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WO2001029227A1 - Le tonneau beta de la lipoxygenase agissant sur les corps lipidiques - Google Patents

Le tonneau beta de la lipoxygenase agissant sur les corps lipidiques Download PDF

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Publication number
WO2001029227A1
WO2001029227A1 PCT/EP2000/009912 EP0009912W WO0129227A1 WO 2001029227 A1 WO2001029227 A1 WO 2001029227A1 EP 0009912 W EP0009912 W EP 0009912W WO 0129227 A1 WO0129227 A1 WO 0129227A1
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Prior art keywords
nucleic acid
acid sequence
organism
sequence
seq
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PCT/EP2000/009912
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German (de)
English (en)
Inventor
Helmut Kindl
Christian May
Ivo Feussner
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Basf Aktiengesellschaft
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Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to AU10235/01A priority Critical patent/AU781120B2/en
Priority to CA002388305A priority patent/CA2388305A1/fr
Priority to JP2001532210A priority patent/JP2003512058A/ja
Priority to EP00971351A priority patent/EP1222282A1/fr
Publication of WO2001029227A1 publication Critical patent/WO2001029227A1/fr
Priority to NO20021851A priority patent/NO20021851L/no

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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)
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a method for targeting proteins involved in the biosynthesis of lipids or fatty acids in liposomes or lipid bodies.
  • the invention relates to a method for producing fatty acids and lipids in an oil-producing organism.
  • the invention also relates to nucleic acid sequences which code for a polypeptide and which is composed of a combination of nucleic acid sequences of a biosynthetic nucleic acid sequence of the fatty acid or lipid metabolism with a nucleic acid sequence which codes for the targeting of proteins.
  • the invention further relates to nucleic acid constructs containing these nucleic acid sequences, vectors and transgenic organisms which contain the nucleic acids, nucleic acid constructs and / or vectors.
  • Triacylgycerides are stored in particularly highly specialized tissues, for example in endosperm or cotyledons. Cells of this type contain lipid bodies as compartments that store the fat [Murphy, DJ, Prog. Lipid Res. , 32, 1993: 247-280; Huang, AH C, curr. Opinion Struct.
  • PLA a patatin-like protein [May. C. et al., Biochim. Biophys. Acta, 1393, 1998: 267-276]) plays a crucial role in the initiation of the mobilization process by the phospholipid monolayer Lipid body destroyed [Noll, F. et al. , J. Struct. Biol., 1999: submitted].
  • This object was achieved by a method for targeting proteins involved in the biosynthesis of lipids or fatty acids in liposomes or lipid bodies, characterized in that the nucleic acids coding for the proteins are combined with one of the following sequences in a common protein coding sequence:
  • nucleic acid sequences which are derived from the nucleic acid sequence shown in SEQ ID NO: 1 as a result of the degenerate genetic code
  • nucleic acid sequence shown in SEQ ID NO: 1 which code for polypeptides with the amino acid sequences shown in SEQ ID NO: 2 and have at least 60% homology at the amino acid level
  • a nucleic acid sequence with the sequence shown in SEQ ID NO: 3 or the amino terminal part of the coding region of this sequence and
  • the method according to the invention makes it possible to specifically direct proteins which are advantageously involved in the fatty acid and / or lipid metabolism to the site of the desired synthesis.
  • Proteins can be directed in the method according to the invention by introducing the nucleic acid sequence into a eukaryotic organism.
  • the organisms are grown in a suitable medium.
  • nucleic acid sequence according to the invention as described below or at least one nucleic acid construct is placed in an oil-producing organism.
  • a process for the production of fatty acids or lipids characterized in that at least one nucleic acid sequence according to the invention or at least one nucleic acid construct is placed in an oil-producing organism, this organism is attracted and that oil contained in the organism is isolated.
  • a process for the production of fatty acids characterized in that at least one nucleic acid sequence according to the invention or at least one nucleic acid construct is placed in an oil-producing organism, this organism is attracted and that oil contained in the organism is isolated and the fatty acids are released.
  • Method as described above characterized in that the organism is a plant or a eukaryotic microorganism.
  • Culturing the organism as described above means the cultivation of plants as well as the cultivation of eukaryotic microorganisms such as yeasts, fungi, ciliates, algae, animal or plant cells or cell groups.
  • eukaryotic microorganisms such as yeasts, fungi, ciliates, algae, animal or plant cells or cell groups.
  • organisms for the process are plants such as arabidopsis, barley, wheat, rye, oats, maize, soybeans, rice, cotton, sugar beet, tea, carrots, peppers, canola, sunflower, flax, hemp, potatoes, triticale, tobacco, tomatoes , Rapeseed, coffee, tapioca, manioc, arrowroot, tagetes, alfalfa, peanut, castor, coconut, oil palm, safflower (Carthamus tinetorius), lettuce and the various tree, nut and wine species, or cocoa bean, microorganisms such as yeasts such as Yarrowia or Saccharomyces; Mushrooms called Mortierella, Saprolegnia, Traustochytrium or Pythium, algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • Organisms which can naturally synthesize oils in large quantities such as microorganisms such as yeasts such as Yarrowia lypolytica or Saccharomyces cereviseae, fungi such as Mortierella alpina, Pythium insidiosum or plants such as Arabidopsis thaliana, soybeans, oilseed rape (Braccica napus), coconut, oil palm, canola, are preferred.
  • Dyer safflower (Carthamus tinetorius), castor oil, calendula, flax (Linium usitatissimum), borage, peanut, cocoa bean or sunflower, soybean, rape or sunflower are particularly preferred.
