology comparisons of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SeQ ID NO: 8.
  • HMG-CoA reductases and HMG-CoA reductase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 7 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 which encode proteins containing the amino acid sequence of the HMG-CoA reductase of the sequence SEQ ID NO: 8 are introduced into organisms to increase the HMG-CoA reductase activity.
  • 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: 7 is introduced into the organism.
  • (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: 9 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 code for proteins containing the amino acid sequence of (E) - 4- Hydroxy-3-methylbut-2-enyl-diphosphate reductase of the sequence SEQ ID NO: 10.
  • 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: 9 is introduced into the organism.
  • nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 12 or one of these sequences by substitution, insertion or deletion 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: 12, and which has the enzymatic property a (1-deoxy-D-xylose-5-phosphate synthase.
  • (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 , easily by homology comparisons of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SeQ ID NO: 12 find.
  • (1-deoxy-D-xylose-5-phosphate synthase 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: 11 whose genomic sequence is not known, as described above, can easily be found by hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the (1-deoxy-D-xylose-5 Phosphate synthase of sequence SEQ ID NO: 12.
  • 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: 11 is introduced into the organism.
  • 1-deoxy-D-xylose-5-phosphate reductoisomerase genes are preferably used nucleic acids which encode proteins containing the amino acid sequence SEQ ID NO: 14 or one of these sequences by substitution, insertion or deletion of 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: 14, 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 Homology comparisons of the amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SeQ ID NO: 14 easy to find.
  • 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: 13 from different organisms, their genomic Sequence is not known, as described above, can be easily found by hybridization and PCR techniques in a manner known per se.
  • nucleic acids are introduced into organisms which code for proteins containing the amino acid sequence of the 1-deoxy-D-xylose-5-phosphate Reductoisomerase of sequence SEQ ID NO: 14.
  • 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: 13 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: 16 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and having an identity of at least 30%, preferably at least 50%, more preferably at least 70%, more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 16, and which have the enzymatic property of an isopentenyl-D-isomerase.
  • 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: 16 easy to find.
  • Further examples of isopentenyl-D-isomerases and isopentenyl-D-isomerase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 15 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: 16.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons which are frequently used in accordance with the organism-specific codon usage are preferably used for this.
  • 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: 15 is introduced into the organism.
  • the geranyl diphosphate synthase genes used are preferably nucleic acids which encode proteins, the amino acid sequence SEQ ID NO: 18 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: 18, and which has the enzymatic property of a geranyl diphosphate Have 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 amino acid sequences or the corresponding back-translated nucleic acid sequences from databases with the SeQ ID NO: 18 easy to find.
  • geranyl diphosphate synthases and geranyl diphosphate synthase genes can also be derived, for example, starting from the sequence SEQ ID NO: 17 from different organisms whose genomic sequence cannot be determined. is, as described above, easy to find 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 geranyl diphosphate synthase of the sequence SEQ ID NO: 18.
  • Suitable nucleic acid sequences can be obtained, for example, by back-translating the polypeptide sequence in accordance with the genetic code.
  • codons which are frequently used in accordance with the organism-specific codon usage are preferably used for this.
  • 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: 17 is introduced into the organism.
  • nucleic acids which encode proteins, containing the amino acid sequence SEQ ID NO: 20 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: 20, and which have the enzymatic property of a farnesyl diphosphate synthase ,
  • famesyl diphosphate synthases and famesyl 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: 20 easy to find.
  • farnesyl diphosphate synthases and famesyl diphosphate synthase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 19 from various organisms whose genomic sequence is not known, as described above, by means of hybridization and PCR techniques easy to find in a manner known per se.
  • nucleic acids which encode proteins containing the amino acid sequence of the farnesyl diphosphate synthase of the sequence SEQ ID NO: 20 are introduced into organisms in order to increase the famesyl diphosphate synthase activity.
  • 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: 19 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: 22 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%, even more preferably at least 90%, most preferably at least 95% at the amino acid level with the sequence SEQ ID NO: 22, 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: 21 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 code for proteins containing the amino acid sequence of the geranyl-geranyl- Diphosphate synthase 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.
  • nucleic acids encoding proteins are preferably used as phytoene synthase genes, containing the amino acid sequence SEQ ID NO: 24 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: 24, 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: 24.
  • phytoene synthases and phytoene synthase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 23 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: 24. Suitable nucleic acid sequences are, for example, by back-translating the
  • 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: 23 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: 26 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: 26, 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: 26.
  • phytoene desaturases and phytoene desaturase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 25 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: 26 are introduced into organisms in order 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: 25 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: 28 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: 28, 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: 28 easy to find.
  • zeta-carotene desaturases and zeta-carotene desaturase genes can also be obtained, for example, starting from the sequence SEQ ID NO: 119 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 encode proteins containing the amino acid sequence of the zeta-carotene desaturase 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.
  • 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: 119 is introduced into the organism.
  • nucleic acids which encode proteins are preferably used as CrtlSO genes, comprising the amino acid sequence SEQ ID NO: 30 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: 30, 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: 30.
  • CrtlSO and CrtlSO genes can also be easily found, 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 techniques in a manner known per se.
  • nucleic acids which encode proteins containing the amino acid sequence of the CrtlSO of the sequence SEQ ID NO: 30 are introduced into organisms to increase the CrtlSO 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 SEQJD NO: 29 is introduced into the organism.
  • the FtsZ 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 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: 32, 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: 32.
  • FtsZn and FtsZ genes can also be easily found, 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 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: 32
  • 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: 31 is introduced into the organism.
  • the preferred MinD genes are nucleic acids encoding 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 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: 34, and which have the enzymatic property of a MinD.
  • MinDn and MinD 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: 34.
  • MinDn and MinD genes can also easily 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 in a manner known per se find.
  • nucleic acids are introduced into organisms which encode proteins containing the amino acid sequence of the MinD 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.
  • 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.
  • 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 gap filling 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.
  • nucleic acids encoding a ketolase containing the amino acid sequence SEQ. ID. NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 70% at the amino acid level with the sequence SEQ. ID. NO.
  • 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 encoding a phytoene desaturase, nucleic acids encoding a zeta-carotene
  • 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 of the effect genes or more than three effect genes
  • 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.
  • 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, for example, bacteria of the genus Escherichia, which contain, for example, crt genes from Erwinia, as well Bacteria that are able to synthesize xanthophylls on their own, such as, for example, bacteria of the genus Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc or cyanobacteria of the genus Synechocystis.
