WO2004007732A1

WO2004007732A1 - Cloning and characterization of a lipoxygenase from phaeodactylum tricornutum

Info

Publication number: WO2004007732A1
Application number: PCT/EP2003/007354
Authority: WO
Inventors: Ivo Feussner; Toralf Senger; Cornelia Göbel
Original assignee: Basf Plant Science Gmbh
Priority date: 2002-07-11
Filing date: 2003-07-09
Publication date: 2004-01-22
Also published as: DE10231589A1; AU2003280984A1

Abstract

The invention relates to a method for producing hydroperoxy fatty acids by oxidizing polyunsaturated fatty acids comprising at least two double bonds in the fatty acid molecule, particularly polyunsaturated fatty acids such as arachidonic acid or eicosapentaenoic acid. The invention relates to the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism, having an increased hydroperoxy fatty acid content due to the expression of a lipoxygenase from Phaeodactylum tricornutum. Also disclosed are expression cassettes containing a nucleic acid sequence, a vector, and organisms containing at least one nucleic acid sequence or an expression cassette.

Description

Cloning and characterization of a lipoxygenase from Phaseodactylum tricornutum

description

The present invention relates to a process for the preparation of hydroperoxy fatty acids by oxidation of polyunsaturated fatty acids with at least two double bonds in the fatty acid molecule, in particular by oxidation of polyunsaturated fatty acids such as arachidonic acid or eicosapentaenoic acid. The invention relates to the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism with an increased content of hydroperoxy fatty acids due to the expression of a lipoxygenase from Phaeodactylum tricornutum.

In addition, the invention relates to expression cassettes containing a nucleic acid sequence, a vector and organisms containing at least one nucleic acid sequence or an expression cassette.

Lipoxygenases (linoleate: oxygen oxidoreductases, EC 1.12.11.13; LOX) are enzymes which are ubiquitously widespread in the animal and animal kingdom [AR Brash, J. Biol Chem, 274: 23679-23682, 1999]. They contain one non-haem iron atom per mole of enzyme and catalyze the regio- and stereoselective dioxygenation of polyunsaturated fatty acids, whereby their hydroperoxide derivatives are formed in the S configuration [p. Rosahl, Z Naturforsch, 51: 123-138, 1996]. The first lipoxygenase was described by Andre and Hou in 1932 as an enzyme which caused the oxidation of fatty acids and the breakdown of carotenoids [Andre et al., Lieb CR Acad Sei (Paris), 194: 645-647, 1932]. The enzymatic activity of lipoxygenases was used in the food industry even before they were discovered. For example, wheat flour was bleached by reacting carotenoids contained therein by lipoxygenases, which were the active ingredient of added soy flour [JN Siedow, Annu Rev Plant Physiol Plant Mol Biol, 42: 145-188, 1991]. In the 1980s, the physiological and especially the biochemical properties were elucidated [Galliard et al., The Biochemistry of Plants, p. 131-161, Academic Press, London, 1980; DF Hildebrand, Physiol. Plant, 76 ^ 249-253, 1989]. A first comprehensive summary of the catalytic mechanism was made in 1991 [HW Gardner, Biochim Biophys Acta, 1084: 221-239, 1991]. In particular, the availability of the first data from the structural analyzes by X-ray diffraction of animal [Gillmor et al., Nat. Struct. Biol., 4: 1003-1009, 1998] and vegetable [Boyington et al., Biochem. Soc. Trans, 21: 744-748, 1993; Minor et al., Biochemistry, 35: 10687-10701, 1996; Skrzypczak-Jankum et al., Proteins-Structure Function and Genetics, 29: 15-31, 1997] Lipoxygenases helped to create models which Attempting to explain substrate and position specificity [Feussner et al .; Enzymes in Lipid Modification, p. 309-336, WileyNCH, Weinheim, 2000]. To date, sequences encoding lipoxygenases have been known from over sixty species of plants and animals [AR Brash, J. Biol Chem, 274: 23679-23682, 1999]. A microbial lipoxygenase from the fungus Gaeumannomyces graminis was first isolated [Sugio et al., Lipoxygenase, 2002; WO 02/20730]. In mammals they convert arachidonic acid, among other things, and form the precursors of regulatory compounds, such as leukotrienes and lipoxins, which play an important role in inflammatory and immune reactions [Kühn et al., FEBS Lett, 449: 7-11, 1999]. In plants, the hydroperoxides formed by the lipoxygenases serve as substrates for at least seven enzyme families [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 002]. There are four metabolic pathways that are relatively well characterized: peroxy genase, hydroperoxide, lyase, all oxide synthase, and the divinyl ether synthase pathway. The implementation of the epoxy alcohol synthases and by reductases is less well characterized, as is the hydroperoxidase activity of the lipoxygenases [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 002]. However, despite intensive studies, little is still known about the physiological function of many plant lipoxygenases [AR Brash, J. Biol Chem, 274: 23679-23682, 1999].

Plant lipoxygenases are enzymes that have a molecular weight between 90 and 115 kDa. They belong to the family of dioxygenases and catalyze the incorporation of molecular oxygen into a (1Z, 4Z) diene system of unsaturated fatty acids [AR Brash, J. Biol Chem, 274: 23679-23682, 1999]. The resulting hydroperoxides of the fatty acids have an S configuration and have a (1Z, 3E) diene system (FIG. 1). Most studies on the reaction mechanism have been carried out on the soybean lipoxygenase isoform 1 [Feussner et al., Enzymes in Lipid Modification, pages 309-336, Wiley-VCH, Weinheim, 2000; HW Gardner, Biochim Biophys Acta, 1084: 221-239, 1991; Veldink et al., Studies in Natural Products Chemistry, p. 559-589, 1991]. When converting linoleic acid and α-oleolenic acid, the most common substrates of lipoxygenases in plants, the oxygen can be introduced at two different positions. After initial hydrogen abstraction by the non-hemic iron atom on the carbon atom Cn of these fatty acids, a [+2] or [-2] rearrangement of the pentadienyl radical can take place. An exception to the hydrogen acceptor is the fungus Gaeumannomyces graminis, in which manganese is found in the active center of the catalytically active lipoxygenase instead of the divalent iron atom [Sugio et al., Lipoxygenase, 2002; WO 02/20730]. After oxygenation and the reduction of the peroxy radical to the hydroperoxide, either linoleic acid produces either (13S, 9Z, 11E) -13-hydroperoxy-9,11-octadienoic acid (13-HPODE, [+2] rearrangement) or ( 9S, 10E, 12Z) -9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE, [-2] rearrangement ([Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000], Figure 2). Since the formation of the ratio of these positional isomers occurs specifically for each enzyme, a distinction is made between 9- and 13-lipoxygenase in plants [HW Gardner, Biochim Biophys Acta, 1084: 221-239, 1991]. A further subdivision of plant lipoxygenases is based on sequence homologies at the cDNA level. Type 1 lipoxygenases do not have a chloroplasticidal signal peptide, in contrast to type 2 lipoxygenases [Shibata et al, Plant Miol Biol Reporter, 12 (2): S41-S42, 1994].

As already mentioned, in addition to the enzyme lipoxygenase itself, six further enzyme families have been described which further implement the hydroperoxide derivatives of fatty acids. Four main metabolic pathways have been characterized to date ([E. Blέe, Prog. Lipid Res., 37: 33-72, 1998; Feussner et al., Trends Plant Sei, 6: 262-267, 2001], Figure 3, thick arrows):

1. The Peroxygenase Route [E. Blee, Prog. Lipid Res., 37: 33-72, 1998] earlier hydroperoxide isomerase): Peroxygenase produces hydroxy, epoxy and ß-hydroxyepoxy fatty acids from the hydroperoxides of the fatty acids. The epoxy groups can be reduced to hydroxyl groups, so that di- and trihydroxy fatty acids are formed [M. Hamberg, J Lipid Mediators Cell Signal,

12: 283-292, 1995].

2. The Allen Oxide Synthase Pathway (AOS, formerly hydroperoxide dehydratase [HW Gardner, Biochim Biophys Acta, 1084: 221-239, 1991]: The Allen oxide synthase belongs to the family of cytochrome P ₄₅₀ enzymes [Song et al., Proc Natl

Acad Sei USA, 90: 8519-8523, 1993], subfamily CYP74A. Here the hydroperoxides are converted into unstable allenoxides, which break down into α- and γ-ketols. In the implementation of 13-HPOTE, apart from this decay to the ketols, a further reaction pathway can be labeled which leads to the formation of jasmonic acid, the so-called jasmonate cascade [Vick et al., Biochem

Biophys Res Commun, 111: 470-477, 1983]. In this case, 13-HPOTE initially creates an unstable allen oxide, which is converted to (9S, 13S, 15Z) -12-oxo-9,13,15-phytodienonic acid (cis - [+] - 12-oxo-PDA) by the highly specific allen oxide cyclase ) is cyclized [Ziegler et al., Lipids, 34: 1005-1015, 19.99]. The 12-oxo-PDA-Δ ⁰ reductase then catalyzes the double bond of the cyclopentanone ring. This reaction is dependent on NAPH [Schaller et al., Planta, 210: 979-984, 2000]. The carboxylic acid side chain is shortened by three subsequent β-oxidation steps and (3R, 7S) -jasmonic acid is formed, which spontaneously epimerizes to (3R, 7R) -jasmonic acid [Sembdner et al., Annu Rev Plant-Physiol Plant Mol Biol, 44: 569- 589, 1993]. Both connections and their Methyl esters are biologically active. They have phytohormone-like properties and have a stimulating or inhibiting effect on plant development processes such as germination and senescence. The question of whether jasmonate acts as a hormonal regulator of senescence or as a stress signal that can also trigger senescence has not yet been clarified [Wastemack et al., Trends

Plant Sei, 2 (8): 302-307, 1997].

3. The hydroperoxide lyase route (HPL, formerly hydroperoxide isomerase [Zimmermann et al., Plant Physiol, 46: 445-453, 1970]: The enzyme is widespread in plants [K. Matsui, Belgian Journal of Botany, 131: 50-62, 1998], also has a cytochrome P ₄₅₀ and forms the subfamilies CYP74B and CYP74C. The peroxides of linoleic and α-linolenic acid are converted into the saturated aldehyde hexanal or unsaturated (3Z) aldehydes and ω- Oxo derivatives of fatty acids cleaved [K. Matsui, Belgian Journal of Botany, 131: 50-62, 1998] Alcohol dehydrogenases can be used to convert the (3Z) -aldehydes into (3Z) -alcohols, which in turn are then used with short-chain Carboxylic acids can form volatile esters, or the aldehydes can be isomerize to their corresponding (2E) aldehydes before they are reduced (see p. 5) .These substances resulting from the HPL reaction are responsible for the characteristic smell of plants and fruits [A. Hatanake, Food Rev Int, 12: 303-

350, 1996].

4. The divinyl ether synthase route (DES): The enzyme activity of DES, the formation of divinyl ethers from the fatty acid hydroperoxides, was described as early as the 1970s [Gilliard et al., Biochem, 129: 743-753, 1972]. The products appear to play a role in the defense against pathogenic bacteria and fungi [Weber et al., Plant Cell, 11: 485-493, 1999]. DES was first isolated from Lycopersicum esculentum at the end of 2000 [Itoh et al., J Biol Chem, 276 (5): 3620-3627, 2001].

In addition to these four main metabolic pathways, three further, less well characterized enzyme activities are known.

1. The hydroperoxide reductase includes involved in the degradation of polyene fatty acids in the storage lipids in a large number of oilseeds [Feussner et al., Trends

Plan Sei, 6: 262-267, 2001]. The mechanism is not fully understood, but glutathione involvement is discussed [Feussner et al., Advances in Plant Lipid Research, p. 311-333, 1998]. 2. The Lipoxygenase-Catalyzed Peroxidase Reaction (Ketodiene-Generating Way): In the absence of oxygen, lipoxygenases can catalyze the homeolytic cleavage of the OO bond. The resulting alkoxy radicals can then rearrange to form ketoconjudic fatty acids, with other derivatives being able to form to a lesser extent [Kühn et al., Biochemistry, 30: 10269-

10273, 1991; Kuehn et al., Eicosanoids, 4: 9-14, 1991].

3. The Epoxy Alcohol Synthase Pathway (EAS): As an alternative to the peroxygenase pathway, the conversion of (9S) hydroperoxides by an EAS has been described. The characterization of the enzyme was successful from the leaves of potatoes

[M. Hamberg, Lipids, 34: 1131-1142, 1999]. In this catalysis, α- and ß-hydroxyepoxy derivatives of the fatty acids result from the intramolecular rearrangement of their hydro (pero) xides, but with a different absolute configuration than in the case of peroxygenase. The EAS path has so far only been found in Solanaceae, and the oxylipins which result from this could play a role in the defense against pathogens [Göbel et al., J. Biol. Chem, 276: 6267-6273, 2001].

The further conversion of the aldehydes, which resulted from the HPL reaction, should also be mentioned. Thus, they can be isomerized by a (3Z): 2E enal isomerase [Phillips et al., Phytochem, 18: 401-404, 1979]. Reaction with lipoxygenases that would lead to the formation of hydroxyaldehydes has also been proposed [Gardner et al., Plant Physiol., 116: 1359-1366, 1998]. However, this implementation is not considered to be assured. Working from two other groups assumes autoxidative formation more like vertebrates [Noordermeer et al.,

Biochem Biophys Res Commun, 277 (1): 112.116, 2000; Schneider et al., J Biol Chem, 276 (20831-20838), 2001]. Subsequent reactions result in the ω-oxo acid, (9Z) -12-oxo-9-dodecenoic acid formed in the reaction of 13-HPOTE, which is spontaneously converted into traumatic acid by oxidation [Zimmermann et al., Plant Physiol, 63: 536-541, 1979]. Both substances are called wound hormones because they may be involved in the wound response of plants.

Lipoxygenase isoforms are found in most plant cells. However, the expression of lipoxygenase depends on the developmental and environmental conditions of the plant [p. Rosahl, Z Naturforsch, 51: 123-138, 1996]. They can be soluble or bound to membranes. Soluble lipoxygenases are found, for example, in the cytosol of seedlings and ripening seeds [Vernooy-Gerritsen et al., Plant Physiol, 76: 1070-1080, 1984], in later stages of development, for example in leaves, also in vacuoles [Tranbarger et al., Plant Cell , 3: 973-987, 1991], in the cell nucleus [Feussner et al., Plant J, 7: 949-957, 1995] and in chloroplast [Bell et al., Proc NatI Acad Sei USA, 92: 8675-8676; 1995, Feussner et al., Plant J, 7: 949-957, 1995]. The membrane-bound lipoxygenases were also found in the most diverse compartments of the plant cells. They can be found on the plasma membrane [Nellen et al., Z Naturforsch, 50 (c): 29-36, 1995], on the membrane of the lipid bodies of seedlings [Feussner et al., FEBS Lett, 298: 223-225, 1992; Radetzky et al., Planta, 191: 166-172, 1993] and on the envelope and thylakoid membrane of the chloroplasts of leaves, flowers and fruits [Blee et al., Plant Physilogy, 110 (2): 445-454, 1996; Bowsher et al., Plant Physiol, 100: 1802-1807, 1992].

The following possible physiological functions are discussed for lipoxygenases:

1. in plant development

The 15-epoxygenases of the vertebrates are known to be involved in the membrane degradation process [Schewe et al., TIBS, 16: 369-373, 1991]. Based on this, plant lipoxygenases are also believed to cause loss of membrane integrity through the conversion of lipids to lipid hydroperoxides [Thompson et al., Prog Lipid Res, 37: 119-141, 1998]. Another hypothesis is that the destruction of the membranes in the seedling enables the transport of storage substances to the embryo [D.F. Hildebrand, Current Topics in Plant Biochemistry and Physiology, 7: 201-219, 1988]. Furthermore, participation or triggering of the mobilization of storage fats is proposed [Feussner et al., Trends Plant Sei, 6: 262-267, 2001], since the lipid body in cucumber, sunflower, flax, anise and A membrane-bound lipoxygenase could be identified in soybean seedlings.

After binding to the membrane of the lipid body [Feussner et al., Biol Chem Hoppe-Seyler, 376: 51, 1995], the enzyme from cucumber showed an increase in the enzyme activity and a change in the regiospecificity. It is the first plant lipoxygenase to be shown to be able to react directly with esterified linoleic or linolenic acid [Feussner et al., Proc NatI Acad Sei

USA, 92: 11849-11853, 1995]. A possible function of this reaction could be the triggering of the catabolism of unsaturated fatty acids esterified in lipids [Feussner et al., Trends Plant Sei, 6: 262-267, 2001]. Another possibility of the role of lipoxygenases in seedling development could be through protection against pathogens through the production of antimicrobial

Substances are given [A. Slusarenko, Lipoxygenase and Lipoxygenase Pathway Enzymes, p. 176-197, 1996, AOCS Press Champaign II]. 2. with the herbal stress response

From the products of the lipoxygenase reaction, further enzymatic reactions can give rise to compounds with regulatory properties such as jasmonic acid and traumatin [Wasternack et al., Trends Plant Sei, 2 (8): 302-307, 1997]. The wound hormone traumatin seems to proliferate cells

[Zimmermann et al., Plant Physiol., 63: 536-541, 1979] at the wounded sites within the plant. Jasmonic acid or its methyl ester appears to be formed as a signal transduction molecule of the lance when wounded or infected with pathogens [Creelman et al., Ann Rev Plant Physiol Plant Mol Biol, 48: 355-381, 1997]. They induce enzymes that are important for a defense response [p. Rosahl et al., Z Naturforsch, 51: 123-138, 1996]. In the case of pathogen attack, the plants react by activating defense genes, the synthesis of antimicrobial compounds such as phytotoxins, and hypersensitive cell death to develop resistance. Here, too, lipoxygenases seem to play a role [A. Slusarenko, Lipoxygenase and Lipoxygenase Pathway Enzymes, p. 176-197, 1996, AOCS Press Champaign II].

