WO2001029227A1

WO2001029227A1 - The beta-barrel of the lipid body lipoxygenase

Info

Publication number: WO2001029227A1
Application number: PCT/EP2000/009912
Authority: WO
Inventors: Helmut Kindl; Christian May; Ivo Feussner
Original assignee: Basf Aktiengesellschaft
Priority date: 1999-10-21
Filing date: 2000-10-10
Publication date: 2001-04-26
Also published as: DE19950921A1; CN1382217A; NO20021851D0; AU781120B2; JP2003512058A; AU1023501A; CA2388305A1; EP1222282A1; NO20021851L

Abstract

The invention relates to an isolated nucleic acid sequence which codes for a polypeptide and consists of a combination of the nucleic acid sequences of a biosynthesis nucleic acid sequence of the fatty acid or lipid metabolism and the following nucleic acids: a) a nucleic acid sequence having the sequence as represented in SEQ ID NO: 1, b) nucleic acid sequences which derive from the degenerated genetic code of the nucleic acid sequence as represented in SEQ ID NO: 1, c) derivatives of the nucleic acid sequence as represented in SEQ ID NO: 1, said derivatives coding for polypeptides with the amino acid sequence as represented in SEQ ID NO: 2 and being provided with at least 60 % homology in the amino acid level, d) a nucleic acid sequence with the sequence as represented in SEQ ID NO: 3 or the aminoterminal component of the coding area pertaining to said sequence.

Description

THE BETA BARREL OF LIPID BODY LIPOXYGENASE

description

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.

Oil fruits play an important role in human nutrition. The endogenous stores of triacylgyceride-storing plants are broken down in germinating seeds, so that de novo formation of new tissue is possible even in the absence of light [Rees et al. , Biochim. Biophys. Acta, 385, 1975: 145-156; Kindl, H., Biochimie, 75, 1993: 225-230]. Triacylglycerides 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. Biol., 4, 1994: 493-498]. ^{^"FOR"} the beginning of germination are degraded the lipid bodies and their content and provide fatty acids for Glyoxysomes ready responsible for the beta-oxidation of fatty acids [Kindl, H., Z. Naturforschende, C 52, 1997. 1-8 ]. In cucumber cotyledons, phospholipase A ₂ (PLA) [May. C. et al. , Biochim. Biophys. Acta, 1393, 1998: 267-276] and a certain isoform of lipoxygenase [lipid body lipoxygenase, LBLOX) [Feussner, I. et al. , Planta, 198, 1998: 288-293; Höhne, M. et al. , Eur. J. Biochem. , 241, 1996: 6-11.] Synthesized in the step of triacylglyceride mobilization and transported to the lipid bodies. 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]. Then the Lipoxygenase the modification of the acyl residues in the triacylglycerides, which are mainly linoleyl groups [Feussner, I. et al. , FEBS Lett., 431, 1998: 433-436]. Finally, after reduction and the action of a specific lipase dependent on hydroxyoctadecadienoyl residues, S-13-hydroxyoctadecadienoate is released from the lipid bodies into the cytosol [Feussner, I. et al. , Proc. Natl. Acad. Be. USA, 92, 1995: 11849-11853] and then degraded by the glyoxysomes [Kindl, H., Biochimie, 75, 1993: 225-230; Kindl, H., Z. Naturforsch., C 52, 1997: 1-8].

In the course of lipid mobilization, a set of newly synthesized proteins is transported to the surface of the lipid body [Sturm, A. et al. , Eur. J. Biochem. , 150, 1985: 461-468], including LBLOX and PLA. Previous works [May. C. et al. , Biochim. Biophys. Acta, 1393, 1998: 267-276; Feussner, I. et al. , Planta, 198, 1998: 288-293] have shown that LBLOX and PLA only last for a short period of time, e.g. H. at the beginning of lipid mobilization, transiently synthesized. The processes involved in transporting these proteins within the plant cell are still completely unclear.

The task was therefore to understand the signals of the intracellular transport and to use them for targeting proteins.

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:

a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,

b) 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,

c) derivatives of the 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, d) 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

and introduces the obtained sequence into a eukaryotic organism.

In the present work, evidence for a post-translational transfer of LBLOX and PLA to lipid bodies was found. It was shown that an N-terminal domain of the lipid body LOX, which is folded as a β-barrel, is responsible for the membrane binding properties of the enzyme. This domain of the protein encoded by the nucleic acid sequence according to the invention, which has a membrane targeting structure, can be used in the method according to the invention for protein targeting of foreign proteins to lipid bodies, for example in oil seeds.

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. For this purpose, the organisms are grown in a suitable medium.

Further objects of the invention are a method for targeting proteins involved in the biosynthesis of lipids or fatty acids in liposomes or lipid bodies, characterized in that at least one 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.

Examples of 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. All stages of the purification of the saturated or unsaturated fatty acids can be used, ie from crude extracts of the fatty acids to fully purified fatty acids are suitable for the production of the aforementioned products. In an advantageous embodiment form, 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.

Depending on the host organism, the organisms used in the processes are grown or cultivated in a manner known to those skilled in the art. 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.

After culturing, the lipids are usually obtained from the organisms. For this purpose, 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.

To obtain the free fatty acids from the triglycerides, they are usually saponified, as described above.

Another subject matter of the invention are isolated 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:

a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,

b) Nucleic acid sequences which differ as a result of the degenerate genetic code from that shown in SEQ ID NO: 1

Derive nucleic acid sequence,

c) derivatives of the 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,

d) 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.

These 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.

The nucleic acid sequence (s) used in the method according to the invention (the singular should include the plural for the application and vice versa) or fragments thereof can advantageously be used for the isolation of further genomic sequences via homology screening.

10 The derivatives mentioned can be isolated, for example, from other eukaryotic organisms such as fungi, yeasts or plants such as specifically mosses.

Furthermore, derivatives or functional derivatives include

15 sequence in SEQ ID NO: 1 to understand, for example, 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 ,

20 The homology was calculated over the entire amino acid range. The program PileUp, BESTFIT, GAP, TRANSLATE or BACKTRANSLATE (= part of the program package UWGCG, Wisconsin Package, Version 10.0-UNIX, January 1999, Genetics Computer Group, Inc., Deverux et al., Nucleic. Acid Res., 12

25 1984: 387-395) (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153). The 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%

30 identical. The 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

Such 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.

10

Under standard conditions, for example, depending on the nucleic acid, temperatures between 42 and 58 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in

15 To understand the presence of 50% formamide such as 42 ° C in 5 x SSC, 50% formamide. The hybridization conditions for DNA: DNA hybrids are advantageously 0.1 × SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C. For DNA: RNA hybrids, the hybridization

20 conditions advantageous at 0.1 x SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C. These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of

25 50% in the absence of formamide. The experimental conditions for DNA hybridization are in relevant textbooks of genetics such as Sambrook et al. , "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be described according to formulas known to the person skilled in the art, for example depending on

30 calculate the length of the nucleic acids, the type of hybrid or the G + C content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids

35 Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

40 Furthermore, 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.

45 In addition, 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.

The 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. In general, 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. These plant preferred codons can be determined from the highest protein frequency codons expressed in most interesting plant species. An example of Corynebacterium glutamicum is given in: Wada et al. (1992) Nucleic Acids Res.

20: 2111 to 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 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.