  • Preferred bacteria are Escherichia coli, Erwinia herbicola, Erwinia uredovora, Agrobacterium aurantiacum, Alcaligenes sp. PC-1, Flavobacterium sp. strain R1534, the Cyanobacterium Synechocystis sp. PCC6803, Paracoccus marcusii or Paracoccus carotinifaciens.
  • yeasts are Candida, Saccharomyces, Hansenula, Pichia or Phaffia. Particularly preferred yeasts are Xanthophyllomyces dendrorhous or Phaffia rhodozyma.
  • Preferred 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 plants selected from the families Amaranthaceae, Amaryllidaceae, Apocynaceae ceae, Asteraceae, Balsaminaceae, Begonia-, Berberidaceae, Brassicaceae.Cannabaceae, Caprifoliaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Cruciferae, Euphorbiaceae, Fabaceae, Gentianaceae, Geraniaceae , Graminae, llliaceae, Labiatae, Lamiaceae, Leguminosae, Liliaceae, Linaceae, Lobeliaceae, Malvaceae, Oleaceae, Orchidaceae, Papaveraceae, Plumbaginaceae, Poaceae, Polemoniaceae, Primulacaceae, Roseaeaeae, Rosunceae ceae, Vitaceae and Violacea
  • 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
  • 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., for example 20 ° C. to 40 ° C., and a pH of about 6 to 9.
  • a cultivation temperature of at least about 20 ° C., for example 20 ° C. to 40 ° C., and a pH of about 6 to 9.
  • genetically modified microorganisms preferably first culturing the microorganisms in the presence of oxygen and in a complex medium, such as, for example, 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 is.
  • a complex medium such as, for example, TB or LB medium
  • an inducible promoter is preferred.
  • the cultivation is carried out after induction of ketolase expression in presence of oxygen, for example 12 hours to 3 days.
  • ketocarotenoids are isolated from the harvested biomass in a manner known per se, for example by extraction and, if appropriate, further chemical or physical purification processes, such as, for example, precipitation methods, crystallography, thermal separation processes, such as rectification processes or physical separation processes, such as 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 by 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 ketocarotenoids are obtained in the process according to the invention in the form of their mono- or diesters with fatty acids.
  • Some detected fatty acids are, for example, 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 are also called petals.
  • genetically modified plants are used which have the highest expression rate of a ketolase in flowers.
  • the gene expression of the ketolase takes place under the control of a flower-specific promoter.
  • the nucleic acids described above, as described in detail below are introduced into the plant in a functionally linked manner with a flower-specific promoter in a nucleic acid construct.
  • 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.
  • Cyclase activity described wherein the altered ß-cyclase activity is caused by a ß-cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • Increasing other activities such as hydroxylase activity, 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, famesyl diphosphate 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 used analogously corresponding effects occur.
  • the transformation can take place individually or through multiple constructs.
  • the transgenic plants are preferably produced by transforming the starting plants, using a nucleic acid construct which contains the nucleic acids described above, coding for a ketolase and coding for a .beta.-cyclase, which are functionally linked to one or more regulation signals which relate to transcription and translation in plants ensure, wherein the nucleic acid encodes a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the transgenic plants are preferably produced by transforming the starting plants using two nucleic acid constructs.
  • a nucleic acid construct contains at least one nucleic acid described above, encoding a ketolase, which is functionally linked to one or more regulatory signals which ensure transcription and translation in plants.
  • the second nucleic acid construct contains at least one nucleic acid described above, encoding a ⁇ -cyclase, which are functionally linked to one or more regulatory signals which ensure transcription and translation in plants, the nucleic acid encoding a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has ..
  • 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 regulatory nucleic acid sequences which control the expression of the effect genes in the host cell.
  • an expression cassette comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and optionally further regulatory elements which are operatively linked to the coding sequence of the effect gene in between 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.
  • nucleic acid constructs, expression cassettes and vectors for plants and methods for producing transgenic plants and the transgenic plants themselves are described below by way of example.
  • sequences which are preferred, but not limited to, for operative linking are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil bodies or other compartments and translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).
  • any promoter which can control the expression of foreign genes in plants is suitable as the promoter of the expression cassette.
  • Constuent promoter means those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development.
  • a plant promoter or a plant virus-derived promoter is preferably used.
  • Particularly preferred is the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228), the 19S CaMV promoter (US 5,352,605; WO 84/02913; Benfey et al.
  • TPT triose-phosphate translocator
  • Another suitable constitutive promoter is the pds promoter (Pecker et al. (1992) Proc. Natl. Acad. Be USA 89: 4962-4966) or the "Rubisco small subunit (SSU)" promoter (US 4,962,028), the LeguminB Promoter (GenBank Acc. No. X03677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), the Ubiquitin 1 promoter (Christensen et al.
  • the expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the effect genes in the plant can be controlled at a specific point in time.
  • a chemically inducible promoter such as the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), a promoter induced by salicylic acid (WO 95/19443), a promoter induced by benzenesulfonamide (EP 0 388 186) , a tetracycline-inducible promoter (Gatz et al.
  • an abscisic acid-inducible promoter (EP 0 335 528) or an ethanol- or cyclohexanone-inducible promoter (WO 93 / 21334) can also be used.
  • promoters that are induced by biotic or abiotic stress such as the pathogen-inducible promoter of the PRP1 gene (Ward et al.
  • 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
  • wound inducible promoters such as that of the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wunl and wun2 genes ( US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin gene (McGurl et al. (1992) Science 225: 1570-1573), des WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Ekeikamp et al. (1993) FEBS Letters 323: 73-76), the MPI gene (Corderok et al. (1994) The Plant J 6 (2): 141-150) and the like.
  • suitable promoters are, for example, fruit-ripening-specific promoters, such as the fruit-ripening-specific promoter from tomato (WO 94/21794, EP 409 625).
  • Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.
  • promoters are particularly preferred which ensure expression in tissues or parts of plants in which, for example, the biosynthesis of ketocarotenoids or their precursors takes place.
  • promoters with specificities for the anthers, ovaries, petals, sepals, flowers, leaves, stems, seeds and roots and combinations thereof are preferred.
  • Tuber-, storage-root or root-specific promoters are, for example, the patatin promoter class I (B33) or the promoter of the cathepsin D inhibitor from cardiac toffel.
  • Leaf-specific promoters are, for example, the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al. ( 1989) EMBO J 8: 2445-2451).
  • Flower-specific promoters are, for example, the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593), the AP3 promoter from Arabidopsis thaliana, 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.
  • 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-
  • Seed-specific promoters are, for example, the ACP05 promoter (acyl carrier protein gene, W09218634), 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 corn promoters End1 and End2 (WO 0011177).
  • ACP05 promoter acyl carrier protein gene, W09218634
  • 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
  • SBP promoter from Vicia faba
  • An expression cassette is preferably produced by fusing a suitable promoter with at least one of the effects described above.
  • ne 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, as described, for example, in T. Maniatis, EF Fritsch and J.
  • nucleic acids encoding a plastid transit peptide ensure localization in plastids and in particular in chromoplasts.
  • Expression cassettes 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 are split off enzymatically from the effect gene product part after translocation of the effect genes 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 examples include the transit peptide of 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 Brooks, L, Mullineaux, P (1988) An expression casette for targeting foreign proteins into the chloroplasts. Nucl. Acids Res. 16: 11380).
  • IPP-2 plastid isopentenyl pyrophosphate isomerase-2
  • rbcS ribulose bisphosphate carboxylase
  • nucleic acids according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural nucleic acid constituents, and can consist of different heterologous gene segments from different organisms.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp within the regulatory areas, often less than 60 bp, but at least 5 bp.
  • the promoter can be native or homologous as well as foreign or heterologous to the host plant.
