WO2004007732A1 - Clonage et caracterisation d'une lipoxygenase extraite de phaeodactylum tricornutum - Google Patents

Clonage et caracterisation d'une lipoxygenase extraite de phaeodactylum tricornutum Download PDF

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WO2004007732A1
WO2004007732A1 PCT/EP2003/007354 EP0307354W WO2004007732A1 WO 2004007732 A1 WO2004007732 A1 WO 2004007732A1 EP 0307354 W EP0307354 W EP 0307354W WO 2004007732 A1 WO2004007732 A1 WO 2004007732A1
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acid
sequence
nucleic acid
lipoxygenase
plant
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Ivo Feussner
Toralf Senger
Cornelia Göbel
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Basf Plant Science Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to a process for the preparation of hydroperoxy fatty acids by oxidation of polyunsaturated fatty acids with at least two double bonds in the fatty acid molecule, in particular by oxidation of polyunsaturated fatty acids such as arachidonic acid or eicosapentaenoic acid.
  • the invention relates to the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism with an increased content of hydroperoxy fatty acids due to the expression of a lipoxygenase from Phaeodactylum tricornutum.
  • the invention relates to expression cassettes containing a nucleic acid sequence, a vector and organisms containing at least one nucleic acid sequence or an expression cassette.
  • Lipoxygenases (linoleate: oxygen oxidoreductases, EC 1.12.11.13; LOX) are enzymes which are ubiquitously widespread in the animal and animal kingdom [AR Brash, J. Biol Chem, 274: 23679-23682, 1999]. They contain one non-haem iron atom per mole of enzyme and catalyze the regio- and stereoselective dioxygenation of polyunsaturated fatty acids, whereby their hydroperoxide derivatives are formed in the S configuration [p. Rosahl, Z Naturforsch, 51: 123-138, 1996].
  • the first lipoxygenase was described by Andre and Hou in 1932 as an enzyme which caused the oxidation of fatty acids and the breakdown of carotenoids [Andre et al., Lieb CR Acad Sei (Paris), 194: 645-647, 1932].
  • the enzymatic activity of lipoxygenases was used in the food industry even before they were discovered.
  • wheat flour was bleached by reacting carotenoids contained therein by lipoxygenases, which were the active ingredient of added soy flour [JN Siedow, Annu Rev Plant Physiol Plant Mol Biol, 42: 145-188, 1991].
  • Lipoxygenases helped to create models which Attempting to explain substrate and position specificity [Feussner et al .; Enzymes in Lipid Modification, p. 309-336, WileyNCH, Weinheim, 2000]. To date, sequences encoding lipoxygenases have been known from over sixty species of plants and animals [AR Brash, J. Biol Chem, 274: 23679-23682, 1999].
  • a microbial lipoxygenase from the fungus Gaeumannomyces graminis was first isolated [Sugio et al., Lipoxygenase, 2002; WO 02/20730]. In mammals they convert arachidonic acid, among other things, and form the precursors of regulatory compounds, such as leukotrienes and lipoxins, which play an important role in inflammatory and immune reactions [Kühn et al., FEBS Lett, 449: 7-11, 1999]. In plants, the hydroperoxides formed by the lipoxygenases serve as substrates for at least seven enzyme families [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 002].
  • Plant lipoxygenases are enzymes that have a molecular weight between 90 and 115 kDa. They belong to the family of dioxygenases and catalyze the incorporation of molecular oxygen into a (1Z, 4Z) diene system of unsaturated fatty acids [AR Brash, J. Biol Chem, 274: 23679-23682, 1999]. The resulting hydroperoxides of the fatty acids have an S configuration and have a (1Z, 3E) diene system (FIG. 1).
  • either linoleic acid produces either (13S, 9Z, 11E) -13-hydroperoxy-9,11-octadienoic acid (13-HPODE, [+2] rearrangement) or ( 9S, 10E, 12Z) -9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE, [-2] rearrangement ([Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000], Figure 2).
  • Peroxygenase Route (E. Blee, Prog. Lipid Res., 37: 33-72, 1998] earlier hydroperoxide isomerase): Peroxygenase produces hydroxy, epoxy and ß-hydroxyepoxy fatty acids from the hydroperoxides of the fatty acids. The epoxy groups can be reduced to hydroxyl groups, so that di- and trihydroxy fatty acids are formed [M. Hamberg, J Lipid Mediators Cell Signal,
  • the Allen Oxide Synthase Pathway (AOS, formerly hydroperoxide dehydratase [HW Gardner, Biochim Biophys Acta, 1084: 221-239, 1991]:
  • the Allen oxide synthase belongs to the family of cytochrome P 450 enzymes [Song et al., Proc Natl
  • the hydroperoxide lyase route (HPL, formerly hydroperoxide isomerase [Zimmermann et al., Plant Physiol, 46: 445-453, 1970]: The enzyme is widespread in plants [K. Matsui, Belgian Journal of Botany, 131: 50-62, 1998], also has a cytochrome P 450 and forms the subfamilies CYP74B and CYP74C.
  • the peroxides of linoleic and ⁇ -linolenic acid are converted into the saturated aldehyde hexanal or unsaturated (3Z) aldehydes and ⁇ - Oxo derivatives of fatty acids cleaved [K.
  • Alcohol dehydrogenases can be used to convert the (3Z) -aldehydes into (3Z) -alcohols, which in turn are then used with short-chain Carboxylic acids can form volatile esters, or the aldehydes can be isomerize to their corresponding (2E) aldehydes before they are reduced (see p. 5) .
  • These substances resulting from the HPL reaction are responsible for the characteristic smell of plants and fruits [A. Hatanake, Food Rev Int, 12: 303-
  • DES divinyl ether synthase route
  • the hydroperoxide reductase includes involved in the degradation of polyene fatty acids in the storage lipids in a large number of oilseeds [Feussner et al., Trends
  • Lipoxygenase isoforms are found in most plant cells. However, the expression of lipoxygenase depends on the developmental and environmental conditions of the plant [p. Rosahl, Z Naturforsch, 51: 123-138, 1996]. They can be soluble or bound to membranes.
  • Soluble lipoxygenases are found, for example, in the cytosol of seedlings and ripening seeds [Vernooy-Gerritsen et al., Plant Physiol, 76: 1070-1080, 1984], in later stages of development, for example in leaves, also in vacuoles [Tranbarger et al., Plant Cell , 3: 973-987, 1991], in the cell nucleus [Feussner et al., Plant J, 7: 949-957, 1995] and in chloroplast [Bell et al., Proc NatI Acad Sei USA, 92: 8675-8676; 1995, Feussner et al., Plant J, 7: 949-957, 1995].
  • the membrane-bound lipoxygenases were also found in the most diverse compartments of the plant cells. They can be found on the plasma membrane [Nellen et al., Z Naturforsch, 50 (c): 29-36, 1995], on the membrane of the lipid bodies of seedlings [Feussner et al., FEBS Lett, 298: 223-225, 1992; Radetzky et al., Planta, 191: 166-172, 1993] and on the envelope and thylakoid membrane of the chloroplasts of leaves, flowers and fruits [Blee et al., Plant Physilogy, 110 (2): 445-454, 1996; Bowsher et al., Plant Physiol, 100: 1802-1807, 1992].
  • the 15-epoxygenases of the vertebrates are known to be involved in the membrane degradation process [Schewe et al., TIBS, 16: 369-373, 1991]. Based on this, plant lipoxygenases are also believed to cause loss of membrane integrity through the conversion of lipids to lipid hydroperoxides [Thompson et al., Prog Lipid Res, 37: 119-141, 1998]. Another hypothesis is that the destruction of the membranes in the seedling enables the transport of storage substances to the embryo [D.F. Hildebrand, Current Topics in Plant Biochemistry and Physiology, 7: 201-219, 1988].
  • Jasmonic acid or its methyl ester appears to be formed as a signal transduction molecule of the lance when wounded or infected with pathogens [Creelman et al., Ann Rev Plant Physiol Plant Mol Biol, 48: 355-381, 1997]. They induce enzymes that are important for a defense response [p. Rosahl et al., Z Naturforsch, 51: 123-138, 1996].
  • pathogen attack the plants react by activating defense genes, the synthesis of antimicrobial compounds such as phytotoxins, and hypersensitive cell death to develop resistance.
  • lipoxygenases seem to play a role [A. Slusarenko, Lipoxygenase and Lipoxygenase Pathway Enzymes, p. 176-197, 1996, AOCS Press Champaign II].
  • Diatoms are unicellular algae that occur in oceanic and fresh water phytoplankton, but also in biofilms and solid substrates. They are more closely related to brown and gold algae than to green algae, moss and higher plants. In contrast to the latter, diatoms form chrysolaminarin (a ß-1, 3-glucan) instead of starch (a ß-1, 4-glucan). In general, assimilation products such as oils (in special oil vacuoles) as well as chrysolaminarin and volutin are deposited outside the chromatophores f www.kieselalaen.coml. Another point of differentiation is the presence of chlorophyll c instead of chlorophyll b.
  • Chromophores that contain chlorophyll b are called chloroplasts.
  • the chromophores in diatoms are enveloped by four instead of two uniform membranes.
  • the two inner ones are the actual chromatophore membranes, the two outer ones represent a fold of the endoplasmic reticulum.
  • Chromatophores of this blueprint are found not only in Bacillariophyceen (silica layers) but also in Chrysophyceen, Xanthophyceen, Chloromonadophyceen and Phaeophyceen, with which they are combined to form the Heterozziphyta (Chrysophyta), are deposited [www.kieselalQen.com1.
  • Diatoms are the most important primary source in the marine food chain. They also contribute up to 25% globally to the primary production of biomaterial, with a corresponding share in oxygen production [Scala et al., Cell. Mol Life Sei, 58: 1666-1673, 2001]. Due to their outstanding role as a primary marine source, they are under immense herbivorous pressure. Recent research has shown that lipoxygenases are involved in a defense mechanism against herbivorous organisms in phytoplankton [Miralto et al., Nature, 402: 173-176, 1999; Pohnert et al., Nat. Pro, Rep., 19: 108-122, 002].