  • the organisms obtained in the process according to the invention advantageously contain saturated or unsaturated fatty acids in the form of bound fatty acids, i.e. the unsaturated fatty acids are predominantly in the form of their mono-, di- or triglycerides, glycolipids, lipoproteins or phospholipids such as oils or lipids or otherwise Ester or amide bound fatty acids.
  • Free fatty acids are also contained in the organisms in the form of the free fatty acids or in the form of their salts.
  • the organisms obtained by cultivation in the process according to the invention and the saturated or unsaturated fatty acids contained in them can be used directly, for example, for the production of pharmaceutical preparations, agrochemicals, animal feeds or foods or after isolation from the organisms.
  • the bound fatty acids can be released from, for example, the oils or lipids, for example via basic hydrolysis, for example with NaOH or KOH. These free fatty acids can be used directly in the mixture obtained or after further purification for the production of pharmaceutical preparations, agrochemicals, animal feeds or foods.
  • the bound or free fatty acids can also be used for transesterification or esterification, for example with other mono-, di- or triglycerides or glycerol, in order to increase the proportion of unsaturated fatty acids in these compounds, for example in the triglycerides.
  • Microorganisms such as bacteria, fungi, ciliates, plant or animal cells 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 iron, Manganese, magnesium salts and possibly vitamins contains, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C, depending on the organism, oxygen or in the absence of oxygen.
  • the pH of the nutrient liquid can be kept at a fixed value, i.e.
  • the pH is regulated during cultivation or the pH is not regulated and changes during cultivation.
  • the cultivation can be batch-wise, semi-batch wise or continuous. Nutrients can be added at the start of the fermentation or fed semi-continuously or continuously. Cultivation on solid media is also possible.
  • Plants are generally regenerated after transformation and then grown or grown as usual. This can be done in the greenhouse or outdoors.
  • the lipids are usually obtained from the organisms.
  • the organisms can first be digested after harvesting or used directly.
  • the lipids are advantageously mixed with suitable solvents such as apolar
  • Solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol extracted 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 done 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 purification using chromatographic processes, distillation or crystallization is also possible.
  • nucleic acid sequences which code for a polypeptide and which are composed of a combination of the nucleic acid sequences of a biosynthetic nucleic acid sequence of the fatty acid or lipid metabolism with one of the following nucleic acids:
  • nucleic acid sequences according to the invention enable proteins to be targeted in the method according to the invention.
  • Derivatives are, for example, functional homologs of the proteins encoded by SEQ ID NO: 1 or their biological activity, that is to say proteins which have the same biological reactions as those controlled by SEQ ID NO: 1. These genes also enable advantageous targeting of proteins. Under biological activity, directing proteins is advantageous of proteins that are fatty acid and / or Lipid metabolism is involved to understand within the cell.
  • nucleic acid sequence (s) used in the method according to the invention can advantageously be used for the isolation of further genomic sequences via homology screening.
  • the derivatives mentioned can be isolated, for example, from other eukaryotic organisms such as fungi, yeasts or plants such as specifically mosses.
  • derivatives or functional derivatives include
  • allelic variants which have at least 60% homology at the derived amino acid level, advantageously at least 70% homology, preferably at least 80% homology, particularly preferably at least 85% homology, very particularly preferably 90% homology ,
  • amino acid sequence derived from the nucleic acids mentioned can be found in sequence SEQ ID NO: 2. Homology is to be understood as identity, i.e. the amino acid sequences are at least 60%
  • sequences according to the invention are at least 50% homologous, preferably at least 60%, particularly preferably 70%, very particularly preferably at least 80% at the nucleic acid level.
  • Allelic variants include in particular functional variants, 35 which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1, the biological activity of the derived synthesized proteins being retained, that is to say these proteins still have the ability of Protein carryings. 40
  • DNA sequences can be isolated 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, from other eukaryotes such as, for example, the one mentioned above. These DNA sequences hybridize to the sequences mentioned under standard conditions. Short oligonucleotides are advantageously used for hybridization. used from 20 to 50 nucleotides in length. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization. These standard conditions vary depending on the nucleic acid used: oligonucleotide, 5 longer fragment or complete sequence or depending on the type of nucleic acid DNA or RNA used for the hybridization. For example, the melting temperatures for DNA: DNA 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.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • DNA RNA hybrids, the hybridization
  • derivatives homologs of the sequence SEQ ID No: 1 are understood to mean, for example, 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 has been changed such that the gene expression and / or the protein expression is changed, preferably increased. Derivatives are also to be understood as variants that were changed at the 3 'end.
  • nucleic acid sequences which code for the proteins according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components, and can consist of different heterologous 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 heterologous genes.
  • plant preferred codons can be determined from the highest protein frequency codons expressed in most interesting plant species.
  • Corynebacterium glutamicum is given in: Wada et al. (1992) Nucleic Acids Res.
  • Functionally equivalent sequences which code for the proteins according to the invention 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 biological activity of the proteins.
  • Functional equivalents thus include naturally occurring variants of the sequences described here as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.
  • artificial DNA sequences are suitable as long as, as described above, they have the desired property, for example targeting in the fatty acid and / or lipid metabolism. auxiliaries.
  • Such artificial DNA sequences can be determined, for example, by back-translating proteins constructed using molecular modeling, which have the biological activity, or 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 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.
