EP1831358A2 - Procede de production d'acides gras a longues chaines multi-insatures dans des organismes transgeniques - Google Patents

Procede de production d'acides gras a longues chaines multi-insatures dans des organismes transgeniques

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Publication number
EP1831358A2
EP1831358A2 EP05821795A EP05821795A EP1831358A2 EP 1831358 A2 EP1831358 A2 EP 1831358A2 EP 05821795 A EP05821795 A EP 05821795A EP 05821795 A EP05821795 A EP 05821795A EP 1831358 A2 EP1831358 A2 EP 1831358A2
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
acyl
fatty acids
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05821795A
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German (de)
English (en)
Inventor
Petra Cirpus
Jörg BAUER
Ernst Heinz
Amine Abbadi
Jelena Kirsch
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BASF Plant Science GmbH
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BASF Plant Science GmbH
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Application filed by BASF Plant Science GmbH filed Critical BASF Plant Science GmbH
Priority to EP09179432.1A priority Critical patent/EP2180046B1/fr
Publication of EP1831358A2 publication Critical patent/EP1831358A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • the present invention relates to a process for producing polyunsaturated fatty acids in an organism by introducing into the organism nucleic acids encoding polypeptides having acyl-CoA: lysophospholipid acyltransferase activity.
  • these nucleic acid sequences may optionally be used together with others
  • Nucleic acid sequences encoding polypeptides of the fatty acid or lipid metabolism are expressed in the transgenic organism.
  • the invention furthermore relates to the nucleic acid sequences according to the invention, nucleic acid constructs containing the nucleic acid sequences according to the invention, vectors comprising the nucleic acid sequences according to the invention and / or the nucleic acid constructs and transgenic organisms containing the aforementioned nucleic acid sequences, nucleic acid constructs and / or vectors.
  • Another part of the invention relates to oils, lipids and / or fatty acids prepared by the process according to the invention and their use.
  • Fatty acids and triglycerides find a variety of uses in the
  • polyunsaturated fatty acids are added to the baby formula to increase the nutritional value.
  • Polyunsaturated ⁇ -3 and ⁇ -6 fatty acids represent an important component of animal and human food. Due to the composition of human food that is customary today, an addition of polyunsaturated ⁇ -3 fatty acids, which occur mainly in fish oils , to Food especially important.
  • DHA docosahexaenoic acid
  • the polyunsaturated ⁇ -3 fatty acids are also considered to have a positive effect on the blood cholesterol level and thus on the possibility of preventing heart disease.
  • ⁇ -3 fatty acids By adding these ⁇ -3 fatty acids to the diet, the risk of heart disease, stroke or hypertension can be significantly reduced.
  • inflammatory, especially chronic inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can be positively influenced by ⁇ -3-fatty acids. They are therefore added to foods, especially dietary foods, or are used in medicines.
  • ⁇ -6 fatty acids such as arachidonic acid tend to have a negative effect on these diseases in these rheumatic diseases due to our usual food composition.
  • ⁇ -3 and ⁇ -6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as the prostaglandins derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid, and the thromboxanes and leukotrienes derived from arachidonic acid and eicosapentaenoic acid.
  • Eicosanoids which are formed from ⁇ -6 fatty acids (so-called PG 2 series), usually promote inflammatory reactions, while eicosanoids from ⁇ -3 fatty acids (so-called PG 3 series) have little or no pro-inflammatory effect.
  • polyunsaturated fatty acids are referred to as PUFAs, PUFAs, LCPUFAs or LCPUFAs
  • PUFA polyhydric fatty acid
  • PUFA long-chain unsaturated fatty acid
  • the free fatty acids are advantageously prepared by saponification.
  • Common natural sources of these fatty acids are fish such as herring, salmon, sardine, perch, eel, carp, trout, halibut, mackerel, zander or tuna, and algae.
  • ⁇ 6-desaturases are described in WO 93/06712, US 5,614,393, WO 96/21022, WO00 / 21557 and WO 99/27111, during their use for the production of fatty acids in transgenic organisms in WO98 / 46763, WO98 / 46764 and WO98 / 46765.
  • the expression of various desaturases as in WO99 / 64616 or WO98 / 46776 and formation of polyunsaturated fatty acids is also described and claimed.