3. As messenger substances, lipoxygenases play a critical role in the synthesis of signal substances

(Pheromones) of brown and diatoms [Pohnert et al., Nat. Per. Rep., 19: 108-122, 2002]. Through these messengers e.g. male spermatozoids are directed to the immobile female gametes, which release them. Products of the lipoxygenase-HPL pathway also play a role in the defense of brown diatoms (see next paragraph).

Diatoms are unicellular algae that occur in oceanic and fresh water phytoplankton, but also in biofilms and solid substrates. They are more closely related to brown and gold algae than to green algae, moss and higher plants. In contrast to the latter, diatoms form chrysolaminarin (a ß-1, 3-glucan) instead of starch (a ß-1, 4-glucan). In general, assimilation products such as oils (in special oil vacuoles) as well as chrysolaminarin and volutin are deposited outside the chromatophores f www.kieselalaen.coml. Another point of differentiation is the presence of chlorophyll c instead of chlorophyll b. Chromophores that contain chlorophyll b (in higher plants, mosses, etc.) are called chloroplasts. The chromophores in diatoms are enveloped by four instead of two uniform membranes. The two inner ones are the actual chromatophore membranes, the two outer ones represent a fold of the endoplasmic reticulum. Chromatophores of this blueprint are found not only in Bacillariophyceen (silica layers) but also in Chrysophyceen, Xanthophyceen, Chloromonadophyceen and Phaeophyceen, with which they are combined to form the Heterokontophyta (Chrysophyta), are deposited [www.kieselalQen.com1. Diatoms are the most important primary source in the marine food chain. They also contribute up to 25% globally to the primary production of biomaterial, with a corresponding share in oxygen production [Scala et al., Cell. Mol Life Sei, 58: 1666-1673, 2001]. Due to their outstanding role as a primary marine source, they are under immense herbivorous pressure. Recent research has shown that lipoxygenases are involved in a defense mechanism against herbivorous organisms in phytoplankton [Miralto et al., Nature, 402: 173-176, 1999; Pohnert et al., Nat. Pro, Rep., 19: 108-122, 002]. They are thought to be formed from highly unsaturated eicosanoids (arachidonic acid - ERA, eicosapentaenoic acid - EPA) via the lipoygenase-H PL pathway, aldehydes, which have proven to be the effective defense substances. These aldehydes reduce the success in hatching oar crabs, which make up 90% of phytoplankton. Since most diatoms are surrounded by a highly structured cell wall with embedded silicates, they also play a key role in the bio-geochemical cycle of silicates [Treger et al., Science, 268: 375-379, 1995]. Diatoms are used commercially for a variety of purposes, such as forage in all forms of water cultivation, as a source of polyunsaturated fatty acids (PUFAs) or as a component of pharmaceutical articles [Apt et al., J. Phycol, 35: 215- 226, 1999].

Phaeodactylum tricornutum, a unicellular silica-free algae, is one of the most widely used model systems for studying the ecology, physiology, biochemistry, and molecular biology of diatoms [Apt et al., Mol Gen Genet, 252 (5): 572 -9, 1996]. It differs significantly from higher plants in that up to 30% of the total fatty acid component is the highly unsaturated fatty acid EPA and 11% docosahexanoic acid (DHA) [Schobert et al., Plant Physiol, 66: 215-219, 1980]. 40% EPA was already obtained under optimized cultivation conditions [Yongmanitchai et al., AppI Environ Mierobiol, 57 (2): 419-25, 1991]. P. tricornutum became all the more attractive by establishing a stable transformation protocol, which made many new studies on diatoms possible. The availability of this technique has thus demonstrated a sensor system which is used by P. tricornutum to react to relevant environmental influences [Faiciatore et al., Science, 288: 2363-2366, 2000]. This technique also made it possible to introduce a gene that codes for a glucose transporter. As a result, photoautotrophic diatom was converted into a heterotrophic organism, which opens up new possibilities for large-scale fermentation for commercial use [Zaslavskaia et al., Science, 292 (5524): 2073-5, 2001]. The presence of these PUFAs, which are rare in the plant kingdom, such as EPA and DHA, makes Phaeodactylum tricornutum an interesting object of study for enzymes that are involved in the synthesis and degradation of these fatty acids.

It was a goal of this work to isolate the first lipoxygenase from an alga. Since the spectrum of PUFAs in diatoms differs fundamentally from the higher realms, it can be expected that PUFA-metabolizing enzymes have different substrate specificities than enzymes from higher plants. Therefore, after successful isolation, the substrate specificity for Pt lipoxygenase-1 should be determined with substrates that do not occur in higher plants. For example, no lipoxygenase is known in the plant kingdom, the preferred substrate of which is ERA, EPA or DHA.

As described, oxygenated fatty acids, the so-called oxylipins, are of great importance in the plant wound and defense response. The first step in oxylipin synthesis is the formation of hydroperoxide derivatives of polyunsaturated fatty acids. This leads to the formation of hydroperoxy fatty acids, which are important starting materials for plant biosynthesis and are further implemented through a variety of enzyme reactions. They are, for example, raw materials for jasmonic acid and jasmonic acid methyl ester, which act as phytohormones within the plant. Products of the lipoxygenase reaction are involved, for example, in the regulation of development processes (germination, tuber formation, senescence) through the formation of leaf aldehydes and leaf alcohols or in wound reactions and defense against pathogens through the synthesis of signaling molecules and phytoalexins. They are also involved in the herbal stress response. For example, traumatin and tramatinic acid probably contribute to the wound response by promoting cell division, or jasmonates and their octadecane precursors induce the expression of proteinase inhibitors locally in the damaged tissue after mechanical wounding and insect damage. They are also involved in the synthesis of antimicrobial metabolites.

Therefore, there is still a great need for new and more suitable genes which code for enzymes which are involved in the biosynthesis of hydroperoxy fatty acids and which mediate pathogen protection in Rance. It is also advantageous that these nucleic acid sequences make it possible to convert regioselectively polyunsaturated fatty acids into specific hydroperoxy fatty acids.

In order to be able to produce the widest possible range of functionalized fatty acids, there is therefore a great need for specific lipoxygenases which make it possible to produce specific hydroperoxy fatty acids.

It was therefore the object of a process for the preparation of hydroperoxy fatty acids by oxidation of polyunsaturated fatty acids with at least two double bonds in the fatty acid molecule in one organism, advantageously in one eukaryotic organism preferred to develop in a plant. Furthermore, the task was to provide advantageous nucleic acid sequences for the aforementioned method. This object was achieved by the isolated nucleic acid sequences according to the invention which code for polypeptides with lipoxygenase activity, selected from the group: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, b) nucleic acid sequences which result from the degenerate genetic Let codes be derived from the coding sequence contained in SEQ ID NO: 1, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1 which are suitable for

Encode polypeptides with lipoxygenase activity and have at least 40% homology at the amino acid level with SEQ ID NO: 2.

Surprisingly, it was found that a lipoxygenase from diatoms, advantageously from Phaeodactylum tricornutum, is particularly specific for the conversion of arachidonic acid or eicosapentaenoic acid if they are advantageously expressed in a heterologous system. This lipoxygenase can advantageously be used to produce hydroperoxy fatty acids in plants, non-human animals or microorganisms.

The advantageous lipoxygenase from Phaeodactylum tricornutum works in a pH range from 5 to 9, preferably in a pH range from 6 to 8.5, particularly preferably in a pH range from 6.5 to 8, very particularly preferably in a pH - Range from 7 to 8. The pH optimum of the enzymatic reaction is pH 8.2. The lipoxygenase according to the invention shows a regiospecificity for the hydroperoxy formation of linoleic acid at the C atom 13 (13-LOX) and has a chloroplastic signal sequence.

This distinguishes the lipoxygenase found, in addition to the increased regiospecificity and activity, from the enzymes of the prior art.

The term “lipoxygenase” in the sense of the invention encompasses proteins, as well as their homologues, derivatives or analogs, which form hydroperoxy fatty acids from polyunsaturated fatty acids with at least two double bonds, which are advantageous as preferred polyunsaturated fatty acids arachidonic acid (= ERA) and / or eicosapentaenoic acid (= EPA ), but other polyunsaturated fatty acids are also substrates for the enzymatic reaction of lipoxygenase.

In a further embodiment, derivatives of the nucleic acid molecule according to the invention, reproduced in SEQ ID NO: 1, encode proteins with at least 60%, advantageously with at least 70%, preferably at least 75% and more preferably at least 80%, 85%, 90%, 95% and most preferably at least 96%, 97%, 98%, 99% or more identity to full amino acid sequence of SEQ ID NO: 2. The identity was established over the entire amino acid or. Nucleic acid sequence range calculated. The PileUp program was used for the sequence comparisons (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J Mol. Biol. 48; 443-453 (1970) and Smith and Waterman (Adv. AppI. Math. 2; 482-489 (1981)], which are contained in the GCG software package [Genetics Computer Group, 575 Science Drive, Madison , Wisconsin, USA 53711 (1991)] The sequence homology values given above in percent were determined with the program Gap over the entire sequence range with the following settings: Gap Weight: 8, Length Weight: 2 for proteins and Gap Weight: 50, Length Weight: 3 for nucleic acid sequences It is clear to the person skilled in the art that when comparing homologies of nucleic acid sequences and not identities with one another, the percentages for homology obtained with the algorithms are slightly higher than the aforementioned percentages for identity. The invention also encompasses nucleic acid moles which differ from (and parts of) the nucleotide sequences shown in SEQ ID NO: 1 due to the degenerate genetic code and thus encode the same lipoxygenase as that encoded by the nucleotide sequence shown in SEQ ID NO: 1. In addition to the coding region (see SEQ ID NO: 1), SEQ ID NO: 3 also shows the 5 'and 3' region of the nucleic acid sequence. SEQ ID NO: 4 shows the corresponding protein sequence.

In addition to the lipoxygenase nucleotide sequence shown in SEQ ID NO: 1, those skilled in the art will recognize that DNA sequence polymorphisms that lead to changes in the amino acid sequences of lipoxygenase may exist within a population. These genetic polymorphisms in the lipoxygenase gene can exist between individuals within a population due to natural variation. These natural variants usually cause a variance of 1 to 5% in the nucleotide sequence of the lipoxygenase gene. All and all of these nucleotide variations and the resulting amino acid polymorphisms in lipoxygenase, which are the result of natural variation and do not change the functional activity of lipoxygenase, are intended to be included in the scope of the invention.

In the following, polyunsaturated fatty acids (PUFAS) are to be understood as meaning double or polyunsaturated fatty acids which have double bonds. The double bonds can be conjugated or non-conjugated.

The nucleic acid sequence according to the invention or fragments thereof can advantageously be used to isolate further genomic sequences via homology screening. The derivatives mentioned can advantageously be isolated, for example, from other organisms in eukaryotic organisms such as compounds such as mosses, diatoms or fungi from other diatoms.

Allelic variants include, in particular, functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1, the enzymatic activity of the derived synthesized proteins being retained.

Such DNA sequences can be isolated from other eukaryotes such as those mentioned above starting from the DNA sequence described in SEQ ID NO: 1 or parts of these sequences, for example using conventional hybridization methods or the PCR technique. These DNA sequences hybridize to the sequences mentioned under standard conditions. For the hybridization, it is advantageous to use short oligonucleotides, for example of the conserved regions, which can be determined by comparison with other lipoxygenase genes in a manner known to the person skilled in the art. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization. These standard conditions vary depending on the nucleic acid used: oligonucleotide, 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.

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 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 conditions are advantageously 0.1 × SSC and temperatures between approximately 30 ° C. to 55 ° C., preferably between approximately 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 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant textbooks of genetics, such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be made according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G- + C content.For more information on hybridization, one skilled in the art can read the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids 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.

Derivatives homologs of the sequence SEQ ID NO: 1 are furthermore to be understood, for example, as eukaryotic homologs, shortened sequences, single-stranded DNA of the coding and non-coding DNA sequence or RNA of the coding and non-coding DNA sequence.

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 have been changed such that the gene expression and / or the protein expression is changed, preferably increased. Derivatives are also to be understood as variants which have been changed at the 3 'end.

The nucleic acid sequence (s) according to the invention which code for a lipoxygenase can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA constituents and consist of different heterologous lipoxygenase gene segments from different organisms. 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 heterogeneous genes. These codons preferred by Rance can be determined from codons with the highest protein frequency, which are expressed in most interesting plant species. An example of Corynebacterium glutamicum is given in: Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). Such experiments can be carried out using standard methods and are known to the person skilled in the art.

Functionally equivalent sequences which code for the lipoxygenase gene are those derivatives of the sequence according to the invention which, despite a different nucleotide sequence, still have the desired functions, that is to say the enzymatic activity and specific selectivity of the proteins. Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a lance.

In addition, artificial DNA sequences are suitable as long as they have the desired property, as described above, for example the synthesis of hydroperoxy fatty acids in oils, lipids or as free fatty acids in the eukaryotic organisms mediate advantageously in the plants by overexpression of the lipoxygenase gene in crop plants. Such artificial DNA sequences can have, for example, back-translation of proteins constructed using molecular modeling, lipoxygenase activity, or can be determined by in vitro selection. Possible techniques for the in vitro evolution of DNA to change or improve the DNA sequences are described in Patten, PA et al., Current Opinion in Biotechnology 8, 724-733 (1997) or in Moore, JC et al., Journal of Molecular Biology 272, 336-347 (1997). Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable. The specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other known genes of the lance to be transformed.

Sequences which code for fusion proteins are to be mentioned as further suitable equivalent nucleic acid sequences, part of the fusion protein being a lipoxygenase polypeptide or a functionally equivalent part thereof. The second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect lipoxygenase expression (e.g. myc-tag or his-tag). However, this is preferably a regulatory protein sequence, such as e.g. a signal sequence for the ER or a transit peptide that directs the lipoxygenase protein to the desired site of action.

The isolated nucleic acid sequences according to the invention advantageously originate from diatoms such as the Bacillariophyta strain with the classes Centrales, Cosciniodiscophyceae, Fragilariophyceae, Pennales or Bacillariophyceae, preferably from the Centrales or Pennales orders, more preferably from the subordinates Araphidineae, Raphididineaeae, especially Monor preferably from the families Naviculaceae, Cymbellacease, Entomoneidaceae, Nitzschiaceae, Epithemiaceae, Auriculaceae and Surirellaceae or very particularly preferably from the genera Amphora, Cymbella and Phaeodactylum. Advantageous nucleic acid sequences originate from the genera and species Phaeodactylum tricornutum, Cymbella microcephala, Cymbella leptoceros, Cymbella naviculiformis, Cymbella prostrata, Cymbella sileslaca, Cymbella sinuata, Cymbella trugidula, Cymbella tumida, Encyonema Reformeraeaumumum, Encyonema Reformeraeaumum, Encyonema Reformeaumiau Amphora ovalis or Amphora exigua. The lipoxygenase genes can advantageously be combined in the method according to the invention with other genes of fatty acid biosynthesis. Examples of such genes are the acyltransferases, further desaturases or elongases.

The amino acid sequences according to the invention are to be understood as proteins which contain an amino acid sequence shown in the sequence SEQ ID NO: 2 or a sequence obtainable therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues, the enzymatic activities of the in SEQ ID NO: 2 protein is retained or is not significantly reduced, that is, the ability to form hydroperoxy fatty acids is retained or is not significantly reduced. Not significantly reduced is understood to mean all enzymes which still have at least 10%, preferably 20%, particularly preferably 30% of the enzymatic activity of the starting enzyme. For example, certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.). For example, arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues 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.

Derivatives are also to be understood as functional equivalents which, in particular, also include natural or artificial mutations of an originally isolated sequence coding for lipoxygenesis, which furthermore show the desired function, that is to say its enzymatic activity and substrate selectivity is not significantly reduced. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, for example, the present invention also encompasses those nucleotide sequences which are obtained by modification of the lipoxygenase nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also the insertion of further restriction enzyme interfaces.

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

The nucleic acid sequence can advantageously be a DNA or cDNA sequence, for example. Coding sequences suitable for insertion into an expression cassette according to the invention are, for example, those which code for a lipoxy genes with the sequences described above and which give the host the ability to overproduce hydroperoxy fatty acids in free form or bound in oils or lipids. These sequences can be of homologous or heterologous origin.

The expression cassette according to the invention (= 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 derivatives and which is advantageously functionally linked to one or more regulation signals to increase gene expression and which control the expression of the coding sequence in the host cell. These regulatory sequences are said to be targeted Enable expression of genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed and / or overexpressed after induction, or that it is expressed and / or overexpressed immediately. 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 in front of the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased. 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 regulatory sequence was mutated so 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 can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or Terminators The lipoxygenase gene can be contained in one or more copies in the expression cassette (= gene construct).