  • Biosynthesis genes of fatty acid and / or lipid metabolism may be mentioned as components of the nucleic acids according to the invention, such as advantageously a sequence which codes for proteins from the following group of proteins:
  • nucleic acids which code for one of the following proteins: fatty acid acyl transferase (s), ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 9-desaturase, ⁇ 12-desaturase, ⁇ 15-desaturase and / or a fatty acid elongase
  • nucleic acid sequences mentioned code for so-called fusion proteins, part of the fusion protein being a polypeptide with the sequence mentioned in SEQ ID NO: 2 or a functionally equivalent part thereof.
  • the second part of the fusion protein can e.g. be another polypeptide with enzymatic activity such as the above proteins.
  • the nucleic acids can advantageously be combined with other genes of fatty acid biosynthesis.
  • genes of fatty acid biosynthesis examples include acetyltransferases, further desaturases or elongases of unsaturated or saturated fatty acids as described in WO 00/12720.
  • acetyltransferases further desaturases or elongases of unsaturated or saturated fatty acids as described in WO 00/12720.
  • NADH cytochrome B5 reductases is advantageous, which can absorb or release reduction equivalents.
  • proteins used in the method 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 5 more amino acid residues, the biological activity of the protein shown in SEQ ID NO: 2 is retained or is not significantly reduced. Not significantly reduced is to be understood as meaning all proteins which are still at least 10%, preferably 20%, particularly preferred
  • amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
  • arginine residues against lysine residues valine residues against isoleucine
  • amino acids 15 residues or aspartic acid residues exchanged 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 the proteins according to the invention, which furthermore contain the desired one
  • Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
  • nucleotide sequences are also included in the present invention.
  • nucleotide sequence coding for the protein comprises which is obtained by modification of the nucleotide sequence coding for the protein.
  • the aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also be the insertion of further restriction enzyme interfaces.
  • nucleic acid sequences can also be introduced directly into the host organism.
  • the nucleus The acid sequence can advantageously be, for example, a DNA or cDNA sequence. Coding sequences suitable for insertion into an expression cassette are, for example, those which enable the protein targeting according to the invention. These sequences can be of homologous or heterologous origin.
  • These regulatory sequences are intended to enable targeted expression of the 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 before 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
  • 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, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences.
  • 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 plants or fungi are suitable as promoters in the nucleic acid construct.
  • a plant promoter or promoters which originate, for example, 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, lpp, lac, lpp-lac, lacl ⁇ - T7, T5, T3 -, gal-, trc-, ara-, SP6-, ⁇ -P R - or contained in the ⁇ -P L promoter, which are advantageously used in gram-negative bacteria.
  • the expression cassette can also contain a chemically inducible promoter, by means of which the expression of the exogenous 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 (W093 / 21334) promoter.
  • Further 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 phosphoribosylpyrophosphate amidotransferase from Glycine max (see also Genbank accession number U87999) or a node-specific promoter as in EP 249676 can advantageously be used.
  • Those plant promoters which express in tissues are particularly advantageous tu? a ⁇ P - H 3 0 t ⁇ r 0 C ⁇ ⁇ -_. cn FP P. S 3 P ⁇ ⁇ ⁇ SU iQ ⁇ .
  • ⁇ SU 3 3> ⁇ 3 o 13 H- o cn o rt 3 rt o 3 X 3 1 o • - 'oo 1 P F- ⁇ 3 P 13 ⁇ o ⁇ cn ⁇ oo • Ü P ⁇ - ⁇ r ⁇ i cn ⁇ H- ff.
  • Synthetic genes advantageous of fatty acid biosynthesis, which enable an increased synthesis.
  • the genes for the ⁇ 15-, ⁇ 12-, ⁇ 9-, ⁇ 6-, ⁇ 5-, ⁇ 4-desaturase, the various hydroxylases, the acyl-ACP-thioesterases, ⁇ -ketoacyl synthases or ⁇ -ketoacyl reductases may be mentioned.
  • the desaturase genes are advantageously used in the nucleic acid construct.
  • DNA fragments can be manipulated in order to obtain a nucleotide sequence which is expediently read in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter can be native or homologous as well as foreign or heterologous to the host 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 gene used in the method according to the invention and a region for the transcriptional termination. Different termination areas are interchangeable.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or corresponding functional equivalents.
  • a nucleic acid construct is produced by fusing a suitable promoter with a suitable nucleic acid sequence according to the invention and a polyadenylation signal according to common recombination and
  • the DNA sequence coding for a lipid body lipoxygenase from cucumber contains all the sequence features which are necessary in order to achieve a localization correct for the location of the fatty acid, lipid or oil biosynthesis. Therefore no further targeting sequences per se are necessary. However, such a location can be desirable and advantageous and can therefore be artificially changed or reinforced, so that such fusion constructs are also a preferred advantageous embodiment of the invention.
  • Sequences which ensure targeting in plastids are particularly preferred. Under certain circumstances, targeting in other compartments (ref .: Kermode, Crit. Rev. Plant Sei. 15, 4 (1996), 285-423) can also be carried out, for example, in the vacuole, in the mitochondrion, in the endoplasmic reticulum (ER) , Peroxisomes, lipid bodies or due to a lack of such operative sequences to remain in the compartment of formation, the cytosol, may be desirable.
  • the nucleic acid sequences coding for proteins 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, if appropriate, further regulatory elements which are operatively linked to the DNA coding sequence in between.
  • 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 in the expression of the coding sequence.
  • the preferred sequences for the operative linkage are targeting sequences to ensure subcellular localization.
  • An expression cassette can contain, for example, a constitutive promoter (preferably the USP or napin promoter) and the gene to be expressed.