In addition, 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:

Acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] - desaturase (s), acyl-ACP-thioesterase (s), fatty acid acyl transferase (s), fatty acid synthase (s), fatty acid -Hydroxylase (s), acetyl-coenzyme A carboxylase (s), acyl-coenzyme A oxidase (s), fatty acid desaturase (s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allen oxide synthases, hydroperoxide lyases and / or fatty acid elongase (s). These are advantageously nucleic acids which code for one of the following proteins: fatty acid acyl transferase (s), Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ9-desaturase, Δ12-desaturase, Δ15-desaturase and / or a fatty acid elongase

The 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.

In the method according to the invention, the nucleic acids can advantageously be combined with other genes of fatty acid biosynthesis. Examples of such genes are acetyltransferases, further desaturases or elongases of unsaturated or saturated fatty acids as described in WO 00/12720. For in-vivo and especially in-vitro synthesis, the combination with, for example, NADH cytochrome B5 reductases is advantageous, which can absorb or release reduction equivalents. The 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

10 30% of the biological activity of the parent protein. For example, certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.). For example, arginine residues against lysine residues, valine residues against isoleucine

15 residues or aspartic acid residues exchanged for glutamic acid residues. However, 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.

20

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

25 Show function, which means that their biological activity is not significantly reduced. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, for example, such nucleotide sequences are also included in the present invention

30 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.

35

Functional equivalents are also those variants whose function is weakened (= not significantly reduced) or enhanced (= not significantly reduced) or enhanced (= biological activity is stronger than the activity of the parent protein, i.e. activity is higher than 100) compared to the original gene or gene fragment %, preferably higher than 110%, particularly preferably higher than 130%).

The above-mentioned nucleic acid sequences used in the method according to the invention are advantageously inserted into an expression cassette (= nucleic acid construct) for introduction into a host organism. However, the 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.

An expression cassette (= nucleic acid construct or fragment) is the sequence mentioned in SEQ ID NO: 1, which is to be understood as the result of the genetic code and / or its functional or non-functional derivatives, which advantageously with one or more regulatory signals to increase the Gene expression were functionally linked and which control the expression of the coding sequence in the host cell. 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. For example, these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new regulatory sequences or instead of these sequences, 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. However, 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

Regulation sequence mutated in such a way that regulation no longer takes place and / or gene expression is increased. These modified promoters can also be brought in front of the natural gene to increase the activity in the form of partial sequences (= promoter with parts of the nucleic acid sequences according to the invention). 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 nucleic acid sequences according to the invention can be contained in one or more copies in the expression cassette (= gene construct). Other genes that are also possibly expressed and which are advantageously involved in fatty acid biosynthesis can also be found in one or more copies are present in the expression cassette.

As described above, the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA.

In principle, all 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-, λ-P _R - or contained in the λ-P _L promoter, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFOC, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters such as CaMV / 35S [Franck et al. , Cell 21 (1980) 285-294], SSU, OCS, lib4, STLS1, B33, nos (= nopalin synthase promoter) or in the ubiquitin promoter. 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

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H- o Ξ cn Φ Ω i »3 ^ P o ιf => cn P Φ o 3 H • o <P. z \ p. Φ rt e Φ cn Φ 3 _^^ 1 φ G rt o 1 φ PP cn cn P Pi φ C Ω H- CΛ 3 Φ p. Pi Φ o 3 Φ F- P- yo K t - σ. Ξ $, NH Φ ff F- Φ 3 o Φ o

& Φ SU o φ u cn H- CΛ Φ 3 • xl Pi P- cn P- ff 3 3 cn 3 P F- ^ FP » _^ -. P- Φ F- 3 SU 3 PPP et P- 3 O ff rr 3 rr N 3 3 1 3 rt Φ Φ Pi S cn l Φ d Φ Φ -— 'G φ 3 Ω Φ lh 1 Pi IQ rt φ IQ

Φ p ff Φ rr Φ φ P: αi cn Φ ^r ϋ P Φ d 3 Φ Φ ti Ω 03 3 P »iQ Pi _^ CΛ P- Φ 3 rr 13 $ u Φ Φ F- cn SU

Φ rt rr 3 Φ 3 φ ^ rt ri Φ lh ω F> o φ Φ P d Φ αi φ Φ 03 3 rt Φ P <£ P- 3 cn 3 in Φ Φ cn * - 3 rt LΠ SU o cn Φ P - o Φ Pi Pi F- 3 »P- 3 PP ^ ff rt OZ oo SU: H Φ Φ

P P £ ff - -. • σ ≤ Φ o C SU 3 3 o p. Φ φ 3 cn φ ≤ Φ to P- Φ c ^ φ ff Ω F- G 3 3 ff ff cn 3 3 cn Φ Φ II Φ O 3 (U * -. Cn o U 3 w Φ cn 3 p, 1 ^ P- O .. 3 dd P os; Φ Φ O CΛ o ^ P SU F-

F- P- Pd P 10 N 3 ^. rr Φ cn p, o. Ω ω ^r ϋ oo 00 SU Φ 3? R 13 rt th o 3

O 3 Φ Pi o Ω F- αi Φ. <0 t> _^ oo 3 rt ^ P. Φ Pi αi P to cn Φ tl P 13 Φ 1 o Pi Φ rt ff o P- ff IQ Φ ff Φ lh ^ -_ 3 ^ o 3 cn $ u rt ^r ϋ o φ 3 Φ Ω ^ o φ ιt ^ ff P ^» PP rt d 13 P Φ F- Φ PO dd PP 3 F- H ¹ o SU so TJ rt ff lh ff cn cn 03 H t 3 3 1 SU Lπ cn φ o 3 p. P 03 φ cn ff ff 3 F- "cn V I- ¹ ff o SU rt Φ rt Φ φ d 0 ^r ϋ 03 to 3 3 iQ o rt 13 3 rt Φ φ IQ $ U co Φ OOM H- o N ι -h cn) U »s: rt to rt 1 td P φ - ff o P ¹ 3 ö F Φ P rt d P

F- ω rr o ff 3 ff O o cn H P- P- φ 3 ^r ϋ o P Φ Φ ooo • φ rt o Φ o Φ M 3 o Z lh 03

03 iQ F- Φ cn Φ 3 t P o Ω 3 N iQ Φ F- o P to 1 3 F- θ: o P- rt P tJd 3 F- Φ rt

• 0 φ o 3 fo ^ Φ Ό Φ <Pi P- Pi <Φ o IQ cn N P- - Φ ^r ϋ o P- P 03 3 PP ¹ 3 o rf ^ s; o O 3 Φ

F- 3 3 Φ P t _ (-! Rr. Cn 3 H- Φ cn Φ 0 3 3 Φ Φ 3 3 P rt 3 Φ Ό 3 Φ ^ Φ P Φ 1 F- rt

Φ SU: 3 3><3 o 13 H- o cn o rt 3 rt o 3 X 3 1 o • - 'oo 1 P F- Φ 3 P 13 Φ o φ cn Λ oo • Ü P ι- <r → i cn Ω H- ff. Φ rt SU Ω (P 3 P ^r ϋ Φ 3 co cn o lh PPP ¹ rt Φ cn Φ P- O er P Φ SU Φ Φ (U 3 φ • _ P- o CΛ th Ω Φ φ Φ oo P o co Φ P F- o ff Φ 3 SU 3

≤ 3 Φ φ ff ^ Φ ≤ 3 3 3 SU H Φ H p, φ Φ H 3 P 03 P rt SU o ff P LΠ ff φ 3 3 Φ 3 Pi rt φ P 3 ω O rt ≤ Φ th W w 3 rt Φ Φ o F, P- 03 φ od 3 φ (U cn P- PP Pi o F- Φ rt