  • 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: transeript mapping and DNA sequence. J Mol Appl Genet.
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as Transitions and transversions can be used in w ⁇ ro mutagenesis, "primer repair", restriction or ligation.
  • 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 pTiACH ⁇ (Gielen et al., EMBO J. 3 ( 1984), 835 ff) or functional equivalents.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • Methods known per se for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation can be used for this purpose.
  • 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
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711) or particularly preferably pSUN2, pSUN3, pSUN4 or pSUN5 (WO 02/00900).
  • Agrobacteria transformed with an expression plasmid can be used in a known manner to transform plants, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the fused expression cassette 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.
  • the expression cassettes can be cloned into suitable vectors which allow 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 and increased or caused ⁇ -cyclase activity is described in more detail, the changed ⁇ -cyclase activity being caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • 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 / or Min D activity can be carried out analogously using the corresponding effect genes.
  • nucleic acids described above encoding a ketolase, ⁇ -hydroxylase or ⁇ -cyclase
  • nucleic acids encoding an HMG-CoA reductase nucleic acids encoding an (E) -4-hydroxy-3-methylbut-2-enyl-diphosphate
  • 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 one Geranyl diphosphate synthase, nucleic acids encoding a farnesyl diphosphate synthase, nucleic acids encoding a geranyl geranyl diphosphate synthase, nucleic acids encoding a phytoene synthase, encoding nucleic acids linseed acids encoding a phytoene synthase, nucleic acids encoding a phytoene desaturase, nucleic acids encoding a zeta-carotene desaturase, nucleic acids encoding a crtlSO protein, nucleic acids encoding an isopenteny
  • 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 and translation enhancers, enhancers, polyadenylation signals and the like.
  • 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 SP02 or the yeast promoters ADC1, MFa, AC, P-60, CYC1, GAPDH.
  • inducible promoters such as, for example, light-inducible and in particular temperature-inducible, is particularly preferred Promoters, such as the P r P r promoter.
  • 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.
  • 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 are also known to all those skilled in the art.
  • vectors such as phages, viruses such as SV40, CMV, baculovirus and adevirus, transposons, IS elements, phasmids, cosmids, and linear or circular 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
  • 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
  • Vectors and methods of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol .. 3: 2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • recombinant constructs according to the invention described above are advantageously introduced and expressed in a suitable host system.
  • Common cloning and transfection methods known to the person skilled in the art such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used to bring the nucleic acids mentioned into expression in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997.
  • marker genes which are also contained in the vector or in the expression cassette.
  • marker genes are genes for antibiotic resistance and for enzymes which catalyze a coloring reaction which stains the transformed cell. These can then be selected using automatic cell sorting.
  • Microorganisms successfully transformed with a vector and carrying an appropriate antibiotic resistance gene can be selected using appropriate antibiotic-containing media or nutrient media.
  • Marker proteins that are presented on the cell surface can be used for selection by means of affinity chromatography.
  • the invention further relates to the genetically modified, non-human organisms, the genetic modification being the activity of a ketolase
  • ⁇ -cyclase activity increased after C or caused after D is caused by a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the ketolase activity is increased (according to A) or caused (according to B) compared to the wild type, preferably by increasing the gene expression of a nucleic acid encoding a ketolase.
  • the gene expression of a nucleic acid encoding a ketolase is increased by introducing nucleic acids encoding ketolases into the organism.
  • the transgenic organisms according to the invention therefore have at least one further ketolase gene compared to the wild type.
  • any ketolase gene that is to say any nucleic acids encoding a ketolase, can be used for this purpose.
  • Preferred nucleic acids encoding a ketolase are described above in the method according to the invention.
  • the ⁇ -cyclase activity is preferably increased or caused, as described above, by increasing the gene expression compared to the wild type of nucleic acids, coding for a ⁇ -cyclase 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 70% Amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the gene expression of a nucleic acid coding for a ⁇ -cyclase is increased by introducing into the organism at least one nucleic acid coding for a ⁇ -cyclase 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has.
  • the transgenic organisms according to the invention therefore have at least one further ⁇ -cyclase gene compared to the wild type.
  • any ⁇ -cyclase gene that is to say any nucleic acid encoding a ⁇ -cyclase, 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 70% at the amino acid level with the sequence SEQ. ID. NO. 2 has to be used.
  • genetically modified organisms additionally have an increased or caused hydroxlase activity compared to the wild-type organism. 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.
  • HMG-CoA reductase activity selected from the group HMG-CoA reductase activity
  • E 4-hydroxy-3
  • organisms are preferably understood to mean organisms which, as wild-type or starting organisms, naturally or by genetic complementation and / or reorganization of the metabolic pathways, are capable of producing carotenoids, in particular ⁇ -carotene and / or zeaxanthin and / or neoxanthine and / or violaxanthin and / or to produce lutein.
  • Further preferred organisms already have hydroxylase activity as wild-type or starting organisms and are therefore capable of producing zeaxanthin as wild-type or starting organisms.
  • Preferred organisms are plants or microorganisms, such as bacteria, yeasts, algae or fungi.
  • Both bacteria can be used as bacteria that are able to synthesize xanthophylls due to the introduction of genes of the carotenoid biosynthesis of a carotenoid-producing organism, such as bacteria of the genus Escherichia, which contain, for example, crt genes from Erwinia, as well as bacteria. which are capable of synthesizing xanthophylls, such as, for example, bacteria of the genus Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc or cyanobacteria of the genus Synechocystis.
  • Preferred bacteria are Escherichia coli, Erwinia herbicola, Erwinia uredovora, Agrobacterium aurantiacum, Alcaligenes sp. PC-1, Flavobacterium sp. strain R1534, the Cyanobacterium Synechocystis sp. PCC6803, Paracoccus marcusii or Paracoccus carotinifaciens.
  • yeasts are Candida, Saccharomyces, Hansenula, Pichia or Phaffia. Particularly preferred yeasts are Xanthophyllomyces dendrorhous or Phaffia rhodozyma.
  • Preferred 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 tricomatum, 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.
  • Particularly preferred plants are plants selected from the families amateur ranthaceae.Amaryllidaceae, Apocynaceae, Asteraceae, Balsaminaceae Begoniaceae, Berberidaceae, Brassicaceae.Cannabaceae, Caprifoliaceae, Caryophyllaceae, Chemischen nopodiaceae, Compositae, Cucurbitaceae, Cruciferae, Euphorbiaceae, Fabaceae, Gentianaceae, Geraniaceae , Graminae, liliaceae, 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 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, Forsythie, Fremontia, Gazania, Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillaea, Helenium, Helianthus, Hepatica , Heracleum, Hisbiscus, Heliopsis, Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kenya, Laburnum, Lathyrus, Leontodon, Lilium
  • 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, whereby the genetically modified plant contains at least one transgenic nucleic acid encoding a ketolase.
  • transgenic plants, their reproductive material and their plant cells, tissue or parts, in particular their fruits, seeds, flowers and petals are another object of the present invention.
  • 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 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, a changed content of the preferred ketocarotenoids, without the total carotenoid content necessarily having to be increased.
  • the genetically modified plants according to the invention have an increased astaxanthin content compared to the wild type.
  • an increased content is also understood to mean a caused content of ketocarotenoids or astaxanthin.
  • the sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from Licor (distributed by MWG Biotech, Ebersbach) according to the Sanger's method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
  • the DNA required for the NOST ketolase from Nostoc sp. PCC 7120 coded was by means of PCR from Nostoc sp. PCC 7120 (strain of the "Pasteur Culture Collection of Cyanobacterium”) amplified.
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8,000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (2 ml volume). After adding 100 ⁇ l Proteinase K (concentration: 20 mg / ml), the cell suspension was incubated for 3 hours at 37 ° C. The suspension was then extracted with 500 ⁇ l of phenol. After centrifugation at 13,000 rpm for ⁇ minutes, the upper, aqueous phase was transferred to a new 2 ml Eppendorf reaction vessel.
  • the extraction with phenol was repeated 3 times.