  • the expression cassette is advantageously used for expression in a prokaryotic or eukaryotic host organism, for example a microorganism such as a fungus or a plant, in a vector such as a plasmid, for example
  • Suitable plasmids are for example in E. coli pLG338, pACYCl84, pBR series such as pBR322, pUC series such as pUC18 or pUC19, Mll3mp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 -Bl , ⁇ gtll or pBdCI, in Streptomyces pIJlOl, pIJ364, pIJ702 or pIJ361, in Bacillus pUBllO, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in mushrooms pALSl, pIL2 or pBBllzector, etc.
  • Advantageous Yeast vectors are, for example, 2 ⁇ M, pAG-1, YEp6, YEpl3 or pEM-BLYe23.
  • Examples of algae or plant vectors are pLGV23, pGHlac + , pBINl9, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, L., 1988)
  • the abovementioned vectors or derivatives of the abovementioned 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.
  • 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 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 of the expression cassette as a vector or the nucleic acid sequences according to the invention.
  • the 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 each vector or several genes together in different vectors, the different vectors can be introduced simultaneously or successively.
  • the vector advantageously contains at least one copy of the nucleic acid sequences according to the invention and / or the expression cassette.
  • 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.
  • pRT transformation vector
  • Fusion vectors used in prokaryotes frequently use inducible systems with and without fusion proteins or fusion oligopeptides, it being possible for these fusions to take place both N-terminally and also cterally or other usable domains of a protein.
  • Such fusion vectors generally serve: i.) To increase the expression rate of the RNA ii.) To increase the achievable protein synthesis rate, iii.) To increase the solubility of the protein, iv. ) or to simplify purification by means of a binding sequence that can be used for affinity chromatography.
  • proteolytic cleavage sites are also introduced via fusion proteins, which enables a part of the fusion protein to be split off, also for purification.
  • recognition sequences for proteases are e.g. Factor Xa, thrombin and enterokinase.
  • Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Ine; Smith, DB and Johnson, KS (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
  • Further examples of E. coli expression vectors are pTrc [Amann et al.
  • yeast vectors for use in yeast are pYep-Secl (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 ( Schultz et al.,
  • insect cell expression vectors can also be used advantageously, e.g. for expression in Sf 9 cells. 20 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).
  • plant cells or algal cells can advantageously be used for gene expression.
  • plant 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 30 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 named in: Seed, B.
  • 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
  • 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.
  • 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 are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene cannon - the so-called particle bombardment method -, electroporation, the incubation of dry embryos in DNA-containing solution, micro-injection and that by Agrobacterium mediated gene transfer.
  • the methods mentioned are described, for example, in B. Jenes et al. , Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev.
  • 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 for transforming plants, in particular crop plants, such as tobacco plants, for example, by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the transformation of plants with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res.
  • Agrobacteria transformed with an expression vector as described above 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, triticale, rice, Sunflower, flax, hemp, potato, tobacco, tomato, coffee, cocoa, tea, carrot, paprika, rapeseed, tapioca, cassava, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species, especially oil containing crops such as soy, peanut, castor, borage, linseed, sunflower, canola, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinetorius) or cocoa bean are used, for example by bathing wounded leaves or pieces of leaf in an agrobacterial solution and then cultivated in suitable media.
  • test plants such as Arabidops
  • the genetically modified plant 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 S.D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer can be found.
  • all organisms which are able to synthesize fatty acids, especially unsaturated fatty acids or are suitable for the expression of recombinant genes are advantageously suitable as organisms or host organisms for the nucleic acids used, the nucleic acid construct used or the vector used.
  • plants such as Arabidopsis, Asteraceae such as Calendula or crop plants such as Brassica, Linium, soybean, peanut, castor bean, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinetorius) or cocoa bean, microorganisms such as yeasts, for example the genera Yarrowia or Saccharomyces, fungi, for example the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, cyano-bacteria, ciliates, algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • yeasts for example the genera Yarrowia or Saccharomyces
  • fungi for example the genus Mortierella, Saprolegnia or Pythium
  • bacteria such as the genus Escherichia, cyano-bacteria, ciliates, algae or protozoa
  • transgenic animals are also suitable as host organisms, for example C. elegans.
  • Useful host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the expression of the nucleic acid sequences according to the invention can take place specifically in the leaves, in the seeds, in the tubers or in other parts of the plant.
  • Such fatty acids, oils or lipids overproducing transgenic plants, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention.
  • a preferred subject of the invention are transgenic plants, for example crop plants such as corn, oats, rye, wheat, barley, corn, rice, soybeans, sugar beet, canola, triticale, sunflower, flax, hemp, tobacco, tomato, coffee, cocoa, tea, carrot, Paprika, rapeseed, tapioca, cassava, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species, potato, in particular oil-containing crops, such as soybean, peanut, castor bean, borage, flax, sunflower, canola, tree - wool, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinetorius) or cocoa bean, test plants such as Arabidopsis or other plants such as moss or algae containing a functional nucleic acid sequence according to the invention or a functional expression cassette.
  • Function means that a biologically active protein is formed.
  • the expression cassette or the nucleic acid sequences according to the invention containing a nucleic acid sequence according to the invention can also be used to transform the above-mentioned organisms such as bacteria, cyanobacteria, filamentous fungi, ciliates, animals or algae with the aim of increasing the content of fatty acids, oils or lipids.
  • Preferred transgenic organisms are yeasts, fungi or plants, particularly preferably plants.