F- 3 rr ≤ 3 cn ^r ao rt SU H- X cn rt 3 3 SU (U _^ PPP cn oo lh P- Φ -—. D rt tn 3 F- cn cd (3- Φ d F- ι_π ≤ i ff 3 • o ff cn Ω Pi F- • Ö rt cn r 3 lh 3 0 • d P- o 3 φ 3 (U F- O H- o SU 1 Φ SU: cn ff P- F- P φ rt lh Öd o F- PP φ iQ P F- F- 3

H rr rr H 3 t Φ (-: 3 - \ Ω φ cn d P- Φ 3 d 3 Φ t d: F P P 3 tu d: .. d = 03 03 Φ φ c P "P

Φ Φ rr Φ Φ l *> ι- (o _^ - _^ Φ cn oo <1 rt 1 cn> ϋ l P Φ f 1 3 PP ff 03 IQ ff * Φ Φ

§ F- P Φ P F- <to Pi 3 rr LΠ o G 3 cn 3 φ ff 0 Ω Φ Ω P Cd 03 $ rϋ iQ F- φ Φ Pi ω φ P rt 3

3 3 Φ 3 Φ φ fu o ιi l- ¹ CΛ cn P- Pi 3 φ P Φ Φ Pd oos>! 03 d P Φ Φ 3 LΠ Ω 3 3 Φ s; F- * Φ

£ cn w P o ri σy ^> Α o Φ π N 3 P- 3 Φ 3 Pi o ^ P- o lh o yo Φ (U: P Φ 3 o Z 3 φ SU: Φ cn Ω CD: 3 "- ^ r → 11 SU 3 p. cn d cn 3 cn P- o Φ 3 o 3 d: 3 1 3 α t ⁾ P- φ. P-

F- 0 P Φ Φ d F- ^ P. Pi - 'to c Φ O: rt iQ 1 03 rt P o td f »o ff d: o cn Φ Φ 13 03 Φ P. n- P F- 3 P rr Φ t Φ - to p cn ^| Q Φ rt Ω o SU ff rt PP ι P $ u ff φ 03 H F- φ Φ iQ 3 Φ Φ rr i cn cn cn ff Φ N Φ H ^ P Φ P o P 0 Φ SU oro - ^ - • o P- d SU 0) 3 ff Φ

P 3 _^ 1 Φ H- _^ - _^ P Φ: «s; 3 P- 3 Φ P, Φ • α rt d 03 P Φ α rt. P <cn P 3 cn φ

Φ Φ Φ M co F φ - _^ SU CD: Φ P φ cn cn 3 rt 3 Φ F- Φ SU iQ φ φ Φ ff F- ^> _

P ¹ F- ö P. 3 • 0 - Φ cn ⁾ SU Ό ff 3 d 3 P- N o SU 2, S3 cn rt φ o P 3 φ 03 Φ ω H- O F- F- H- rt .. IQ ^ - ^ 3 cn Φ 3 Φ F- S P- P 1 Φ φ d SU 0 Φ Φ cn Φ ι-h H 1 cn Ό rt

F- Φ ff Φ Φ 3 t .. p 1 3 Pi 3 Φ 3 Φ Ω P- d cn • 0 rt F- P- d: F- G o P- rt

0 IQ Φ [0 rr F- F- 1 03 Φ 3 03 F- d rr o ff 3 CΛ 3 φ 1 Φ 3 Φ F- H- Φ Φ 3 N 3 3 3 Φ 3 P 1 13 P 3 3 3 cn 3 Φ 03 1 φ PP rt 1 03

3 Φ φ 1

Synthetic genes advantageous of fatty acid biosynthesis, which enable an increased synthesis. For example, the genes for the Δ15-, Δ12-, Δ9-, Δ6-, Δ5-, Δ4-desaturase, the various hydroxylases, the acyl-ACP-thioesterases, β-ketoacyl synthases or β-ketoacyl reductases may be mentioned. The desaturase genes are advantageously used in the nucleic acid construct.

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the expression cassette according to the invention and the method according to the invention as described above. In addition, synthetic promoters can also be used advantageously.

Various 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. To connect the DNA fragments (= nucleic acids according to the invention) to one another, 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. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. In general, 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.

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, vifcro mutagenesis, primer repair, restriction or ligation can be used. With suitable manipulations, such as restriction, chewing back or filling in of overhangs for blunt ends, complementary ends of the fragments can be made available for the ligation. The attachment of the specific ER retention signal SEKDEL (Schouten, A. et al., Plant Mol. Biol. 30 (1996), 781-792) can be important for an advantageous high expression, the average expression level is thus tripled to quadrupled , Other retention signals, which occur naturally in plant and animal proteins located in the ER, can also be used to construct the cassette.

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

Cloning techniques such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al. , Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

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. Examples of reporter genes are antibiotic or herbicide resistance genes, hydrolase genes, fluorescence protein genes, bioluminescence genes, sugar or nucleotide metabolism genes or biosynthesis genes such as the Ura3 gene, the Ilv2 gene, the luciferase gene, the β-galactosidase gene, the gfp gene glucose 6-phosphate-phosphatase gene, the ß-glucuronidase gene, ß-lactamase gene, the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene or the BASTA (= gluphosinate resistance) gene. 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.

According to a preferred embodiment, 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

Phage or other DNA inserted, which enables optimal expression of the genes in the host organism. 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 , λgtll 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. , [(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488] and by van den Hondel, CAMJJ et al. [(1991) "Heterologous gene expression in filamentous fungi] and in More Gene Manipulations in Fungi [JW Bennet & LL Lasure, eds., P. 396-428: Academic Press: San Diego] and in" Gene transfer Systems and vector development for filamentous fungi "[van den Hondel, CAMJJ & Punt, PJ (1991) in: Applied Molecular Genetics of Fungi, Peberdy, JF et al., eds., p. 1-28, Cambridge University Press: Cambridge]. Advantageous Yeast vectors are, for example, 2μM, 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. Elsevier, Amsterdam-New York- Oxford, 1985, ISBN 0 444 904018. Gee Ignited plant vectors are described, inter alia, 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 which replicate in E. coli and Agrobacterium.

In addition to plasmids, 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.

In a further embodiment of the vector, 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. In a further advantageous embodiment, the nucleic acid sequence according to the invention can also be introduced into an organism on its own.

If, in addition to the nucleic acid sequence according to the invention, further genes are to be introduced into the organism, they 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.

For example, 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.

Alternatively, a recombinant vector (= expression vector) can also be transcribed and translated in vitro, e.g. by using the T7 promoter and the T7 RNA polymerase.

Expression 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. Frequently, proteolytic cleavage sites are also introduced via fusion proteins, which enables a part of the fusion protein to be split off, also for purification. Such 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. Further examples of 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, 5 Amsterdam, The Netherlands].

Further advantageous 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.,

10 (1987) Gene 54: 113-123), and pYES derivatives (Invitrogen

Corporation, San Diego, CA). Vectors for use in filamentous mushrooms 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,

15 J.F. Peberdy, et al. , eds., p. 1-28, Cambridge University Press: Cambridge.

Alternatively, 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).

Furthermore, plant cells or algal cells can advantageously be used for gene expression. Examples of 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.

Furthermore, the 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.

35 (1987) Nature 329: 840 or Kaufman et al. (1987) EMBO J. 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

40 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.