  • aldehydes which have proven to be the effective defense substances. These aldehydes reduce the success in hatching oar crabs, which make up 90% of phytoplankton. Since most diatoms are surrounded by a highly structured cell wall with embedded silicates, they also play a key role in the bio-geochemical cycle of silicates [Treger et al., Science, 268: 375-379, 1995].
  • Diatoms are used commercially for a variety of purposes, such as forage in all forms of water cultivation, as a source of polyunsaturated fatty acids (PUFAs) or as a component of pharmaceutical articles [Apt et al., J. Phycol, 35: 215- 226, 1999].
  • PUFAs polyunsaturated fatty acids
  • Phaeodactylum tricornutum a unicellular silica-free algae, is one of the most widely used model systems for studying the ecology, physiology, biochemistry, and molecular biology of diatoms [Apt et al., Mol Gen Genet, 252 (5): 572 -9, 1996]. It differs significantly from higher plants in that up to 30% of the total fatty acid component is the highly unsaturated fatty acid EPA and 11% docosahexanoic acid (DHA) [Schobert et al., Plant Physiol, 66: 215-219, 1980].
  • DHA docosahexanoic acid
  • oxygenated fatty acids As described, oxygenated fatty acids, the so-called oxylipins, are of great importance in the plant wound and defense response.
  • the first step in oxylipin synthesis is the formation of hydroperoxide derivatives of polyunsaturated fatty acids. This leads to the formation of hydroperoxy fatty acids, which are important starting materials for plant biosynthesis and are further implemented through a variety of enzyme reactions. They are, for example, raw materials for jasmonic acid and jasmonic acid methyl ester, which act as phytohormones within the plant.
  • Products of the lipoxygenase reaction are involved, for example, in the regulation of development processes (germination, tuber formation, senescence) through the formation of leaf aldehydes and leaf alcohols or in wound reactions and defense against pathogens through the synthesis of signaling molecules and phytoalexins. They are also involved in the herbal stress response.
  • traumatin and tramatinic acid probably contribute to the wound response by promoting cell division, or jasmonates and their octadecane precursors induce the expression of proteinase inhibitors locally in the damaged tissue after mechanical wounding and insect damage. They are also involved in the synthesis of antimicrobial metabolites.
  • nucleic acid sequences according to the invention which code for polypeptides with lipoxygenase activity, selected from the group: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, b) nucleic acid sequences which result from the degenerate genetic Let codes be derived from the coding sequence contained in SEQ ID NO: 1, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1 which are suitable for
  • a lipoxygenase from diatoms advantageously from Phaeodactylum tricornutum, is particularly specific for the conversion of arachidonic acid or eicosapentaenoic acid if they are advantageously expressed in a heterologous system.
  • This lipoxygenase can advantageously be used to produce hydroperoxy fatty acids in plants, non-human animals or microorganisms.
  • the advantageous lipoxygenase from Phaeodactylum tricornutum works in a pH range from 5 to 9, preferably in a pH range from 6 to 8.5, particularly preferably in a pH range from 6.5 to 8, very particularly preferably in a pH - Range from 7 to 8.
  • the pH optimum of the enzymatic reaction is pH 8.2.
  • the lipoxygenase according to the invention shows a regiospecificity for the hydroperoxy formation of linoleic acid at the C atom 13 (13-LOX) and has a chloroplastic signal sequence.
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • derivatives of the nucleic acid molecule according to the invention encode proteins with at least 60%, advantageously with at least 70%, preferably at least 75% and more preferably at least 80%, 85%, 90%, 95% and most preferably at least 96%, 97%, 98%, 99% or more identity to full amino acid sequence of SEQ ID NO: 2.
  • the identity was established over the entire amino acid or. Nucleic acid sequence range calculated.
  • the PileUp program was used for the sequence comparisons (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J Mol. Biol.
  • the invention also encompasses nucleic acid moles which differ from (and parts of) the nucleotide sequences shown in SEQ ID NO: 1 due to the degenerate genetic code and thus encode the same lipoxygenase as that encoded by the nucleotide sequence shown in SEQ ID NO: 1.
  • SEQ ID NO: 3 also shows the 5 'and 3' region of the nucleic acid sequence.
  • SEQ ID NO: 4 shows the corresponding protein sequence.
  • lipoxygenase nucleotide sequence shown in SEQ ID NO: 1 DNA sequence polymorphisms that lead to changes in the amino acid sequences of lipoxygenase may exist within a population. These genetic polymorphisms in the lipoxygenase gene can exist between individuals within a population due to natural variation. These natural variants usually cause a variance of 1 to 5% in the nucleotide sequence of the lipoxygenase gene. All and all of these nucleotide variations and the resulting amino acid polymorphisms in lipoxygenase, which are the result of natural variation and do not change the functional activity of lipoxygenase, are intended to be included in the scope of the invention.
  • polyunsaturated fatty acids are to be understood as meaning double or polyunsaturated fatty acids which have double bonds.
  • the double bonds can be conjugated or non-conjugated.
  • the nucleic acid sequence according to the invention or fragments thereof can advantageously be used to isolate further genomic sequences via homology screening.
  • the derivatives mentioned can advantageously be isolated, for example, from other organisms in eukaryotic organisms such as compounds such as mosses, diatoms or fungi from other diatoms.
  • Allelic variants include, in particular, functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1, the enzymatic activity of the derived synthesized proteins being retained.
  • DNA sequences can be isolated from other eukaryotes such as those mentioned above starting from the DNA sequence described in SEQ ID NO: 1 or parts of these sequences, for example using conventional hybridization methods or the PCR technique. These DNA sequences hybridize to the sequences mentioned under standard conditions. For the hybridization, it is advantageous to use short oligonucleotides, for example of the conserved regions, which can be determined by comparison with other lipoxygenase genes in a manner known to the person skilled in the art. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • RNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between approximately 30 ° C. to 55 ° C., preferably between approximately 45 ° C.
  • homologs of the sequence SEQ ID NO: 1 are furthermore to be understood, for example, as eukaryotic homologs, shortened sequences, single-stranded DNA of the coding and non-coding DNA sequence or RNA of the coding and non-coding DNA sequence.
  • homologs of the sequence SEQ ID NO: 1 are to be understood as derivatives such as promoter variants. These variants can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired. Furthermore, the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.
  • Derivatives are also advantageously to be understood as variants whose nucleotide sequence in the range -1 to -2000 before the start codon have been changed such that the gene expression and / or the protein expression is changed, preferably increased. Derivatives are also to be understood as variants which have been changed at the 3 'end.
  • nucleic acid sequence (s) according to the invention which code for a lipoxygenase can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA constituents and consist of different heterologous lipoxygenase gene segments from different organisms.
  • synthetic nucleotide sequences with codons are generated which are preferred by the corresponding host organisms, for example plants. This usually leads to optimal expression of the heterogeneous genes.
  • These codons preferred by Rance can be determined from codons with the highest protein frequency, which are expressed in most interesting plant species.
  • Corynebacterium glutamicum is given in: Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). Such experiments can be carried out using standard methods and are known to the person skilled in the art.
  • Functionally equivalent sequences which code for the lipoxygenase gene are those derivatives of the sequence according to the invention which, despite a different nucleotide sequence, still have the desired functions, that is to say the enzymatic activity and specific selectivity of the proteins.
  • Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a lance.
  • artificial DNA sequences are suitable as long as they have the desired property, as described above, for example the synthesis of hydroperoxy fatty acids in oils, lipids or as free fatty acids in the eukaryotic organisms mediate advantageously in the plants by overexpression of the lipoxygenase gene in crop plants.
  • Such artificial DNA sequences can have, for example, back-translation of proteins constructed using molecular modeling, lipoxygenase activity, or can be determined by in vitro selection. Possible techniques for the in vitro evolution of DNA to change or improve the DNA sequences are described in Patten, PA et al., Current Opinion in Biotechnology 8, 724-733 (1997) or in Moore, JC et al., Journal of Molecular Biology 272, 336-347 (1997).
  • Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable.
  • the specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other known genes of the lance to be transformed.
  • Sequences which code for fusion proteins are to be mentioned as further suitable equivalent nucleic acid sequences, part of the fusion protein being a lipoxygenase polypeptide or a functionally equivalent part thereof.
  • the second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect lipoxygenase expression (e.g. myc-tag or his-tag).
  • this is preferably a regulatory protein sequence, such as e.g. a signal sequence for the ER or a transit peptide that directs the lipoxygenase protein to the desired site of action.
  • the isolated nucleic acid sequences according to the invention advantageously originate from diatoms such as the Bacillariophyta strain with the classes Centrales, Cosciniodiscophyceae, Fragilariophyceae, Pennales or Bacillariophyceae, preferably from the Centrales or Pennales orders, more preferably from the subordinates Araphidineae, Raphididineaeae, especially Monor preferably from the families Naviculaceae, Cymbellacease, Entomoneidaceae, Nitzschiaceae, Epithemiaceae, Auriculaceae and Surirellaceae or very particularly preferably from the genera Amphora, Cymbella and Phaeodactylum.
  • diatoms such as the Bacillariophyta strain with the classes Centrales, Cosciniodiscophyceae, Fragilariophyceae, Pennales or Bacillariophyceae, preferably from the Centrales or Pennales orders, more preferably from the subordinates Araph
  • nucleic acid sequences originate from the genera and species Phaeodactylum tricornutum, Cymbella microcephala, Cymbella leptoceros, Cymbella naviculiformis, Cymbella prostrata, Cymbella sileslaca, Cymbella sinuata, Cymbella gruidula, Cymbella tumida, Encyonema Reformeraeaumumum, Encyonema Reformeraeaumum, Encyonema Reformeaumiau Amphora ovalis or Amphora exigua.
  • the lipoxygenase genes can advantageously be combined in the method according to the invention with other genes of fatty acid biosynthesis. Examples of such genes are the acyltransferases, further desaturases or elongases.