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 radicals or fungi are suitable as promoters in the expression cassette. In particular, a plant promoter or promoters derived from a plant virus are preferably used. Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl ^ 'T7, T5, Contain T3, gal, trc, ara, SP6, λ-P _R - or in the λ-P promoter, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFα, 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, Iib4, STLS1, B33, nos (= nopaline synthase promoter) or in the ubiquitin promoter. The expression cassette can also be a chemical contain inducible promoter, by means of which the expression of the exogenous lipoxygenase gene in the organisms can advantageously be controlled in the plants at a specific point in time. Such advantageous plant promoters are, for example, the PRP1 promoter [Ward et al., Plant.Mol. Biol. 22 (1993), 361-366], a benzene sulfonamide-inducible (EP 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2,397-404), a salicylic acid-inducible promoter ( WO 95/19443), an abscisic acid-inducible (EP335528) or an ethanol or cyclohexanone-inducible (WO93 / 21334) promoter. 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 phosphoribosyl pyrophosphate amidotransferase from Glycine max (see also Genbank Accession Number U87999) or a node-specific promoter as in EP 249676 can advantageously be used. Plant promoters which ensure expression in tissues or plant parts / organs in which fatty acid biosynthesis or its precursors take place, such as, for example, in the endosperm or in the developing embryo, are particularly advantageous. Particular mention should be made of advantageous promoters which ensure seed-specific expression, for example the USP promoter or derivatives thereof, the LEB4 promoter, the phaseolin promoter or the napin promoter. The particularly advantageous USP promoter or its derivatives listed according to the invention mediate gene expression very early in seed development (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67). Further advantageous seed-specific promoters which can be used for monocotyledon and dicotyledonous plants are the promoters suitable for dicotyledons, such as the Napingen promoter from rapeseed (US Pat. No. 5,608,152), the oleosin promoter from Arabidopsis (WO 98/45461), the phaseolin Promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the leguminous B4 promoter (LeB4, Baeumlein et al., Plant J., 2, 2, 1992: 233-239) or promoters suitable for monocotyledons such as the promoters the promoters of the lpt2 or lpt1 gene from barley (WO 95/15389 and WO 95/23230) or the promoters of the barley Hordein gene, the rice glutelin gene, the rice oryzin gene , the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene or the rye secalin gene, which are described in WO99 / 16890 , Furthermore, promoters are particularly preferred which ensure expression in tissues or plant parts in which, for example, the biosynthesis of fatty acids, oils and lipids or their precursors takes place. Promoters that ensure seed-specific expression should be mentioned in particular. The promoter of the napin gene from rapeseed (US 5,608,152), the USP promoter from Vicia faba (USP = unknown seed protein, Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67) should be mentioned, the oleosin gene from Arabidopsis (WO98 / 45461), the phaseolin promoter (US 5,504,200) or the promoter of the legumin B4 gene (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9) , Also to be mentioned Promoters such as that of the barley lpt2 or lpt1 gene (WO95 / 15389 and

WO 95/23230) which mediate seed-specific expression in monocotyledonous plants.

As described above, the expression cassette (= gene construct, nucleic acid construct) can also contain further genes which are to be introduced into the organisms. These genes can be under separate regulation or under the same regulatory region as the lipoxygenase gene. These genes are, for example, further biosynthesis genes advantageous in fatty acid biosynthesis, such as biosynthesis genes for fatty acid or lipid metabolism, which enable increased synthesis, selected from the group acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] - Desaturase (s), acyl-ACP-thioesterase (s), fatty acid acyl transferase (s), acyl-CoA: lysophospholipid 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, triacylglycerol lipases, allen oxide synthases, hydroperoxide lyases or fatty acid elongase (s). For example, the genes for the Δ-15, Δ-12, Δ-9, Δ-6, Δ-5 desaturase, β-ketoacyl reductases, β-ketoacyl synthases, elongases or the various hydroxylases and Called acyl-ACP thioesterases. Desaturase and elongase genes are advantageously used in the nucleic acid construct. Genes selected from the group Δ-4-desaturase-, Δ-5-desaturase-, Δ-6-desaturase-, Δ-8-desatase-, Δ-9-desaturase-, Δ-12-desaturase-, Δ-5-elongase, Δ-6-elongase or Δ-9-elongase used in the 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 below. In addition, synthetic promoters can also be used advantageously.

When preparing an expression cassette, various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame. 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 within the regulatory areas, often less than 60 bp, but at least 5 bp. The promoter can be native or homologous as well as foreign or heterologous to the host organism, for example to the host plant. The expression cassette contains in the 5'-3 'transcription direction the promoter, a DNA sequence which codes for a lipoxygenase gene and a region for the transcriptional termination. Different termination areas are interchangeable. Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as transitions and transversions are possible, v / fro mutagenesis, primer repair, restriction or ligation can be used. With suitable manipulations, such as restriction, -chewing-back- or filling of overhangs for -bluntends-, complementary ends of the fragments can be provided for the ligation.

Of importance for an advantageous high expression can i.a. the attachment of the specific ER retention signal SEKDEL (Schouten, A. et al., Plant Mol. Biol. 30 (1996), 781-792), the average expression level is 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 vegetable polyadenylation signals, preferably those which essentially comprise T-DNA polyadenylation signals

Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHδ (Gielen et al., EMBO J.3 (1984), 835 ff) or corresponding functional equivalents.

An expression cassette is produced by fusing a suitable promoter with a suitable lipoxygenase DNA sequence and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

When preparing an expression cassette, various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.

The nucleic acid sequences according to the invention are advantageously cloned together with at least one reporter gene into an expression cassette which is introduced into the organism via a vector or directly into the genome. This reporter gene should enable easy detection via a growth, fluorescence, chemo, bioluminescence or resistance assay or via a photometric measurement. 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 α-galactosidase gene, the gfp gene 2-deoxyglucose-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 optionally further regulatory elements which are operatively linked to the intervening coding sequence for the lipoxygenase DNA sequence. An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence. The sequences preferred for the operative linkage are targeting sequences to ensure subcellular localization in plastids, advantageously in chloroplasts. Targeting sequences to ensure subcellular localization in the mitochondrion, in the endoplasmic reticulum (= ER), in the cell nucleus, in oil cells or other compartments can also be used if required, and translation enhancers such as the 5 'guide sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).

An expression cassette can contain, for example, a constitutive promoter (preferably the USP or napin promoter), the gene to be expressed and the ER retention signal. The amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as the ER retention signal.

For expression in a prokaryotic or eukaryotic host organism, for example a microorganism such as a fungus or a lance, the expression cassette is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host organism. Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR series such as pBR322, pUC series such as pUC18 or PUC19, M113mp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-lI ¹¹³ -B1, λgt11 or pBdCI, in Streptomyces plJ101, plJ364, plJ702 or plJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in mushrooms pALSI, pll_2 or pBBz116 ., [(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 promoters are, for example, 2μM, pAG-1, YEp6, YEp13 or pEMBLYe23. Examples of algae or plant promoters are pLGV23, pGHIac ⁺ , pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, L, 1988). The above-mentioned vectors or derivatives of the above-mentioned vectors represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford , 1985, ISBN 0444 904018). Suitable plant vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119. Advantageous vectors are so-called shuttle vectors or binary vectors that replicate in E. coli and Agrobacterium.

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 can be 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 the expression cassette as a vector or the nucleic acid sequences according to the invention.

What is advantageous here is the so-called co-transformation in its various configurations for introducing the nucleic acid sequence according to the invention into the host organism.

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 one vector, the different vectors being introduced simultaneously or successively can.

The vector advantageously contains at least one copy of the nucleic acid sequences according to the invention and / or the expression cassette according to the invention. 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, for example by using the T7 promoter and the T7 RNA polymerase.

Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Ine; Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein, or protein A.

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, Amsterdam, Netherlands].

Further advantageous vectors for use in yeast are pYepSed (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives (Invitrogen Corporation, San Diego, CA). Vectors for use in filamentous fungi are described in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge.

Alternatively, insect cell expression vectors can also be used advantageously, e.g. for expression in Sf 9 cells. These are e.g. the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

Furthermore, lanceol cells or algae cells can advantageously be used for gene expression. Examples of lance expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721.

Furthermore, the nucleic acid sequences according to the invention can be expressed in mammalian cells. Examples of corresponding expression vectors are pCDM8 and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBOJ. 6: 187-195). Promoters to be used are preferably of viral origin, e.g. Promoters of polyoma, adenovirus 2, cytomegalovirus or simian virus 40. Further prokaryotic and eukaryotic expression systems are mentioned in chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

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 FM Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by DM Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or Guthrie et al. See Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press.

The transfer of foreign genes into the genome of a plant is called transformation. The methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods include protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - the so-called particle bombardment method, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and gene transfer mediated by Agrobacterium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. 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 to transform plants, in particular crop plants, such as e.g. of tobacco plants can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media. The transformation of kingdoms with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known, inter alia, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

Agrobacteria transformed with an expression vector according to the invention can also be used in a known manner to transform plants such as test plants such as Arabidopsis or crop plants such as cereals, corn, oats, rye, barley, wheat, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, Potato, tobacco, tomato, carrot, paprika, rapeseed, tapioca, manioc, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species, in particular of oil-containing crops, such as soybean, peanut, castor oil, sunflower, corn, Cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean are used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media. The genetically modified Lancel cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by SD Kung and R. Wu, Potrykus or Höfgen and Willmitzer. In principle, suitable as transgenic organisms or host organisms for the nucleic acid according to the invention, the expression cassette or the vector are all organisms which are able to synthesize fatty acids, especially unsaturated fatty acids or are suitable for the expression of recombinant genes such as microorganisms, non-human animals or Plants beneficial all crops. Examples include cultivated plants such as soybean, peanut, castor oil, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, Yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae or protozoa such as dinoflagellates such as Crypthecodinium. Organisms which can naturally synthesize oils in large amounts, such as fungi such as Mortierella alpina, Pythium insidiosum or plants such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, calendula, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, are particularly preferred soy, rapeseed, sunflower, flax, calendula or Saccharomyces cerevisiae. In principle, transgenic animals are also suitable as host organisms, for example C. elegans.

A transgenic organism or transgenic plant within the meaning of the invention is understood to mean that the nucleic acids used in the method are not in their natural place in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously. However, as mentioned, transgene also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been changed. Transgenic is preferably to be understood as meaning the expression of the nucleic acids according to the invention at a non-natural location in the genome, that is to say that the nucleic acids are homologous or preferably heterologous. Preferred transgenic organisms are fungi such as Mortierella or plants such as crop plants, advantageously as oil crop plants.

"Transgene" means, for example, with respect to a nucleic acid sequence, an expression cassette or a vector containing a nucleic acid sequence which codes for the lipoxygenase or its derivatives, or an organism transformed with this nucleic acid sequence, an expression cassette or a vector, all such constructions which have been obtained by genetic engineering methods which either a) the lipoxygenase nucleic acid sequence, or b) a genetic control sequence functionally linked to the lipoxygenase nucleic acid sequence, for example a promoter, or c) (a) and (b) are not in their natural, genetic environment or modified by genetic engineering methods where the modification can be, for example, substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. Natural genetic environment means the natural chromosomal locus in the organism of origin or the presence in a genomic library. In the case of a genomic library, the natural, genetic environment of the nucleic acid sequence is preferably at least partially preserved. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, very particularly preferably at least 5000 bp.

Useful host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology l 85, Academic Press, San Diego, CA (1990).

Expression strains that can be used, 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.

Another object of the invention relates to the use of an expression cassette containing DNA sequences coding for a lipoxygenase gene or DNA sequences hybridizing therewith for the transformation of plant cells, tissues or plant parts. The aim of the use is to increase the content of hydroperoxy fatty acids in these organisms.

Depending on the choice of the promoter, the expression of the lipoxygenase gene can take place specifically in the leaves, in the seeds, in the tubers or in other parts of the plant. Such transgenic plants that overproduce lipoxygenase, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention. A preferred subject matter according to the invention are transgenic plants containing a nucleic acid sequence according to the invention, an expression cassette or a vector containing the nucleic acid sequence according to the invention which codes for a lipoxygenase.

The expression cassette or the nucleic acid sequences according to the invention containing a lipoxygenase gene sequence can also be used to transform the above-mentioned organisms such as bacteria, cyanobacteria, yeasts, filamentous fungi, ciliates and algae with the aim of increasing the content of hydroperoxy fatty acids. The invention relates, as described, to transgenic compounds, transformed with an expression cassette containing a lipoxygenase gene sequence or DNA sequences hybridizing therewith, and transgenic cells, tissues, parts and propagation material of such plants. Transgenic crops, such as barley, wheat, rye, oats, corn, soybeans, rice, cotton, sugar beet, rapeseed and canola, sunflower, flax, hemp, potatoes, tobacco, tomato, rapeseed, tapioca, cassava, are particularly preferred , Arrowroot, alfalfa, lettuce and the various tree, nut and wine species.

Plants in the sense of the invention are mono- and dicotyledonous plants, mosses or algae. Another object of the invention is a process for the preparation of hydroxyperoxy fatty acids by oxidation of polyunsaturated fatty acids with at least two double bonds in the fatty acid molecule, characterized in that at least one nucleic acid sequence according to the invention described above (SEQ ID NO: 1 and derivatives) or at least one gene construct according to the invention ( = Nucleic acid construct or expression construct) advantageously brings an oil-producing organism such as a plant or a eukaryotic microorganism into an organism, attracts this organism (cultivation) or cultivates and isolates the oil contained in the organism and, if necessary, releases the hydroperoxy fatty acids contained in the oil. The fatty acids can be released from the oils or lipids, for example by basic hydrolysis, for example with aqueous NaOH or KOH or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol, or by enzymatic elimination. They can then be isolated by, for example, phase separation and subsequent acidification using, for example, H ₂ SO ₄ . The fatty acids can also be released directly without the workup described above.

In the process according to the invention, transgenic plants are advantageously used as organisms, such as oil-containing plants. These plants contain the hydroperoxy fatty acids synthesized in the process according to the invention and can advantageously be marketed directly without the oils, lipids or fatty acids synthesized having to be isolated. Plants in the process according to the invention include whole plants and all parts of plants, plant organs or parts of plants such as leaf, stem, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, calli, kotelydones, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that are derived from the transgenic plant and / or can be used to produce the transgenic plant. The semen comprises all parts of the semen such as the seminal shell, epidermal and sperm cells, endosperm or embyro tissue. However, the compounds produced in the process according to the invention can also advantageously be isolated from the organisms in the form of their oils, fats, lipids and / or free fatty acids. Hydroperoxy fatty acids produced by this method can be harvested by harvesting the organisms either from the culture in which they grow or from the field. This can preferably be done by pressing or extracting the body parts Plant seeds are made. The oils, fats, lipids and / or free fatty acids can be obtained by cold pressing or cold pressing without the addition of heat by pressing. So that the plant parts, especially the seeds, can be more easily broken down, they are crushed, steamed or roasted beforehand. The seeds pretreated in this way can then be pressed or with

Solvents such as warm hexane can be extracted. The solvent is then removed again. In the case of microorganisms, these are extracted directly after harvesting, for example, without further work steps, or extracted after digestion using various methods known to the skilled worker. In this way, more than 96% of the compounds produced in the process can be isolated. The products thus obtained are then processed further, that is to say refined. For example, the plant mucus and turbidity are removed first. The so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid such as phosphoric acid. The free fatty acids are then removed by treatment with a base, for example sodium hydroxide solution. The product obtained is washed thoroughly with water to remove the lye remaining in the product and dried. In order to remove the dyes still contained in the product, the products are subjected to bleaching with, for example, bleaching earth or activated carbon. Finally, the product is still deodorized with steam, for example.

The hydroperoxy fatty acids produced in the process are advantageously obtained in the form of their oils, lipids or fatty acids or fractions thereof.

In this bound form, they can also be synthesized with the lipoxygenases according to the invention from the corresponding bound fatty acids. Free fatty acids are also implemented.

The term "oil", "lipid" or "fat" is understood to mean a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat has a high proportion of free or advantageously esterified hydroperoxy fatty acid (s). The proportion of esterified hydroperoxy fatty acids is preferably approximately 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80% or more. Depending on the starting organism, the proportion of different fatty acids in the oil or fat can fluctuate.

The hydroperoxy fatty acids produced in the process are, for example, hydroperoxy fatty acids which are bound in sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.

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 are usually in a liquid medium containing a carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as Contains iron, manganese, magnesium salts and possibly vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C with oxygen. The pH of the nutrient liquid can be kept at a fixed value, that is to say it can be regulated during cultivation or not. The cultivation can be batch-wise, semi-batch wise or continuous. Nutrients can be introduced at the beginning of the fermentation or fed semi-continuously or continuously.

After transformation, plants are first regenerated as described above and then grown or cultivated as usual. 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 extracted with suitable solvents such as apolar solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol at temperatures between 0 ° C. to 80 ° C., preferably between 20 ° C. to 50 ° C. The biomass is usually extracted with an excess of solvent, for example an excess of solvent to biomass of 1: 4. The solvent is then removed, for example by distillation. The extraction can also be carried out with supercritical CO ₂ . After extraction, the remaining biomass can be removed, for example, by filtration.

The crude oil obtained in this way can then be further purified, for example by removing turbidity by adding polar solvents such as acetone or chloroform and then filtering or centrifuging. Further cleaning via columns is also possible. To obtain the free fatty acids from the triglycerides, they are usually saponified as described above.

Further objects of the invention are:

- Process for transforming a lance, characterized in that expression cassettes according to the invention containing a lipoxygenase gene sequence from diatoms or DNA 'sequences which hybridize therewith are introduced into a lanceol cell, into callus tissue, a whole lance or protoplasts of plants.

Proteins containing the amino acid sequences shown in SEQ ID NO: 2.