  • the DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol.
  • the DNA pellet was dried at room temperature, taken up in 25 ⁇ l of water and dissolved with heating to 65 ° C.
  • the nucleic acid encoding a ketolase from Nostoc PCC 7120 was determined by means of a "polymerase chain reaction” (PCR) from Nostoc sp. PCC 7120 using a sense-specific primer (NOSTF, SEQ ID No. 79) and an antisense specific primers (NOSTG SEQ ID No. 80).
  • 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. 79 and SEQ ID No. 80 resulted in an 805 bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 81).
  • the amplificate was cloned into the PCR cloning vector pGEM-T (Promega) and the clone pNOSTF-G was obtained.
  • This clone pNOSTF-G was therefore used for the cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • the cloning was carried out by isolating the 799 bp Sphl fragment from pNOSTF-G and ligating into the SphI-cut vector pJIT117.
  • the clone that is the ketolase from Nostoc sp. PCC 7120, in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide, is called pJNOST.
  • Example 2 Construction of the plasmid pMCL-CrtYlBZ idi / gps for the synthesis of zeaxanthin in E. coli
  • PMCL-CrtYlBZ / idi / gps was constructed in three steps using the intermediate stages pMCL-CrtYlBZ and pMCL-CrtYlBZ / idi.
  • the plasmid pMCL200 compatible with high-copy-number vectors was used as the vector (Nakano, Y., Yoshida, Y., Yamashita, Y. and Koga, T .; Construction of a series of pACYC-derived plasmid vectors; Gene 162 ( 1995), 157-158).
  • Example 2.1 Construction of pMCL-CrtYlBZ
  • the biosynthetic genes crtY, crtB, crtl and crtZ come from the bacterium Erwinia uredovora and were amplified by PCR.
  • Erwinia uredovora genomic DNA (DSM 30080) was prepared by the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig) as part of a service.
  • the PCR reaction was carried out according to the manufacturer's instructions (Röche, Long Template PCR: Procedure for amplification of 5-20 kb targets with the expand long template PCR system).
  • the PCR conditions for the amplification of the Erwinia uredovora biosynthesis cluster were as follows:
  • PCR amplification with SEQ ID No. 82 and SEQ ID No. 83 resulted in a fragment (SEQ ID NO: 84) which is responsible for the genes CrtY (protein: SEQ ID NO: 85), CrtI (protein: SEQ ID NO: 86), crtB (protein: SEQ ID NO: 87) 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 Hindill, the resulting Sall / Hindlll fragment isolated and transferred by ligation into the Sall / Hindlll cut vector pMCL200.
  • the Sall / Hindlll fragment from pCR2.1-CrtYIBZ cloned in pMCL 200 is 4624 bp long, codes for the genes CrtY, CrtI, crtB and CrtZ and corresponds to the sequence from positions 2295 to 6918 in D90087 (SEQ ID No. 84).
  • the gene CrtZ is transcribed against the reading direction of the genes CrtY, CrtI and CrtB by means of its endogenous promoter.
  • the resulting clone is called pMCL-CrtYlBZ.
  • Example 2.2 Construction of pMCL-CrtYlBZ / idi
  • the gene ⁇ (isopentenyl diphosphate isomerase; IPP isomerase) was amplified from E. coli by means of PCR.
  • the nucleic acid encoding the entire idi gene with idi promoter and ribosome binding site was extracted from E. coli by means of "polymerase chain reaction” (PCR) using a sense-specific primer (5'-idi SEQ ID No. 88) and an antisense-specific primer (3'-idi SEQ ID No. 89) was amplified.
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 88 and SEQ ID No. 89 resulted in a 679 bp fragment coding for a protein consisting of the entire primary sequence (SEQ ID No. 90).
  • 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 / oY gene in the vector pMCL-CrtYlBZ.
  • the cloning was carried out by isolating the Xhol / Sall fragment from pCR2.1-idi and ligating into the Xhol / Sall cut vector pMCL-CrtYlBZ.
  • the resulting clone is called pMCL-CrtYlBZ / idi.
  • Example 2.3 Construction of pMCL-CrtYlBZ / idi / gps
  • the gene gps (geranylgeranyl pyrophosphate synthase; GGPP synthase) was amplified from Archaeoglobus fulgidus by means of PCR.
  • the DNA of Archaeoglobus fulgidus was prepared by the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig) as part of a service.
  • the PCR conditions were as follows: The PCR for the amplification of the DNA, which codes for a GGPP synthase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • Sequencing of the clone pCB-gps confirmed a sequence for the GGPP synthase from A. fulgidus, which differs from the published sequence AF120272 in one nucleotide.
  • the second codon of the GGPP synthase was changed by inserting an Ncol site in the grps gene.
  • CTG (position 4-6) codes for leucine.
  • SEQ ID No. 92 and SEQ ID No. In 93 this second codon was changed to GTG, which codes for valine.
  • the clone pCB-gps was therefore used for the cloning of the gps gene into the vector pMCL-CrtYlBZ / idi.
  • the cloning was carried out by isolating the Kpnl / Xhol fragment from pCB-gps and ligation into the Kpnl and Xhol cut vector pMCL-CrtYlBZ / idi.
  • the cloned Kpnl / Xhol fragment (SEQ ID No.
  • GGPP synthase carries the Prm16 promoter together with a minimal 5 'UTR sequence of rbcL, the first 6 codons of rbcL, which extend the GGPP synthase N-terminally and 3 'from the gps gene the psbA sequence.
  • the N-terminus of the GGPP synthase thus has the changed amino acid sequence Met-Thr-Pro-Gln-Thr-Ala-Met instead of the natural amino acid sequence with Met-Leu-Lys-Glu (amino acid 1 to 4 from AF120272) -Val-Lys- Glu.
  • the recombinant GGPP synthase starting with Lys in position 3 (in AF120272), is identical and has no further changes in the amino acid sequence.
  • the rbcL and psbA sequences were based on a reference according to EibI et al. (Plant J. 19. (1999), 1-13).
  • the resulting clone is called pMCL-CrtYlBZ / idi / gps.
  • the plasmid pMCL-CrtYlBZ idi / gps was constructed to produce E. co // strains which enable the synthesis of zeaxanthin in high concentration.
  • the plasmid carries the genes crtY, crtB, crtl and crtY from Erwinia uredovora, the gene gps (for geranylgeranyl pyrophoshate synthastase) from Archaeoglobus fulgidus and the gene idi (isopentenyl diphosphate isomerase) from E. coli. Limiting steps for a high accumulation of carotenoids and their biosynthetic precursors were eliminated with this construct. This was previously reported by Wang et al.
  • E. coli TOP10 Cultures of E. coli TOP10 were transformed in a manner known per se with the two plasmids pNOSTF-G and pMCL-CrtYlBZ / idi / gps and cultured in LB medium at 30 ° C. and 37 ° C. overnight. Ampicillin (50 ⁇ g / ml), chloramphenicol (50 ⁇ g / ml) and isopropyl- ⁇ -thiogalactoside (1 mmol) were also added overnight in a conventional manner.
  • the cells were extracted with acetone, the organic solvent was evaporated to dryness and the Ca Rotinoids separated by HPLC on a C30 column. The following process conditions were set.
  • Detection 300 - 500 nm
  • the spectra were determined directly from the elution peaks using a photodiode array detector.
  • the isolated substances were identified by their absorption spectra and their retention times in comparison to standard samples.
  • an E.co// strain which contains a ketolase from Haematococcus pluvialis Flotow em. Will expressed.
  • the cDNA which is responsible for the entire primary sequence of the ketolase from Haematococcus pluvialis Flotow em. Will is coded amplified and cloned into the same expression vector according to Example 1.
  • the cDNA coding for the ketolase from Haematococcus pluvialis was amplified by means of PCR from a Haematococcus pluvialis (strain 192.80 from the "Collection of algal cultures of the University of Göttingen") suspension culture.
  • RNA For the preparation of total RNA from a suspension culture of Haematococcus pluvialis (strain 192.80), which was exposed to indirect daylight at room temperature in Haematococ cus medium (1.2 g / l sodium acetate, 2 g / l yeast extract, 0.2 g / l MgCI2x6H20, 0.02 CaCI2x2H20; pH 6.8; after autoclaving, 400 mg / l L-asparagine, 10 mg / l FeS04xH20) had been grown the cells are harvested, frozen in liquid nitrogen and pulverized in a mortar.
  • Haematococ cus medium 1.2 g / l sodium acetate, 2 g / l yeast extract, 0.2 g / l MgCI2x6H20, 0.02 CaCI2x2H20; pH 6.8; after autoclaving, 400 mg / l L-asparagine, 10 mg / l FeS04
  • RNA For the cDNA synthesis, 2.5 ⁇ g of total RNA were denatured for 10 min at 60 ° C., cooled on ice for 2 min and using a cDNA kit (ready-to-go-you-prime beads, Pharmacia Biotech) according to the manufacturer's instructions rewritten into cDNA using an antisense specific primer PR1 (gcaagctcga cagctacaaa cc).
  • the nucleic acid encoding a kematolase from Haematococcus pluvialis was amplified by means of a polymerase chain reaction (PCR) from Haematococcus pluvialis using a sense-specific primer PR2 (gaagcatgca gctagcagcg acag) and an antisense-specific primer PR1.
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the cDNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ml reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the PCR amplification with PR1 and PR2 resulted in a 1155 bp fragment consisting encodes a protein consisting of the entire primary sequence: gaagcatgca gctagcagcg acagtaatgt tggagcagct taccggaagc gctgaggcac 60 tcaaggagaa ggagaaggag gttgcaggca gctctgacgt gtgtaca tgggcgaccc 120 agtactcgct tccgtcagag gagtcagacg cggccccc gggactgaag aatgcctaca 180 agccaccaccacc ttccgacaca aagggcatca caatggcgct agctcatc ggctctggg 240 ccgcagtgttt c
  • the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) and the clone pGKET02 was obtained.
  • This clone was used for the expression of Haematococcus pluvialis ketolase.
  • the transformation of the E. coli strains, their cultivation and the analysis of the carotenoid profile were carried out as described in Example 3.
  • Table 1 shows a comparison of the bacterially produced amounts of carotenoids:
  • Table 1 Comparison of the bacterial ketocarotenoid synthesis when using two different ketolases, the NOST ketolase from Nostoc sp. PCC7120 (Example 1) and the Haematococcus pluvialis ketolase (Example 4). Amounts of carotenoids are in ng / ml culture fluid.