45 In principle, 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.

For microorganisms, 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 F.M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by D.M. Glover et al., DNA cloning

Vol.l, (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. Guide to Yeast Genetics and Molecular

Biology, Methods in Enzymology, 1994, Acade ic Press.

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. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). 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. (1988) 16, 9877 or is known, inter alia, from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press, 1993, pp. 15-38. 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.

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.

In principle, 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. Examples are 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. Preference is given to organisms which can naturally synthesize oils in large amounts, such as yeasts such as Saccharomyces cereviseae or Yarrowia lypolytica, fungi of the genera Mortierella, Traustochytrium or Pythium such as Mortierella alpina, Pythium insidiosum or plants such as Brassica napus, Linium usitatissimum, soybean, rapeseed, coconut Oil palm, safflower, castor, calendula, peanut, cocoa bean or sunflower, soybean, rapeseed, sunflower, castor, mortierella or pythium are particularly preferred. In principle, 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).

Usable expression strains e.g. those which have a lower protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.

Depending on the choice of the promoter, 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.

Examples:

Plant material and cell fractionation

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].

Plasmid construction and preparation of proteins

To produce deletion forms of the LBLOX, 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. .

<ξ rr σy • 0 iQ α cn tf> P P α Pi d g ff 2 C

Φ P. p P 1 π p Φ 3 φ P φ φ Φ φ 3 φ Φ su d

P φ P tu ω P tn • o o 03 P Pi rt iQ n P z 3 P 3 F i d: Φ ff rr rt rt Φ ff SU ff. φ rt φ »- 'ω Φ ff P M Su 3 Φ rt P. a

3 F- x F P rt £ P F- 3 03 P- φ o p Pi

P cn P P O P. rt cn o 3 rt o 3 3 φ. d O 3 X. P cn SU: σy 3 P. P. P

3 ff ff F- 1 <φ Ω rt Φ r 13 3 dd iQ rt co tsi O cn ω co σy 3 P φ X

13 φ co d 3 • _ 3 *>. s P o φ o

<cn SU P ^ 3 P ω l su ιl rt β P o o rt rt rt • O o F P F 3 F Φ rt P ff

3 - SU rt <K O 3 .— * 03 X <ω dd F- SU rt φ rt d H X Φ φ 13 F o F n F 3 3 3

TJ 3 F- 3 H O: X P F <ff O Φ 3 o 1 O P-

O F- 3 P rt

3 ^* W o n> PX rr X Φ ^

ΓΛ rt 1 iQ 0 rt F 3 P co d F> Φ. SU P SU d

13 rr SU: Φ (B P Φ O 3 3 bd LΠ P ff rt 3 co

Φ B ^* 3 cn ** XFS iQ F 3 P iQ l Ό 1 dd o O o rt φ o

1 cn P $ u d co F P- 3 X d 3 Z 3 3 <^ co Φ H • 0 3 co O SU F- d = 3 o 3 o

O rt rt o iQ P ^> X - - rt P Φ (→ ff ≥; P 3 r → b ff F- Φ Φ P rr 3 Φ cn d Φ

Φ X 3 rt rt <F- P- F- Φ P rt F 3 O X 3 d <^ d 1 0 3 3 cn Cd F 3 σ.

3 ^' μ. 13 SU 3 o Φ tl F 03 dd F- Φ 2 ___ • •

P. rt P od H ¹ φ ω PO Φ F rt o 3> o 3. F F- fu π 13 SU XP o rr rt cn (

PO rt • <: rt _^ 3 dd 3 x Φ ff P o> Φ X Pi 3 ι

Φ 1 Φ rt rt F rr P P o ff yo> P- P t)

P ι-3 F- Φ P M ff O 3 Φ rt o rt * • σ. Ω Π Φ P rt

P 3 3 d SU XP. 3 opens '- _'

13 SU <P Ό F- SU Mι Z ιP> ιo O ^

F 3, - • <: Φ rt 3 IO Φ rt 3 d: cn tu oo co ff

Pi P- M 3 o P P φ rt 03 SU

1 X $ u SU z, <→ 3 rt l iQ Φ cn P 3 <3 t → n P cn r Φ rt <≤ P P φ o 1 O P p. cn ö 3 Φ p u> 3 3 3 rt 3 b

S • ö 3 rr Pi 03 F x P • 0 φ d Φ 3 F rt P d F- ω 03 rt P ff α ff o P W i 03

F- rr 3 o F o φ d «Φ Z ff 3 o 13 μ. F d 0 3 iQ n o p. P φ 3 H>: P- cn P rt O

3 3 13 ff X Ö 3 o Φ PF 3 F 0 ^r i X rt ff <Φ _* -_ $ u $ u 3 rt rt dd dd rt o>

SU o SU o 3 <iO O d $ J F- cn F F Φ 1

P P rt 3 φ μ. d ff p Φ o o O>. O

SU • ö P- rt F- P P 3 Pi X 3 X 3 P 3

«3 ff Φ SU« Ω P d Φ> 1 cn o o P ~ o to φ O iQ H Φ 3 3 _? * Co 3 ff

3 rt 1 ιl 3 1 Φ od iQ co ff cn Φ rt. 13 P ^> 13 3 rt P <φ X

P rt • ö o • P d o & Z 3 P

0 F- SU M SU Φ rt cn 3 3 SU o φ

H o cn ff P> 3 P P ff Pi 3 P 3 φ Ό o

H 3 φ Φ vo cn d φ ι_ LΠ o φ SU: et F- rt

Φ 3 Φ P 3 od SU cn 3 d cn 3 d P σι SU 0 3 H SU: PO Φ o B φ ^"

P 3 o 0 ⁾ .. rr ff P. 1 tn od φ P 3 3 rt φ rt cn Φ o PPP •

Φ φ o cn 3 Φ Φ φ OP 3 cn P 03 ff

rt

T7 promoters obtained [May. C. et al. , Biochim. Biophys. Acta 1393, 1998: 267-276].

Transfection of tobacco

Using the vector pBI121 (Clontech), which contains the 35S-KNA promoter of the cauliflower mosaic virus, the coding region of the β-glucuronidase gene and the NOS terminator, the construct pBI121ΔGUS 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 pBI121ΔGUS. The product, pBI121ΔGUS-LBL0X, was either used for transformation in Agrobacterium tumefaciens or further modified to pBI121ΔGUS-LBLOX-HA. The construction of the latter plas id was carried out using a single Sacl site behind nucleotide 252 of pCSLBLOX-221. After cleavage with Sacl and

Dephosphorylation, 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 pBI121ΔGUS-LBLOX or pBI121ΔGUS-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:

1229-1231] used. 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.

Preparation of HA-labeled LBLOX by expression in tobacco

Leaves of homozygous kanamycin-resistant tobacco lines containing the pBI121ΔGUS-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. Fractions that corresponded to the 100 kDa range and analyzed by Western blots using anti-HA antiserum were collected. 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.

10 Radioactive labeling of proteins using in vitro synthesis

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.

Labeling of proteins using in vivo protein synthesis

20

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.

Administration of ⁴⁵ Ca ²⁺ and analysis of the lipid body fraction

30 Two g of cotyledons which had been collected from cucumber seedlings grown in the dark at 26 ° C. for 2.5 days (= T) were cut into pieces and in the dark for 3 hours with 6 MBq ⁴⁵ Ca ²⁺ (8 GBq / mmol) incubated. After homogenization by crushing with a scalpel in the presence of buffer A, the homogenate became

35 centrifuged at 2000 x g for 25 min. After removing the top layer containing lipid bodies and decanting from the sediment, the extract was subjected to centrifugation at 100,000 x g for 1 hour.