The invention is illustrated by the following examples: Examples

a) Materials

Polyunsaturated fatty acids such as arachidonic acid (= ERA), docosapentaenoic acid (= DPA), docosahexaenoic acid (= DHA), linoleic acid, α-linolenic acid and γ-oleolenic acid were used by Cayman Chemicals, USA.

All other chemicals used were obtained from Merck (Darmstadt), Roth (Karlsruhe), Sigma (Deisenhofen) or Serva (Heidelberg).

The products of J.T. Baker (Phillipsburg, N.J., USA) used in HPLC quality.

b) vectors

Vectors used:

PQE30 ™ is from Qiagen, Hilden pGem-T is from Promega, Madison, USA

c) bacteria

The following bacteria were used:

E. coli XL1-Blue [Bullock et al., Bio Techniques, 5: 376-378, 1987 recA1 endA1 gyrA96 thi-1hsdR17 supE44 relA1 lac [F proAB lac ^q Z

MIS TnlOCTef)]

E. coli SG13009 [pREP4] [Gottesman et al., J Bacteriol, 148 (1): 265-73, 1981] Nal ^s Str ^s rif ^s lac ^" ara gal ^" mtl ^" F ^" recA ⁺ uvr ⁺

d) antibiotics

The following antibiotics were used for the selection: Carbenicillin stock solution: 100 mg / ml, final concentration: 100 μg / ml (Roth, Karlsruhe)

Kanamycin stock solution: 50 mg / ml, final concentration: 25 μg / ml

(Dichefa, Netherlands) e) Antibodies and conjugates

The following antibodies were used:

anti-CsLb-Lipoxygenase Antibody Rabbit, Dr. I. Feussner, Gatersleben [Haus et al., Planta, 210: 708-714, 2000]

anti-rabbit IgG alkaline

Phosphate (ALP) conjugate Röche Diagnostics Grenzach

f) PCR protocol

• 10 cycles

94 ° C 30 sec

52-57 ° C 30 sec

72 ° C 2 min

• 25 cycles

94 ° C 30 sec

47-52 ° C 30 sec

72 ° C 2 min, increment δ sec

• 1 cycle

72 ° C 5 min

Example 1: Colony PCR

A colony PCR was carried out to identify the bacterial colonies which contain the desired fragment in the plasmid DNA after transformation. Cell material from a colony was suspended in 10 μl of water using a toothpick.

The above-mentioned [PCR protocol (f)] PCR reaction mixture was prepared by adding 10 × buffer, MgCl ₂ , dNTP, Tfl polymerase and the corresponding primer. The same program was run and the same primers used to amplify the fragment. If a primer was used for the amplification, which also has a binding site in the vector, this primer has been replaced by vector-specific primers. If the fragment was not cloned directionally in this case (eg in pGEM-T), colony PCR was carried out for both directions with the corresponding vector-specific primers. For the identification of bacterial colonies, which contain DNA constructs in the vector, which were composed of more than one PCR fragment, the 5 'primer of the fragment located at the 5' end and the 3 'primer of the fragment located at the 3' End fragment was used.

Example 2: EXPAND ™ high fidelity PCR

If a low error rate was essential in PCR, e.g. for the DNA sequence of the recombinant Pt lipoxygenase, the amplification of DNA fragments was carried out using the "EXPAND ™ high fidelity PCR system" according to the manufacturer's instructions. This system contains a mixture of the thermostable Taq DNA polymerase and the also thermostable Pwo DNA polymerase, which also has a 3'-5 'exonuclease activity, and only 30 cycles were used instead of 35. Both served to keep the error rate low in order to minimize mutations in the to obtain amplifying DNA.

Example 3: Ligation of DNA

DNA fragments were linked to vector DNA by ligation using T4 DNA ligase. Terminal 5'-phosphate groups and 3'-hydroxyl groups are bonded to form phosphorus diester bonds. The cofactor of this reaction is ATP. In order to reduce the intramolecular or intermolecular ligation of the vector, the DNA fragment was used in a three-fold excess compared to the vector DNA. The ligation reaction took place at 4 ° C. overnight. The ligation was carried out in a volume of 10 μl or 20 μl.

Basically, PCR-amplified DNA fragments were first cloned in pGEM-T. Fragments amplified with Tfl or "EXPAND ™ High Fidelity" have a 3'-adenine overhang which hybridizes with the 3'-thymine overhang of the pGEM-T vector. This is one of the reasons why the ligation efficiency of this vector is relatively high pGEM-T offers the possibility of blue-white selection with IPTG and X-Gal.

Fragments which were cut out from vectors via interfaces for restriction endonucleases (example 5) have been ligated with a correspondingly pre-cut vector. The approach given in Table 1 for ligation using restriction sites was varied in accordance with the given DNA concentration in order to achieve an insert: vector concentration of around 1 μM: 0.3 μM in the approach. With a hypothetical ligation efficiency of 100%, this approach yields around 15 fmol / μl ligation product. Table 1: Ligation of vector and donor DNA

Ligation mix 5 μl 2x ligation buffer, 3 μl DNA (fragment), 1 μl vector,

(pGEM-T vector) 1 μl T4 DNA ligase (1 U / μl)

Ligation mix 2 μl 10x ligation buffer, 5-9 μl DNA (fragment),

(Restriction sites) 1-3 μl vector, 2 μl T4-DNA-ugase (1 U / μl), ad 20 μl H ₂ O

Example 4: Transformation of E. coli

In the course of cloning DNA fragments, the E. coli strains XL1-Blue and SG 13009 were transformed. 100 μl of competent E. coli cells were thawed on ice and about 5 fmol of DNA were added. This corresponds to 10 ng of a 3000 bp DNA construct or 17 ng of a 5000 bp DNA construct. After 20 min incubation in ice, the bacteria were exposed to a heat shock at 42 ° C for 50 s. The mixture was then left on ice for 5 min, 900 μl of LB medium were added and the mixture was cultured at 37 ° C. with shaking for 1 h. The bacteria were plated on selection medium according to their resistance and incubated overnight at 37 ° C.

Example 5: Cleavage of DNA with restriction endonucleases [Sambrook et al.,

Molecular cloning: A loboratory manual, Cold Spring Habor Laboratory Press, New York, 1989]

Restriction nucleases cleave double-stranded DNA in a sequence-specific manner by hydrolysis of covalent bonds. 4 to 8 bp long palindromic sequences are recognized and cut. Cleavages with the restriction endonucleases used in this work result in ends that carry a 3 'or 5' overhang.

Restrictions were carried out to control bacterial colonies which are suitable for the cloning strategy (see Example 28) and for obtaining DNA fragments for the ligation (Example 3). Cutting out the DNA fragments in two steps. After the restriction with the first enzyme, the reaction mixture was cleaned and only then was the restriction with the second enzyme. This was done in order to increase the yield of correctly cleaved DNA by cleaving each enzyme in the optimal buffer with 100% activity without unspecific side reaction (“star activity”). The control of bacterial colonies that were suitable for the cloning strategy was carried out Only one enzyme was used per batch in order to obtain a clear statement for each enzyme.Table 2 shows the batch cleavage approaches used, and the optimum buffer was the reaction buffer Buffer used according to the manufacturer's instructions. The restriction mixture was incubated at 37 ° C. for about 12 h. Table 2: Cleavage of DNA with restriction endonucleases

Restriction mixture 1 μl 10x reaction buffer, 2 μl plasmid DNA (approx. 0.6 μg), for screening 0.21 μl restriction enzyme, 1.8 μl H ₂ O

preparative 5 μMOx reaction buffer, 30 μl plasmid DNA (approx. 9 μg),

Restriction mixture 2 μl restriction enzyme, 13 μl H ₂ O

Example 6: Separation of DNA in Agarose Gels [Sambrook et al., Molecular cloning: A loboratory manual, Cold Spring Harbor Laboratory Press, New York, 1989]

DNA fragments of 0.2 to 4 kb could be separated effectively using a 1.5% agarose gel. For this purpose, 1.5% (w / v) agarose was dissolved in TAE buffer by boiling in the microwave, cooled to 60 ° C. and poured into a horizontal gel carrier. The polymerization was cooled for at least 30 minutes. The DNA samples were mixed with 0.1 volume of sample buffer and separated electrophoretically at a voltage of 120 V. The S ARTLADDER standard (Eurogentec, Seraing, Belgium) with DNA fragments of a defined size was used as the size marker. In order to visualize the DNA bands in the gel, the agarose gel was then incubated for 15 to 20 min in an ethidium bromide bath (2 μg / ml) and decolorized in distilled water for about 5 min. The ethidium bromide attached to the DNA fluoresces when irradiated with UV light (312 nm). The agarose gels were photographed for documentation and evaluation.

Example 7: Mini preparation of plasmid DNA

The plamide mini preparation was carried out either with the "QLAprep Plasmid MiniPrep Kit" (Qiagen, Hilden) or with the "NucleoSpin® Plasmid MiniPrep Kit" (Macherey-Nagel, Düren), each according to the method specified by the manufacturer. These methods are based on the binding of DNA to silica gel membranes at high concentrations of chaotropic salts. Elution is carried out with low-concentration salt buffers or with water. With this method, the DNA is separated from other cell components and preserved in high purity. Example 8: DNA fragment isolation from agarose gels

The gel band of the DNA fragment to be purified was cut out cleanly under UV light with a scalpel and cleaned with the “GFT ™ PCR DNA and Gel Band Purification Kif according to the manufacturer's instructions (Amersham Pharmacia Biotech, UK). In order to avoid mutations in the DNA fragments, care was taken when isolating the fragments that the UV irradiation time was only very short.

Example 9: Sequence analysis

The sequences were analyzed with HUSAR (DKFZ, Heidelberg) using the fragment assembly programs that are part of the WISCONCIN package version 10.2 (Actylrys, formerly Genetics Computer Group (GCG), Madison, Wisc.). Sequences were loaded into the project, components of the cloning vector and phage bank vector were removed semi-automatically and then assembled into sequences (Contig's), sequence comparison against the databases SWISSPROT, TREMBL, PIR and OWL was carried out using the BLASTX2 algorithm [Altschul et al., Nucleic Acids Res, 25 (17): 3389-3402, 1997].

A phylogenetic family tree was created with the PHYLIP ₃₅ program. On the basis of a CLUSTALX sequence comparison of a large number of known lipogenase proteins, one hundred series of point mutations were created using SEQBOOT (random seed number: 5). The known lipoxygenase proteins are divided into three family tree segments, namely the type 1 13-LOX, type 1 9-LOX and type 2 13-LOX family tree. PROTPARS creates the pedigrees of the one hundred point mutation series by calculating the evolutionary distances between the proteins. With CONSENS, the most probable is calculated from the one hundred family trees. The removal of two proteins in the phylogenetic family tree is based on the number of mutations that are necessary at the DNA level to match the sequence of one protein to the other. The genetic code is taken into account. For example, three point mutations are necessary for a Gln / Cys exchange. The different mutation rates of organisms are not included.

Example 10: Expression of the recombinant protein

The expression of the recombinant protein took place in the E. coli strain SG 13009. The expression clones were initially incubated up to an OD ₆₀₀ of 0.6 to 0.8 in LB medium (with cabenicillin and kanamycin) at 37 ° C. After induction of the expression with IPTG (final concentration: 1 mM), the bacteria were cultivated at 10 ° C. for 2 or 7 days. Example 11: Cell disruption

The E. coli cells were centrifuged at 4000 xg and 4 ° C for 20 min and taken up in lysine buffer (50 mM Tris.HCI, 10% glycerol, 0.1% Tween, 0.5 M NaCI, pH 7.5) ( 7 ml lysine buffer per 250 ml cell culture). The cells were disrupted using ultrasound (ten times 1 min, 50% power, 50% pulse). The cell debris was centrifuged at 4000 x g, 4 ° C for 30 min. The supernatant with the soluble proteins was removed and stored at 4 ° C. The activity of the recombinantly produced lipoxygenase was analyzed with this lysate.

Example 12: Activity test

With linoleic acid as the substrate, the lipoxygenase was tested for activity. In contrast to the substrate with its isolated double bonds, the hydro (pero) xides formed have an absorption maximum at 234 nm due to their conjugated diene system. Due to the high self-absorption of the cell lysate, this determination was not carried out spectrophotometically. The products of the fatty acid conversion were therefore carried out by means of normal phase HPLC (see Example 15). The reaction was carried out according to the protocol given below at pH 6 and 8.

Example 13: Determination of the pH optimum

800 μl of the enzyme solution (see Example 11) were mixed with 1.2 ml of pH buffer (100 mM Na phosphate, pH 5.5 to 8; 100 mM TrisHCI, pH 8 to 9.5) and 1 μl (250 μg ) Added linoleic acid. The reaction took place for 30 min in an open 15 ml vessel at room temperature (= 23 ^C C RT). The resulting hydroperoxide derivatives were reduced by adding a crumb (2 mm ³ ) sodium borohydride to the corresponding hydroxide derivatives of the fatty acids. After 2 min, the mixture was acidified to pH 3 with 100 μl of glacial acetic acid in order to stop the reduction and to convert the hydroxides of the fatty acids into the non-ionic form for the subsequent extraction. In order to isolate the products, extraction was carried out with methanol and chloroform [Bligh et al., Can J Biochem Physiol, 37: 911-917, 1959]. With an aqueous phase / methanol / chloroform ratio of 1: 1: 1 (v / v / v). After centrifugation at 4000 xg for 10 min to separate the phases, the chloroform phase was transferred to a 1.5 ml vessel, completely evaporated in a nitrogen stream and then taken up in the appropriate solvent for HPLC. Example 14: Determination of substrate specificity

The conversion took place as described above (Example 13) at pH 8.2 with 250 μg substrate in each case. The different substrates were dissolved in ethanol in an uneven concentration. The resulting deviating volumes of substrate were compensated for by adding pure ethanol in order to level out an influence of the ethanol on the activity of the possibly membrane-associated enzyme.

Example 15: Normal phase HPLC

The position isomers formed were analyzed with devices from AGILENT SERIES IIOO (Hewlett-Packard, Waldbronn) using normal phase columns (50 x 4.6 mm, 3 μm LUNA SILICA (2), Phenomenex, Aschaffenburg, or 150 x 2, 1 mm, 5 μm, ZORBAX RX-SIL, Hewlett Packard, Waldbronn) and a diode array detector were analyzed. When using the LUNA SiucA column, a mixture of hexane / isopropanol / TCA (99: 1: 0.02, v / v / v, isocratic flow 1 ml / min, 20 min) was used as the eluent, whereas the ZORBAX RX-SIL column a mixing ratio of 98: 2: 0.02 (v / v / v, isocratic flow 250 μl / min, 20 min) was used. Since a higher selectivity was required for the investigation of the hydroxides of the long-chain fatty acids, hexane / iso-propanol / TCA in a ratio of 99: 1: 0 was used on the ZORBAX RX-SIL column for the investigation of the substrate specificity for all substrates , 02 (v / v / v, isocratic flow 250 μml / min, 60 min). Standards from Cayman Chemicals (USA) were used to identify the hydroxy fatty acids formed. Inactive hydroxide derivatives were identified by comparison with autooxidation products (example 17) and by GC / MS (example 18). The data acquisition and evaluation was carried out with the software CHEMSTATION FOR LC ₃ D [Rev. A.08.01 (783)] from Agilent Technologies, Waldbronn.

Example 16: Chiral-phase HPLC

The fatty acid hydroxides purified by normal-phase HPLC were equipped with a chiral-phase column (150 × 2.1 mm, 5 μm Daicel Chemical Industries Ltd.CHIRACEL OD-H, Merk, Darmstadt) using the system described above (Example 15) ), examined for their enantiomer composition. Hexane / iso-propanol / TCA (95: 5: 0.2, v / v / v, isocratic flow 100 μl / min, 20 min) was used as the eluent for the hydroxides of linoleic acid and α-linoleic acid, and for the hydroxide Derivatives of y-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid were used in the solvent 98: 2: 0.02 (v / v / v, isocratic flow 100 μl / min, 60 min ). The data- recording and evaluation was carried out with the software CHE STATION FOR LC ₃ D [Rev. A.08.01 (783)] from Agilent Technologies Waldbronn.

Example 17: Autooxidation of fatty acids

200 μg of the fatty acids were taken up in 100 μl of methanol, 800 μl of a 100 mM TrisHCI buffer, pH 9.9, a spatula tip of iron (II) sulfate was added and the solution was incubated at 80 ° C. for 1 h. Air was blown into the samples every 10 minutes. After cooling to room temperature, a crumb (“2 mm ³ ) sodium borohydride was added to reduce the hydroperoxides

Fatty acids to the corresponding hydroxide derivatives. This reaction is stopped by acidification with 100 μl glacial acetic acid to pH 3 after 2 min and the hydroxides of the fatty acids are converted into the uncharged form for the following extraction. The extraction was carried out twice with 1 ml of n-heptane. The samples were centrifuged to separate the phases. The two organic phases were combined, completely evaporated in a stream of nitrogen and taken up in the corresponding HPLC mobile phase.

Example 18: Derivatization of hydroxy fatty acids and GC / MS analyzes

For the GC / MS analysis, hydroxy fatty acids are derivatized to increase their volatility and thermal stability and to reduce polarity [W.W. Christie, Lipids, 33 (4): 343-53, 1998]. This increases the gas chromatographic separation and improves the detectability. The derivatization of hydroxy fatty acids to their methyl ester trimethylsilyl ethers allows the compound to be identified and the position of the hydroxy group to be determined [Wollard et al., J Chromatogr, 306: 1-21, 1984].