  • amino acid sequences according to the invention are to be understood as proteins which contain an amino acid sequence shown in the sequence SEQ ID NO: 2 or a sequence obtainable therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues, the enzymatic activities of the in SEQ ID NO: 2 protein is retained or is not significantly reduced, that is, the ability to form hydroperoxy fatty acids is retained or is not significantly reduced. Not significantly reduced is understood to mean all enzymes which still have at least 10%, preferably 20%, particularly preferably 30% of the enzymatic activity of the starting enzyme. For example, certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
  • arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • one or more amino acids can also be interchanged, added or removed in their order, or several of these measures can be combined with one another.
  • Derivatives are also to be understood as functional equivalents which, in particular, also include natural or artificial mutations of an originally isolated sequence coding for lipoxygenesis, which furthermore show the desired function, that is to say its enzymatic activity and substrate selectivity is not significantly reduced. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
  • the present invention also encompasses those nucleotide sequences which are obtained by modification of the lipoxygenase nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also the insertion of further restriction enzyme interfaces.
  • the nucleic acid sequence can advantageously be a DNA or cDNA sequence, for example.
  • Coding sequences suitable for insertion into an expression cassette according to the invention are, for example, those which code for a lipoxy genes with the sequences described above and which give the host the ability to overproduce hydroperoxy fatty acids in free form or bound in oils or lipids. These sequences can be of homologous or heterologous origin.
  • These regulatory sequences are said to be targeted Enable expression of genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed and / or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present in front of the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased.
  • the gene construct can also have a simpler structure, that is to say no additional regulation signals have been inserted in front of the nucleic acid sequence or its derivatives, and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence was mutated so that regulation no longer takes place and / or gene expression is increased.
  • the gene construct can also advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or Terminators
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase 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.
  • promoters which can advantageously control the expression of foreign genes in organisms in radicals or fungi are suitable as promoters in the expression cassette.
  • a plant promoter or promoters derived from a plant virus are preferably used.
  • Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl ⁇ 'T7, T5, Contain T3, gal, trc, ara, SP6, ⁇ -P R - or in the ⁇ -P promoter, which are advantageously used in gram-negative bacteria.
  • the expression cassette can also be a chemical contain inducible promoter, by means of which the expression of the exogenous lipoxygenase gene in the organisms can advantageously be controlled in the plants at a specific point in time.
  • Such advantageous plant promoters are, for example, the PRP1 promoter [Ward et al., Plant.Mol. Biol. 22 (1993), 361-366], a benzene sulfonamide-inducible (EP 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2,397-404), a salicylic acid-inducible promoter ( WO 95/19443), an abscisic acid-inducible (EP335528) or an ethanol or cyclohexanone-inducible (WO93 / 21334) promoter.
  • plant promoters are, for example, the promoter of the cytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245), the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max (see also Genbank Accession Number U87999) or a node-specific promoter as in EP 249676 can advantageously be used. Plant promoters which ensure expression in tissues or plant parts / organs in which fatty acid biosynthesis or its precursors take place, such as, for example, in the endosperm or in the developing embryo, are particularly advantageous.
  • advantageous promoters which ensure seed-specific expression
  • the USP promoter or derivatives thereof for example the USP promoter or derivatives thereof, the LEB4 promoter, the phaseolin promoter or the napin promoter.
  • the particularly advantageous USP promoter or its derivatives listed according to the invention mediate gene expression very early in seed development (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67).
  • Further advantageous seed-specific promoters which can be used for monocotyledon and dicotyledonous plants are the promoters suitable for dicotyledons, such as the Napingen promoter from rapeseed (US Pat. No.
  • the oleosin promoter from Arabidopsis (WO 98/45461), the phaseolin Promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the leguminous B4 promoter (LeB4, Baeumlein et al., Plant J., 2, 2, 1992: 233-239) or promoters suitable for monocotyledons such as the promoters the promoters of the lpt2 or lpt1 gene from barley (WO 95/15389 and WO 95/23230) or the promoters of the barley Hordein gene, the rice glutelin gene, the rice oryzin gene , the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene or the rye secalin gene, which are described in WO99 /
  • Promoters that ensure seed-specific expression should be mentioned in particular.
  • genes for the ⁇ -15, ⁇ -12, ⁇ -9, ⁇ -6, ⁇ -5 desaturase, ⁇ -ketoacyl reductases, ⁇ -ketoacyl synthases, elongases or the various hydroxylases and Called acyl-ACP thioesterases.
  • Desaturase and elongase genes are advantageously used in the nucleic acid construct.
  • DNA fragments When preparing an expression cassette, 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 organism, for example to the host plant.
  • the expression cassette contains in the 5'-3 'transcription direction the promoter, a DNA sequence which codes for a lipoxygenase gene and a region for the transcriptional termination. Different termination areas are interchangeable.
  • 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 are possible, v / fro mutagenesis, primer repair, restriction or ligation can be used. With suitable manipulations, such as restriction, -chewing-back- or filling of overhangs for -bluntends-, complementary ends of the fragments can be provided for the ligation.
  • Preferred polyadenylation signals are vegetable polyadenylation signals, preferably those which essentially comprise T-DNA polyadenylation signals
  • 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 corresponding functional equivalents.
  • An expression cassette is produced by fusing a suitable promoter with a suitable lipoxygenase DNA sequence and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
  • 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 nucleic acid sequences according to the invention are advantageously cloned together with at least one reporter gene into an expression cassette which is introduced into the organism via a vector or directly into the genome.
  • This reporter gene should enable easy detection via a growth, fluorescence, chemo, bioluminescence or resistance assay or via a photometric measurement.
  • These genes enable the transcription activity and thus the expression of the genes to be measured and quantified easily. This enables genome sites to be identified that show different levels of productivity.
  • 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 intervening coding sequence for the lipoxygenase DNA sequence.
  • An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.
  • the sequences preferred for the operative linkage are targeting sequences to ensure subcellular localization in plastids, advantageously in chloroplasts.
  • An expression cassette can contain, for example, a constitutive promoter (preferably the USP or napin promoter), the gene to be expressed and the ER retention signal.
  • a constitutive promoter preferably the USP or napin promoter
  • the amino acid sequence KDEL lysine, aspartic acid, glutamic acid, leucine
  • KDEL lysine, aspartic acid, glutamic acid, leucine
  • the expression cassette is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host organism.
  • a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host organism.
  • Suitable plasmids are, for example, in E.
  • Yeast promoters are, for example, 2 ⁇ M, pAG-1, YEp6, YEp13 or pEMBLYe23.
  • Examples of algae or plant promoters are pLGV23, pGHIac + , pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, L, 1988).
  • the above-mentioned vectors or derivatives of the above-mentioned vectors represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford , 1985, ISBN 0444 904018).
  • Suitable plant vectors are described in "Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Chap. 6/7, p.71-119.
  • Advantageous vectors are so-called shuttle vectors or binary vectors that replicate in E. coli and Agrobacterium.
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally. Chromosomal replication is preferred.
  • the expression cassette according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized plasmid or only the expression cassette as a vector or the nucleic acid sequences according to the invention.
  • nucleic acid sequence according to the invention can also be introduced into an organism on its own.
  • nucleic acid sequence according to the invention can all be introduced into the organism together with a reporter gene in a single vector or each individual gene with a reporter gene in one vector, the different vectors being introduced simultaneously or successively can.
  • the vector advantageously contains at least one copy of the nucleic acid sequences according to the invention and / or the expression cassette according to the invention.
  • the plant expression cassette can be transformed into the transformation vector pRT ((a) Toepfer et al., 1993, Methods Enzymol., 217: 66-78; (b) Toepfer et al. 1987, Nucl. Acids. Res. 15: 5890 ff. ) to be built in.
  • Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Ine; Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein, or protein A.
  • GST glutathione S-transferase
  • E. coli expression vectors are pTrc [Amann et al., (1988) Gene 69: 301-315] and pET vectors [Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, Netherlands].
  • vectors for use in yeast are pYepSed (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives (Invitrogen Corporation, San Diego, CA).
  • Vectors for use in filamentous fungi are described 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., P. 1-28, Cambridge University Press: Cambridge.
  • insect cell expression vectors can also be used advantageously, e.g. for expression in Sf 9 cells. These are e.g. the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • lanceol cells or algae cells can advantageously be used for gene expression.
  • lance expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721.
  • nucleic acid sequences according to the invention can be expressed in mammalian cells.
  • Examples of corresponding expression vectors are pCDM8 and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBOJ. 6: 187-195).
  • Promoters to be used are preferably of viral origin, e.g. Promoters of polyoma, adenovirus 2, cytomegalovirus or simian virus 40. Further prokaryotic and eukaryotic expression systems are mentioned in chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the nucleic acids according to the invention, the expression cassette or the vector can be introduced into organisms, for example in plants, by all methods known to the person skilled in the art.
  • the person skilled in the art can use the corresponding textbooks from Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by FM Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by DM Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or Guthrie et al. See Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods include protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - the so-called particle bombardment method, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and gene transfer mediated by Agrobacterium.
  • the methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol.
  • 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).
  • Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as e.g. of tobacco plants can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the transformation of kingdoms with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl.
  • Agrobacteria transformed with an expression vector according to the invention can also be used in a known manner to transform plants such as test plants such as Arabidopsis or crop plants such as cereals, corn, oats, rye, barley, wheat, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, Potato, tobacco, tomato, carrot, paprika, rapeseed, tapioca, manioc, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species, in particular of oil-containing crops, such as soybean, peanut, castor oil, sunflower, corn, Cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean are used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • test plants such as Arabidopsis or crop plants
  • crop plants such as cereals, corn, oats
  • the genetically modified Lancel cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by SD Kung and R. Wu, Potrykus or Höfgen and Willmitzer.
  • the expression cassette or the vector are all organisms which are able to synthesize fatty acids, especially unsaturated fatty acids or are suitable for the expression of recombinant genes such as microorganisms, non-human animals or Plants beneficial all crops.