40 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.

45 Preparation of liposomes

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

10 0.6: 1.0. The efficiency of deriving by dialysis in the Amicon chamber equipped with a PMIO membrane was checked [Yamazaki, N. et al. , Methods Enzymol. 242, 1994: 56-65]. The size of the vesicles was determined under the microscope by comparison with the size (10 ± 0.5 μm) of MonoQ beads (Pharmacia)

15 determined. For certain experiments, triacylglycerides were used

(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

20 10 ml of methanol / chloroform (1: 1) were dissolved. After removal of the solvent in vacuo, the residue was dialyzed according to [Yamazaki, N. et al. , Methods Enzymol. 242, 1994: 56-65]. In each case, the vesicles obtained by flotation were analyzed by DSC. The diameter of the

25 liposomes was in the range of 1 μm.

Binding experiments in combination with the flotation assay

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 μg 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.

40

Binding experiments with lipid bodies or microsomes were carried out in an analogous manner. All densities in the sucrose gradients are given in correlation with the sucrose concentration (w / w).

45 Immunological procedures

The antisera against LBLOX [Sturm, A. et al. , Eur. J. Biochem. 150, 1985: 461-468], PLA [May. C. et al. , Biochim. Biophys. Acta 5 1393, 1998: 267-276] and isocitrate lyase [Frevert, J. & Kindl, H. Eur. J. Biochem., 92, 1978: 35-43] were prepared in rabbits. Monoclonal antibodies to GST (Pharmacia) and the epitope from the hemagglutinin protein of the human influenza virus (Boehringer) were also used. Immunoprecipitation of the radioactively labeled enzymes was carried out under standard conditions (method 1) by adding 1 μg of the corresponding purified protein to the mixture with 20 μl 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 μl 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.

Other assays

5 In order to compare the protein structures, limited proteolysis in the gel was carried out [Höhne, M. et al. , Eur. J. Biochem. 241, 1996: 6-11]. The DSC analysis of the lipid was carried out on silica gel G (Merck) using methanol-chloroform-water, 65: 25: 4, as the solvent system. 0

ERGEBNIISE

To examine the sequence of steps required for the transfer of LBLOX from the ribosome to its final cellular destination, the first step was to determine which pools in the cell cross the LBLOX protein. Second, the targeting structure, sequence or domains responsible for the transfer were localized.

0 Investigations with LBLOX in vivo and in vitro to distinguish between co-translational transport into the ER and post-translational transport to lipid bodies

No differences were observed between the molecular mass of LBLOX translated in 5 vitro and the cellular forms membrane-bound to the ER or to lipid bodies (FIG. 1A). This shows that the transport to the membranes with no visible chemical modifications take place. Both a co-translational transport to the ER and the subsequent transfer into the lipid body as well as a post-translational transport to the lipid body via a cytosolic pool were likely mechanisms with which LBLOX can reach its final position in the cell. First, in vitro translation of LBLOX mRNA was carried out under co-translational transport conditions in the presence of microsomes from dog pancreas or microsomes from cucumber cotyledons. Second, a similar protocol was used to test whether radioactively labeled LBLOX translate was also transported to the membranes when the translation and the addition of microsomes were done sequentially (Fig. 1B). In the latter case, the ribosomes were removed from the newly synthesized protein by centrifugation before the addition of microsomes. After renewed isolation of ER-enriched membranes by means of flotation, only a part of the translated protein remained at the position at which the suspension had been filled in in the gradient (No. 12). Some LBLOX migrated to densities that corresponded to 33% sucrose (Fr. No. 11) or 30% sucrose (Fr. No. 9). Small amounts of LBLOX were found at the position of the smooth ER (Fr. No. 5-7).

Comparison of the amount of LBLOX radioactivity in the membranes that had been reisolated by gradient centrifugation in a series of experiments showed that the use of the arrangement for post-translational transport to microsomes gave the same or slightly higher amounts of membrane-bound LBLOX than the use of the protocol for co-translational transport (data not shown). When the migration behavior of the LBLOX primary translatate and the LBLOX reisolated from the membranes after flotation were compared exactly, no discernible differences in the molecular mass were found.

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.

The results obtained by examining in vitro transfer from LBLOX to microsomes (Fig. 1B) were confirmed by analyzing the in vivo situation by following the sequence of events that occurred when LBLOX was transferred to lipid bodies. By using pulse-chase experiments we determined the pools that were traversed by the newly synthesized LBLOX on its way to the lipid bodies. We wanted to find out whether the fraction of the ER membranes or the cytosol was the pool in which the labeled LBLOX appeared first.

Figure 2A (lane 2) 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. Using 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.

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. However, it should be emphasized that 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).

The capacity of binding to microsome membranes was also shown for LBLOX under another in vivo condition. In a heterologous system, i.e. H. green tobacco leaves that expressed LBLOX from cucumber cotyledons, the high expression of LBLOX led to the binding of considerable amounts of LBLOX to microsomes. By subfractionation of the 100,000 x g sediment using a sucrose density gradient flotation, we showed that practically all of the LBLOX previously sedimented by centrifugation at 100,000 x g remained bound to the flotated membranes. The peak of the membrane-bound LBLOX (Fig. 2, Part D) matched the marker proteins for ER membranes.

To expand our chain of evidence for a direct post-translational transfer of LBLOX to the membranes of lipid bodies, we examined the affinity of soluble LBLOX for microsome membranes in vitro. In a mixing experiment, we added a mixture of radioactive cytosolic proteins, which had been isolated from cotyledons after pulse labeling, (prep. A) to a supernatant of an extract, which had been prepared from unlabelled cotyledons and centrifuged at 2000 xg (prep. B). This procedure should enable the radioactive LBLOX, which was contained as a precursor in the cytosolic and membrane-free preparation (preparation A), to post-translationally on microsome, lipid bodies and other membranes that are present in the non-radioactive extract were to bind (prep. B). After a brief incubation, we isolated microsomes as potential acceptor membranes for radioactive LBLOX (Fig. 3A) and glyoxysomes as controls (Fig. 3B). The electrophoretic analysis showed that the cytosolic LBLOX was bound to microsomes even under these conditions, which were similar to the in vivo situation. 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. For example, 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

In order to test whether 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. First, 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.

The results of the liposome experiment (Fig. 4) show the clear difference between the membrane affinity of cucumber LBLOX and that of soybean LOX-1. While the two proteins previously assigned to the cucumber lipid bodies, namely LBLOX and PLA, were bound to the liposomes almost quantitatively, the cytosolic LOX forms of the cucumber and soybean

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

Taking into account the lack of proteolytic processing and the data obtained from the binding studies, it can be hypothesized that domains of a folded protein are responsible for the transfer to the membranes. Such a domain was likely when the amino acid sequence of LBLOX was converted into a structure based on the crystal structure data obtained for soybean LOX-1 at a resolution of 1.4 Å [Frevert, J. & Kindl, H., Eur J. Biochem. 92, 1998: 35-43]. In contrast to soybean LOX-1, cucumber LBLOX not only showed a ß-barrel structure shortly downstream of the N-terminal section, but also had a rather unique arrangement of glutamyl residues in this domain, which is used to coordinate Ca ²⁺ could be (Fig. 5). This in turn can indicate a relationship between the LBLOX-ß-barrel and the members of Ca ²⁺ -dependent membrane-binding domains.