  • Example 5 Amplification of a DNA encoding the entire primary sequence of the NP196 ketolase from Nostoc punctiforme ATCC 29133
  • the DNA which codes for the NP196 ketolase from Nostoc punctiform ATCC 29133 was amplified by means of PCR from Nostoc punctiform ATCC 29133 (strain of the "American Type Culture Collection").
  • the bacterial cells were pelleted from a 10 ml liquid culture by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred into an Eppendorf reaction vessel (2 ml volume). After adding 100 QCI 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 13000 rpm for 5 minutes, the upper, aqueous phase was transferred to a new 2 ml Eppendorf reaction vessel.
  • the extraction with phenol was repeated 3 times.
  • the DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol.
  • the DNA pellet was temperature dried, in 25 ⁇ l
  • the nucleic acid encoding a ketolase from Nostoc punctiform ATCC 29133 was synthesized by means of a "polymerase chain reaction” (PCR) from Nostoc punctiform ATCC 29133 using a sense-specific primer (NP196-1, SEQ ID No. 100) and an antisense -specific primer (NP196-2 SEQ ID No. 101).
  • 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. 100 and SEQ ID No. 101 resulted in a 792 bp fragment that codes for a protein consisting of the entire primary sequence (NP196, SEQ ID No. 102).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNP196 was obtained.
  • This clone pNP196 was therefore used for the cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
  • pJIT117 was modified by the 35S terminator using the OCS terminator (octopine synthase) of the Ti plasmid pTi15955 from Agrobacterium tumefaciens (database entry X00493 from position 12.541-12.350, Gielen et al. (1984) EMBO J. 3 835-846) was replaced.
  • OCS terminator octopine synthase
  • the DNA fragment containing the OCS terminator region was PCR-isolated using the plasmid pHELLSGATE (database entry AJ311874, Wesley et al. (2001) Plant J. 27 581-590, isolated from E. coli by standard methods) and the primer OCS-1 (SEQ ID No. 133) and OCS-2 (SEQ ID No. 134).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the octopine synthase (OCS) terminator region (SEQ ID No. 106), was carried out in a 50 ⁇ l reaction mixture, which contained:
  • the PCR was carried out under the following cycle conditions:
  • Sequencing of the clone pOCS confirmed a sequence which corresponds to a sequence section on the Ti plasmid pTi 15955 from Agrobacterium tumefaciens (database entry X00493) from positions 12,541 to 12,350.
  • the cloning was carried out by isolating the 210 bp Sall-Xhol fragment from pOCS and ligation into the Sall-Xhol cut vector pJIT117.
  • This clone is called pJO and was therefore used for the cloning into the expression vector pJONPI 96.
  • the cloning was carried out by isolating the 782 bp Sphl fragment from pNP196 and ligating into the SphI cut vector pJO.
  • the clone that contains the NP196 ketolase from Nostoc punctiforme in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJONP196.
  • Example 6 Production of expression vectors for the constitutive expression of the NP196 ketolase from Nostoc punctiforme ATCC 29133 in Lycopersicon esculentum and Tagetes erecta.
  • NP196 ketolase from Nostoc punctiforme in L. esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase, database entry AB011474 position 70127 to 69493;
  • WO03 / 006660 from Arabidopsis thaliana.
  • the FNR gene begins at base pair 69492 and is annotated with "ferredoxin-NADP + reductase”. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the DNA fragment containing the FNR promoter region from Arabidopsis thaliana was PCR-analyzed using genomic DNA (isolated from Arabidopsis thaliana according to standard methods) and the primers FNR-1 (SEQ ID No. 107) and FNR-2 (SEQ ID No. 108).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the FNR promoter fragment FNR (SEQ ID No. 109), was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 652 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (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 positions 70127 to 69493.
  • This clone is called pFNR and was therefore used for the cloning into the expression vector pJONP196 (described in Example 5).
  • the cloning was carried out by isolating the 644 bp Smal-Hindlll fragment from pFNR and ligating into the Ecl136ll-Hindlll cut vector pJONP196.
  • the clone which contains the promoter FNR instead of the original promoter d35S and the fragment NP196 in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJOFNR: NP196.
  • the expression vector MSP105 contains fragment FNR promoter the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the Nostoc punctiforme NP196 ketolase, fragment OCS terminator (192 bp) the polyadenylation signal from the octopine synthase.
  • MSP106 To produce the Tagetes expression vector MSP106, the 1,839 bp EcoRI-Xhol fragment from pJOFNR: NP196 was ligated with the EcoRI-Xhol cut vector pSUN5.
  • MSP106 contains fragment FNR promoter the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the nostoc punctiform NP196 ketolase, fragment OCS terminator (192 bp) the polyadenylation signal of octopine synthase.
  • NP196 ketolase from Nostoc punctiforme in L. esculentum and Tagetes erecta was carried out with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240: 709-715). The expression was carried out under the control of the flower-specific promoter EPSPS from Petunia hybrida (database entry M37029: nucleotide region 7-1787; Benfey et al. (1990) Plant Cell 2: 849-856).
  • the DNA fragment which contains the EPSPS promoter region (SEQ ID No. 112) from Petunia hybrida was PCR-analyzed using genomic DNA (isolated from Petunia hybrida according to standard methods) and the primers EPSPS-1 (SEQ ID No. 110) and EPSPS -2 (SEQ ID No. 111).
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which contains the EPSPS promoter fragment (database entry M37029: nucleotide region 7-1787), was carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR was carried out under the following cycle conditions:
  • the 1773 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods and the plasmid pEPSPS was obtained.
  • Sequencing of the clone pEPSPS confirmed a sequence consisting only of two deletions (bases ctaagtttcagga in position 46-58 of sequence M37029; bases aaaaatat in positions 1422-1429 of sequence M37029) and the base changes (T instead of G in position 1447 of sequence M37029 ; A instead of C in position 1525 of sequence M37029; A instead of G in position 1627 of sequence M37029) differs from the published EPSPS sequence (database entry M37029: nucleotide region 7-1787).
  • the two deletions and the two base changes at positions 1447 and 1627 of sequence M37029 were reproduced in an independent amplification experiment and thus represent the actual nucleotide sequence in the Petunia hybrida plants used.
  • the clone pEPSPS was therefore used for the cloning into the expression vector pJONP196 (described in Example 5).
  • the cloning was carried out by isolating the 1763 bp SacI-HindIII fragment from pEPSPS and ligation into the SacI-HindIII cut vector pJ0NP196.
  • the clone that contains the EPSPS promoter instead of the original d35S promoter is called pJOESP: NP196.
  • This expression cassette contains the fragment NP196 in the correct orientation as an N-terminal fusion with the rbcS transit peptide.
  • the expression vector MSP107 contains fragment EPSPS the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the Nostoc punctiform NP196 ketolase (fragment OCS terminator) bp) the polyadenylation signal of octopine synthase.
  • An expression vector for the Agrobacterium -mediated transformation of the EPSPS-controlled NP196 ketolase from Nostoc punctiforme in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00900).
  • the expression vector MSP108 contains fragment EPSPS the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), coding for the nostoc punctiform NP196 ketolase, fragment OCS terminator 192 bp) the polyadenylation signal of octopine synthase.
  • the DNA encoding the NP195 ketolase from Nostoc punctiform ATCC 29133 was amplified by PCR from Nostoc punctiform ATCC 29133 (strain of the "American Type Culture Collection"). The preparation of genomic DNA from a suspension culture of Nostoc punctiforme ATCC 29133 was described in Example 5.
  • the nucleic acid encoding a Nostoc punctiform ATCC 29133 ketolase was synthesized by means of a "polymerase chain reaction” (PCR) from Nostoc punctiform ATCC 29133 using a sense-specific primer (NP195-1, SEQ ID No. 113) and an antisense-specific one Primers (NP195-2 SEQ ID No. 114) amplified.
  • PCR polymerase chain reaction
  • the PCR conditions were as follows:
  • the PCR for the amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence, was carried out in a 50 ⁇ l reaction mixture in which hold was:
  • the PCR was carried out under the following cycle conditions:
  • PCR amplification with SEQ ID No. 113 and SEQ ID No. 114 resulted in an 819 bp fragment which codes for a protein consisting of the entire primary sequence (NP195, SEQ ID No. 115).
  • the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNP195 was obtained.
  • Primer confirmed a sequence identical to the DNA sequence 55.604-56.392 of database entry NZ_AABC010001965, except that T at position 55.604 was replaced by A to create a standard ATG start codon.
  • This nucleotide sequence was reproduced in an independent amplification experiment and thus represents the nucleotide sequence in the Nostoc punctiforme ATCC 29133 used.
  • This clone pNP195 was therefore used for the cloning into the expression vector pJO (described in Example 5).
  • the cloning was carried out by isolating the 809 bp Sphl fragment from pNP195 and ligation into the SphI-cut vector pJO.
  • the clone which contains the NP 95 ketolase from Nostoc punctiforme in the correct orientation as an N-terminal translational fusion with the rbcS transit peptide is called pJONP195.
  • Example 9 Production of expression vectors for the constitutive expression of NP195-ketolase from Nostoc punctiforme ATCC 29133 in Lycopersicon esculentum and Tagetes erecta.