  • Examples include cultivated plants such as soybean, peanut, castor oil, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, Yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • fungi for example the genus Mortierella, Saprolegnia or Pythium
  • bacteria such as the genus Escherichia
  • Yeasts such as the genus Saccharomyces
  • cyanobacteria ciliates
  • algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • Organisms which can naturally synthesize oils in large amounts such as fungi such as Mortierella alpina, Pythium insidiosum or plants such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, calendula, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, are particularly preferred soy, rapeseed, sunflower, flax, calendula or Saccharomyces cerevisiae.
  • transgenic animals are also suitable as host organisms, for example C. elegans.
  • transgenic organism or transgenic plant within the meaning of the invention is understood to mean that the nucleic acids used in the method are not in their natural place in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously.
  • transgene also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been changed.
  • Transgenic is preferably to be understood as meaning the expression of the nucleic acids according to the invention at a non-natural location in the genome, that is to say that the nucleic acids are homologous or preferably heterologous.
  • Preferred transgenic organisms are fungi such as Mortierella or plants such as crop plants, advantageously as oil crop plants.
  • Transgene means, for example, with respect to a nucleic acid sequence, an expression cassette or a vector containing a nucleic acid sequence which codes for the lipoxygenase or its derivatives, or an organism transformed with this nucleic acid sequence, an expression cassette or a vector, all such constructions which have been obtained by genetic engineering methods which either a) the lipoxygenase nucleic acid sequence, or b) a genetic control sequence functionally linked to the lipoxygenase nucleic acid sequence, for example a promoter, or c) (a) and (b) are not in their natural, genetic environment or modified by genetic engineering methods where the modification can be, for example, substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues.
  • Natural genetic environment means the natural chromosomal locus in the organism of origin or the presence in a genomic library.
  • the natural, genetic environment of the nucleic acid sequence is preferably at least partially preserved.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, very particularly preferably at least 5000 bp.
  • Another object of the invention relates to the use of an expression cassette containing DNA sequences coding for a lipoxygenase gene or DNA sequences hybridizing therewith for the transformation of plant cells, tissues or plant parts.
  • the aim of the use is to increase the content of hydroperoxy fatty acids in these organisms.
  • transgenic plants that overproduce lipoxygenase, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention.
  • a preferred subject matter according to the invention are transgenic plants containing a nucleic acid sequence according to the invention, an expression cassette or a vector containing the nucleic acid sequence according to the invention which codes for a lipoxygenase.
  • the expression cassette or the nucleic acid sequences according to the invention containing a lipoxygenase gene sequence can also be used to transform the above-mentioned organisms such as bacteria, cyanobacteria, yeasts, filamentous fungi, ciliates and algae with the aim of increasing the content of hydroperoxy fatty acids.
  • the invention relates, as described, to transgenic compounds, transformed with an expression cassette containing a lipoxygenase gene sequence or DNA sequences hybridizing therewith, and transgenic cells, tissues, parts and propagation material of such plants.
  • Transgenic crops such as barley, wheat, rye, oats, corn, soybeans, rice, cotton, sugar beet, rapeseed and canola, sunflower, flax, hemp, potatoes, tobacco, tomato, rapeseed, tapioca, cassava, are particularly preferred , Arrowroot, alfalfa, lettuce and the various tree, nut and wine species.
  • Plants in the sense of the invention are mono- and dicotyledonous plants, mosses or algae.
  • the fatty acids can be released from the oils or lipids, for example by basic hydrolysis, for example with aqueous NaOH or KOH or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol, or by enzymatic elimination. They can then be isolated by, for example, phase separation and subsequent acidification using, for example, H 2 SO 4 .
  • the fatty acids can also be released directly without the workup described above.
  • transgenic plants are advantageously used as organisms, such as oil-containing plants. These plants contain the hydroperoxy fatty acids synthesized in the process according to the invention and can advantageously be marketed directly without the oils, lipids or fatty acids synthesized having to be isolated.
  • Plants in the process according to the invention include whole plants and all parts of plants, plant organs or parts of plants such as leaf, stem, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, calli, kotelydones, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that are derived from the transgenic plant and / or can be used to produce the transgenic plant.
  • the semen comprises all parts of the semen such as the seminal shell, epidermal and sperm cells, endosperm or embyro tissue.
  • the compounds produced in the process according to the invention can also advantageously be isolated from the organisms in the form of their oils, fats, lipids and / or free fatty acids.
  • Hydroperoxy fatty acids produced by this method can be harvested by harvesting the organisms either from the culture in which they grow or from the field. This can preferably be done by pressing or extracting the body parts Plant seeds are made.
  • the oils, fats, lipids and / or free fatty acids can be obtained by cold pressing or cold pressing without the addition of heat by pressing. So that the plant parts, especially the seeds, can be more easily broken down, they are crushed, steamed or roasted beforehand. The seeds pretreated in this way can then be pressed or with
  • Solvents such as warm hexane can be extracted.
  • the solvent is then removed again.
  • these are extracted directly after harvesting, for example, without further work steps, or extracted after digestion using various methods known to the skilled worker. In this way, more than 96% of the compounds produced in the process can be isolated.
  • the products thus obtained are then processed further, that is to say refined.
  • the plant mucus and turbidity are removed first.
  • degumming can be carried out enzymatically or, for example, chemically / physically by adding acid such as phosphoric acid.
  • the free fatty acids are then removed by treatment with a base, for example sodium hydroxide solution.
  • the product obtained is washed thoroughly with water to remove the lye remaining in the product and dried.
  • the products are subjected to bleaching with, for example, bleaching earth or activated carbon.
  • the product is still deodorized with steam, for example.
  • hydroperoxy fatty acids produced in the process are advantageously obtained in the form of their oils, lipids or fatty acids or fractions thereof.
  • oil lipid
  • fat is understood to mean a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat has a high proportion of free or advantageously esterified hydroperoxy fatty acid (s).
  • the proportion of esterified hydroperoxy fatty acids is preferably approximately 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80% or more. Depending on the starting organism, the proportion of different fatty acids in the oil or fat can fluctuate.
  • the hydroperoxy fatty acids produced in the process are, for example, hydroperoxy fatty acids which are bound in sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
  • Microorganisms are usually in a liquid medium containing a carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as Contains iron, manganese, magnesium salts and possibly vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C with oxygen.
  • the pH of the nutrient liquid can be kept at a fixed value, that is to say it can be regulated during cultivation or not.
  • the cultivation can be batch-wise, semi-batch wise or continuous. Nutrients can be introduced at the beginning of the fermentation or fed semi-continuously or continuously.
  • the lipids are usually obtained from the organisms.
  • the organisms can first be digested after harvesting or used directly.
  • the lipids are advantageously extracted with suitable solvents such as apolar solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol at temperatures between 0 ° C. to 80 ° C., preferably between 20 ° C. to 50 ° C.
  • the biomass is usually extracted with an excess of solvent, for example an excess of solvent to biomass of 1: 4.
  • the solvent is then removed, for example by distillation.
  • the extraction can also be carried out with supercritical CO 2 . After extraction, the remaining biomass can be removed, for example, by filtration.
  • the crude oil obtained in this way can then be further purified, for example by removing turbidity by adding polar solvents such as acetone or chloroform and then filtering or centrifuging. Further cleaning via columns is also possible.
  • polar solvents such as acetone or chloroform
  • Further cleaning via columns is also possible.
  • free fatty acids from the triglycerides they are usually saponified as described above.
  • PQE30 TM is from Qiagen
  • Hilden pGem-T is from Promega, Madison, USA
  • Kanamycin stock solution 50 mg / ml, final concentration: 25 ⁇ g / ml
  • a colony PCR was carried out to identify the bacterial colonies which contain the desired fragment in the plasmid DNA after transformation.
  • Cell material from a colony was suspended in 10 ⁇ l of water using a toothpick.
  • PCR reaction mixture was prepared by adding 10 ⁇ buffer, MgCl 2 , dNTP, Tfl polymerase and the corresponding primer. The same program was run and the same primers used to amplify the fragment. If a primer was used for the amplification, which also has a binding site in the vector, this primer has been replaced by vector-specific primers. If the fragment was not cloned directionally in this case (eg in pGEM-T), colony PCR was carried out for both directions with the corresponding vector-specific primers. For the identification of bacterial colonies, which contain DNA constructs in the vector, which were composed of more than one PCR fragment, the 5 'primer of the fragment located at the 5' end and the 3 'primer of the fragment located at the 3' End fragment was used.
  • thermostable Taq DNA polymerase contains a mixture of the thermostable Taq DNA polymerase and the also thermostable Pwo DNA polymerase, which also has a 3'-5 'exonuclease activity, and only 30 cycles were used instead of 35. Both served to keep the error rate low in order to minimize mutations in the to obtain amplifying DNA.
  • DNA fragments were linked to vector DNA by ligation using T4 DNA ligase. Terminal 5'-phosphate groups and 3'-hydroxyl groups are bonded to form phosphorus diester bonds.
  • the cofactor of this reaction is ATP.
  • the DNA fragment was used in a three-fold excess compared to the vector DNA. The ligation reaction took place at 4 ° C. overnight. The ligation was carried out in a volume of 10 ⁇ l or 20 ⁇ l.
  • PCR-amplified DNA fragments were first cloned in pGEM-T. Fragments amplified with Tfl or "EXPAND TM High Fidelity" have a 3'-adenine overhang which hybridizes with the 3'-thymine overhang of the pGEM-T vector. This is one of the reasons why the ligation efficiency of this vector is relatively high pGEM-T offers the possibility of blue-white selection with IPTG and X-Gal.