  • Transgenic organisms are understood to mean organisms which contain a foreign nucleic acid from another organism which codes for a protein used in the method according to the invention.
  • Transgenic organisms are also understood to mean organisms that contain a nucleic acid that comes from the same organism, this nucleic acid being contained as an additional gene copy or not in the natural one Nucleic acid environment of the nucleic acid sequence according to the invention is included.
  • Transgenic organisms are also organisms in which the natural 3 'and / or 5' region of the nucleic acid according to the invention has been changed by targeted genetic engineering changes compared to the starting organism.
  • Transgenic organisms in which a foreign DNA has been introduced are preferred.
  • Transgenic plants are particularly preferred.
  • Transgenic plants are understood to mean individual plant cells and their cultures, such as callus cultures on solid media or in liquid culture, parts of plants and whole plants.
  • Cucumber seeds were germinated in the dark at 26 ° C for 2 or 4 days as indicated for each preparation.
  • the cotyledons were harvested and homogenized using the method described previously [Kindl, H. et al. , Methods Enzymol., 96, 1983: 700-715] were cut with a scalpel. After removing cell debris and differential centrifugation, the sediments of centrifugation at 10,000 xg or centrifugation at 100,000 xg were sedimented in a sucrose gradient according to [Sturm, A. et al., FEBS Lett., 160, 1983: 165-168] or floats.
  • a crude lipid body fraction was obtained by subjecting the supernatant to a short (10 min) centrifugation at 2,000 x g, centrifugation for 30 min at 10,000 x g and removing the lipid layer formed in the upper section of the centrifuge tube. The further cleaning of the lipid bodies was carried out by gently suspending the lipid layer and repeated flotation [Sturm, A. et al. , Eur. J. Biochem. , 150, 1985: 461-468].
  • Tobacco plants were cultivated at 22 ° C under permanent exposure (2000 lux) in magenta boxes.
  • the media for growing transformants were according to [Horsch, R.B. et al., Science 227, 1985: 1229-1231; Jefferson, R.A. et al. , EMBO J., 6, 1987: 3901-3907].
  • CSLBLOX-221 was used in the vector pSport-1 [Höhne, M. et al. , Eur. J. Biochem., 241, 1996: 6-11].
  • An N-terminal deletion was made by digesting CSLBLOX-221 first with Smal / Ndel and then with ffaelll. After ligation in pSport-1 (Life Technologies), this construct, in which the first 80 nucleotides of pCSLBLOX-221 were deleted [Höhne, M. et al. .
  • the construct pBI121 ⁇ GUS was prepared by filling the GUS cassette with Smal and SstI was cut out and a smooth end was produced using T4 DNA polymerase.
  • LBLOX contained in the vector pSport-1 [Höhne, M. et al. , Eur. J. Biochem., 241, 1996: 6-11] was cut out with Smal / BamHI, ligated with a BamHI linker and introduced into the BamHI-cleaved dephosphorylated vector pBI121 ⁇ GUS.
  • HA label a triple hemagglutinin label (HA label), which was provided with a Sacl site, was inserted. Taking into account the 3 -YPYDVPDYA sequences and the linker, the total length of the insert was 30 amino acids.
  • Ag-ro-acterium tumefaciens LB-A4404 was transformed with pBI121 ⁇ GUS-LBLOX or pBI121 ⁇ GUS-LBLOX-HA 3 according to the freeze-thaw method. These bacteria were used to transform leaf disks from Nicotiana tabacum cv. Petit Havana SR-1 according to the established method of Horsch et al. [Science 227, 1985:
  • the rungs were selected on Linsmaier and Skoog medium enriched with 0.5 mg / 1 N-benzylaminopurine, 500 mg / 1 cefotaxime and 75 mg / 1 kanamycin. Kanamycin-resistant plants with increased LOX levels were propagated vegetatively.
  • Leaves of homozygous kanamycin-resistant tobacco lines containing the pBI121 ⁇ GUS-LBLOX-HA 3 construct were in 50 mM Hepes-NaOH, pH 7.4, 5 mM MgCl 2 , 0.5 mM EDTA, 50 mM KCl, 2.5 homogenized mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride and 15% (w / w) sucrose (buffer A) in the presence of polyvinylpyrrolidone (25,000) (Merck). After centrifugation, the supernatant from the 100,000 xg centrifugation was desalted, concentrated and fractionated on a large Biogel A 1.5 column.
  • This mutant LBLOX protein contained an insert of 30 amino acid residues corresponding to a triple HA-5 label behind amino acid residue 68 of the wild-type enzyme and had a theoretical molecular mass of 104 kDa. This difference in size between the wild-type and the recombinant protein was clearly demonstrated by SDS-PAGE.
  • Translation was carried out using purified mRNAs, reticulocyte lysate and [ 35 S] L-methionine in the presence or absence of dog pancreatic microsomes. Alternatively 15 microsomes prepared from cucumber cotyledons were used for co-translational or post-translational transport assays.
  • Radioactive labeling experiments in vivo were performed as short pulse experiments with cotyledons of seedlings germinated for 4 days. Five g of cotyledons were cut into 2 mm pieces and incubated with 8 MBq [ 35 S] L-methionine (40 TBq / mmol) for 15 min 25. The careful homogenization and preparation of subtractions was carried out as described above.
  • the fraction containing the lipid bodies was resuspended in buffer A, adjusted to 30% (w / w) sucrose and covered with buffer A. After centrifugation at 100,000 g for 1 hour, the floated lipid bodies were collected and subjected to various washing processes.