N - 3 F- P. ff ff> G dd F Pi X ff 03 F g X ff 13 d ISl XP φ P IQ odg .3 F »d ZXM g cn WE * * fl g G d 1 03 Φ Φ Φ LΠ SU 3 F- dd SU O: SU: tl): dd p O: F- P 3 Z θ: P- Mι P Φ Pi P p 1 P- P P- θ: 03 1 rt PX 1 d P- 3

Od rt P 03 03 o 3 rt 3 F 03 3 d F P P 3 o P φ P Φ cn 03 Φ cn P 03 t) o P P rt 03 tl Od 03 rt

F SU. rt rt | P> P Φ Pi o p rt P O P TJ Pi rt F- Ό F- cn p: P • 0 i P F- rt tl $ * Φ 3 P P F- rt Pi

F- P P. tu: $ u Φ P d Φ N rt P Φ P P. Φ ff Φ p p P. φ

• os • ^• _ rt Φ X φ φ Φ Φ 3 P o Φ P

P F p rt 3 N 3 3 φ Φ P 1 3 P 3 P- tu Ω P d F- P. rt P.: - P X F- φ P P 3 Φ F- cn P 3 tn

F- φ dd cn F- Pi d <iQ in ff. P. Φ 3 • 3 Ω ö Φ P F- P- 3 Φ O: 3 o 01 Si F- 3 Φ 03 φ cn

PF iQ Z Φ P * Φ cn 3 Φ Φ ff g Pi 3 3 cn IQ φ iQ NHP 03 ff d Φ 3 P- P ¹ * N

X O SU rt 3 F- P- rt F-

§ <φ α P 3 rt dd> PP rt rt Φ 1 • d P 01 P _* * - *. CO 0 pd O θ: ^ XX d 03 P od 3 F- .φ 3 rt P φ Pi P- 1 P i ^" Φ g F F- P- ^ Φ N Ω tl P ^> d CO 3 <o 3

P p> er N P o Φ 3 3 φ 3 o Z (U P- SU 3 «IQ 1 Z P- Ω iQ d P d ff φ φ 3 O o rt d N

Ό P oo F- d SU 3 Pi P: 03 o 3 P P o Φ Φ 03 rt P ts ff rt 03 3 rr 3 τ = 3 Pi P- 3 Φ 3 Φ

Φ P o <Pi P P- d Pi F φ 03 P CD: P 03 φ <3 cn F- Φ P F- ^ F- P. Φ 03 IQ t? 3 pts • d

P φ g Φ Φ d φ Ω d 03 F 03 F- 3 d Mι • o d P- 03 rt P 3 p. P P Φ rr F- _? P F 3 Φ rt

3 3 03 P ff 3 F 03 3 Φ P Φ o P rt rt Φ φ Pi 3 X 3 Pi 3 o 3 Φ o i 3 F- F- 03 03 P

3 o t J rt ^ IQ O F 03 Pi P- Pi Φ 3 P Φ ff P P P d F- α O: P cn P 3 3 Φ F- φ 3 P F P 03 Φ rt P-

IT φ P φ X O φ d 3 P- 01 - rt X F- o d 3 3 F- P Pi 03 • 0 F cn 3 o X O d d P-

P rt 3 SU φ 3 P ü XP 3 Φ Φ Φ F- o 3 1 X rt LΠ iQ P Φ Ό F- 03 P P- F- Φ o Ω 03 rt X cn Ω 3 tu φ rt cn P SU Φ SU > φ iQ P »P ¹ Φ Φ

'S Z Φ pi rt. o φ • d P- P Ω cn p: Φ ff Φ

3 03 P F 0) P 3 ID P- 03 • 0 13 φ P & d P Φ P φ P Φ rt I F- 3 F- cn p: d d P P Ω Φ P

03 F- Pi P «g F- 03 cn Ω rt oo φ F- rt P- 3 P ^ X 3 F- o F 3 Φ Φ Pi Φ Φ ι _ d rr P P- → 3. iQ F- 3 o 1 t) P- F ff Φ rt 03 3 3 rt 3 ö (7- IQ ff F- rt F- 3 X 3 1 PP Φ φ 03 d Φ o 3 Φ 3 rt P- P 3 F - Φ 03 03 Φ F- N _^ -. Tu Φ 01 FPF 1 φ t) O: F Φ rt P 3

Φ ((U t. P rt 3 rt z 3 rt F-

P F- d N Pi α 3 PX 03 F- dd P P- φ Φ P o dd P ff φ o P $ * 3 rt g Φ 3 X Pi P. P- NP 03 rt P- SU P- (U rt cn PF IQ Ό P cn PP 3 »d PF φ F- 03 1 rt p N

P- F- 1 Mι • d θ: F- Φ P. Φ 3 F- o 3 φ rt Φ d F- OP F- PP ¹ X Φ 3 Φ 03 7 = o 03 o rt p rt ff P F-

Φ X rt SU rt F- P o P X d Z Φ 3 Pi Φ 3 Ω Ό X o P P p: O: P F P rt iQ X rt 3 Φ P Φ F- iQ

P SU Φ 03 _^ 3 • 0 SU θ: IQ -J SU Φ rt

3 P cn u Φ XP rt P ^» P t → od? 3 ff!> E ⁾ X φ P Ω PP dd 3 co Φ ff Pi PO Φ Φ rt t 13 rt P- Π Φ O: 03 ff • 0 P. F- F f ^

'S 13 r F- Φ 3 3 3

, rt 3 rr Pi P er F- Ό O Φ Φ P> rt SU 3 P o H 3 P 03 Φ Φ P φ O N P ιi <3 Pi P- 1 Φ

F- _^ SU Φ 3 F- iQ Φ Z 3 o Φ P ¹ F- o 3 W <rt P cn X d P 0 1 o P F- Φ 3 CΛ F- ö iQ 3 cn 3 <. P og rt><Ω rt o ^ d Φ o 3 F 03 3 rt • n 3 d Φ F- P 1 3 O

Φ $ u cn rt PZ Φ φ Φ 03 ff Φ ff ι _ 3 d P 3 P P- ⁽ Q> _ F- P: rt Φ d 3 3 r → rt Φ 3

P d: X P 3 P »3 Pi F- φ F- Φ rt F- P Pi cn F- Ω Φ d • Ö ö rt F- F- 03 F IQ <Φ P p:

F- φ P Pi SU: P SU dd P- φ Φ Pi eff 3 3 03 P P- P F Φ ff ff 03 F- P N rt 3 F- dd 03 o LΠ 3 P Ω 3

3 Φ H 3 P Ω 3 o P φ Pi <iQ Φ F- 3 F or dd rt d P- P. cn P o F 03 3 F ¹ 3 ö Φ iQ <Pi P cn XP ff Φ Ω SU 3 SU o Φ Φ 03 dd 3 cn FX 3 o X P- rt Φ 3 O rt> 03 g

Φ φ SU rr tl F- tu rt Z cn ff 3 cn 3 N H F cn P o F- <P 3 θ: ff Ω F- 3 03 X Φ Ω X ff P