  • Ligation mix 5 ⁇ l 2x ligation buffer, 3 ⁇ l DNA (fragment), 1 ⁇ l vector,
  • the E. coli strains XL1-Blue and SG 13009 were transformed. 100 ⁇ l of competent E. coli cells were thawed on ice and about 5 fmol of DNA were added. This corresponds to 10 ng of a 3000 bp DNA construct or 17 ng of a 5000 bp DNA construct. After 20 min incubation in ice, the bacteria were exposed to a heat shock at 42 ° C for 50 s. The mixture was then left on ice for 5 min, 900 ⁇ l of LB medium were added and the mixture was cultured at 37 ° C. with shaking for 1 h. The bacteria were plated on selection medium according to their resistance and incubated overnight at 37 ° C.
  • Restriction nucleases cleave double-stranded DNA in a sequence-specific manner by hydrolysis of covalent bonds. 4 to 8 bp long palindromic sequences are recognized and cut. Cleavages with the restriction endonucleases used in this work result in ends that carry a 3 'or 5' overhang.
  • Restrictions were carried out to control bacterial colonies which are suitable for the cloning strategy (see Example 28) and for obtaining DNA fragments for the ligation (Example 3). Cutting out the DNA fragments in two steps. After the restriction with the first enzyme, the reaction mixture was cleaned and only then was the restriction with the second enzyme. This was done in order to increase the yield of correctly cleaved DNA by cleaving each enzyme in the optimal buffer with 100% activity without unspecific side reaction (“star activity”). The control of bacterial colonies that were suitable for the cloning strategy was carried out Only one enzyme was used per batch in order to obtain a clear statement for each enzyme.Table 2 shows the batch cleavage approaches used, and the optimum buffer was the reaction buffer Buffer used according to the manufacturer's instructions. The restriction mixture was incubated at 37 ° C. for about 12 h. Table 2: Cleavage of DNA with restriction endonucleases
  • Restriction mixture 1 ⁇ l 10x reaction buffer, 2 ⁇ l plasmid DNA (approx. 0.6 ⁇ g), for screening 0.21 ⁇ l restriction enzyme, 1.8 ⁇ l H 2 O
  • DNA fragments of 0.2 to 4 kb could be separated effectively using a 1.5% agarose gel.
  • 1.5% (w / v) agarose was dissolved in TAE buffer by boiling in the microwave, cooled to 60 ° C. and poured into a horizontal gel carrier. The polymerization was cooled for at least 30 minutes.
  • the DNA samples were mixed with 0.1 volume of sample buffer and separated electrophoretically at a voltage of 120 V.
  • the S ARTLADDER standard Eurogentec, Seraing, Belgium
  • DNA fragments of a defined size was used as the size marker.
  • the agarose gel was then incubated for 15 to 20 min in an ethidium bromide bath (2 ⁇ g / ml) and decolorized in distilled water for about 5 min.
  • the agarose gels were photographed for documentation and evaluation.
  • the plamide mini preparation was carried out either with the "QLAprep Plasmid MiniPrep Kit” (Qiagen, Hilden) or with the "NucleoSpin® Plasmid MiniPrep Kit” (Macherey-Nagel, Düren), each according to the method specified by the manufacturer. These methods are based on the binding of DNA to silica gel membranes at high concentrations of chaotropic salts. Elution is carried out with low-concentration salt buffers or with water. With this method, the DNA is separated from other cell components and preserved in high purity.
  • Example 8 DNA fragment isolation from agarose gels
  • the gel band of the DNA fragment to be purified was cut out cleanly under UV light with a scalpel and cleaned with the “GFT TM PCR DNA and Gel Band Purification Kif according to the manufacturer's instructions (Amersham Pharmacia Biotech, UK). In order to avoid mutations in the DNA fragments, care was taken when isolating the fragments that the UV irradiation time was only very short.
  • sequences were analyzed with HUSAR (DKFZ, Heidelberg) using the fragment assembly programs that are part of the WISCONCIN package version 10.2 (Actylrys, formerly Genetics Computer Group (GCG), Madison, Wisc.). Sequences were loaded into the project, components of the cloning vector and phage bank vector were removed semi-automatically and then assembled into sequences (Contig's), sequence comparison against the databases SWISSPROT, TREMBL, PIR and OWL was carried out using the BLASTX2 algorithm [Altschul et al., Nucleic Acids Res, 25 (17): 3389-3402, 1997].
  • a phylogenetic family tree was created with the PHYLIP 35 program.
  • SEQBOOT random seed number: 5
  • the known lipoxygenase proteins are divided into three family tree segments, namely the type 1 13-LOX, type 1 9-LOX and type 2 13-LOX family tree.
  • PROTPARS creates the pedigrees of the one hundred point mutation series by calculating the evolutionary distances between the proteins. With CONSENS, the most probable is calculated from the one hundred family trees.
  • the removal of two proteins in the phylogenetic family tree is based on the number of mutations that are necessary at the DNA level to match the sequence of one protein to the other.
  • the genetic code is taken into account. For example, three point mutations are necessary for a Gln / Cys exchange. The different mutation rates of organisms are not included.
  • Example 10 Expression of the recombinant protein
  • the expression of the recombinant protein took place in the E. coli strain SG 13009.
  • the expression clones were initially incubated up to an OD 600 of 0.6 to 0.8 in LB medium (with cabenicillin and kanamycin) at 37 ° C. After induction of the expression with IPTG (final concentration: 1 mM), the bacteria were cultivated at 10 ° C. for 2 or 7 days.
  • Example 11 Cell disruption
  • the E. coli cells were centrifuged at 4000 xg and 4 ° C for 20 min and taken up in lysine buffer (50 mM Tris.HCI, 10% glycerol, 0.1% Tween, 0.5 M NaCI, pH 7.5) ( 7 ml lysine buffer per 250 ml cell culture). The cells were disrupted using ultrasound (ten times 1 min, 50% power, 50% pulse). The cell debris was centrifuged at 4000 x g, 4 ° C for 30 min. The supernatant with the soluble proteins was removed and stored at 4 ° C. The activity of the recombinantly produced lipoxygenase was analyzed with this lysate.
  • lysine buffer 50 mM Tris.HCI, 10% glycerol, 0.1% Tween, 0.5 M NaCI, pH 7.5
  • the lipoxygenase was tested for activity.
  • the hydro (pero) xides formed have an absorption maximum at 234 nm due to their conjugated diene system. Due to the high self-absorption of the cell lysate, this determination was not carried out spectrophotometically.
  • the products of the fatty acid conversion were therefore carried out by means of normal phase HPLC (see Example 15). The reaction was carried out according to the protocol given below at pH 6 and 8.
  • the position isomers formed were analyzed with devices from AGILENT SERIES IIOO (Hewlett-Packard, Waldbronn) using normal phase columns (50 x 4.6 mm, 3 ⁇ m LUNA SILICA (2), Phenomenex, Aillesburg, or 150 x 2, 1 mm, 5 ⁇ m, ZORBAX RX-SIL, Hewlett Packard, Waldbronn) and a diode array detector were analyzed.
  • the fatty acid hydroxides purified by normal-phase HPLC were equipped with a chiral-phase column (150 ⁇ 2.1 mm, 5 ⁇ m Daicel Chemical Industries Ltd.CHIRACEL OD-H, Merk, Darmstadt) using the system described above (Example 15) ), examined for their enantiomer composition.
  • Hexane / iso-propanol / TCA (95: 5: 0.2, v / v / v, isocratic flow 100 ⁇ l / min, 20 min) was used as the eluent for the hydroxides of linoleic acid and ⁇ -linoleic acid, and for the hydroxide Derivatives of y-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid were used in the solvent 98: 2: 0.02 (v / v / v, isocratic flow 100 ⁇ l / min, 60 min ). The data- recording and evaluation was carried out with the software CHE STATION FOR LC 3 D [Rev. A.08.01 (783)] from Agilent Technologies Waldbronn.
  • hydroxy fatty acids are derivatized to increase their volatility and thermal stability and to reduce polarity [W.W. Christie, Lipids, 33 (4): 343-53, 1998]. This increases the gas chromatographic separation and improves the detectability.
  • the derivatization of hydroxy fatty acids to their methyl ester trimethylsilyl ethers allows the compound to be identified and the position of the hydroxy group to be determined [Wollard et al., J Chromatogr, 306: 1-21, 1984].
  • a quarter of the turnover of the batches for the substrate determination was purified by means of normal phase HPLC.
  • the speculative hydroxides of the fatty acids (absorption at 234 to 237 nm) were collected, completely evaporated in a stream of nitrogen and taken up in 400 ⁇ l of methanol.
  • 10 ⁇ l of the EDAC stock solution (100 mg / ml) were added and shaken at room temperature for 2 h.
  • the mixture was shaken twice with 1 ml of n-hexane.
  • the hexane phases were evaporated in a stream of nitrogen.
  • Carrier gas helium linear flow rate 1, 2 ml / min
  • Separation column 30 mx 0.25 mm HP-5MS (Crosslinked 5% PH ME Siloxane); 0.25 ⁇ m film thickness; Temperature program: 60 ° C (injection temperature) ⁇ 25 ° C / min ⁇ 300 ° C (1 min) ⁇ 10 ° C / min ⁇ 270 ° C (10 min) MS Aligent 5973 Network ionization energy 70 eV Scanning speed 1 scan / s
  • the cells were disrupted as described in Example 11, the cell debris centrifuged off and the supernatant incubated overnight at 4 ° C. with "TALON® Metal Affinity Resin".
  • the binding capacity is 5 mg / ml bed volume the protein supernatant, which was obtained from 500 ml of cell culture, was used in 250 to 500 ⁇ l of TALON®, and the column material was then placed on an empty column (TJ Baker, Phillipsburg, NJ, USA). Buffer (50 mM, 300 mM NaCl, pH 8.0) was washed in.
  • simple bed volumes of Na phosphate buffer were used
  • the protein was determined using the “Bio-Rad protein assay” from Bio-Rad (Hercules, USA) according to the manufacturer's instructions using a BSA calibration line.
  • Example 21 SDS.polyacrylamide gel electrophoresis (SDS-PAGE [U.K. Laemmli, Nature, 227 (259): 680-5, 1970], modified method)
  • the protein samples were mixed directly with sample buffer or precipitated with TCA (trichloroacetic acid).