  • Liposomes were made according to [Woodle, M C. & Papahadjopoulos, D., Methods Enzymol. 171, 1989: 193-217] from a crude soybean-5 lecithin mixture (Sigma) or from defined dilinoleoylphosphatidylcholine, with or without addition of the serine derivative, as unilamellar vesicles. Routinely, using detergent solubilization and removal, the molar ratio of dilinoleoylphosphatidylcholine to sodium cholate was
  • Trilinolein built into the liposomes, so that phospholipid-covered lipid droplets were obtained which are comparable to "black” lipid bodies. This preparation was initiated by adding 200 mg soybean lecithin and 500 mg trilinolein
  • 25 liposomes was in the range of 1 ⁇ m.
  • a liposome suspension corresponding to 1 mg lecithin in 150 mM 30 NaCl, 50 mM Tris-HCl, pH 8.0 was incubated for 10 min either with 4 ⁇ g unlabelled protein or with the supernatant of a reticulocyte lysate translation mixture. After adjusting to 42% (w / w) sucrose, the suspension was transferred to a 12 ml centrifuge tube. A linear sucrose density gradient of 37-26% (w / w) sucrose was layered on the sample. The flotation of the protein-covered liposomes was carried out by centrifugation for 6 hours at 100,000 g. After fractionation, protein analysis was carried out using SDS-PAGE and immune labeling.
  • Immunoprecipitation of the radioactively labeled enzymes was carried out under standard conditions (method 1) by adding 1 ⁇ g of the corresponding purified protein to the mixture with 20 ⁇ l antiserum before the precipitation. After standing for 12 hours at 20 ° C and 20 hours at 4 ° C, the precipitate was sedimented by centrifugation at 3000 x g. The sediment was washed at least 5 times and then dissolved in SDS. For the direct precipitation of other proteins (method 2), the antigen was not further diluted, but incubated for 6 hours with 2 ⁇ l antiserum and then mixed with protein A-Sepharose. After transferring the mixture to a small vessel 0 and washing extensively, the antigen was eluted together with the IgG using 100 mM acetic acid.
  • the first step was to determine which pools in the cell cross the LBLOX protein.
  • the targeting structure, sequence or domains responsible for the transfer were localized.
  • PLA mRNA obtained by in vifcro transcription from pPAT291 was also translated and the translation product, i.e. H. radiolabelled phospholipase was incubated with microsomes. After flotation in a sucrose gradient, the subtractions were analyzed by SDS-PAGE and fluorography (FIG. IC). Fluorography showed that a large part of the translation product had bound to microsome membranes.
  • Figure 2A shows that the majority of the pulse labeled LBLOX was obtained in the fraction containing the cytosol. A small but significant amount of the radioactive LBLOX was continuously found in the microsomes and also in microsome fractions which were further purified by flotation in sucrose gradients (FIG. 2A). In order to rule out the possibility that small percentages of all newly synthesized proteins contaminated the ER-containing fraction, we analyzed the amount of proteins in the microsome fraction that were either cytosolic or as an artifact in the cytosol-containing supernatant of a 100,000 g Centrifugation were released.
  • isocitrate lyase as a strongly expressed cucumber protein at this stage of development, the presence of this protein in the microsome fraction and in the soluble supernatant was determined by immunoprecipitation. 2B shows that, in contrast to LBLOX, isocitrate lyase was practically absent in the ER preparation.
  • FIG. 2A The data shown in FIG. 2A show that LBLOX crosses the cytosol as the first pool on its way to the lipid bodies. It is also evident that LBLOX has membrane binding properties and is partially protected from proteolysis when bound to the ER membrane. Despite areas in the LBLOX molecule that have affinity for membranes, a large part of the LBLOX in vitro is accessible for chemical modification and could therefore protrude into the cytosol in vivo. Part C of FIG. 2 provides an indication of the extent to which the part of LBLOX that is bound to microsome membranes is accessible for proteolytic degradation.
  • the LOX isoform which can be detected by Western blot analysis on microsomes isolated from cotyledons, is structurally identical to the LOX isoform bound to lipid bodies. This was demonstrated by comparing the fragment pattern after limited proteolysis (data not shown).
  • the type of binding of LBLOX to the microsome membrane corresponds to that of a peripherally bound membrane protein. Washing with 100 mM MgCl 2 removed more than 90% of the labeled LBLOX.
  • the binding of LBLOX to the microsomes in the presence of a low salt buffer and also under the conditions of repeated gradient centrifugation was quite stable. The stable binding was also shown by the fact that LOX on microsomes was only partially accessible for proteolysis (FIG. 2, part C).
  • LBLOX was undetectable in the glyoxysome fraction which was subjected to a final purification by sucrose gradient flotation. Soluble LBLOX thus binds to membranes, but not evenly to all membranes.
  • the affinity of LBLOX for glyoxysome membranes (Fig. 3B) is at least an order of magnitude lower than the demonstrated affinity for microsomes (Fig. 3A, lane 3).
  • the binding of LBLOX in cucumber cotyledons therefore requires a high degree of selectivity, since other organelles are not labeled with LBLOX and are not modified for degradation.
  • Cucumber LBLOX and soybean LOX-1 differ in their affinity for liposomes
  • LBLOX has an intrinsic affinity for membrane lipids regardless of specific protein-protein interactions
  • the binding studies were expanded and included liposomes as acceptor membranes.
  • the experiments were carried out using crude soybean lecithin as a source of phosphatidylcholine. Subsequently, phosphatidylcholine with different degrees of purity was also used in combination with phosphatidylserine for the production of liposomes. The size of the liposomes was checked by comparison with MonoQ beads as the standard.