A quarter of the turnover of the batches for the substrate determination (example 14) was purified by means of normal phase HPLC. The speculative hydroxides of the fatty acids (absorption at 234 to 237 nm) were collected, completely evaporated in a stream of nitrogen and taken up in 400 μl of methanol. For the methylation, 10 μl of the EDAC stock solution (100 mg / ml) were added and shaken at room temperature for 2 h. After adding 200 μl of 100 mM Tris HCl, pH 7.5 for phase formation, the mixture was shaken twice with 1 ml of n-hexane. After combining, the hexane phases were evaporated in a stream of nitrogen. After the methylation product had been taken up in 3 μl acetonitrile, 1 μl BSTFA-Regisil (1%) was added for the silylation and the GC / MS analysis was carried out. The GC / MS analyzes were carried out under the following analysis conditions: GC: Agilent 6890 series

Inj. Volume 4 μl (manual)

Carrier gas helium, linear flow rate 1, 2 ml / min

Separation column 30 mx 0.25 mm HP-5MS (Crosslinked 5% PH ME Siloxane); 0.25 µm film thickness; Temperature program: 60 ° C (injection temperature) → 25 ° C / min → 300 ° C (1 min) → 10 ° C / min → 270 ° C (10 min) MS Aligent 5973 Network ionization energy 70 eV Scanning speed 1 scan / s

The data acquisition and evaluation was carried out with the software ENHANCED CHEMSTATION G1701CA version C.00.00 from Aligent Technologies, Waldbronn.

Example 19: Purification of Pt lipoxygenase

To purify the proteins, the cells were disrupted as described in Example 11, the cell debris centrifuged off and the supernatant incubated overnight at 4 ° C. with "TALON® Metal Affinity Resin". According to the manufacturer, the binding capacity is 5 mg / ml bed volume the protein supernatant, which was obtained from 500 ml of cell culture, was used in 250 to 500 μl of TALON®, and the column material was then placed on an empty column (TJ Baker, Phillipsburg, NJ, USA). Buffer (50 mM, 300 mM NaCl, pH 8.0) was washed in. In order to determine the optimal cleaning conditions, simple bed volumes of Na phosphate buffer were used

(50 mM, 150 mM NaCl, pH 8.0) with increasing imidazole concentration (10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 250 mM and 300 mM imidazole). Each elution step was completely collected, 10% glycerol was added and the mixture was frozen at -80 ° C. After protein determination [M.M. Bradford, Anal Biochem, 72: 248-54, 1976], 4 μg protein were applied in each case to an SDS protein gel and a Western blot was carried out to determine which fraction (s) the purified protein was in ,

Elution under optimized conditions is carried out with three times with a single bed volume of Na phosphate buffer (50 mM, 150 mM NaCl, pH 0.8) with 125 mM imidazole. Subsequent elution with 300 and 500 mM imidazole was carried out to ensure that nothing of the protein to be purified actually remains on the TALON®. This was examined again using SDS-PAGE and Western blot. Example 20: Protein Determination [MM Bradford, Anal Biochem, 72: 248-54, 1976]

The protein was determined using the “Bio-Rad protein assay” from Bio-Rad (Hercules, USA) according to the manufacturer's instructions using a BSA calibration line.

Example 21: SDS.polyacrylamide gel electrophoresis (SDS-PAGE [U.K. Laemmli, Nature, 227 (259): 680-5, 1970], modified method)

A mini gel apparatus from Bio-Rad, Hercules, USA, was used for the SDS-PAGE. The protein samples were mixed directly with sample buffer or precipitated with TCA (trichloroacetic acid). For this purpose, 1 ml of sample was incubated with 250 μl of 30% TCA for 30 min on ice, then centrifuged for 10 min at 14,000 × g and the precipitate obtained was washed again with water. After centrifugation and the removal of the aqueous supernatant, the precipitate was dried and dissolved in "Roti®-Load 1" sample buffer. Before application, the samples were centrifuged at 15000 xg for 3 min. Alternatively, the precipitate was mixed with very small amounts of protein (= 4 μg) after TCA precipitation directly in "Roti®-Load 1" sample buffer and 1 N NaOH added until the yellow color changed to blue. The samples were separated electrophoretically in an 8% polyacrylamide gel (separating gel: 2.15 ml water, 1, 2 ml acrylamide / methylbisacrylamide (30% / 0.8%), 1, 1 ml 4 x separating gel buffer, 14 μl APS ( 50%), 3.5 μl TEMED; stacking gel: 0.87 ml water, 0.24 ml acrylamide / methylbisacrylamide (30% / 0.8%), 0.375 ml 4 x collecting gel buffer, 6 μl APS, 3 μl (50% )). The sample was run into the collection gel for 25 min at 12.5 mA per gel. The separation of the proteins in the separating gel was carried out at a current of 25 mA per gel for 40 min or until the bromophenol blue front had reached the end. The separated proteins were stained in the gel.

Example 22: Coomasia staining of protein gels

The staining of proteins in SDS polyacrylamide gels was stained according to the manufacturer's instructions with the GELCODE® Blue Stain Reagent from Pierce (Rockford, IL, USA). Example 23: Silver staining of protein gels [Blum et al .; Electrophoresis, 8: 93-99, 1987]

For silver staining, the gel was washed three times for 30 min with ethanol / acetic acid / water (30: 5: 65, v / v / v) [for all buffers and washing steps up to / incl. 18 MΩ water was used in the development.] and then washed three times with water for 10 min. Sensitization was carried out by incubation with 0.02% Na thiosulfate solution (freshly prepared) for 1 min, after which it was washed twice with water for 1 min. After impregnation with silver nitrate solution (0.025% formaldehyde, 0.0125% AgNO ₃ ) for 30 minutes, care was taken to wash with water for a maximum of 15 seconds (5 to 15 seconds). Incubation with developer solution (3% potassium carbonate, 0.01% formaldehyde, 0.001% Na thiosulfate) continued until the protein gel was sufficiently colored (1 to 10 min). Was stopped by repeated rinsing with 1% acetic acid, whereupon once with dist. Water was washed.

Example 24: Western blot

After electrophoretic separation in an 8% protein gel [U.K. Laemmli, Nature, 227 (259): 680-5, 1970] the proteins were transferred onto a nitrocellulose membrane using transfer buffers (mini-gel apparatus from Bio-Rad, Hercules, USA; 75 min at 65 V). The membrane was blocked for 4 h at room temperature with 1% ovalbumin solution (PBST) and then briefly washed with PBST. The primary antibody (anti-CsIb-epoxygenase (1: 1000) and anti-his (1: 2500)) was diluted with PBST (1% BSA) and incubated for 1 h with the membrane. The membrane was then washed three times in PBST for 10 min. For the colorimetric detection, the corresponding ALP conjugate was diluted 1: 5000, incubated with the membrane for 1 h, washed twice in PBST for 5 min, once with PBS for 5 min and once in AP buffer for 10 min. NBT / BCIP was used diluted in AP buffer according to the manufacturer's instructions.

Example 25: Isolation of Phaeodactylum tricornutum Lipoxygenase

Isolation of the sequence of a putative lipoxygenase from the diatom Phaeodactylum tricornutum by 5'-RACE. SEQ ID NO: 5 shows the consensus sequence of all 5 'RACE fragments. The isolated RNA of the diatom P. tricornutum was in the form of a “Lambda Zap II Express” cDNA library, into which it was cloned in a directional manner. The underlying vector was pBK-CVM. The complete sequence was then cloned into a bacterial expression vector The following biochemical characterization of the recombinant active enzyme included the determination of the pH optimum and the substrate specificity Hydroxy fatty acids were not available as standard substances, they were characterized as combined methyl ester and trimethylsilyl ether derivatives by means of GC / MS analysis. Purification for the recombinant enzyme was also started.

Example 26: Cloning

A cDNA library was created from the P. tricornutum diatom. Furthermore, an in vivo excision was carried out, the plasmids were recovered and transformed into E. coli DH1 OB. Then plasmid DNA was obtained automatically and random sequencing was carried out using the chain termination method. 8400 clones were sequenced at the 5 'end, combined into 3400 non-redundant sequences (Contig's) and annotated. Subsequent analysis of the database created in this way identified two clones via their 5 'sequence which showed homologies to plant lipoxygenases. One of them was the lipoxygenase from P. tricornutum (LOX2: R: 1 (PtLOXI) described in this work. Clone Pt001077095r) showed the greatest homology to lipoxygenase with 42% at the DNA level in the sequenced region of 800 bp of the 5 'end - 1 from Pisum sativum. The 3 'end of this cDNA clone was then sequenced to verify this finding (FIG. 4, gray area). In the range of 170 bp above the 3 'end, the homology was 55% to the same lipoxygenase from Pisum sativum. At the same time, this sequence comparison showed that presumably around 500 bp of the open reading frame (ORF) was missing at the 5 'end.

Example 27: Sequence extension by 5'-RACE

The gene-specific primer Pt-LOX2_5RACE (1) (5'-GAG CCC CTG.TCT TCT CGG TAT TG) was derived from the known sequence in the 5 'region. 5'-RACE with this gene-specific and vector-specific primer 3 (5'-GCT CGA AAT TAA CCC TCA CTA AAG GG) gave the fragments of different lengths shown in FIG. 4, which extend the known sequence by around 650 bp in 5'- Extended direction. The dark gray highlighted sequences of clone Pt001077095r resulted from the random sequencing of 8400 clones from a cDNA bank of the diatom P. tricornutum. The sequences highlighted in light gray were obtained using 5'-RACE. The arrows indicate the primers mentioned above. The upper curve shows the quality of the sequence information, which is defined by the number and the extent to which the sequences match. A further sequence extension with the primer PtLOX2_5RACE (2) (5'-CAA TAT CGA TCA AAC CTC GCT AC), which binds 677 bp upwards from the first 5'-RACE primer, could not be achieved. Based on sequence comparisons with plant lipoxygenases However, it was assumed that the sequence information obtained would nevertheless be sufficient to derive primers with which the complete DNA sequence of the PtLOXI from the underlying phage library can be amplified and cloned. However, since this did not succeed, the complete sequence was composed of two overlapping fragments. As a prerequisite for the development of the cloning strategy, the cDNA clone that had been identified by means of random sequencing (Pt001077095r) was first completely sequenced.

Example 28: Obtaining the complete cDNA sequence

A 1336 bp region (fragment 1) representing the 5 'end of the PtLOX sequence was primed using A (5'-GGJ_ACC ATG ATG CTC AAC CGG TTG AC) and B (5'-AAA TTC CCG AGC AAA CTC GT) amplified from the P. tricornutum cDNA library (see FIG. 4). With primer A, which contains the start codon ATG, a restriction site for Kpnl was introduced. The second fragment of the gene was present in the vector pBK-CMV in the form of the clone with which the two ESTs were obtained. In the overlapping region of 787 bp of the two fragments is the restriction site for Sal I that cuts uniquely in the sequence. To insert the restriction site for Hind III at the 3 'end of the second fragment with primer C, this fragment was primed M13-rev (5'-GGA AAC AGC TAT GAC CAT G) and the primer C (5'-CCC AAG CTT CTA TAT GGT GAT GCT GTT GGG CAC) were amplified by PCR. Both fragments were first cloned into the vector pGEM-T and then assembled into unique gene sequences via unique restriction sites introduced by means of PCT in pQE30. Fragment 2 was ligated to the expression vector pQE30 via the restriction sites for Sal I and Hind III. After the corresponding restriction digest, this construct was used using the restriction sites for Kpn I and Sal I with fragment! ligated. In order to keep the risk of introducing a mutation as low as possible, two or three bacterial colonies were used for the cloning experiments, which contained fragment 1 and 2, respectively. After all of the clones tested were active (Example 30), the clone with the number ([52-1] +43) -1-1 was designated PtLOXI and all further tests were carried out with this clone.

Example 29: Sequence analysis of PtLOXI

The complete cDNA clone was then sequenced, the known sequence (overview in FIG. 4) being confirmed. With the help of the BLASτX2 algorithm, the highest homology to AtLOX3, a 13-LOX of type 2, from Arabidopsis thaliana was found for the complete PtLOXI sequence with 43%. This result was confirmed by the phylogenetic parent structure created as part of this work. PtLOXI's ORF consists of 938 amino acids and has a calculated molecular weight of 105 kDa. Possible glycosylations in the original organism P. tricornutum were not taken into account. The calculated isoelectric point is pH 6.69. The beginning of the protein sequence with two methionines is unusual. The first methionine is considered the translation start codon and was used for cloning. The cDNA is 66 bp longer in the 5 'direction than the sequence region used for the cloning. Since there is no starting methionine in this region, it was considered an untranslated region at the 5 'end. With the complete sequence of PtLOXI an analysis with the program PSORT on HUSAR was carried out regarding possible signal sequences. As this database does not take into account chloroplastic signal sequences, only a 50% probability of plastid localization was found. However, the sequence comparison of the PtLOXI with other known lipoxygenase sequences had shown that the PtLOXI belongs to the group of 13-lipoxygenases of type 2, for which a chloroplastic signal sequence is characteristic. However, it can be seen from the phylogenetic family tree that this assignment is burdened with a high degree of uncertainty, since PtLOXI is approximately the same distance to 13- and 9-sipoxygenases of type 2 there. In addition, some lipoxygenases are misclassified in this family tree, as for example LOX1: Cs: 1, LOX1: Cs: 3 and

LOX1: Hv: 3 and 13-epoxygenases and not 9-lipoxygenases. Nevertheless, plastid localization can be discussed for the RLOX1. Due to lower security modes, an analysis with other databases was not carried out.

Compared to other plant lipoxygenases, PtLOXI shows some interesting differences at the level of the protein sequence. In the active center of lipoxygenases, three determinants have been determined that have an influence on the substrate and position specificity, whereby. the BORNGRÄBER and SLOANE determinants are to be emphasized (summarized in [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley, -VCH, Einheim, 2000]). A highly conserved arginine was identified at the bottom of the binding pocket in all plant lipoxygenases, which plays a critical role in the conversion of the lipid body 12-lipoxygenases from cucumber into a 9-lipoxygenase (R758 of the CsIbLOX [Hornung et al., Proc NatI Acad Sei USA, 96 (7): 4192-4197, 1999]. This arginine occurs regardless of the substrate and position specificity and precedes an aparagin in more than 90% of the cases, with the exception of two histidine in PtLOXI which coincide with this asparagine The amino acid, on the other hand, is conserved in the sequence comparison with the SLOANE determinant [Sloane et al., Nature, 354: 149-152, 1991]. At this point there is a small amino acid in 9-lipoxygenases like valine and a large one like phenylalanine in 13-epoxygenases, with the exception of the ones above mentioned LOX1: Cs: 1 and LOX1: Cs: 3 (H instead of F) and LOX1: Hv: 3 (V instead of F). This discrepancy may be due to the fact that in these cases no "classic" 13-lipoxygenases are present [Feussner et al., Annu. Rev, Plant. Biol., 53: 275-297, 2002]. However, the voluminous one lies in PtLOXI Amino acid phenylalanine, as expected for a 13-lipoxygenase At the position in the sequence comparison that coincides with the BoRNGRÄBER determinant, it is more difficult to find conserved differences between 13- and 9-lipoxygenases, usually a combination of hydrophobic ones and hydrophilic amino acids, which are separated from the small amino acid glycine or the hydrophobic amino acid cysteine (e.g. S515, G516, V517 in LOX1: Nt: 1) There are conserved differences between the individual groups with regard to the combination of hydrophobic / hydrophilic amino acids. PtLOXI differs from all other lipoxygenases in that it separates two hydrophobic amino acids from the hydrophilic amino acid threonine, the C-terminus is highly conserved as with all other lipoxygenases, with the small difference that threonine is in the penultimate position instead of being, as with all other lipoxygenases.

Example 30: Biochemical characterization of PtLOXI

First, it was checked whether the recombinant enzyme was active. Since linoleic acid is a general substrate for lipoxygenases [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002], the activity of the enzyme was first determined against this fatty acid. Due to the high self-absorption of the crude extract after disruption of the E. coli cells, this determination was not made using a spectophotometer. The products of the fatty acid conversion were analyzed by normal phase HPLC. It was shown that all the clones tested were active, since 13-HODE in an S configuration was formed when reacting with linoleic acid. Six different combinations were obtained in the composition of the 5 'and 3' region of the sequence during the cloning (Example 28). Two clones of each combination were tested.

The pH at which the enzyme shows the maximum activity was then determined. The question of whether the enzyme's product and region specificity changes in this pH range was also investigated. It was also examined which other fatty acids are accepted as substrates by PtLOXI. On the one hand, the structure of the resulting hydroxy fatty acids was analyzed in more detail and, on the other hand, the preferred substrate of this enzyme was determined. Example 31: pH optimum

The pH optimum was determined by means of an end point determination using normal phase HPLC for the conversion of linoleic acid. Cell digests (Example 11) of protein expression at 10 ° C. were used for the analyzes. The lysates were used immediately after their preparation in order not to reduce the stability of the proteins by freezing and thawing the samples. The cell disruption was incubated with linoleic acid and the resulting hydroperoxide derivatives were reduced to the corresponding hydroxides with sodium borohydride, which were analyzed after the acidic extraction using normal-phase HPLC (SP-HPLC). The areas of the integrated HODE signals at 234 nm and the linoleic acid at 202 nm were evaluated. From FIG. 6 it can be seen that the maximum activity of the PtLOXI for linoleic acid is achieved at pH values between pH 8.0 and pH 8.4.