PP 03 O Φ o φ P tu ff ff cn φ zp SU P o Ό Φ X 3 O Φ cn P 0 ff <rt tl P ¹ α cn Φ

Φ 1 P P iQ cn P P- • 0 F- X F- P 3 rt X P F- Z 3 t) • d ff Φ 03 P Φ d P Ω P X 03

P SU: rt P rt rt φ F- - 3 o 3 Pi Φ iQ Pi F-> o Ω P φ P ¹ Φ P Φ Φ N 3 ts o 3 Φ rt P 0

3 (Φ Φ P- Φ 03 iQ cn Pi P F- Pi rt φ F- o O rt ff H F- 03 3 o P d P rt rt P tQ cn 3 <X>

M IQ P cn Φ 03 er 3 P. Pi P- d o 3 P - 3 03 3 σ. 03 rt 03 rt rr o>> _ P Φ ff rt P F- φ cn P

Φ IQ 3 P Φ P- F- Φ rt 3 • α o • o ^ F-. Φ φ p: F Φ ff d P Ω P- F- P ff o rt rt o

P Φ F- F rt ι _ P ¹ Φ 3 P- iQ ff ts Φ ≤ 3 F- 3 dd F- φ cn Φ P ff 3 Φ P F- -3 rt d P rr

F- d rt 3 03 P P- <o & tu ö P- p ≤ cn H 3 P (F 3 P 3 P 3 ^ 03 cn o φ 3 d Φ

N 3 P- tu F SU et X M «(U σ 03 rt P- φ P p 3 P F- O P n rr rt 3 cn Ω Pi P- F-

F- iQ 3 O p 3 Φ tu odd Φ Ω ff Φ F- PPP iQ X iQ P ¹ ff Φ d. Φ 1 O Φ 3

Φ iQ Φ X P 03 3 3 3 3 03 3 F- cn N rt φ

B ^" d Pi P dp: Φ φ F- 3 cn 13 Pi Pi P 1

3 1 Pi P ¹ rt F- 03 PP Φ Φ F- Φ 3 Φ P Φ P d 3 X rt Ω PP 3 Si 1 P F- Φ rt ι _

NS 3 Pl g Φ SU p. N rt Φ 03 F- 1 Φ 3 Ω P F- o ff P- Φ iQ Φ rt 0 Φ 3 _^ P ff F- Φ o. rt b <F- PN F- Φ rt 13 M 3 FP ff Φ rt 3 3 Φ 3 F- P. Φ Φ 3 _^ P rt PP

SU Φ Ω F- tu Φ Φ d Φ Ω P rr ^ rt P- P- ff CΛ P- N X F- rt Φ cn F- P Φ rt g iQ

P ff φ F rt <; P 3 X F- ff Φ o • d • Ü Φ Φ P • ö 3 d F d rt IQ {_. 01 03 Φ 3 3 F- Φ 1 F- Φ

03 rt X dd μ. P N er IQ Φ F- <rt φ N F- P P- d Φ 03 dd P Φ Φ rt Φ d 03 3. rt Φ rt

3 <: F <rt P- Φ rt 3 Ω o cn P d P ⁽ Q φ PPFPP ff 3 F- ^» _ N 3 Φ F-

F P- 3 ooo P. 3 Pi 3 Φ Pi ff 3 F- X φ Φ 01 3 O F- Φ d 3 d rt FP Φ F- g P 3 dd rt o 3 03 X 3 0 iQ Φ 3 Pi 3 F 0:

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By extending this type of in vitro experiment, it was found that the transfer to the lipid bodies is done in a Ca ²⁺ independent manner (data not shown). Despite this result, the possibility was investigated that the Ca ²⁺ -dependent binding of proteins to the lipid body surface could ultimately lead to the formation of a Ca ²⁺ -enriched protein layer which surrounds the lipid bodies. To investigate the uptake of Ca ²⁺ together with proteins such as LBLOX and PLA, we examined the formation of Ca ²⁺ -covered lipid bodies by administering ⁵ CaCl ₂ to cotyledons and then isolating cell structures. After reflotation, washing and treatment with 100 mM Na ₂ CO 50, 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. These data do not agree with the hypothesis that Ca ^{2+ is} a prerequisite for the binding of LBLOX to the surface of the lipid body.

A significant change in LBLOX's ß-barrel eliminates its ability to bind to liposomes

To further characterize the type of interaction between the lipid body surface and the N-terminal β-barrel structure of LBLOX, it was investigated whether a modified β-barrel structure also binds to liposomes. For this purpose, a recombinant protein (LBL0X-HA) was produced which, compared to wild-type LBLOX, contained a triple hemagglutinin label, inserted between amino acid residue 70 and 71 of the LBLOX. This construction interrupts the β-barrel with a section of 30 amino acid residues.

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.

DISCUSSION

Few isoforms of lipoxygenase bind to or integrate into the membranes of various organelles. In these cases, the function of lipoxygenase can target the modification of the corresponding membrane. Expression of 15-LOX in reticulocytes Z Φ

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whether the LBLOX binding due to its interaction with an integral membrane protein as a partner, e.g. B. with the oleosin as an anchor, or whether the function of the LBLOX-ß-barrel with the function of a C2 domain binding to the phospholipid membrane, 5 as for Synaptotagmin [Rizo, J. & Sudhof, TCJ Biol. Chem. 273, 1998: 15879-15882] and cytosolic phospholipase C-Delta [Perisic, 0. et al. , J. Biol. Chem. 273, 1998: 1596-1604] or phospholipase A ₂ [Xu, GY et al. , J. Mol. Biol. 280, 1998: 485-500]. This kind of comparison with

10 C2 domains requires a thorough study of a possible effect of Ca ²⁺ either on the membrane binding properties of LBLOX or on the structure of LBLOX as found for 5-lipoxygenase [Hammarberg, T. & Radmark, 0., Biochem. 38, 1999: 4441-4447]. In our experiments with lipid bodies, one

15 ⁴⁵ Ca ²⁺ coating of lipid bodies was found, but no Ca ²⁺ dependence of the binding of LBLOX constructs was observed. In order to unambiguously demonstrate the presence or absence of a Ca ⁺ -mediated bond, the experimental protocols for future studies must be used in various ways.

20 can be refined. Previous experiments [Busch, MB et al. , Eur. J. Cell Biol. 60, 1993: 88-100] with root tissue using energy filter electron microscopy demonstrated that the lipid body surface is covered by a zone with a high Ca ²⁺ content.

25

We should remember that the comparison of the primary amino acid sequences and the putative secondary structures of LOX forms shows that LBLOX is not only characterized by the formation of a ß-barrel structure that is exposed to exposed glutamyl residues.

30 is permitted, but also by an N-terminal extension of 30 amino acid residues, which is not present in cytosolic LOX isoforms [Höhne, M. et al. , Eur. J. Biochem., 241, 1996: 6-11; Rosahl, S., Z. Naturforsch. C 51, 1996: 123-138]. It is therefore likely that both types of interaction

35 play a crucial role, 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.

0 Our experiment with the mutant LBLOX protein, which differs from the wild type only by a significant change in the ß-barrel, i.e. by inserting three repetitions of an HA label, showed loss of binding to lipid bodies or membranes. This means that the intact ß-barrel as

45 essential for binding to lipid bodies, regardless of whether the most N-terminal region contributes to further selectivity.

Targeting signals as unfolded areas of 7 amino acid residues have been found when proteins are transported in mitochondria, chloroplasts or the ER. Here, in the case of the binding of a protein to already existing lipid bodies, 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.