  • LOX-1 has no affinity for the lipid phase.
  • Control experiments with either a typical cytosolic protein or with a mixture containing LBLOX and cytosolic proteins and prepared from cucumber cotyledons showed that the membrane affinity of LBLOX is quite unique (data not shown).
  • the N-terminal region including the ß-barrel structure is essential for the binding of LBLOX to lipid bodies
  • the lipid body fraction contained 50 kBq 45 Ca 2+ , which corresponds to approximately 1 n ol.
  • the same preparation had 2 nmol LBLOX.
  • Further treatment with 100 mM unlabeled CaCl 2 removed 90% of the radioactivity from the lipid bodies, whereas the majority of the LBLOX remained bound to the lipid bodies.
  • LBL0X-HA recombinant protein
  • the liposome experiments shown in FIG. 7 summarize the evidence that the N-terminal ß-barrel of LOX alone is sufficient for their binding to membranes and the destruction of the ß-barrel inactivates the transfer.
  • Wild-type LBLOX and the ß-barrel fusion protein (GST-LOX) bind to the liposomes practically quantitatively, the insertion of a peptide into the barrel structure removes the membrane affinity.
  • the N-terminal extension as a specific motif and the ß-barrel, which is in the amino acid sequence following the N-terminal extension, as a general means of increasing membrane affinity.
  • Targeting signals as unfolded areas of 7 amino acid residues have been found when proteins are transported in mitochondria, chloroplasts or the ER.
  • a domain that has already been folded into a certain form can bring about membrane binding.
  • Another part has to be postulated which mediates the selectivity of the binding of LBLOX to lipid bodies. If there is a large amount of LBLOX, in addition to lipid bodies, the ER and the Golgi vesicles are also covered with LBLOX. However, if the intracellular amount of LBLOX decreases, the LBLOX molecules mainly occupy the surface of lipid bodies.
  • Part A shows a comparison of the migration behavior of LBLOX (lane 1) produced by vifcro translation, LBLOX isolated from microsomes (lane 2) and LBLOX (lane 3) isolated from lipid bodies.
  • LBLOX LBLOX isolated from microsomes
  • LBLOX LBLOX isolated from lipid bodies
  • LBLOX LBLOX isolated from lipid bodies
  • LBLOX LBLOX isolated from lipid bodies
  • the membrane bound lipid body lipoxygenase (Part B) or phospholipase (Part C) obtained after flotation are shown on the left.
  • Lane 1 at B and C corresponds to the upper portion of the sucrose gradient
  • lane 12 (at B) and lane 13 (at C) correspond to the bottom and represent non-membrane-bound (non-floating) proteins that remain in place on which the incubation mixture was layered under the gradient before centrifugation.
  • Fig. 2 Results of the pulse labeling of proteins in cotyledons, which indicate a weak binding of LBLOX to microsomes, but a significant LBLOX pool in the cytosol.
  • Lane 1 (microsomes) and lane 2 (“cytosol”) show fluorograms, whereas lanes 3 and 4 show the protein stains. Lane 1 (and the corresponding protein stain in Lane 3) shows the absence of contamination of isocitrate lyase in the microsomes.
  • Part C Treatment of labeled microsomes (analyzed as in Part A, lane 1) with proteinase K. After proteolysis, phenylmethylsulfonyl fluoride and 1 ⁇ g of the corresponding cold protein were added and immunoprecipitation was carried out. Lanes 1 through 4 show fluorograms: untreated microsomes in lane 1; untreated cytosol in lane 2; treated microsomes in lane 3; treated cytosol in lane 4.
  • Part D Localization of the cucumber LBLOX on microsomes from transgenic tobacco plants. After the flotation of the membranes in a linear sucrose density gradient, subtractions were analyzed using immunoblot. Lane 1 corresponds to the upper section of the centrifuge tube (23% sucrose), lane 3 (32% sucrose), lane 5 (39% sucrose), lane 6 (41% sucrose) and lane 7 (sample filled with 43% sucrose).
  • Fig. 3 In vitro experiments showing that radioactively labeled cytosolic LBLOX weakly binds to microsome membranes (part A) but practically does not bind to glyoxysomes (part B).
  • Part A 1 g of cotyledons was incubated with 9 MBq [ 35 S] L-methionine for 3 hours. A 100,000 g supernatant containing radiolabelled cytosolic LBLOX was then prepared. This preparation was mixed with a homogenate made from untreated cotyledons. The ER / Golgi fraction was isolated by subsequent gradient centrifugation. An aliquot (1/20) of the ER / Golgi fraction (lane 1) and an aliquot (1/20) of the re-isolated cytosol (lane 2) and a large aliquot (1/2) of the ER / Golgi fraction (lane 3 ) were subjected to SDS-PAGE and fluorography.
  • Part B In a similar mixing experiment, isolated unlabeled glyoxysomes were incubated with the radioactive cytosol prepared in vivo-labeled cotyledons. The subsequent rice isolation of the glyoxysomes and glyoxysome membranes was carried out by flotation in a sucrose gradients performed. For this purpose, the incubation mixture was adjusted to 60% (w / w) sucrose. A gradient (56 to 38% sucrose) was layered on the sample. After centrifugation at 27,000 rpm for 15 hours in a Beckman SW-28 rotor, the fractions were analyzed by SDS-PAGE and fluorography. The position of the marked LBLOX in the fluorogram is indicated by an arrow.