  • TCA trichloroacetic acid
  • 1 ml of sample was incubated with 250 ⁇ l of 30% TCA for 30 min on ice, then centrifuged for 10 min at 14,000 ⁇ g and the precipitate obtained was washed again with water. After centrifugation and the removal of the aqueous supernatant, the precipitate was dried and dissolved in "Roti®-Load 1" sample buffer. Before application, the samples were centrifuged at 15000 xg for 3 min.
  • the samples were separated electrophoretically in an 8% polyacrylamide gel (separating gel: 2.15 ml water, 1, 2 ml acrylamide / methylbisacrylamide (30% / 0.8%), 1, 1 ml 4 x separating gel buffer, 14 ⁇ l APS ( 50%), 3.5 ⁇ l TEMED; stacking gel: 0.87 ml water, 0.24 ml acrylamide / methylbisacrylamide (30% / 0.8%), 0.375 ml 4 x collecting gel buffer, 6 ⁇ l APS, 3 ⁇ l (50% )).
  • the sample was run into the collection gel for 25 min at 12.5 mA per gel.
  • the separation of the proteins in the separating gel was carried out at a current of 25 mA per gel for 40 min or until the bromophenol blue front had reached the end.
  • the separated proteins were stained in the gel.
  • the gel was washed three times for 30 min with ethanol / acetic acid / water (30: 5: 65, v / v / v) [for all buffers and washing steps up to / incl. 18 M ⁇ water was used in the development.] and then washed three times with water for 10 min.
  • Sensitization was carried out by incubation with 0.02% Na thiosulfate solution (freshly prepared) for 1 min, after which it was washed twice with water for 1 min. After impregnation with silver nitrate solution (0.025% formaldehyde, 0.0125% AgNO 3 ) for 30 minutes, care was taken to wash with water for a maximum of 15 seconds (5 to 15 seconds).
  • the corresponding ALP conjugate was diluted 1: 5000, incubated with the membrane for 1 h, washed twice in PBST for 5 min, once with PBS for 5 min and once in AP buffer for 10 min. NBT / BCIP was used diluted in AP buffer according to the manufacturer's instructions.
  • RNA of the diatom P. tricornutum was in the form of a “Lambda Zap II Express” cDNA library, into which it was cloned in a directional manner.
  • the underlying vector was pBK-CVM.
  • the complete sequence was then cloned into a bacterial expression vector
  • the following biochemical characterization of the recombinant active enzyme included the determination of the pH optimum and the substrate specificity Hydroxy fatty acids were not available as standard substances, they were characterized as combined methyl ester and trimethylsilyl ether derivatives by means of GC / MS analysis. Purification for the recombinant enzyme was also started.
  • a cDNA library was created from the P. tricornutum diatom. Furthermore, an in vivo excision was carried out, the plasmids were recovered and transformed into E. coli DH1 OB. Then plasmid DNA was obtained automatically and random sequencing was carried out using the chain termination method. 8400 clones were sequenced at the 5 'end, combined into 3400 non-redundant sequences (Contig's) and annotated. Subsequent analysis of the database created in this way identified two clones via their 5 'sequence which showed homologies to plant lipoxygenases. One of them was the lipoxygenase from P. tricornutum (LOX2: R: 1 (PtLOXI) described in this work.
  • Clone Pt001077095r showed the greatest homology to lipoxygenase with 42% at the DNA level in the sequenced region of 800 bp of the 5 'end - 1 from Pisum sativum.
  • the 3 'end of this cDNA clone was then sequenced to verify this finding (FIG. 4, gray area).
  • the homology was 55% to the same lipoxygenase from Pisum sativum.
  • this sequence comparison showed that presumably around 500 bp of the open reading frame (ORF) was missing at the 5 'end.
  • the gene-specific primer Pt-LOX2_5RACE (1) (5'-GAG CCC CTG.TCT TCT CGG TAT TG) was derived from the known sequence in the 5 'region.
  • 5'-RACE with this gene-specific and vector-specific primer 3 (5'-GCT CGA AAT TAA CCC TCA CTA AAG GG) gave the fragments of different lengths shown in FIG. 4, which extend the known sequence by around 650 bp in 5'- Extended direction.
  • the dark gray highlighted sequences of clone Pt001077095r resulted from the random sequencing of 8400 clones from a cDNA bank of the diatom P. tricornutum.
  • the sequences highlighted in light gray were obtained using 5'-RACE.
  • the arrows indicate the primers mentioned above.
  • the upper curve shows the quality of the sequence information, which is defined by the number and the extent to which the sequences match.
  • a further sequence extension with the primer PtLOX2_5RACE (2) (5'-CAA TAT CGA TCA AAC CTC GCT AC), which binds 677 bp upwards from the first 5'-RACE primer, could not be achieved.
  • PtLOX2_5RACE (2) 5'-CAA TAT CGA TCA AAC CTC GCT AC
  • the sequence information obtained would nevertheless be sufficient to derive primers with which the complete DNA sequence of the PtLOXI from the underlying phage library can be amplified and cloned.
  • the complete sequence was composed of two overlapping fragments.
  • the cDNA clone that had been identified by means of random sequencing (Pt001077095r) was first completely sequenced.
  • a 1336 bp region (fragment 1) representing the 5 'end of the PtLOX sequence was primed using A (5'-GGJ_ACC ATG ATG CTC AAC CGG TTG AC) and B (5'-AAA TTC CCG AGC AAA CTC GT) amplified from the P. tricornutum cDNA library (see FIG. 4).
  • primer A which contains the start codon ATG
  • Kpnl was introduced.
  • the second fragment of the gene was present in the vector pBK-CMV in the form of the clone with which the two ESTs were obtained.
  • In the overlapping region of 787 bp of the two fragments is the restriction site for Sal I that cuts uniquely in the sequence.
  • this fragment was primed M13-rev (5'-GGA AAC AGC TAT GAC CAT G) and the primer C (5'-CCC AAG CTT CTA TAT GGT GAT GCT GTT GGG CAC) were amplified by PCR. Both fragments were first cloned into the vector pGEM-T and then assembled into unique gene sequences via unique restriction sites introduced by means of PCT in pQE30. Fragment 2 was ligated to the expression vector pQE30 via the restriction sites for Sal I and Hind III. After the corresponding restriction digest, this construct was used using the restriction sites for Kpn I and Sal I with fragment! ligated.
  • PtLOXI's ORF consists of 938 amino acids and has a calculated molecular weight of 105 kDa. Possible glycosylations in the original organism P. tricornutum were not taken into account. The calculated isoelectric point is pH 6.69. The beginning of the protein sequence with two methionines is unusual.
  • the first methionine is considered the translation start codon and was used for cloning.
  • the cDNA is 66 bp longer in the 5 'direction than the sequence region used for the cloning. Since there is no starting methionine in this region, it was considered an untranslated region at the 5 'end.
  • LOX1 Hv: 3 and 13-epoxygenases and not 9-lipoxygenases. Nevertheless, plastid localization can be discussed for the RLOX1. Due to lower security modes, an analysis with other databases was not carried out.
  • PtLOXI shows some interesting differences at the level of the protein sequence.
  • three determinants have been determined that have an influence on the substrate and position specificity, whereby. the BORNGR ⁇ BER and SLOANE determinants are to be emphasized (summarized in [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley, -VCH, Einheim, 2000]).
  • PtLOXI differs from all other lipoxygenases in that it separates two hydrophobic amino acids from the hydrophilic amino acid threonine, the C-terminus is highly conserved as with all other lipoxygenases, with the small difference that threonine is in the penultimate position instead of being, as with all other lipoxygenases.
  • the pH optimum was determined by means of an end point determination using normal phase HPLC for the conversion of linoleic acid.
  • Cell digests (Example 11) of protein expression at 10 ° C. were used for the analyzes.
  • the lysates were used immediately after their preparation in order not to reduce the stability of the proteins by freezing and thawing the samples.
  • the cell disruption was incubated with linoleic acid and the resulting hydroperoxide derivatives were reduced to the corresponding hydroxides with sodium borohydride, which were analyzed after the acidic extraction using normal-phase HPLC (SP-HPLC).
  • SP-HPLC normal-phase HPLC
  • the areas of the integrated HODE signals at 234 nm and the linoleic acid at 202 nm were evaluated. From FIG. 6 it can be seen that the maximum activity of the PtLOXI for linoleic acid is achieved at pH values between pH 8.0 and pH 8.4.
  • Linoleic acid was reduced in substrate excess for 30 min with enzyme extract and the hydroperoxides formed were reduced to the hydroxides. After acid extraction, the substrate product spectrum was analyzed using SP-HPLC. The sum of the area of all integrated HODE signals at 234 nm was related to the area of the integrated linoleic acid signal at 202 nm. The representation in FIG. 6 shows the average and the standard error for two experiments.
  • Example 32 Analysis of the regiospecificity towards linoleic acid
  • a characteristic feature of lipoxygenases is their regiospecificity when introducing molecular oxygen [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000]. Since linoleic acid is a substrate for lipoxygenases both in the animal kingdom and in the plant kingdom and, in contrast, arachidonic acid does not occur in the plant kingdom or only occurs in storage lipids, plant lipoxygenases are classified in terms of the position specificity of the oxygenation of linoleic acid [H.W. Gardner, Biochim Biophys Acta, 1084: 221-239, 1991]. Oxygen can be introduced either at carbon atom 9 (9-lipoxygenase) or 13 (13-lipoxygenase) of linoleic acid.
  • Linoleic acid was incubated in excess substrate for 30 min with crude extract enzyme and the hydroperoxides formed were reduced to the hydroxides. After acid extraction, the substrate product spectrum was analyzed using SP-HPLC. The integrated signals of 9-HODE and 13-HODE at 234 nm were compared to the integrated linoleic acid signal at 20 nm. The representation in FIG. 7 shows the average and the standard error for two experiments.
  • the ratio of 13 to 9 HODE is also summarized in Table 3.