Figure legends

Part A (fluorogram) 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. For lanes 2 and 3, the corresponding cellular subtractions from cotyledons after protein labeling were prepared in vivo using [ ³⁵ S] methionine. Parts B and C show fluorograms showing the post-translational binding to isolated cucumber microsomes. Cucumber microsomes were incubated with the radioactively labeled proteins produced in reticulocyte lysates using either LBLOX mRNA or PLA mRNA and then purified by flotation in a density gradient. 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, whereas 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.

After a short period (15 min) used to administer the radioactive amino acid as a precursor to the cotyledons, one line fractionation was performed and two

Proteins were isolated from the cellular subtractions by immunoprecipitation. Isolation of the radioactive proteins from the Solubilized subtractions are carried out after the addition of 1 μg of the corresponding cold protein (LBLOX or isocitrate lyase, ICL) as a carrier by adding antiserum and direct precipitation (see methods). After electrophoresis and protein staining (lane 3: microsomes; lane 4: cytosol), the fluorogram (lanes 1 and 2) in part A showed that LOX was marked strongly (lane 2) in the cytosol and much weaker in the microsomes (lane 1). Part B: As a control, the distribution of isocitrate lyase between these two fractions was determined. 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 μg 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.

1 μg of the corresponding proteins was incubated in 200 μl buffer with an amount of liposomes corresponding to 1 mg phosphatidylcholine. After 30 min the mixture was adjusted to 42% sucrose and layered in a sucrose gradient. The

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. In the upper part of the figure, 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 α-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.

Figure 6. In vitro binding of the GST-LBLOX244 fusion protein to isolated lipid bodies.

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

Flotation separated from the soluble proteins. Aliquots of both fractions and the original crude lipid body fraction (lanes 3 and 6) were analyzed by SDS-PAGE and subsequent immunoblotting using anti-LBLOX antiserum. Lanes 2 and 5: Lipid bodies obtained by flotation: Lanes 1 and 4: re-isolated soluble proteins. The arrow pointing to the band at 51 kDa shows the fusion protein which has bound to lipid bodies in addition to the endogenous LBLOX. Lanes 1-3: protein staining; Lanes 4-6: immune labeling.

Fig. 7. Binding of LBLOX constructs and recombinant proteins with wild-type and modified β-barrel to liposomes.

After incubation of the purified recombinant proteins with liposomes, the liposomes were reisolated from the incubation mixture by means of flotation in a sucrose gradient. The behavior of wild-type lipid body LOX (LBLOX) was compared to that of a fragment containing the N-terminal half of LBLOX (LBLOX-Δ504), with the fusion protein with the β-barrel (GST-LOX) and with an LBLOX , whose N-terminal ß-barrel had been changed significantly (LBLOX-HA ₃ ). The figure shows the analysis of gradient fractions by means of SDS-PAGE and immune labeling using anti-LBLOX antiserum. Abbreviations. Hemagglutinin, HA; Glutathione-S-transferase, GST; Lipid body lipoxygenase, LBLOX; Lipoxygenase, LOX; Phospholipase A ₂ , PLA.

Enzymes. Glutathione-S-transferase (EC 2.5.1.18); Isocitrate lyase (EC 4.1.3.1); Lipoxygenase (EC 1.13.11.12); Phospholipase A ₂ (EC 3.1.4.3).

Claims

claims

1. Isolated nucleic acid sequence which codes for a polypeptide and which consists of a combination of the nucleic acid sequences

Biosynthetic nucleic acid sequence of the fatty acid or lipid metabolism is composed with one of the following nucleic acids:

a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,

2. Isolated nucleic acid sequence according to claim 1, characterized in that a sequence of the following protein groups is used as the biosynthetic gene nucleic acid sequence of the fatty acid or lipid metabolism: acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] - Desaturase (s), acyl-ACP thioesterase (s), fatty acid acyl transferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl-coenzyme A carboxylase (s), acyl -Coenzyme A oxidase (s), fatty acid desaturase (s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allen oxide synthases, hydroperoxide lyases or fatty acid elongase (s).

3. Isolated nucleic acid sequence according to claim 1 or 2, characterized in that a sequence of the following protein groups is used as the biosynthetic gene nucleic acid sequence of the fatty acid or lipid metabolism:

Fatty acid acyl transferase (s), Δ4-desaturase, Δ5-desaturase,

Δ6-desaturase, Δ9-desaturase, Δ12-desaturase, Δ15-desaturase or a fatty acid elongase.

4. Isolated nucleic acid sequence according to claims 1 to 3, characterized in that the derivatives mentioned under (c) at the amino acid level have a homology of 70%, preferably 80%, particularly preferably 90% over the entire range of in

5 SEQ ID NO: 2 sequence shown (program PileUp, J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153).

5. amino acid sequence encoded by a nucleic acid sequence 10 according to claim 1.

6. A nucleic acid construct containing a nucleic acid sequence according to claim 1, wherein the nucleic acid sequence is linked to one or more regulation signals.

15

7. Use of a nucleic acid sequence according to claim 1 or a nucleic acid construct according to claim 6 for the production of transgenic plants.

8. Vector containing a nucleic acid sequence according to claim 1 or a nucleic acid construct according to claim 6.

9. The vector of claim 8, wherein the vector is linear or circular DNA, phages, viruses, transposons,

25 IS elements, phasmids, phagemids, cosmids or plasmids.

10. Organism containing at least one nucleic acid sequence according to claim 1, at least one nucleic acid construct according to

30 Claim 6 or at least one vector according to Claim 8.

11. The organism of claim 10, wherein the organism is a eukaryotic organism.

35 12. Organism according to claim 10 or 11, wherein the organism is a plant, a eukaryotic microorganism or an animal.

13. Organism according to claims 10 to 12, wherein the organism is a plant, a fungus or a yeast.

14. Organism according to claims 10 to 13, wherein the organism is Yarrowia lypolytica, Saccharomyces

45 cereviseae, Traustochytrium, Arabidopsis thaliana, Brassica napus or Linium usitatissimum.

15. Transgenic plant containing a nucleic acid sequence according to claim 1 or a nucleic acid construct according to claim 6.

16. A method for targeting proteins involved in the biosynthesis of lipids 5 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: 10 a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,

b) nucleic acid sequences which are the result of the

Derive 15 degenerate genetic codes from the nucleic acid sequence shown in SEQ ID NO: 1,

c) Derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which for polypeptides with the sequence shown in SEQ ID NO: 2

Code 20 amino acid sequences and at least

Have 60% homology at the amino acid level,

d) a nucleic acid sequence with the sequence shown in SEQ ID NO: 3 or the amino terminal part of the

25 coding region of this sequence, and

and introduces the obtained sequence into a eukaryotic organism.

17. A method for targeting proteins involved in the biosynthesis of lipids or fatty acids in liposomes or lipid bodies, characterized in that at least one nucleic acid sequence according to claim 1 or at least one nucleic acid construct according to claim 6 in an oil

35 producing organism brings.

18. A process for the production of fatty acids or lipids, characterized in that at least one nucleic acid sequence according to claim 1 or at least one nucleic acid construct 40 according to claim 6 is brought into an oil-producing organism, attracts this organism and that oil contained in the organism is isolated.

19. A process for the production of fatty acids, characterized in that at least one nucleic acid sequence according to claim 1 or at least one nucleic acid construct according to claim 6 is brought into an oil-producing organism, attracts this organism and that oil contained in the organism is isolated and the fatty acids are released.

20. The method according to claims 16 to 19, characterized in that the organism is a plant or a eukaryotic microorganism.