Linoleic acid was reduced in substrate excess for 30 min with enzyme extract and the hydroperoxides formed were reduced to the hydroxides. After acid extraction, the substrate product spectrum was analyzed using SP-HPLC. The sum of the area of all integrated HODE signals at 234 nm was related to the area of the integrated linoleic acid signal at 202 nm. The representation in FIG. 6 shows the average and the standard error for two experiments.

Example 32: Analysis of the regiospecificity towards linoleic acid

A characteristic feature of lipoxygenases is their regiospecificity when introducing molecular oxygen [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000]. Since linoleic acid is a substrate for lipoxygenases both in the animal kingdom and in the plant kingdom and, in contrast, arachidonic acid does not occur in the plant kingdom or only occurs in storage lipids, plant lipoxygenases are classified in terms of the position specificity of the oxygenation of linoleic acid [H.W. Gardner, Biochim Biophys Acta, 1084: 221-239, 1991]. Oxygen can be introduced either at carbon atom 9 (9-lipoxygenase) or 13 (13-lipoxygenase) of linoleic acid.

The regioisomers formed were again separated by means of SP-HPLC after the reduction of the hydroperoxides to the corresponding hydroxides [Kühn et al., Anal Biochem, 160: 24-34, 1987]. Standards of the respective hydroxide derivatives were compared with one another before or after the measurements of the cell disruption extracts and the retention times of the signals. The ratio of 9- to 13-HODE was determined by integrating the respective signals at 234 nm. In Figure 7 the ratio of total product to substrate is formed for each pH value and the respective proportion of 13- and 9-HODE in the total product is also shown.

Linoleic acid was incubated in excess substrate for 30 min with crude extract enzyme and the hydroperoxides formed were reduced to the hydroxides. After acid extraction, the substrate product spectrum was analyzed using SP-HPLC. The integrated signals of 9-HODE and 13-HODE at 234 nm were compared to the integrated linoleic acid signal at 20 nm. The representation in FIG. 7 shows the average and the standard error for two experiments.

The ratio of 13 to 9 HODE is also summarized in Table 3. The prediction from the sequence comparison (Example 29) that PtLOXI is a 13-lipoxygenase was thus confirmed by biochemical data.

By analyzing the enantiomer composition by means of chiral phase HPLC (CP-HPLC), a distinction can be made between the products of enzymatic lipid peroxidation and non-enzymatic autoxidation. The conversion of polyunsaturated fatty acids by vegetable lipoxygenases leads to the formation of the hydroxide derivatives in the S configuration, whereas the enantiomers are formed in a racemic ratio during the autoxidation [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002]. As FIG. 7 shows, more than 90% of the 13-HODE measured in the S configuration is present over the entire pH spectrum investigated, whereas the ratio of the 9-HODE enantiomers is racemic. Since the amount of 9-HODE formed at the different pH values correlates with the enzyme reactivity, the formation of this hydroxide derivative may not be due to autoxidative processes, but results from an enzymatic activity (see FIG. 7 and Table 3). The by-products in the conversion of linoleic acid by lipoxygenases under aerobic conditions are the ketooctadecadienoic acids (KODEs), which have an absorption maximum at 273 nm. In the linoleic acid reaction by PtLOXI, no KODE was formed by PtLOXI.

Table 3: Regiospecificity of PtLOXI compared to linoleic acid. The R / S ratio of 13-HODE was 7% / 93% over the entire pH range, whereas 9-HODE was racemic. These data are part of FIG. 7.

Example 33: Substrate specificity of PtLOXI and analysis of the regioisomers formed

In order to clarify which polyunsaturated fatty acids are accepted as substrates by PtLOXI and which regioisomers preferentially arise, seven different fatty acids were used: linoleic acid (LA), α-linolenic acid (α-LEA), Y-linolenic acid (γ-LEA), arachidonic acid (ERA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). These fatty acids differ in their length and in the number and position of the double bonds (see Table 4). With the exception of LA, several bisallylic methylene groups can be used in these fatty acids for the initial hydrogen abstraction by the non-hemem iron in the reactive center of the lipoxygenases. Due to the [2 +] and [-2] radical rearrangement on each bisallylic methylene group, the oxygen addition can therefore form four hydroperoxide derivatives from α-LEA (9-, 13-, 12-, 16-α-HPOTE) and γ-LEA (6-, 10-, 9-, 13-γ-HPOTE), six from ARA (5-, 9-, 8-, 12-, 11-, 15-HPETE), eight from EPA (5th -, 9-, 8-, 12-, 11-, 15-, 14-, 18- HEPE), eight from DPA (7-, 11-, 10-, 14-, 13-, 17-, 16-, 20-HPDPE) and ten from DHA (4-, 8-, 7-, 11-, 10-, 14-, 13-, 17-, 16-, 20-HPDHE).

In order to obtain a first indication of which of the polyunsaturated fatty acids PtLOXI serve as preferred substrates, the sum of the integrated signals of all hydroxide derivatives which resulted after the reduction of the corresponding hydroperoxides was plotted for each substrate.

The enzyme was incubated with various substrates at pH 8.4 and the product analysis was carried out using SP-HPLC. The sum of the integrated signals of all hydroxide derivatives was plotted for the conversion of each fatty acid. The difference- The extinction coefficients of the hydroxides were not taken into account during the integration. The representation in FIG. 8 shows the average and the standard error of two experiments.

The different extinction coefficients of the hydroxide derivatives (at 234 nm) were not taken into account, since they are not known for all of the hydroxides formed. The comparison of the molar extinction coefficient of HOTE (ε = 27000 l / mol x cm 234 nm) with that of HETE (ε = 27000 l / mol x cm 236 nm) suggests that this value increases with increasing number of double bonds. For this reason, the preferred implementation of ERA over γ-LEA is not quite as pronounced as shown in the diagram. Nevertheless, it can be concluded from this diagram that the C ₂₀ fatty acids arachidonic acid and eicosapentaenoic acid are preferably implemented compared to C ₈ and C ₂₂ fatty acids. The regioisomers of the hydroxides of these fatty acids formed were identified either using standards which were used in the respective HPLC analyzes or by means of GC / MS analyzes (example 18 and example 34). It was found (Table 4) that of the bisallylic methylene groups in question, only one was used for each substrate for the initial removal of hydrogen. This affected the methylene group at position (n-8) in the case of LA and α-LEA and each (n-11) for γ-LEA, ERA, EPA, DPA and DHA. Provided that with 13-lipoxygenases the substrate is immersed in the binding pocket with the methyl end first [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002], it can be estimated that the distance between the hydrogen acceptor and the bottom of the binding pocket is around eleven methylene groups. Table 4 provides an overview of all implementations, which will now be discussed in more detail.

Table 4: Overview of the substrate and region specificity of recombinant PtLOXI

^a 'Structure was not verified by GC / MS

As already treated in Example 32, the conversion of linoleic acid leads to 90% formation of 13-HODE in the S configuration. The chromatogram of the SP-HPLC analysis is shown in FIG. 9. The control implementation was carried out with cell disruption of SG 13009 cells which carried the empty vector. As already explained in Example 32, the chromatogram of this reaction (FIG. 9, lower part) shows that 9-HODE was formed enzymatically and does not result from autoxidation. For the other fatty acids, too, the same amounts of the turnover of the fatty acid with recombinant PtLOXI and the control conversion were analyzed.

For α-LEA as substrate, the proportion of the two possible oxidation products in the [+2] and [-2] positions with respect to the carbon (n-8) in the total hydro (pero) oxide derivatives formed (FIG. 10) is 92 % and is therefore comparable to the sales of LA. The ratio of the [+2] to [-2] oxidation products drops from 6.7 for 13- / 9-HODE to 2.7 for 13- / 9-α-HOTE. The factor 6.7 / 2.7 «2.5 which can be calculated with this was observed even more frequently when the number of double bonds or the number of methylene groups changed. The conceivable hydroxylation products of α-LEA 12- and 16-α-HOTE, which distance at C ₁ (n-5), have a negligible proportion of the hydro (pero) xy derivatives of α-LEA.

In γ-LEA in Figure 11, the bisallylic methylene group in position (n-11) was preferentially attacked by the hydrogen acceptor, which leads to the formation of 10- and 6-γ-HOTE. The methylene group at position (n-8), in which the hydro (pero) oxide derivatives 13- and 9-α-HOTE are formed, play a role with a total share of around 17% of the total hydro (pero) oxide derivatives subordinate role. But here too the ratio is in favor of the [+2] -hydro (pero) oxide derivative. The ratio of the main products 10- to 6-γ-HOTE is comparable to that of 13- to 9-α-HOTE from α-LEA. For LA, α-LEA and γ-LEA, the respective [-2] hydro (pero) oxide derivative is racemic.

In arachidonic acid (FIG. 12), the hydro (peroxidation) takes place at position [+2] and to a small extent at position [-2] with respect to the bisallylic methylene group at position (n-11). The ratio of hydro (pero) xylation at position [+2] to [-2] is clearly shifted towards [+2] compared to α- or γ-LEA. Here we observe what has already been mentioned and was later observed again: When comparing two fatty acids with regard to the methylene group from which hydrogen was abstracted, the introduction of a double bond in the "[- X]" position, that is to say at the carboxy end, led to a shift in hydro (pero) xylation in favor of the [+2] product, as well as the introduction of two methylene groups in the "[+ X]" position (at the methyl end). Conversely, there is a shift in favor of the [-2] product if a double bond "[+ X]" position has been introduced (as already observed for Y and α-LEA), or two methylene groups in ,, [- X] "position (the carboxyl end) was added. With regard to the [+2] to [-2] ratio, an increase by a factor of 2 to 4 was observed in the first case and a decrease by a factor of 2 to 4 in the second case. This observation is purely empirical and must be discussed. It should be emphasized that only characteristics regarding the position of the bisallylic methylene group in question are described here, which do not in any way reflect the biochemical introduction of methylene groups or double bonds. Compared to α- and γ-LEA, arachidonic acid is two methylene groups longer in the "[+ X]" position in terms of the bisallylic methylene groups used (x4) and a double bond is added in the "[- X]" position (x4) , This results in an increase in the ratio of the [+2] to [-2] -hydro (pero) oxide derivatives 12-HETE to 8-HETE of 4 x 4 = 16 compared to the corresponding products in γ-LEA; 2.8 x 16 = 44.8. The measured ratio of 12-HETE to 8-HETE is 46.3 (97%). The main product 12-HETE was unexpectedly only 79% in S configuration, although the comparative analysis in Figure 8 suggests that ERA or EPA are the preferred substrates. The secondary product 8-HETE was in S configuration.

According to the explanations above, the introduction of a double bond at the methyl end in EPA lowers the ratio of [+2] to [-2] of the hydroxide derivatives formed with respect to the bisallylic methylene group compared to ERA. As the only exception in the series, the extent of the reduction by a factor of sixteen cannot be explained here, since the double bond just mentioned led to a change by a factor of 2 to 4 in all other cases (LA / α-LEA, γ-LEA / ERA, DPA / DHA). The ratio of 12- to 8-HEPE is 46.3 / (4 x 4) = 2.9 * 3.01 (75.1%: 24.9%). Both hydroxide derivatives are almost 100% in the S configuration.

The extension by two methylene groups on the carboxy side with respect to the bisallylic methylene group (n-9) of DPA (FIG. 14) is expected to lower the ratio of the oxidation products at position [+2] to [-2] compared to EPA. The factor of 3 is also within the range of what has always been observed with the exception above. 14- to 10-HDPE was formed in the ratio 3 / (3) = 1 (51%: 49%). As with arachidonic acid, the proportion of the S configuration in the main product (here 14-HDPE) is surprisingly low at 68%.

Comparing the implementation of DHA (Figure 15) with that of DPA also fits well into the picture: The additional double bond on the carboxy side of the (n-9) methylene group favors the formation of the [+2] product, hence of 14 -HDHE. Compared to the (51%: 49%) ratio of 14- to 10-HDPE, the ratio of 14- to 10-HDHE is increased by a factor of 2.9 and is therefore 2.9 = 74.3%: 25.7 %. For the main product 14-HDHE, a splitting of the signal of the S-position isomer was found in the CP analysis. An almost identical chromatogram was also observed in one of four chiral phase analyzes of 12-HEPE, therefore the R to S ratio of 14-HDHE in Table 4 was calculated on the basis of the signals R and Si in FIG. 15 , If necessary, this analysis would have to be carried out again.

In the case of arachidonic acid, the above-mentioned (Example 32) by-products were detected under aerobic conditions, namely the keto acids, to an extremely small extent of 1.2% at 273 nm. They also occurred in the implementation of DPA and DHA with around 7.0% each. Example 34: GC / MS analysis of the fatty acid hydroxides

The products of the implementation of PtLOXI with La, α-LEA and γ-LEA could be clearly identified with standards by the normal phase HPLC. Of the possible hydroxides, 10-γ-HOTE was not available as a standard. The comparison with the autoxidation products of the γ-LEA also did not lead to a reliable identification. For this reason, in collaboration with Ms. Dr. C. Göbel (IPK, Gaterieben) GC / MS analyzes performed. For this purpose, the hydroxides of the fatty acids were converted into their combined methyl ester and trimethylsilyl ether derivatives in order to increase their volatility and thermal stability and to reduce the polarity [W.W. Christie, Lipids, 33 (4): 343-53, 1998]. This increases the gas chromatographic separation and improves the detectability. The derivatized fatty acids had a retention time between 16 min and 20 min in the analyzes. The mass spectra shown below were recorded in this area of the chromatogram. Usually a characteristic fragment of the decay of the compound was detected in the mass spectrum, with additional signals the molecular weight of the unfragmented compound, the M-15 signal (without methyl group) and the M-31 signal (without methoxy group) being detected. The possible decay products are shown with their molecular weights in the respective diagram. The characteristic decay allows the compound to be identified and the position of the hydroxy group to be determined [Woollard et al., J Chromatogr, 306: 1-21, 1984].

As can be seen in FIG. 16, it was thus confirmed on the basis of the characteristic masses (shown in the diagram) that the combination of the γ-LEA conversion in question was 10-γ-HOTE. 8-HETE, for which no clear statement could be made after the HPLC analysis, was also verified in this way (FIG. 17).

With the exception of the putative 10-HDHE, all hydroxides of the fatty acids EPA, DPA, DHA could be determined. Figure 18 and Figure 19 show the mass spectra of the identified products of the reaction of PtLOXI with EPA, 12-HEPE and 8-HEPE, respectively. The hydroxides of DPA were found to be 14-HDPE (Figure 20) and 10-HDPE (Figure 21). As the decay pattern becomes more unspecific as the chain length of the compound to be analyzed increases, identification becomes increasingly difficult. So 14-HDHE (Figure 22) could still be identified, due to various limitations that already occurred with 8-HETE, 8HEPE and 10-HDPE, the analysis of 10-HDHE was not successful. Due to mechanistic considerations and the fact that the products from ERA and EPA are the same (12- / 8-HETE, 12- / 8-HEPE) and also 14-HDPE has its equivalent in the form of 14-HDHE, it can be assumed that it is actually 10-HDHE.

Example 35: Purification of the enzyme

The recombinant enzyme should first be purified for a detailed kinetic study of PtLOXI. To this end, PtLOXI was expressed in the vector pQE30 with an N-terminal hexa-histidine tag and then an affinity purification was carried out using TALON®. This cleaning is based on the complexation of the cobalt fixed to the TALON® by histidine. The protein is eluted by competitive displacement of the histidine by imidazole or a chelator of divalent metal ions (e.g. EDTA). Alternatively, elution can be carried out by lowering the pH, since protonated histidine is repelled by the divalent metal.

Example 36: SDS-PAGE and Western blot analysis of cleaning

First the protein was produced in E. coli after adding IPTG for around 60 h at 10 ° C. Then different methods were used to elute the affinity-bound protein: elution with EDTA or imidazole and by lowering the pH. FIG. 23a shows the SDS-PAGE for examining the optimal elution conditions with imidazole.

A SDS gel stained with silver, B Western blot stained with αCsIbLOX / lgG-ALP. The arrow indicates PtLOXI. 1 protein standard, 2 control (lysate from SG 13009 cells transformed with empty vector pQE30), 3 lysate from SG 13009 cells (transformed with PtLOXI in pQE30) before induction with IPTG, 4 lysate from SG 13009 cells (transformed with PtLOXI in pQE30) after induction with IPTG, 5 flow through after affinity binding of the bacterial protein extract to TALON®, 6 washing steps with Na phosphate buffer, 7 to 15 elution with imidazole increasing concentration: 10 mM, 30 mM, 50 mM, 75 mM, 100mM, 125mM, 150mM, 200mM, 300mM.

The protein to be purified has a calculated size of 105 kDa. Several protein bands were clearly visible in this area when the cell disruption was applied after induction and in the fractions of the elution with imidazole (FIG. 23a, arrows). In order to verify that one of the bands in the 105 kDa range was RLOX1, the immunodetection shown in FIG. 23b was carried out. A monospecific antibody was used for this, which was directed against the lipid body lypoxygenase of the cucumber (CsIb-LOC) [Haus et al., Planta, 210: 708-714, 2000]. This antibody exhibits most other plant lipoxygenases due to its high cross-reactivity with other lipoxygenases on. A band just below 105 kDa was also detected in the Western blot (FIG. 23b, lane 5, arrow). The large number of presumably unspecific signals obtained with immunodetection may result from the type of antibody obtained. The same expression system was used for the purified recombinant protein (CsIb-LOX) as for the PtLOXI (vector pQE30 in SG 13009 E. coli cells). For this reason, the antibody fraction obtained with the purified recombinant Cslb lipoxygenase also shows reactivity to other proteins from this system, which were therefore detected in all fractions, including the empty vector purification.