  • the expression of the NP195 ketolase from Nostoc punctiforme in L. esculentum and in Tagetes erecta was carried out under the control of the constitutive promoter FNR (ferredoxin-NADPH-oxidoreductase, database entry AB011474 position 70127 to 69493; WO03 / 006660), from Arabidopsis thaliana.
  • FNR constitutive promoter
  • the FNR gene begins at base pair 69492 and is annotated with "ferredoxin-NADP + reductase”. Expression was carried out using the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240: 709-715).
  • the clone pFNR (described in Example 6) was therefore used for the cloning into the expression vector pJONP 95 (described in Example 8).
  • the cloning was carried out by isolating the 644 bp Sma-Hindill fragment from pFNR and ligation in the Ecl136ll-
  • Hindlll cut vector pJONP195 The clone which contains the promoter FNR instead of the original promoter d35S and the fragment NP195 in the correct orientation as an N-terminal fusion with the rbcS transit peptide is called pJOFNR: NP195.
  • the expression vector MSP109 contains fragment FNR promoter the FNR promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), coding for the nostoc punctiform NP195-Ketolator, fragment (192 bp) the polyadenylation signal from the octopine synthase.
  • the expression vector MSP110 contains fragment FNR promoter the FNR Promoter (635 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), coding for the Nostoc punctiform NP195 ketolase, fragment OCS terminator (192 bp) the polyadenylation signal from octopine synthase.
  • NP195 ketolase from Nostoc punctiforme in L. esculentum and Tagetes erecta was carried out with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240: 709-715). The expression was carried out under the control of the flower-specific promoter EPSPS from Petunia hybrida (database entry M37029: nucleotide region 7-1787; Benfey et al. (1990) Plant Cell 2: 849-856).
  • the clone pEPSPS (described in Example 7) was therefore used for the cloning into the expression vector pJONP195 (described in Example 8).
  • the cloning was carried out by isolating the 1763 bp SacI-HindIII fragment from pEPSPS and ligation into the SacI-HindIII cut vector pJ0NP195.
  • the clone that contains the EPSPS promoter instead of the original d35S promoter is called pJOESP: NP195.
  • This expression cassette contains the fragment NP195 in the correct orientation as an N-terminal fusion with the rbcS transit peptide.
  • An expression vector for the Agrobacterium -mediated transformation of the EPSPS-controlled NP195 ketolase from Nostoc punctiforme ATCC 29133 in L. esculentum was produced using the binary vector pSUN3 (WO02 / 00900).
  • the expression vector MSP111 contains fragment EPSPS the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), coding for the nostoc punctiform NP195 ketolase (fragment OCS terminator) 192 bp) the polyadenylation signal of octopine synthase.
  • An expression vector for the Agrobacterium -mediated transformation of the EPSPS-controlled NP195 ketolase from Nostoc punctiforme in Tagetes erecta was produced using the binary vector pSUN5 (WO02 / 00900).
  • the expression vector MSP112 contains fragment EPSPS the EPSPS promoter (1761 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), coding for the nostoc punctiform NP195 ketolase (fragment OCS terminator) 192 bp) the polyadenylation signal of octopine synthase.
  • the expression of the chromoplast-specific beta-hydroxylase from Lycopersicon esculentum in Tagetes erecta takes place under the control of the flower-specific promoter EPSPS from Petunia (Example 7).
  • EPSPS flower-specific promoter
  • LB3 from Vicia faba is used as the terminator element.
  • the sequence of the chromoplast-specific beta-hydroxylase was generated by RNA isolation, reverse transcription and PCR.
  • genomic DNA from Vicia faba tissue is isolated according to standard methods and used by genomic PCR using the primers PR206 and PR207.
  • the PCR for the amplification of this LB3 DNA fragment is carried out in a 50 ⁇ l reaction mixture which contains:
  • PCR amplification with PR206 and PR207 results in a 0.3 kb fragment which contains the LB terminator.
  • the amplificate is cloned into the cloning vector pCR-BluntII (Invitrogen). Sequencing with the primers T7 and M13 confirm a sequence identical to the sequence SEQ ID: 118. This clone is called pTA-LB3 and is therefore used for the cloning into the vector pJIT117 (see below).
  • RNA from tomato is prepared for the production of the beta-hydroxylase sequence. For this, 100 mg of the frozen, powdered flowers are transferred to a reaction vessel and taken up in 0.8 ml of Trizol buffer (LifeTechnologies). The suspension is extracted with 0.2 ml of chloroform. After centrifugation at 12,000 g for 15 minutes, the aqueous supernatant is removed and transferred to a new reaction vessel and extracted with a volume of ethanol. The RNA is precipitated with a volume of isopropanol, washed with 75% ethanol and the pellet is dissolved in DEPC water (overnight incubation of water with 1/1000 volume of diethyl pyrocarbonate at room temperature, then autoclaved).
  • RNA concentration is determined photometrically.
  • cDNA synthesis 2.5 ⁇ g of total RNA are denatured for 10 min at 60 ° C., cooled on ice for 2 min and using a cDNA kit (ready-to-go-you-prime-beads, Pharmacia Biotech) according to the manufacturer's instructions using an antisense-specific primer (PR215 SEQ ID No. 119) transcribed into cDNA.
  • a cDNA kit ready-to-go-you-prime-beads, Pharmacia Biotech
  • the PCR for the amplification of the VPR203-PR215 DNA fragment which codes for the beta-hydroxylase is carried out in a 50 ⁇ l reaction mixture which contained:
  • the PCR amplification with VPR203 and PR215 results in a 0.9 kb fragment which codes for the beta-hydroxylase.
  • the amplificate is cloned into the cloning vector pCR-BluntII (Invitrogen). Sequencing with primers T7 and M13 confirms a sequence SEQ ID No. 121 identical sequence. This clone is called pTA-CrtR-b2 and is therefore used for cloning into the vector pCSP02 (see below).
  • the EPSPS promoter sequence from petunia is produced by PCR amplification using the plasmid MSP107 (see Example 7) and the primers VPR001 and VPR002.
  • the PCR for the amplification of this EPSPS-DNA fragment is carried out in a 50 ⁇ l reaction mixture, which contains: 1 ul cDNA (prepared as described above)
  • the PCR amplification with VPR001 and VPR002 results in a 1.8 kb fragment which encodes the EPSPS promoter.
  • the amplificate is cloned into the cloning vector pCR-BluntII (Invitrogen). Sequencing with the primers T7 and M13 confirm a sequence identical to the sequence SEQ ID: 124. This clone is called pTA-EPSPS and is therefore used for cloning into the vector pCSP03 (see below).
  • the first cloning step is carried out by isolating the 0.3 kb PR206-PR207 EcoRI-Xhol fragment from pTA-LB3, derived from the cloning vector pCR-BluntII (Invitrogen), and ligation with the EcoRI-Xhol cut vector pJIT117.
  • the clone that contains the 0.3 kb terminator LB3 is called pCSP02.
  • the second cloning step is carried out by isolating the 0.9 kb VPR003-PR215 Eco-Rl-Hindlll fragment from pTA-CrtR-b2, derived from the cloning vector pCR-Bluntll (Invitrogen), and ligation with the EcoRI-Hindlll cut vector pCSP02.
  • the clone that contains the 0.9 kb beta-hydroxylase fragment CrtR-b2 is called pCSP03.
  • the ligation creates a transcriptional fusion between the terminator LB3 and the beta-hydroxylase fragment CrtR-b2.
  • the third cloning step is carried out by isolating the 1.8 kb VPR001-VPR002 Ncol-Sacl fragment from pTA-EPSPS, derived from the cloning vector pCR-BluntII (Invitrogen), and ligation with the Ncol-Sacl cut vector pCSP03.
  • the clone that contains the 1.8 kb EPSPS promoter fragment is called pCSP04.
  • the ligation results in a transcriptional fusion between the EPSPS promoter and the beta hydroxylase fragment CrtR-b2.
  • pCSP04 contains fragment EPSPS fragment (1792 bp) the EPSPS promoter, fragment crtRb2 (929 bp) beta-hydroxylase CrtRb2, fragment LB3 (301 bp) the LB3 terminator.
  • the beta hydroxylase cassette is isolated as a 3103 bp Ecl136II-Xhol fragment.
  • the 3 'ends (30 min at 30 ° C) are filled using standard methods (Klenow fill-in).
  • the expression vector is called pCSEbhyd
  • promoter P76 SEQ ID NO. 125
  • the oligonucleotides were provided with a 5 'phosphate residue during the synthesis.
  • genomic DNA was isolated from Arabidopsis thaliana as described (Galbiati M et al. Funct. Integr. Genomics 2000, 20 1: 25-34).
  • the PCR amplification was carried out as follows:
  • the vector pSun ⁇ is digested with the restriction endonuclease EcoRV and also purified by agarose gel electrophoresis and obtained by gel elution.
  • the purified PCR product is cloned into the vector treated in this way.
  • This construct is called p76.
  • the 1032 bp fragment representing the Arabidopsis promoter P76 was sequenced (Seq ID NO. 131).
  • the terminator 35ST is obtained from pJIT 117 by digestion with the restriction endonucleases Kpnl and Smal.
  • the resulting 969 bp fragment is purified by agarose gel electrophoresis and isolated by gel elution.
  • the vector p76 is also digested with the restriction endonucleases Kpnl and Smal.
  • the resulting 7276bp fragment is purified by agarose gel electrophoresis and isolated by gel elution.
  • the 35ST fragment obtained in this way is cloned into the p76 treated in this way.
  • the resulting vector is called p76_35ST.
  • the Bgene (SEQ ID NO. 128) was isolated by means of PCR using genomic DNA from Lycopersicon esculentum as a template.
  • the oligonucleotides were provided with a 5 'phosphate residue during the synthesis.
  • the genomic DNA was isolated from Lycopersicon esculentum as described (Galbiati M et al. Funct. Integr. Genomics 2000, 20 1: 25-34).
  • the PCR amplification was carried out as follows:
  • the PCR product was purified by agarose gel electrophoresis and the 1665 bp fragment isolated by gel elution.
  • the vector p76_35ST is digested with the restriction endonuclease Smal and also purified by agarose gel electrophoresis and obtained by gel elution.
  • the purified PCR product is cloned into the vector treated in this way.
  • This construct is called pB.