  • Example 33 Substrate specificity of PtLOXI and analysis of the regioisomers formed
  • fatty acids were used: linoleic acid (LA), ⁇ -linolenic acid ( ⁇ -LEA), Y-linolenic acid ( ⁇ -LEA), arachidonic acid (ERA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).
  • LA linoleic acid
  • ⁇ -LEA ⁇ -linolenic acid
  • ⁇ -LEA Y-linolenic acid
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • DPA docosapentaenoic acid
  • DHA docosahexaenoic acid
  • the oxygen addition can therefore form four hydroperoxide derivatives from ⁇ -LEA (9-, 13-, 12-, 16- ⁇ -HPOTE) and ⁇ -LEA (6-, 10-, 9-, 13- ⁇ -HPOTE), six from ARA (5-, 9-, 8-, 12-, 11-, 15-HPETE), eight from EPA (5th -, 9-, 8-, 12-, 11-, 15-, 14-, 18- HEPE), eight from DPA (7-, 11-, 10-, 14-, 13-, 17-, 16-, 20-HPDPE) and ten from DHA (4-, 8-, 7-, 11-, 10-, 14-, 13-, 17-, 16-, 20-HPDHE).
  • the enzyme was incubated with various substrates at pH 8.4 and the product analysis was carried out using SP-HPLC.
  • the sum of the integrated signals of all hydroxide derivatives was plotted for the conversion of each fatty acid.
  • the difference- The extinction coefficients of the hydroxides were not taken into account during the integration.
  • the representation in FIG. 8 shows the average and the standard error of two experiments.
  • Example 32 As already treated in Example 32, the conversion of linoleic acid leads to 90% formation of 13-HODE in the S configuration.
  • the chromatogram of the SP-HPLC analysis is shown in FIG. 9.
  • the control implementation was carried out with cell disruption of SG 13009 cells which carried the empty vector.
  • the chromatogram of this reaction (FIG. 9, lower part) shows that 9-HODE was formed enzymatically and does not result from autoxidation.
  • the same amounts of the turnover of the fatty acid with recombinant PtLOXI and the control conversion were analyzed.
  • the proportion of the two possible oxidation products in the [+2] and [-2] positions with respect to the carbon (n-8) in the total hydro (pero) oxide derivatives formed (FIG. 10) is 92 % and is therefore comparable to the sales of LA.
  • the ratio of the [+2] to [-2] oxidation products drops from 6.7 for 13- / 9-HODE to 2.7 for 13- / 9- ⁇ -HOTE.
  • the factor 6.7 / 2.7 «2.5 which can be calculated with this was observed even more frequently when the number of double bonds or the number of methylene groups changed.
  • the bisallylic methylene group in position (n-11) was preferentially attacked by the hydrogen acceptor, which leads to the formation of 10- and 6- ⁇ -HOTE.
  • the methylene group at position (n-8), in which the hydro (pero) oxide derivatives 13- and 9- ⁇ -HOTE are formed play a role with a total share of around 17% of the total hydro (pero) oxide derivatives subordinate role. But here too the ratio is in favor of the [+2] -hydro (pero) oxide derivative.
  • the ratio of the main products 10- to 6- ⁇ -HOTE is comparable to that of 13- to 9- ⁇ -HOTE from ⁇ -LEA.
  • the respective [-2] hydro (pero) oxide derivative is racemic.
  • arachidonic acid is two methylene groups longer in the "[+ X]" position in terms of the bisallylic methylene groups used (x4) and a double bond is added in the "[- X]" position (x4) ,
  • the measured ratio of 12-HETE to 8-HETE is 46.3 (97%).
  • the main product 12-HETE was unexpectedly only 79% in S configuration, although the comparative analysis in Figure 8 suggests that ERA or EPA are the preferred substrates.
  • the secondary product 8-HETE was in S configuration.
  • Example 32 In the case of arachidonic acid, the above-mentioned (Example 32) by-products were detected under aerobic conditions, namely the keto acids, to an extremely small extent of 1.2% at 273 nm. They also occurred in the implementation of DPA and DHA with around 7.0% each.
  • Example 34 GC / MS analysis of the fatty acid hydroxides
  • the derivatized fatty acids had a retention time between 16 min and 20 min in the analyzes.
  • the mass spectra shown below were recorded in this area of the chromatogram.
  • a characteristic fragment of the decay of the compound was detected in the mass spectrum, with additional signals the molecular weight of the unfragmented compound, the M-15 signal (without methyl group) and the M-31 signal (without methoxy group) being detected.
  • the possible decay products are shown with their molecular weights in the respective diagram.
  • the characteristic decay allows the compound to be identified and the position of the hydroxy group to be determined [Woollard et al., J Chromatogr, 306: 1-21, 1984].
  • the recombinant enzyme should first be purified for a detailed kinetic study of PtLOXI.
  • PtLOXI was expressed in the vector pQE30 with an N-terminal hexa-histidine tag and then an affinity purification was carried out using TALON®. This cleaning is based on the complexation of the cobalt fixed to the TALON® by histidine.
  • the protein is eluted by competitive displacement of the histidine by imidazole or a chelator of divalent metal ions (e.g. EDTA). Alternatively, elution can be carried out by lowering the pH, since protonated histidine is repelled by the divalent metal.
  • Example 36 SDS-PAGE and Western blot analysis of cleaning
  • FIG. 23a shows the SDS-PAGE for examining the optimal elution conditions with imidazole.
  • the arrow indicates PtLOXI.
  • 1 protein standard 2 control (lysate from SG 13009 cells transformed with empty vector pQE30), 3 lysate from SG 13009 cells (transformed with PtLOXI in pQE30) before induction with IPTG, 4 lysate from SG 13009 cells (transformed with PtLOXI in pQE30) after induction with IPTG, 5 flow through after affinity binding of the bacterial protein extract to TALON®, 6 washing steps with Na phosphate buffer, 7 to 15 elution with imidazole increasing concentration: 10 mM, 30 mM, 50 mM, 75 mM, 100mM, 125mM, 150mM, 200mM, 300mM.
  • the protein to be purified has a calculated size of 105 kDa.
  • Several protein bands were clearly visible in this area when the cell disruption was applied after induction and in the fractions of the elution with imidazole (FIG. 23a, arrows).
  • RLOX1 the immunodetection shown in FIG. 23b was carried out.
  • a monospecific antibody was used for this, which was directed against the lipid body lypoxygenase of the cucumber (CsIb-LOC) [Haus et al., Planta, 210: 708-714, 2000]. This antibody exhibits most other plant lipoxygenases due to its high cross-reactivity with other lipoxygenases on.
  • the recombinant protein was expressed under modified conditions. After induction of translation with IPTG, a further incubation took place overnight at 28 ° C and over a period of seven days at 10 ° C. Instead of stepwise elution (example 36), washing was carried out here only with 5 mM imidazole and the protein remaining on the column was then eluted with 125 mM imidazole (see example, 19). The cleaning confirmed that only in the fractions after the cleaning Expression of PtLOXI at 10 ° C. the protein identified as PtLOXI was present (FIG. 24, lane 19, 20, Reil). The Western blot of the expression at 10 ° C. (FIG. 25b) confirms that RLOX1 did not bind to the TALON®, since a band of the correct size was again detected in the flow and in the Na phosphate wash fraction (lanes 19 and 20, Reil).
  • SG 13009 cells were transformed with PtLOXI and, after adding IPTG, expressed at 60 ° C. for about 60 hours.
  • Neither in the cleaning of the cell lysate after the expression of the empty vector nor in the expression of PtLOXI at 28 ° C. is a protein band clearly recognizable in FIG.
  • the arrow indicates PtLOXI.
  • SG 13009 cells were transformed with the following vectors and expressed under the conditions specified in the text after IPTG addition: 1 to 8 pQE2028 ° C, 9 to 12 and 15 to 18 PtLOXI (expression at 28 ° C), 19 to 26 PtLOXI ( Expression at 10 ° C) 1, 9.19 flow rate after affinity binding of the bacterial protein extract to TALON®, 2.10.20 washing step with Na phosphate buffer,
  • Table 5 Analysis of the activity of individual fractions of the preparative purification of PtLOXI. The results for the expression of recombinant PtLOXI at 10 ° C. after addition of IPTG are shown. 40 ⁇ g of substrate were used in each case, incubated for 2 h and the same amounts were analyzed by normal phase PHLC.
  • lipoxygenase was isolated from the diatom P. tricornutum and expressed recombinantly in E. coli. So far, no isolated sequence encoding a diatom lipoxygenase has been described. Compared to vegetable lipoxygenases, lipoxygenase has an exceptional substrate specificity.
  • Type 2 lipoxygenases are lipoxygenases of chloroplastic signal sequence. PtLOXI shows relatively low homology to the representatives of the different groups based on the sequence comparison.
  • the average homology value including all representatives of the 9- and 13-lipoxygenases of type 1, is 36%. It is noteworthy that PtLOXI also has an average of only 33% homology within the group of 13-type 2 lipoxygenases, instead of the 45% otherwise usual among the group members (see Table 6). All of these findings suggest that the cleavage of PtLOXI from the development of lipoxygenases in higher plants occurred relatively early during evolution. This is in line with the evolutionary Distance between diatoms and higher plants, which is also found on other levels.
  • Table 6 Homology of the lipoxygenase groups to one another. Intersections of the group with themselves correspond to the homology of the lipoxygenases within the group.
  • the sequence comparison of known sequences formed the basis for the calculation with GENEDOC. The average of the combination of all sequences which were relevant for the respective comparison was formed (between 130 and 900 combinations). Numbers in percent.
  • Lipid body lipoxygenase plays a role in the mobilization of storage lipids [Feussner et al., Trends Plant Sei, 6: 262-267, 2001] and a chloroplastic lipoxygenase is essential for the synthesis of jasmonic acid [Creelman et al., Ann Rev Plant Physiol Plant Mol Biol, 48: 355-381, 1997].