The result of the SDS-PAGE and the Western blot taken together means that PtLOXI does not bind to the TALON®. A comparative determination of the activity between the individual protein fractions could provide clarification when the same amounts of protein are used. Since there was only enough protein for an SDS gel and a Western blot analysis in this experiment, this purification could not be pursued further. Therefore, a new cleaning was carried out on a preparative scale. It was also eluted with EDTA and by lowering the pH, and consequently the same result was obtained since PtLOXI did not bind to the TALON®.

Example 37: Preparative cleaning

For the preparative purification, the recombinant protein was expressed under modified conditions. After induction of translation with IPTG, a further incubation took place overnight at 28 ° C and over a period of seven days at 10 ° C. Instead of stepwise elution (example 36), washing was carried out here only with 5 mM imidazole and the protein remaining on the column was then eluted with 125 mM imidazole (see example, 19). The cleaning confirmed that only in the fractions after the cleaning Expression of PtLOXI at 10 ° C. the protein identified as PtLOXI was present (FIG. 24, lane 19, 20, Reil). The Western blot of the expression at 10 ° C. (FIG. 25b) confirms that RLOX1 did not bind to the TALON®, since a band of the correct size was again detected in the flow and in the Na phosphate wash fraction (lanes 19 and 20, Reil).

SG 13009 cells were transformed with PtLOXI and, after adding IPTG, expressed at 60 ° C. for about 60 hours. - A SDS gel stained with Coomasie, B Western blot stained with α-CsIbLOX / lgA-ALP. The arrow indicates PtLOXI. 19 flow rate after affinity binding of the bacterial protein extract to TALON®, 20 washing step with Na phosphate buffer, 21 washing step with 5 mM imidazole, 22 to 26 elution with imidazole: 125 mM ^'1 , 125 mM ^"2 , 125 mM ^{" 3} , 300 mM, 500 mM. Neither in the cleaning of the cell lysate after the expression of the empty vector nor in the expression of PtLOXI at 28 ° C. is a protein band clearly recognizable in FIG.

The arrow indicates PtLOXI. SG 13009 cells were transformed with the following vectors and expressed under the conditions specified in the text after IPTG addition: 1 to 8 pQE2028 ° C, 9 to 12 and 15 to 18 PtLOXI (expression at 28 ° C), 19 to 26 PtLOXI ( Expression at 10 ° C) 1, 9.19 flow rate after affinity binding of the bacterial protein extract to TALON®, 2.10.20 washing step with Na phosphate buffer,

3.11.21 washing step with 5 mM imidazole, 13.14 protein standard; Elution with imidazole:

4.12.22 125mM-1, 5.15.23 125mM ^"2 , 6.16.24 125mM ^{" 3} , 7.17.25300mM, 8.18.26 500mM.

As an additional confirmation of the Western blot analyzes, an activity determination for selected fractions for the purification of recombinant PtLOXI, which was expressed at 10 ° C. or at 28 ° C., was carried out with the same amounts of protein (Example 13). The evaluation for the purification of recombinant PtLOX, which was expressed at 10 ° C., is shown in Table 5. The enzyme activity of the expression of PtLOXI at 28 ° C could not be evaluated. The sum of the areas of 13- and 9-HODE was used as a measure of the activity. In order to estimate autoxidative processes, the ratio of 13- to 9-HODE was also given. It was shown that the LOX activity can only be measured in the flow and in the wash fraction. It should be emphasized that the Na phosphate buffer used for washing contained no imidazole. This confirmed that PtLOXI does not bind to TALON®. The active clone PtLOXI was now sequenced in the expression vector pQE30, and when using the vector-specific primers for the outer ends of PtLOXI (3 'and 5' end) the sequencing confirms that the vector pQE30 actually contains the six histidine residues in its contains coding sequence.

Table 5: Analysis of the activity of individual fractions of the preparative purification of PtLOXI. The results for the expression of recombinant PtLOXI at 10 ° C. after addition of IPTG are shown. 40 μg of substrate were used in each case, incubated for 2 h and the same amounts were analyzed by normal phase PHLC.

Overall, the analyzes carried out show that the enzyme obtained could not be obtained either in sufficient purity or in sufficient amounts that are required for the planned kinetic measurements.

discussion

In the course of this work, lipoxygenase was isolated from the diatom P. tricornutum and expressed recombinantly in E. coli. So far, no isolated sequence encoding a diatom lipoxygenase has been described. Compared to vegetable lipoxygenases, lipoxygenase has an exceptional substrate specificity.

The assignment of PtLOXI to the group of 13 lipoxygenases of type 2 is burdened with a high degree of uncertainty, since it is approximately the same distance to 13- and 9-epoxygenases of type 1 in the family tree. Type 2 lipoxygenases are lipoxygenases of chloroplastic signal sequence. PtLOXI shows relatively low homology to the representatives of the different groups based on the sequence comparison.

The average homology value, including all representatives of the 9- and 13-lipoxygenases of type 1, is 36%. It is noteworthy that PtLOXI also has an average of only 33% homology within the group of 13-type 2 lipoxygenases, instead of the 45% otherwise usual among the group members (see Table 6). All of these findings suggest that the cleavage of PtLOXI from the development of lipoxygenases in higher plants occurred relatively early during evolution. This is in line with the evolutionary Distance between diatoms and higher plants, which is also found on other levels.

Table 6: Homology of the lipoxygenase groups to one another. Intersections of the group with themselves correspond to the homology of the lipoxygenases within the group. The sequence comparison of known sequences formed the basis for the calculation with GENEDOC. The average of the combination of all sequences which were relevant for the respective comparison was formed (between 130 and 900 combinations). Numbers in percent.

The intracellular localization is regarded as an indication of the physiological function of lipoxygenases (summarized in [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002]. A simultaneous occurrence of lipoxygenases has been described in

Vacuoles observed in the cytoplasm and plastids. It is believed that the temporal and spatial separation of the activity of different lipoxygenase isoforms forms the basis for following one of the six different reaction pathways for lipoxygenase products [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002]. At least in two cases it was demonstrated that the occurrence of a

Lipid body lipoxygenase plays a role in the mobilization of storage lipids [Feussner et al., Trends Plant Sei, 6: 262-267, 2001] and a chloroplastic lipoxygenase is essential for the synthesis of jasmonic acid [Creelman et al., Ann Rev Plant Physiol Plant Mol Biol, 48: 355-381, 1997].

According to the phylogenetic family tree, PtLOXI apparently belongs to the type 2 lipoxygenases, which are characterized by a chloroplastic signal sequence. However, as discussed in the previous section, the evolutionary distance to all groups of lipoxygenases is approximately the same. In addition, some lipoxygenases are misclassified in the family tree, since LOX1: Cs: 3 and LOX1: Hv: 3 are 13-lipoxygenases and not 9-lipoxygenases, as one could see from the family tree. Since it is therefore not clear whether PtLOXI is actually actually of the LOX2 type or of the LOX1 type, it was examined more closely whether PtLOXI has a signal sequence. In a sequence comparison with a selection of plant lipoxygenases, PtLOXI, like all lipoxygenases of the LOX2 type, shows an N-terminal extension of around 50 AS compared to type 1 lipoxygenases. This speaks strongly for a plastid signal sequence, but since PtLOXI is presumably evolutionarily far away from lipoxygenases of higher levels, this can also have other causes.

Chloroplastidale signal sequences, also called transit peptides, can be very different in terms of their amino acid sequence, but contain certain diagnostic features. They have an exceptionally high level of hydroxylated amino acids (serine, threonine) and are also always positively charged. The pro proteins have an interface for signal peptidase which is conserved to a limited extent in land plants and Chlamydomonas [Lang et al., J. Biol. Chem., 273 (47): 30973-30978, 1998]. Because of this relatively poor identifiability, programs have been developed with which a prediction for these transit peptides can be made.

As already mentioned, due to low security modes, analysis with programs such as CHLOROP [Emanuelsson et al., Protein Sei, 8 (5): 978-84, 1999] or the more modern PCLR [Schein et al., Nucleic Acids Res. 29 ( 16): E82, 2001] for PtLOXI CHLOROP is based on a neural network that has been trained with sequences of proteins, of which the presence of an N-terminal chloroplastic signal sequence is known. Therefore, it has a quite remarkable accuracy in the prediction. A criticism of this program is the difficulty in interpreting the criteria on the basis of which a prediction is made. For this reason, a method was developed which predicts only on the basis of the frequency of different amino acid types in the N-terminal region of the protein [Schein et al., Nucleic Acids Res. 29 (16): E82, 2001]. This results in prediction results that far exceed CHLOROP in the accuracy of the prediction.

These two programs were used to analyze signal sequences for all lipoxygenases. The sequence in the putative cut area of positive candidates was then compared with the known conserved cut area of land plants and with PtLOXI. As expected, a chloroplastid signal sequence was only predicted for 13-lipoxygens of the LOX2 type. As mentioned, land plants have a conserved region in the region of the interface for stromal peptidases. These results were compared to PtLOXI, whereupon a putative interface for stromal signal peptidases was found. Table 7 shows the prediction of PCLR and CHLOROP for some 13-type 2 lipoxygenases, the conserved cut area of land plants and the possible interface of PtLOXI. These findings speak very clearly for plastid localization, which should be confirmed by immunocytochemical studies. Compared to higher plants, Heterocontophyta (Chrysophyta), which also includes diatoms, differ in the remarkable fact that the plastids have four enclosing membranes. This trait is believed to reflect the evolution of these organisms through secondary endosymbiosis by ingesting a photosynthetic eukaryote (originating from primary endosymbiosis) through a eukaryotic heterotrophic host cell [Lang et al., J. Biol. Chem., 273 (47 ): 30973-30978, 1998]. As a result, the inner two membranes appear to correspond to the envelope membranes of the plastids of higher plants, the next membrane is a remnant of the endosymbiotic plasma membrane, and the fourth membrane flows smoothly into the endoplasmic reticulum (ER). It is known that plastid proteins in heterocontophyta have an additional signal sequence for entry into the ER [Lang et al., J. Biol. Chem., 273 (47): 30973-30978, 1998; Bhaya et al., Mol Gen Genet, 229 (3): 400-4, 1991]. However, it is not known how the second membrane is overcome from the ER lumen. Interfaces for eurkaryotic signal peptide ases are usually in the range at positions 15 to 18 of pre-pro proteins by the method of HEIJNE [G. by Heijne, Nucleic Acids Res, 14 (11): 4683-90, 1986]. SIGNALSEQ, which is part of HUSAR, uses the method and predicts an interface for PtLOXI at position 14 (ASAJFR) (P _max = 0.4350). However, since an interface in this area is also predicted with a similar probability for LOX2: At: 1, this finding can only support the thesis to a limited extent that RLOX1 has an ER signal peptide with respect to the transit peptide for plastid localization.

Table 7: Interfaces of stromal peptidases in diatoms and higher plants, interfaces for the LOX2 type of plant lipoxygenases were calculated with CHLOROP and PCLR.

The existence of a signal sequence is inferred for CHLOROP from values greater than 0.5 and for PCLR from 0.42. simple reil, putative cut position calculated by CHLORP; Double arrow, determined by N-terminalβ sequencing. c) Based on sequence homology, fucoxanthin chlorophyll a / c determines binding protein of P. tricornutum [Lang et al., J. Biol. Chem., 273 (47): 30973-

30978, 1998] recognizes the references [Lang et al., J. Biol. Chem., 273 (47): 30973-30978, 1998; Emanuelsson, protein be,

8 (5): 978-84, 1999]

OOddooiinnttella sinensis (diatoms), according to the reference [Lang et al., J. Biol. Chem., 273 (47): 30973-30978,

1998]

Another indication of plastid localization of PtLOXI is the pH optimum of 8.2 (see below).

pH optimum

The pH optima of the PtLOXI was determined by means of normal phase HPLC through the conversion of linoleic acid. The method of determining the final value was used, in which the amount of product formed is determined after a certain time under different conditions. It is unsuitable for the reason that in no case can linear increase in product and decrease in substrate be guaranteed for all conditions, in particular with regard to long sales times and high enzyme activity. For this reason, the optimum curves obtained represent at comparing the activity under different conditions, never the true ratio of the activities. Nevertheless, a good indication of the optimal conditions can be obtained if the amount of enzyme used and the conversion time are chosen accordingly. In comparison to this error, different amounts of enzyme or substrate in the batch (due to pipetting errors) as well as different ionic strength of the buffer, temperature etc. play a minor role. Other methods for determining the optimum pH would be possible via the "online" monitoring of the oxygen consumption in the lipoxygenase reaction via an oxygen electrode or the measurement of the product formation by monitoring the increase in absorption at 234 nm on a spectrophotometer [Axelrod et al., Methods

Enzymol, 71: 441-451, 1981]. About the pH optimum of the enzymes, statements can be made about the localization of the enzymes and thus also conclusions can be drawn about the possible physiological functions. Different pH values in the individual compartments of the plant cell are dependent on the time. Vacuoles and peroxisomes have an acidic environment. In the stroma of the chloroplasts, the pH rises from 7.0 to around 8 during the day, since protons are pumped from the stroma into the thylakoids by the active photosystems in the thylakoid membrane. The pH optimum of RLOX of 8.2 is thus in agreement with the chloroplast plastic signal sequence that was identified for PtLOXI. It also serves as an indication that PtLOXI becomes active during the light reaction. There are also chloroplastic lipoxygenases with an acidic pH optimum (eg LOX2: Hv: 1 (LOX-100) [Kohlmann et al., Eur. J. Biochem., 260: 885-895, 1999]. The pH value drops in the stroma to this level due to stress or damage to the membranes, it is believed that these lipoxygenases become active and can induce further cell degradation (jasmonic acid formation, membrane degradation, etc.). It has been described that products of the LOX- HPL reaction pathways (aldehydes) were formed rapidly after mechanical damage to diatoms, since these aldehydes drastically reduced the success in hatching crayfish, the enemies of diatoms in phytoplankton, suggests a wound-induced defense PtLOXI activity could induce the defense mechanism in P. tricornutum is extremely unlikely since the α-linolenic acid required for synthesis with 0.2% of the fatty acid content [Schobert et al., Pla nt Physiol, 66: 215-219, 1980] is negligible. If PtLOXI plays a role in the wound-induced release of aldehydes, the question of activation arises. As just explained, jasmonic acid as an activator of stress-induced genes can play no role and activation due to the breakdown of the stromal pH gradient can also be ruled out due to the pH optimum of PtLOXI. Thus, RLOX1 seems unlikely to be involved in this defense mechanism. Almost none were found in undamaged cells Degradation products of oxidized fatty acids found [Pohnert et al., Nat. Per. Rep., 19: 108-122, 2002].

cleaning

For precise kinetic studies, it is necessary to use purified enzyme. So the K _{M can} also be determined with unpurified enzyme, but there is an uncertainty factor due to the presence of other proteins in the batch. Since the comparable analysis of the different substrates in FIG. 8 has indicated a preference for ERA and EPA, it would be of interest to determine the K _M and ^ _M for all substrates. This could make a much more precise statement about the substrate specificity of PtLOXI. Purification via the histidine metal complex system is believed to have failed because the recombinant protein does not have the N-terminal sequence of six histidine. The possible cause could be that the translation of the protein did not begin at the location provided in the expression vector, but only at the start methionine of the RLOX1 sequence.

substrate specificity

Plant-based lipoxygenases are classified into 9- and 13-lipoxygenases based on their position-specificity compared to linoleic acid, since linoleic acid is a ubiquitous substrate in plants. The amino acids in the active center of the enzyme are important for the formation of the different products [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000]. Two hypotheses have been developed which attempt to explain the position specificity of lipoxygenases (summarized in [Feussner et al., Enzymes in Lipid Modification, p. 309-336, Wiley-VCH, Weinheim, 2000]. One hypothesis assumes That the available space decides on the position of the oxygen introduction. Thus, the unsaturated fatty acid slides as far as possible into the substrate binding pocket with the methyl end first. The bisallylic methyl group, which is then in close proximity to the hydrogen acceptor, is subsequently attacked, after which a [+2] or [-2] rearrangement of the resulting pentadienyl radical can take place, but it is difficult to explain exactly this rearrangement with this model, which is why a second hypothesis was developed, whereby the orientation of the substrate in the substrate binding pocket is above it is decisive whether oxygen is introduced at the [+2] or [-2] position strat, as with the space model, slide the methyl end into the binding pocket, whereas with 9-lipoxygenases the unsaturated fatty acid slides into the pocket with the carboxyl end first. Part of the bottom of the binding pocket is a highly conserved arginine in all plant lipoxygenases, which is of central importance in the conversion of the lipid body 13-lipoxygenase from cucumber into a 9-lipoxygenase (R758 from CsIbLOX) [Hornung et al., Proc NatI Acid Sei USA, 96 (7): 4192-4197, 1999]. This arginine occurs regardless of substrate and position specificity and is preceded by an asparagine in over 90% of the cases. The only exception among plant lipoxygenases are two histidines instead of the asparagine-arginine tandem in the PtLOXI sequence comparison. It can be assumed that these also form the bottom of the bag for PtLOXI, as this area is otherwise highly conserved. The arginine at the bottom of the binding pocket was unmasked by the H608V mutation of CsIbLOX, whereby the salt bridge between the carboxyl group of the substrate and the arginine greatly reduced the energy of introducing a carboxyl group into a hydrophobic environment [Hornung et al., Proc NatI Acad Sei USA, 96 (7): 4192-4197, 1999]. Even if the pK value of the histidines in the protein cannot be estimated exactly (pK = 6.5), compared to arginine (around pK = 12) it is certainly less likely that they will carry a positive charge. For this reason, it is unlikely that there is a close relative of PtLOXI with 9-lipoxygenase activity that does justice to the orientation-related theory. Since even with this theory this position is masked for a 12-lipoxygenase and therefore does not play a role, this unusual occurrence of histidine instead of arginine does not seem to have any significance for the catalytic mechanism.