  • pB is digested with the restriction endonucleases Pmel and Sspl and the 3906bp fragment containing the promoter P76, Bgene and the 35ST is purified by agarose gel electrophoresis and obtained by gel elution
  • MSP108 (Example 7) is digested with the restriction endonuclease Ecl126ll, purified by agarose gel electrophoresis and obtained by gel elution
  • the purified 3906bp fragment containing the promoter P76, Bgene and the 35ST from pB is cloned into the Vector MSP108 treated in this way.
  • This construct is called pMKP1.
  • Example 13 Production and analysis of transgenic Lycopersicon esculentum plants
  • Transformation and regeneration of tomato plants was carried out according to the published method by Ling and co-workers (Plant Cell Reports (1998), 17: 843-847).
  • kanamycin concentrations 100 mg / L
  • the starting explant for the transformation was cotyledons and hypocotyls, seven to ten day old seedlings of the Microtome line.
  • the culture medium according to Murashige and Skoog (1962: Murashige and Skoog, 1962, Physiol. Plant 15, 473-) with 2% sucrose, pH 6.1 was used for germination. Germination took place at 21 ° C with little light (20 to 100 ⁇ E).
  • the cotyledons were divided transversely and the hypocotyls were cut into sections about 5 to 10 mm long and placed on the medium MSBN (MS, pH 6.1, 3% sucrose + 1 mg / l BAP, 0.1 mg / l NAA), which was loaded with suspension-cultivated tomato cells the day before.
  • the tomato cells were covered with sterile filter paper without air bubbles.
  • the explants were precultured on the medium described for three to five days. Cells from the Agrobacterium tumefaciens LBA4404 strain were individually transformed with the plasmids.
  • the explants were transferred to MSZ2 medium (MS pH 6.1 + 3% sucrose, 2 mg / l zeatin, 100 mg / l kanamycin, 160 mg / l timentin) and for selective regeneration at 21 ° C stored under weak conditions (20 to 100 ⁇ E, light rhythm 16 h / 8 h).
  • MSZ2 medium MS pH 6.1 + 3% sucrose, 2 mg / l zeatin, 100 mg / l kanamycin, 160 mg / l timentin
  • the explants were transferred every two to three weeks until shoots formed. Small shoots could be separated from the explant and rooted on MS (pH 6.1 + 3% sucrose) 160 mg / l timentin, 30 mg / l kanamycin, 0.1 mg / l IAA. Rooted plants were transferred to the greenhouse.
  • MSP107 we got: msp107-1, msp107-2, msp107-3
  • MSP109 we got: mspl 09-1, mspl 09-2, mspl 09-3
  • germination medium MS medium; Murashige and Skoog, Physiol. Plant. 15 (1962), 473-497) pH 5.8, 2% sucrose.
  • Germination takes place in a temperature / light / time interval of 18 to 28 ° G20-200 ⁇ E / 3 to 16 weeks, but preferably at 21 ° C, 20 to 70 mE, for 4 to 8 weeks.
  • the bacterial strain can be grown as follows: A single colony of the corresponding strain is in YEB (0.1% yeast extract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.5% magnesium sulfate x 7 H) 2 0) inoculated with 25 mg / l kanamycin and dressed at 28 ° C for 16 to 20 hours.
  • the bacterial suspension is then harvested by centrifugation at 6000 g for 10 min and resuspended in liquid MS medium in such a way that an OD 6 oo of approximately 0.1 to 0.8 was obtained. This suspension is used for the co-cultivation with the leaf material.
  • the MS medium in which the leaves have been kept is replaced by the bacterial suspension.
  • the leaflets were incubated in the agrobacterial suspension for 30 min with gentle shaking at room temperature.
  • the infected explants are then placed on an MS medium solidified with agar (for example 0.8% plant agar (Duchefa, NL) with growth regulators, such as 3 mg / l benzylaminopurine (BAP) and 1 mg / l indolylacetic acid (IAA).
  • agar for example 0.8% plant agar (Duchefa, NL) with growth regulators, such as 3 mg / l benzylaminopurine (BAP) and 1 mg / l indolylacetic acid (IAA).
  • the orientation of the leaves on the medium is irrelevant: the explants are cultivated for 1 to 8 days, but preferably for 6 days, the following conditions being able to be used: light intensity: 30 to 80 ⁇ mol / m 2 x sec, temperature : 22 to 24 ° C., light / dark change of 16/8 hours, after which the co-cultivated explants are transferred to fresh MS medium, preferably with the same growth regulators, this second medium additionally containing an antibiotic to suppress bacterial growth.
  • Timentin in a concentration of 200 to 500 mg / l is very suitable for this purpose
  • the second selective component is used to select the success of the transformation.
  • Phosphinothricin in a concentration of 1 to 5 mg / l selects very efficiently, but other selective components according to the method to be used are also conceivable.
  • the explants are transferred to fresh medium until shoot buds and small shoots develop, which are then on the same basal medium including timentin and PPT or alternative components with growth regulators, namely, for example, 0.5 mg / l indolylbutyric acid (IBA) and 0.5 mg / l gibberillic acid GA 3 , are transferred for rooting. Rooted shoots can be transferred to the greenhouse.
  • IBA 0.5 mg / l indolylbutyric acid
  • GA 3 gibberillic acid
  • the explants Before the explants are infected with the bacteria, they can be preincubated for 1 to 12 days, preferably 3 to 4, on the medium described above for the co-culture. The infection, co-culture and selective regeneration then take place as described above.
  • the pH value for regeneration (normally 5.8) can be lowered to pH 5.2. This improves the control of agrobacterial growth.
  • Liquid culture medium can also be used for the entire process.
  • the culture can also be incubated on commercially available carriers which are positioned on the liquid medium.
  • MSP108 we got: msp108-1, msp108-2, msp108-3
  • MSP110 we got: mspl 10-1, mspl 10-2, mspl 10-3
  • MSP112 the following was obtained: mspl 12-1, mspl 12-2, mspl 12-3
  • Example 15 Enzymatic lipase-catalyzed hydrolysis of carotenoid esters from plant material and identification of the carotenoids
  • Mortar plant material e.g. petal material (30-100 mg fresh weight) is extracted with 100% acetone (three times 500 ⁇ l; shake for about 15 minutes each). The solvent is evaporated. Carotenoids are then taken up in 495 ⁇ l of acetone, 4.95 ml of potassium phosphate buffer (100 mM, pH 7.4) are added and mixed well. Then about 17 mg of Bile salts (Sigma) and 149 ⁇ l of a NaCl / CaCl 2 solution (3M NaCl and 75 mM CaCl 2 ) are added. The suspension is incubated at 37 ° C for 30 minutes.
  • a NaCl / CaCl 2 solution 3M NaCl and 75 mM CaCl 2
  • a lipase solution 50 mg / ml lipase type 7 from Candida rugosa (Sigma)
  • 595 ⁇ l of lipase solution 50 mg / ml lipase type 7 from Candida rugosa (Sigma)
  • 595 ⁇ l of lipase was added again and incubation was continued for at least 5 hours at 37 ° C.
  • 700 mg Na 2 S0 4 are dissolved in the solution.
  • the carotenoids are extracted into the organic phase by vigorous mixing. This shaking is repeated until the organic phase remains colorless.
  • the petroleum ether fractions are combined and the petroleum ether evaporated. Free carotenoids are taken up in 100-120 ⁇ l acetone. Free carotenoids can be identified on the basis of retention time and UV-VIS spectra using HPLC and C30 reverse phase columns.
  • the Bile salts or bile acid salts used are 1: 1 mixtures of cholate and deoxycholate.
  • the hydrolysis of the carotenoid esters by lipase from Candida rugosa can be achieved after separation by means of thin layer chromatography. For this, 50-100mg of plant material are extracted three times with about 750 ⁇ l acetone. The solvent extract is rotated in a vacuum (elevated temperatures of 40-50 ° C are tolerable). Then add 300 ⁇ l petroleum ether acetone (ratio 5: 1) and good Mixing. Suspended matter is sedimented by centrifugation (1-2 minutes). The upper phase is transferred to a new reaction vessel. The remaining residue is extracted again with 200 ⁇ l of petroleum ether acetone (ratio 5: 1) and suspended matter is removed by centrifugation.
  • the two extracts are combined (volume 500 ⁇ l) and the solvents evaporated.
  • the residue is resuspended in 30 ⁇ l of petroleum ether: acetone (ratio 5: 1) and applied to a thin-layer plate (silica gel 60, Merck). If more than one application is required for preparative-analytical purposes, several aliquots, each with a fresh weight of 50-100 mg, should be prepared in the manner described for thin-layer chromatography separation.
  • the thin-layer plate is developed in petroleum ether-acetone (ratio 5: 1). Carotenoid bands can be identified visually based on their color. Individual carotenoid bands are scraped out and can be pooled for preparative-analytical purposes.
  • the carotenoids are eluted from the silica material with acetone; the solvent is evaporated in vacuo.
  • the residue is dissolved in 495 ⁇ l acetone, 17 mg Bile salts (Sigma), 4.95 ml 0.1 M potassium phosphate buffer (pH 7.4) and 149 ⁇ l (3M NaCl, 75mM CaCl 2 ) are added. After thorough mixing, equilibrate at 37 ° C for 30 minutes.
  • Candida rugosa lipase Sigma, stock solution of 50 mg / ml in 5 mM CaCl 2 . Incubation with lipase takes place overnight with shaking at 37 ° C. After about 21 hours, the same amount of lipase is added again; Incubate again at 37 ° C with shaking for at least 5 hours. Then 700 mg of Na 2 S0 4 (anhydrous) are added; with 1800 ⁇ l of petroleum ether is shaken for about 1 minute and the mixture is centrifuged at 3500 revolutions / minute for 5 minutes. The upper
  • Phase is transferred to a new reaction vessel and the shaking is repeated until the upper phase is colorless.
  • the combined petroleum ether phase is concentrated in vacuo (temperatures of 40-50 ° C are possible).
  • the residue is dissolved in 120 ⁇ l acetone, possibly using ultrasound.
  • the dissolved carotenoids can be separated by means of HPLC using a C30 column and quantified using reference substances.
  • Example 15 The analysis of the samples obtained according to the working instructions in Example 15 is carried out under the following conditions:
  • Some typical retention times for carotenoids formed according to the invention are, for example, violaxanthin 11.7 minutes, astaxanthin 17.7 minutes, adonixanthin 19 minutes, adonirubin 19.9 minutes and zeaxanthin 21 minutes.