  • the lanes correspond to the following fractions (equilibrium densities in brackets): lane 4 (48% sucrose), lane 5 (48.5% sucrose), lane 6 (49% sucrose), lane 7 (50.5% sucrose), Lane 8 (452.5% sucrose), Lane 24 (56% sucrose), Lane 25 (56.5% sucrose), Lane 26 (58% sucrose), Lane 27 (59% sucrose) and Lane 28 (59% sucrose) ).
  • the numbers of fractions 25-28 correspond to the position in the gradient at which the suspension was filled before centrifugation.
  • Lanes 4-5 encompass the glyoxysome membranes and lane 8 contains the glyoxysomes according to the protein profile.
  • Fig. 4 Affinity of LBLOX and PLA expressed in bacteria to liposomes.
  • Flotation was carried out by centrifugation at 100,000 g for 6 hours.
  • the proteins in the subtractions obtained from the gradient were analyzed by SDS-PAGE and immunoblots.
  • Appropriate antisera which had been produced either against LBLOX or against the patatin-like protein, were used for the immune labeling.
  • the rightmost traces always show the soil on which the incubation mixture was layered under the gradient before centrifugation. The flotation thus took place from right to left.
  • Fig. 5 Schematic representation of the LBLOX structure and its N-terminal section (amino acid residues 48-244), which has the ß-barrel.
  • the structure of the LBLOX was calculated based on the crystal structure data obtained for soybean LOX-1 [21] using the primary sequence of the LBLOX.
  • the N-terminus and the ß-barrel consisting exclusively of ß-leaflets are shown somewhat separately at the bottom right.
  • the main part of the LOX, which comprises the active center at the C-terminus, is dominated by ⁇ -helices.
  • the lower part of the figure represents an enlarged view of the N-terminal
  • the 40 7elsmino acid residues of the outermost N-terminus, an extension not found in other LOX structures, are not shown.
  • the broken structure marked with arrows at the bottom right shows a point at which the amino acid sequence of the LBLOX differs considerably from that of the soybean LOX-1.
  • the program used (Swiss 3D model, Expasy-Server) resulted, similar to LOX-1, in a highly flexible loop and thus an undefined structure.
  • This loop consists of 14 amino acid residues in LOX-1, but comprises 20 amino acid residues in LBLOX.
  • the two glutamyl residues E59 and E70 are unique and are only found in LBLOX and not in soybean LOX-1. This site can play a role in the Ca 2+ -coordinated membrane association.
  • the affinity-purified fusion protein GST-LBLOX244 was added to a suspension of lipid bodies in enriched cytosol. After the incubation, the lipid bodies were removed using
  • Fig. 7 Binding of LBLOX constructs and recombinant proteins with wild-type and modified ⁇ -barrel to liposomes.

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Abstract

L'invention concerne une séquence d'acide nucléique isolée qui code pour un polypeptide et qui est constituée d'une combinaison de séquences d'acide nucléique d'une séquence d'acide nucléique de biosynthèse du métabolisme des acides gras ou des lipides et d'un des acides nucléiques suivants: a) séquence d'acide nucléique correspondant au numéro d'identification SEQ ID NO:1; b) séquences d'acide nucléique qui sont dérivées en tant que résultat du code génétique dégénéré de la séquence d'acide nucléique correspondant au numéro d'identification SEQ ID NO:1; c) des dérivés de la séquence d'acide nucléique correspondant au numéro d'identification SEQ ID NO:1 qui codent pour des polypeptides présentant des séquences d'aminoacides correspondant au numéro d'identification de séquence SEQ ID NO:2 et présentent au moins une homologie de 60 % en ce qui concerne les aminoacides; d) une séquence d'acide nucléique comportant la séquence correspondant au numéro d'identification SEQ ID NO:3 ou bien la partie aminoterminale de la région codante de cette séquence.
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JP2001532210A JP2003512058A (ja) 1999-10-21 2000-10-10 脂質体リポオキシゲナーゼのβ−バレル
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TATULIAN SUREN A: "Ca2+-dependent membrane binding of soybean lipoxygenase-1: Possible implication of the N-terminal beta-barrel domain.", FASEB JOURNAL, vol. 12, no. 8, 24 April 1998 (1998-04-24), Meeting of the American Society for Biochemistry and Molecular Biology;Washington, D.C., USA; May 16-20, 1998, pages A1285, XP000982453, ISSN: 0892-6638 *

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WO2002086114A1 (fr) * 2001-04-20 2002-10-31 Novozymes A/S Lipoxygenase
US7264954B2 (en) 2001-04-20 2007-09-04 Novozymes A/S Lipoxygenase
US8263376B2 (en) 2001-04-20 2012-09-11 Novozymes A/S Lipoxygenase
WO2011048119A3 (fr) * 2009-10-20 2011-06-30 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Procédés et moyens pour altérer la biosynthèse des lipides par ciblage de multiples enzymes sur des domaines d'organelles sub-cellulaires
AU2010309840B2 (en) * 2009-10-20 2015-07-16 Bayer Cropscience Nv Methods and means to alter lipid biosynthesis by targeting multiple enzymes to suborganelle domains

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DE19950921A1 (de) 2001-04-26
AU781120B2 (en) 2005-05-05
CA2388305A1 (fr) 2001-04-26
AU1023501A (en) 2001-04-30
EP1222282A1 (fr) 2002-07-17
NO20021851D0 (no) 2002-04-19
NO20021851L (no) 2002-06-03
JP2003512058A (ja) 2003-04-02
CN1382217A (zh) 2002-11-27

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