The amino acid, on the other hand, is conserved, which corresponds to the SLOANE determinant in sequence comparison [Sloane et al., Nature, 354: 149-152, 1991]. At this point there is a small amino acid like valine in 9-lipoxygenases and a big one like phenylalanine in 13-lipoxygenases. An exception are the 13-lipoxygenases LOX1: Cs: 1 and LOX1: Cs: 3 (H instead of F) and LOX1: Hv: 3 (V instead of F). This discrepancy may be due to the fact that in these cases there are no "classic" 13-epoxygenases [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002]. However, PtLOXI does the voluminous amino acid phenylalanine as expected for a 13-epoxygenase.

At the position in the sequence comparison that coincides with the BoRNGRÄBER determinant, it is more difficult to find conserved differences between 13- and 9-lipoxygenases. Usually a combination of hydrophobic and hydrophilic amino acids is found, which are separated from the small amino acid glycine or hydrophobic amino acid cysteine (e.g. S515, G516, V517 in LOX1: Nt: 1). With regard to the combination of hydrophobic / hydrophilic amino acids, there are conserved differences between the individual groups. PtLOXI differs from all others in that Lipoxygenases that separate two hydrophobic amino acids from the hydrophilic amino acid threonine. These two hydrophobic amino acids (isoleucine coincides with V542 from CsIbLOX, alanine with Y544 from CsIbLOX) do not exist at any other lipoxygenase at this point in the sequence comparison, although immediately before and after PtLOXI shows the highest homology with all other lipoxygenases. As these positions were identified as part of the substrate binding pocket and an influence on the position specificity was demonstrated [Feussner et al., Annu. Rev. Plant. Biol., 53: 275-297, 2002], it must be assumed that this difference could influence the way in which the substrate is bound or fixed. Since amino acids of similar character and size are found in two of these positions (before and after the threonine) in a different combination in other lipoxygenases, the nature of this influence is not immediately apparent.

For 13-lipoxygenases, the removal of the bisallylis is in both models

Methylene group from the methyl end of the substrate determines whether the introductory hydrogen abstraction takes place at this point. It was found (Table 4) that of the bisallylic methyl groups in question, only one was used for the initial hydrogen removal for each of the substrates used. This affected the methylene group at position (n-8) in the case of LA and α-LEA and each (n-11) for γ-LEA, ERA, EPA, DPA and DHA. It can thus be estimated that the distance of the hydrogen acceptor from the bottom of the binding pocket is around eleven methylene groups. It is particularly striking in the case of γ-LEA that of the two possible positions of the hydrogen abstraction (C ₈ and Cn) the one in which the substrate immerses maximally in the binding pocket (C ₈ corresponds (n-11)) was used. This is also the case with α-LEA, even if there is still space for three methylene groups to the hypothetical bottom of the binding pocket. The carbon atom (n-11) is always used for all long-chain fatty acids from ERA (20: 4). Based on this observation and through the comparative analysis in FIG. 8, it can be assumed that either ERA or EPA are the preferred substrates of PtLOXI. DPA is unlikely because both products ([+2] and [-2] rearrangement) are formed in a 1: 1 ratio and the amount of product formed decreases again compared to ERA and EPA. The DHA products, on the other hand, are produced in the same ratio as the EPA products, although the amount of products is less than for ERA and EPA. In view of the fact that EPA occurs about three times more than DHA in P: tricornutum [Schobert et al., Plant Physiol, 66: 215-219, 1980], this substrate seems to be preferentially implemented by PtLOX. No rational explanation could be found for the shift in the ratios of the oxidation at [+2] to [-2] when the number of double bonds or methylene groups on either side of or beyond the bisallylic methylene group used. Apparently double bonds on the carboxy side of the bisallylic methylene group used stabilize the [+2] radical, whereas double bonds on the methyl side stabilize the [-2] radical. Since sp ² hybridized carbon has a higher electronegativity than sp ³ hybridized carbon, this result is the opposite of what one would expect from this electron train sp ² hybridized carbon. The shift in the ratio when the number of methylene groups changes while the number of double bonds remains the same cannot be explained by inductive effects of the methylene groups (+). Here, [-2] radicals are apparently stabilized by the introduction of methylene groups on the carboxy side of the bisallylic methylene group used and vice versa.

As an explanation, there remains the possibility of using the binding pocket to fix the substrate (FIG. 26). A double bond changes the structure of the substrate. Assuming that in the case of ERA the substrate at the methyl and carboxyl ends is optimally locked for oxidation at C ₁₂ , the possibility of the bottom of the binding pocket interacting with EPA changes by introducing a double bond at this point. If EPA is less firmly fixed, the change in position during catalysis can adversely affect position specificity. Similar effects may occur with the other substrates. The range from C ₁₈ to C ₇ for γ-LEA (formation of 10-16-γ-HOTE) is identical to the range C ₂₀ to C ₉ of ÄRA (formation of 12 - / - HETE). The difference is the lack of locking at the head end (carboxyl) of γ-LEA due to the binding pocket. With the same effect, ERA (20: 4) and EPA (20: 5) could be optimally locked at the head end, and DPA (22: 5) could make locking impossible by removing the caboxyl group from the entrance of the binding pocket. The introduction of the double bond at this position in DHA (22: 6) could reverse this, since the substrate is restricted ("stiffened") in the degrees of freedom by the double bond. In this context, the stereochemical differences between the main products could also be explained and support this thesis: If the substrate is firmly locked at the methyl and carboxyl ends by interaction with amino acids in the binding pocket, this could explain the almost 100% enantiomeric purity in the case of the implementation of EPA and DHA for both products the interaction at the carboxyl end in DPA could then also result in the lowering of the S to R ratio for 14-HDPE In the case of γ-LEA, the exact same explanation should be given, since the substrate forms 10- / 6-γ- HOTE is so deeply immersed in the binding pocket that the carboxyl end no longer matches the possible determinants of substrate fixation at the head end r binding pocket can interact. In summary, it should be noted that PtLOXI prefers to implement ERA or EPA based on the comparative substrate implementation (Figure 8). If one adds the results of the position specificity and the enantiomer analysis, this speaks for EPA as the preferred substrate. Compared to ERA, the relationship between the main and by-products is clearly shifted in favor of the main product, which is almost enantiomerically pure. In this way, an exceptional substrate specificity was found compared to plant lipoxygenases. Other plant 13-lipoxygenases, which are localized chloroplastid, were able to convert arachidonic acid (eg LOX2: Hv: 1, [Vöros et al., Eur J Biochem, 251: 36-44, 1998] Formed 15-HPETE in S configuration and a number of racemic products, but not 12- and 8-HETE in S configuration.

Ketoconjudic fatty acids were not formed by PtLOX under the conditions of the analysis. CODE are mainly formed at low oxygen partial pressures, but by some plant lipoxygenases, e.g. Lipoxygenase-2 from soybean and lipoxygenase-1 from pea, are known to produce these even in normal oxygen partial pressure in unusually high amounts of up to 50% based on HODE [Axelrod et al., Methods Enzymol, 71: 441-451, 1981; Kuehn et al., Eicosanoids, 4: 9-14, 1991]. The implementation of complex lipids is also known from lipoxygenases from other sources. For example, the lipid body lipoxygenase from cucumber seedlings is able to convert triacylglycerides [Feussner et al., FEBS Lett; 431 (3): 433-436, 1998]. These lipoxygenases are probably involved in the mobilization of storage fats during germination [Feussner et al., Trends Plant Sei, 6: 262-267, 2001]. PtLOX does have oil vacuoles rwww.kieselalαen.coml. but since reproduction takes place through vegetative cell division, the inclusion of lipoxygenases in the mobilization of storage lipids can be ruled out, as in some higher plants. The conversion of phosphatidylcholine can also take place using lipoxygenases. Soybean lipoxygenase-1 first converted this substrate to monohydroperoxy-linoleoyl-phosphatidylcholine and then to dihydroperoxide [Brash et al., Biochemistry, 26: 5465-5471, 1987]. A new oxolipin was also identified in Arabidopsis [Stelmach et al., J Biol Chem, 276 (16): 12832-12838, 2001]. It is a sn-1-O- (12-oxophytodienyl) -sn2-O- (hexadecatrienoyl) monogalactosyl diglyceride. One could, therefore, based on these findings and previous hypotheses

[Feussner et al., Fett-üpid, 100: 146-152, 1998] assume that lipoxygenases accept monogalactosyl diglycerides, the main components of the chloroplast membrane, as substrates and a hydroperoxide group in the fatty acid, which is in the sn1 position, install. In plants there are indications that if such a route exists, jamonic acid is ultimately formed. Although at PtLOXI this is due to the If a small amount of esterified α-linolenic acid is unlikely, a further investigation should also consider complex lipids of the esterified fatty acids such as EPA and AA in the form of phosphatidylcholine and monogalactosyl diglycends.

List of abbreviations

APS ammonium peroxodisulfate ATP adenosine 5'-triphosphate BCIP 5-bromo-4-chloro-3-indoxyl phosphate BLAST Basic LocalAlignment Search Tool - search the next one

Homologues of a sequence by comparison with sequences in different databases bp base pair

BSA Bovine Serum Albumin - Bovine Serum Albumin BSTFA N, O-Bis (trimethylsilyl) trifluoroacetamide cDNA complementary deoxyribonucleic acid

C-terminus carboxy-terminus

DNA deoxyribonucleic acid dNTP deoxynucleotide 5'-triphosphate EDTA ethylenediaminetetraacetic acid

EDAC N-dimethylamino-propyl-N'-ethylcarbodiimide hydrochloride g (symbol) gravitational acceleration g (unit) grams

GC / MS gas chromatograph / mass spectrometer 17-H (P) DHE (17S, 4Z, 7Z, 10Z, 13Z, 15E, 19Z-17-Hydro (pero) xy-4,7,10,13,15,

19-docosahexaenoic acid

14-H (P) DPE (14S, 7Z, 10Z, 12E, 16Z, 19Z) -14-hydro (pero) xy-7,10,12,16,19-docosapentaenoic acid

10-H (P) DPE (10S, 7Z, 11E, 13Z.16Z.19Z) -10-Hydro (pero) xy-7.11, 13,15,19-docosapentaenoic acid

12-H (P) EPE (12S, 5Z, 8Z, 10E, 14Z, 17Z) -12-hydro (pero) xy-5,8, 10,14,17-eicosapentaenoic acid

8-H / P) EPE (8S, 5Z, 9E, 11 E, 14Z, 17Z) -8-hydro (pero) xy-5,9, 11, 14,17-eicosapentaenoic acid -H (P) ETE ( 5S, 6E, 8Z, 11Z, 14Z) -5-hydro (pero) xy-6,8,11,14-eicosatetraenoic acid -H (P) ETE (8S, 5Z, 9E, 11Z, 14Z) -8- Hydro (pero) xy-5,9,11, 14-eicosatetraenoic acid -H (P) ETE (9S, 5Z, 7E, 11Z, 14Z) -9-Hydro (pero) xy-5,7,11, 14 -eicosatetraenoic acid 11-H (P) ETE (11S, 5Z, 8Z, 12E, 14Z) -11-hydro (pero) xy-5,8,12,14-eicosatetraenoic acid

12-H (P) ETE (12S.5Z.8Z, 10E, 14Z) -12-Hydro (pero) xy-5,8, 10, 14-eicosatetraenoic acid 15-H (P) ETE (15S, 5Z, 8Z, 11Z, 13E) -15-hydro (pero) xy-5,8,11,13-eicosatetraenoic acid

HPLC High Performance Liquid Chromatography

9-H (P) ODE (9S, 10E, 12Z) -9-Hydro (pero) xy-10, 12-octadecadienoic acid 13-H (P) ODE (13S, 9Z, 11 E) -13-Hydro (pero) xy-9.11-octadecadienoic acid 9-α H (P) OTE (9S, 10E, 12Z, 15Z) -9-hydro (pero) xy-10,12,15-octadecatrienoic acid

12-α H (P) OTE (12S.9Z.13E, 15Z) -12-hydro (pero) xy-9, 13.15-octadecatrienoic acid 13-α H (P) OTE (13S, 9Z, 11E, 15Z) -13-Hydro (pero) xy-9,11, 15-octadecatrienic acid

16-α H (P) OTE (16S.9Z, 12Z, 14E) -16-hydro (pero) xy-9, 12, 14-octadecatrienoic acid

6-γ H (P) OTE (6S, 7E, 9Z, 12Z) -6-hydro (pero) xy-7,9,12-octadecatrienoic acid

9-γ H (P) OTE (9S, 6Z, 10E, 12Z) -9-hydro (pero) xy-6,10,12-octadecatrienoic acid

10-γ H (P) OTE (10S, 6Z, 8E, 12Z) -10-hydro (pero) xy-6,8, 12-octadecatrienoic acid

13-γ H (P) OTE (13S, 6Z, 9Z, 11E) -13-hydro (pero) xy-6,9,11 -octadecatrienoic acid

IEP isoelectric point

IPTG isopropyl-β-thiogalactopyranoside kDa kilodalton

MOPS 3- (N-morpholino) propanesulfonic acid mRNA messenger ribonucleic acid

NBT sodium nitrotetrazolium blue

N-terminus amino-terminus

OD optical density based on 1 cm cell width

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

P / C / l phenol-chloroform-isoamyl alcohol

PCR polymerase chain reaction pH negative decimal logarithm of the proton concentration

RACE Rapid Amplification of cDNA End - Methods for sequence extension

RNA ribonucleic acid

SDS sodium dodecyl sulfate

SSC sodium sodium chloride TAE Tris Acetate EDTA

TCA trichloroacetic acid

TE Tris-EDTA

TEAA triethylamonium acetate

TEMED N, N, N ', N'-tetramethylene diamine

TENS Tris-EDTA-NaCI-SDS

Tris tris (hydoxymethyl) aminomethane

TMS trimethylsilyl

ORF Open Reading Frame

U unit; Unit of enzyme activity [μmol / min]

UV ultraviolet var. Variety

V "volume", volume w "weight", weight

X-Gal 5-bromo-4-chloro-3-indolyl-β-galactoside

Claims

claims

1. Lipoxygenase from Phaeodactylum tricornutum with a pH optimum of 8.2 and a regiospecificity for the hydroperoxy formation of linoleic acid at the C atom 13 (13-LOX).

2. Isolated nucleic acid sequences which code for polypeptides with lipoxygenase activity, selected from the group: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, b) nucleic acid sequences which, as a result of the degenerate genetic code, differ from that in SEQ Deriving the coding sequence containing ID NO: 1, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which code for polypeptides with lipoxygenase activity and have at least 60% identity at the amino acid level with SEQ ID NO: 2.

3. The isolated nucleic acid sequence according to claim 2, wherein the sequence is derived from diatoms.

4. An isolated nucleic acid sequence according to claim 2 or 3, wherein the sequence originates from Phaeodactylum tricornutum.

5. amino acid sequence encoded by an isolated nucleic acid sequence according to any one of claims 2 to 4.

6. gene construct containing an isolated nucleic acid according to one of the

Claims 2 to 4, wherein the nucleic acid is operably linked to one or more regulatory signals.

7. Gene construct according to claim 6, characterized in that the nucleic acid construct contains additional biosynthesis genes of the fatty acid or lipid metabolism selected from the group acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] desaturase (s) , Acyl-ACP thioesterase (s), fatty acid acyl transferase (s), acyl-CoA: lysophospholipid-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, triacylglycerol lipases, allen oxide synthases, hydroperoxide lyases or fatty acid elongase (s).

8. vector containing a nucleic acid according to claims 2 to 4 or a gene construct according to claim 6.

9. Transgenic non-human organism containing at least one nucleic acid according to claims 2 to 4, a gene construct according to claim 6 or a vector according to claim 8.

10. A transgenic non-human organism according to claim 9, wherein the organism is a microorganism, a non-human animal or a plant.

11. A transgenic non-human organism according to claim 9 or 10, wherein the organism is a plant.

12. A process for the preparation of hydroperoxy fatty acids by oxidation of polyunsaturated fatty acids with at least two double bonds in the fatty acid molecule, the process comprising culturing a transgenic organism which comprises a nucleic acid according to claims 2 to 4, a gene construct according to claim 6 or a vector according to claim 8 encoding a lipoxygenase that has a pH optimum of 8.2 and a regiospecificity for the hydroperoxy formation of linoleic acid

C atom has 13.

13. The method according to claim 12, wherein arachidonic acid or eicosapentaenoic acid are oxidized in the method.

14. The method according to claim 12 or 13, wherein the hydroperoxy fatty acids are isolated from the organism in the form of an oil, lipid or a free fatty acid.

15. The method according to any one of claims 12 to 14, wherein the organism is a microorganism, a non-human animal or a plant.

16. The method according to any one of claims 12 to 15, wherein the organism is a transgenic lance.