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de production de cétocaroténoïdes par culture d'organismes génétiquement modifiés qui présentent une activité de cétolase modifiée et une activité de beta -cyclase modifiée par rapport au type sauvage. L'invention concerne également lesdits organismes génétiquement modifiés, ainsi que leur utilisation comme produits alimentaires et produits fourragers et pour la production d'extraits de cétocaroténoïdes.
EP04741347A 2003-08-18 2004-07-31 Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies Withdrawn EP1658377A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP04741347A EP1658377A1 (fr) 2003-08-18 2004-07-31 Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
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/EP2003/009109 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
PCT/EP2003/009106 WO2004018694A2 (fr) 2002-08-20 2003-08-18 Procede d'obtention de cetocarotinoides dans des organismes genetiquement modifies
PCT/EP2003/009102 WO2004018693A2 (fr) 2002-08-20 2003-08-18 Procede de production de cetocarotenoides dans les petales de plantes
DE102004007622A DE102004007622A1 (de) 2004-02-17 2004-02-17 Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten, nicht-humanen Organismen
EP04741347A EP1658377A1 (fr) 2003-08-18 2004-07-31 Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies
PCT/EP2004/008623 WO2005019467A1 (fr) 2003-08-18 2004-07-31 Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies

Publications (1)

Publication Number Publication Date
EP1658377A1 true EP1658377A1 (fr) 2006-05-24

Family

ID=36204098

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04741347A Withdrawn EP1658377A1 (fr) 2003-08-18 2004-07-31 Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies

Country Status (1)

Country Link
EP (1) EP1658377A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005019467A1 *

Similar Documents

Publication Publication Date Title
ZA200602230B (en) Method for producing ketocarotinoids in genetically modified, non-human organisms
WO2004063366A1 (fr) Procede de preparation de cetocarotinoides par mise en culture d'organismes genetiquement modifies
WO2005019467A1 (fr) Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies
WO2010079032A1 (fr) Production de cétocaroténoïdes dans des plantes
WO2005019460A2 (fr) Promoteurs d'expression de genes dans des tagetes
JP2007502605A6 (ja) 遺伝子的に改変された非ヒト生物におけるケトカロテノイドの製造方法
DE10238980A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in Blütenblättern von Pflanzen
WO2005019461A2 (fr) Nouvelles cetolases et procede de production de cetocarotenoides
DE10253112A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in genetisch veränderten Organismen
EP1658377A1 (fr) Procede de production de cetocarotenoides dans des organismes non humains genetiquement modifies
EP1658372A2 (fr) Nouvelles cetolases et procede de production de cetocarotenoides
DE10238978A1 (de) Verfahren zur Herstellung von Ketocarotinoiden in Früchten von Pflanzen
AU2004267196A1 (en) Method for producing ketocarotinoids in genetically modified, non-human organisms
DE10238979A1 (de) Verfahren zur Herstellung von Zeaxanthin und/oder dessen biosynthetischen Zwischen- und/oder Folgeprodukten
EP1658371A2 (fr) Promoteurs d'expression de genes dans des tagetes

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20060320

AK Designated contracting states

Kind code of ref document: A1

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

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

Effective date: 20070403

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

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20081223