  • PtLOXI apparently belongs to the type 2 lipoxygenases, which are characterized by a chloroplastic signal sequence.
  • the evolutionary distance to all groups of lipoxygenases is approximately the same.
  • some lipoxygenases are misclassified in the family tree, since LOX1: Cs: 3 and LOX1: Hv: 3 are 13-lipoxygenases and not 9-lipoxygenases, as one could see from the family tree. Since it is therefore not clear whether PtLOXI is actually actually of the LOX2 type or of the LOX1 type, it was examined more closely whether PtLOXI has a signal sequence.
  • PtLOXI In a sequence comparison with a selection of plant lipoxygenases, PtLOXI, like all lipoxygenases of the LOX2 type, shows an N-terminal extension of around 50 AS compared to type 1 lipoxygenases. This speaks strongly for a plastid signal sequence, but since PtLOXI is presumably evolutionarily far away from lipoxygenases of higher levels, this can also have other causes.
  • Chloroplastidale signal sequences also called transit peptides
  • the pro proteins have an interface for signal peptidase which is conserved to a limited extent in land plants and Chlamydomonas [Lang et al., J. Biol. Chem., 273 (47): 30973-30978, 1998]. Because of this relatively poor identifiability, programs have been developed with which a prediction for these transit peptides can be made.
  • the inner two membranes appear to correspond to the envelope membranes of the plastids of higher plants, the next membrane is a remnant of the endosymbiotic plasma membrane, and the fourth membrane flows smoothly into the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • Interfaces for eurkaryotic signal peptide ases are usually in the range at positions 15 to 18 of pre-pro proteins by the method of HEIJNE [G. by Heijne, Nucleic Acids Res, 14 (11): 4683-90, 1986].
  • PtLOXI at position 14
  • P max 0.4350
  • Table 7 Interfaces of stromal peptidases in diatoms and higher plants, interfaces for the LOX2 type of plant lipoxygenases were calculated with CHLOROP and PCLR.
  • the pH optima of the PtLOXI was determined by means of normal phase HPLC through the conversion of linoleic acid.
  • the method of determining the final value was used, in which the amount of product formed is determined after a certain time under different conditions. It is unsuitable for the reason that in no case can linear increase in product and decrease in substrate be guaranteed for all conditions, in particular with regard to long sales times and high enzyme activity. For this reason, the optimum curves obtained represent at comparing the activity under different conditions, never the true ratio of the activities. Nevertheless, a good indication of the optimal conditions can be obtained if the amount of enzyme used and the conversion time are chosen accordingly.
  • PtLOXI becomes active during the light reaction.
  • chloroplastic lipoxygenases with an acidic pH optimum (eg LOX2: Hv: 1 (LOX-100) [Kohlmann et al., Eur. J. Biochem., 260: 885-895, 1999].
  • the pH value drops in the stroma to this level due to stress or damage to the membranes, it is believed that these lipoxygenases become active and can induce further cell degradation (jasmonic acid formation, membrane degradation, etc.).
  • Plant-based lipoxygenases are classified into 9- and 13-lipoxygenases based on their position-specificity compared to linoleic acid, since linoleic acid is a ubiquitous substrate in plants.
  • the amino acids in the active center of the enzyme are important for the formation of the different products [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000].
  • Two hypotheses have been developed which attempt to explain the position specificity of lipoxygenases (summarized in [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000].
  • Part of the bottom of the binding pocket is a highly conserved arginine in all plant lipoxygenases, which is of central importance in the conversion of the lipid body 13-lipoxygenase from cucumber into a 9-lipoxygenase (R758 from CsIbLOX) [Hornung et al., Proc NatI Acid Sei USA, 96 (7): 4192-4197, 1999].
  • This arginine occurs regardless of substrate and position specificity and is preceded by an asparagine in over 90% of the cases.
  • the only exception among plant lipoxygenases are two histidines instead of the asparagine-arginine tandem in the PtLOXI sequence comparison.
  • Methylene group from the methyl end of the substrate determines whether the introductory hydrogen abstraction takes place at this point. It was found (Table 4) that of the bisallylic methyl groups in question, only one was used for the initial hydrogen removal for each of the substrates used. This affected the methylene group at position (n-8) in the case of LA and ⁇ -LEA and each (n-11) for ⁇ -LEA, ERA, EPA, DPA and DHA. It can thus be estimated that the distance of the hydrogen acceptor from the bottom of the binding pocket is around eleven methylene groups.
  • DPA is unlikely because both products ([+2] and [-2] rearrangement) are formed in a 1: 1 ratio and the amount of product formed decreases again compared to ERA and EPA.
  • the DHA products are produced in the same ratio as the EPA products, although the amount of products is less than for ERA and EPA.
  • PtLOX PtLOX
  • PtLOXI prefers to implement ERA or EPA based on the comparative substrate implementation ( Figure 8). If one adds the results of the position specificity and the enantiomer analysis, this speaks for EPA as the preferred substrate. Compared to ERA, the relationship between the main and by-products is clearly shifted in favor of the main product, which is almost enantiomerically pure. In this way, an exceptional substrate specificity was found compared to plant lipoxygenases.
  • Ketoconjudic fatty acids were not formed by PtLOX under the conditions of the analysis.
  • CODE are mainly formed at low oxygen partial pressures, but by some plant lipoxygenases, e.g. Lipoxygenase-2 from soybean and lipoxygenase-1 from pea, are known to produce these even in normal oxygen partial pressure in unusually high amounts of up to 50% based on HODE [Axelrod et al., Methods Enzymol, 71: 441-451, 1981; Kuehn et al., Eicosanoids, 4: 9-14, 1991].
  • the implementation of complex lipids is also known from lipoxygenases from other sources.
  • the lipid body lipoxygenase from cucumber seedlings is able to convert triacylglycerides [Feussner et al., FEBS Lett; 431 (3): 433-436, 1998].
  • These lipoxygenases are probably involved in the mobilization of storage fats during germination [Feussner et al., Trends Plant Sei, 6: 262-267, 2001].
  • PtLOX does have oil vacuoles rwww.kieselal ⁇ en.coml. but since reproduction takes place through vegetative cell division, the inclusion of lipoxygenases in the mobilization of storage lipids can be ruled out, as in some higher plants.
  • the conversion of phosphatidylcholine can also take place using lipoxygenases.
  • Soybean lipoxygenase-1 first converted this substrate to monohydroperoxy-linoleoyl-phosphatidylcholine and then to dihydroperoxide [Brash et al., Biochemistry, 26: 5465-5471, 1987].
  • a new oxolipin was also identified in Arabidopsis [Stelmach et al., J Biol Chem, 276 (16): 12832-12838, 2001]. It is a sn-1-O- (12-oxophytodienyl) -sn2-O- (hexadecatrienoyl) monogalactosyl diglyceride.
  • EPE 8-H / P) EPE (8S, 5Z, 9E, 11 E, 14Z, 17Z) -8-hydro (pero) xy-5,9, 11, 14,17-eicosapentaenoic acid -H (P) ETE ( 5S, 6E, 8Z, 11Z, 14Z) -5-hydro (pero) xy-6,8,11,14-eicosatetraenoic acid -H (P) ETE (8S, 5Z, 9E, 11Z, 14Z) -8- Hydro (pero) xy-5,9,11, 14-eicosatetraenoic acid -H (P) ETE (9S, 5Z, 7E, 11Z, 14Z) -9-Hydro (pero) xy-5,7,11, 14 -eicosatetraenoic acid 11-H (P) ETE (11S, 5Z, 8Z, 12E, 14Z) -11-hydro (pero) x
  • IPTG isopropyl- ⁇ -thiogalactopyranoside kDa kilodalton

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Abstract

La présente invention concerne un procédé de production d'acides gras hydroperoxy par oxydation d'acides gras polyinsaturés présentant au moins deux doubles liaisons dans leur molécule d'acide gras, en particulier d'acides gras polyinsaturés tels que l'acide arachidonique ou l'acide eicosapentanoïque. L'invention concerne la production d'un organisme transgénique, de préférence d'une plante transgénique ou d'un micro-organisme transgénique, présentant une teneur élevée en acide gras hydroperoxy, en raison de l'expression d'une lipoxygénase extraite de Phaeodactylum Tricornutum. L'invention concerne en outre des cassettes d'expression contenant une séquence d'acide nucléique, un vecteur et des organismes contenant au moins une séquence d'acide nucléique ou une cassette d'expression.
PCT/EP2003/007354 2002-07-11 2003-07-09 Clonage et caracterisation d'une lipoxygenase extraite de phaeodactylum tricornutum WO2004007732A1 (fr)

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AU2003280984A AU2003280984A1 (en) 2002-07-11 2003-07-09 Cloning and characterization of a lipoxygenase from phaeodactylum tricornutum

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DE10231589A DE10231589A1 (de) 2002-07-11 2002-07-11 Klonierung und Charakterisierung einer Lipoxygenase aus Phaeodactylum tricornutum
DE10231589.2 2002-07-11

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038484A2 (fr) * 1999-11-25 2001-05-31 Basf Plant Science Gmbh Genes de mousse tires de physcomitrella patens, codant des proteines impliquees dans la synthese des acides gras et des lipides polyinsatures

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038484A2 (fr) * 1999-11-25 2001-05-31 Basf Plant Science Gmbh Genes de mousse tires de physcomitrella patens, codant des proteines impliquees dans la synthese des acides gras et des lipides polyinsatures

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MIRALTO A ET AL: "Embryonic development in invertebrates is arrested by inhibitory compound in diatoms", MARINE BIOTECHNOLOGY, (JUL-AUG 1999 ) VOL. 1, NO. 4, PP. 401-402 PUBLISHER: SPRINGER-VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010. ISSN: 1436-2228., XP009021406 *
POHNERT GEORG ET AL: "The oxylipin chemistry of attraction and defense in brown algae and diatoms.", NATURAL PRODUCT REPORTS, (2002 FEB) 19 (1) 108-22., XP009020975 *

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