  • microorganisms for the production of PUFAs are microorganisms such as Thraustochytrien- or Schizochytrien- strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates such as Stylonychia or Colpidium and fungi such as Mortierella, Entomophthora or Mucor.
  • Thraustochytrien- or Schizochytrien- strains such as Phaeodactylum tricornutum or Crypthecodinium species
  • ciliates such as Stylonychia or Colpidium
  • fungi such as Mortierella, Entomophthora or Mucor.
  • mutation and selection of strains with improved production of a particular molecule, such as polyunsaturated fatty acids is a time-consuming and difficult procedure.
  • microalgae such as Phaeodactylum and mosses such as Physcomitrella are unsaturated fatty acids such as linoleic acid or linolenic acid in the form of their acyl CoAs in several
  • LCPUFAs are present in microorganisms and lower plants either exclusively in the form of membrane lipids, as in Physcomitrella and Phaeodactylum, or are present in both membrane lipids and in triacylglycerides, as in Schizochytrium and Moriierella.
  • the incorporation of LCPUFAs into lipids and oils is catalyzed by various acyltransferases and transacylases already known for the incorporation of saturated and unsaturated fatty acids (Slabas (2001) J. Plant Physiology 158: 505-513; Frentzen (1998) Fat / Lipid 100: 161-166; Cases et al. (1998) Proc. Nat.
  • acyltransferases are enzymes of the so-called Kennedy pathway located on the cytoplasmic side of the endoplasmic reticulum membrane system, hereafter referred to as 'ER'.
  • 'ER' cytoplasmic side of the endoplasmic reticulum membrane system
  • membranes of the ER can be isolated as so-called 'microsomal fractions' from various organisms (Knutzon et al., (1995) Plant Physiology 109: 999-1006, Mishra & Kamisaka (2001) Biochemistry 355: 315-322, US 5968791).
  • ER-linked acyltransferases in the microsomal fraction use acyl-CoA as the activated form of the fatty acids.
  • Glycerol-3-phosphate acyltransferase catalyzes the incorporation of acyl groups at the sn-1 position of glycerol-3-phosphate.
  • l-Acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), also called lysophosphatidic acid acyltransferase, hereafter LPAAT, catalyzes the incorporation of acyl groups at the sn-2 position of lysophosphatidic acid, hereinafter abbreviated as LPA.
  • DAGAT diacylglycerol acyltransferase
  • PDAT phospholipid diacylglycerol acyltransferase
  • LPCAT lysophosphatidylcholine acyltransferase
  • LPCAT The enzymatic activity of a LPCAT was first described in rats (Land (1960) J. Biol. Chem. 235: 2233-2237). In plants, there exists a plastidic isoform of LPCAT (Akermoun et al., (2000) Biochemical Society Transactions 28: 713-715) and an ER-linked isoform (Tumaney and Rajasekharan (1999) Biochimica et Biophysica Acta 1439: 47-56; Stobart, Biochemical Society Transactions (2000) 28: 715-7718). LPCATs are involved in the biosynthesis and transacylation of polyunsaturated fatty acids in both animals and plants (Stymne and Stobart (1984) Biochem J.
  • LPCAT lysophospholipid acyltransferase
  • linoleic acid Cl 8: 2 and linolenic acid (Cl 8: 3).
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • genes encoding enzymes of the biosynthesis of LCPUFAs in oilseeds are advantageously introduced and expressed via genetic engineering methods. These are, for example, genes encoding a ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and ⁇ 4-desaturase.
  • ⁇ 6-desaturase genes from the moss Physcomitrella patens and ⁇ 6 elongase genes from P. patens and the nematode C. elegans have already been isolated.
  • SEQ ID NO: 5 which encode a polypeptide having an acyl-CoA3sophospholipid acyltransferase activity
  • nucleic acid sequences which can be deduced as a result of the degenerate genetic code from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; or
  • the polyunsaturated fatty acids prepared in the process according to the invention contain at least two, advantageously three double bonds. Particularly advantageously, the fatty acids contain four or five double bonds.
  • Fatty acids produced in the process advantageously have 16, 18, 20 or 22 C atoms in the fatty acid chain. These fatty acids can be produced as the only product in the process or in a fatty acid mixture.
  • nucleic acid sequences used in the method of the invention are isolated nucleic acid sequences encoding polypeptides having acyl-CoA: lysophospholipid acyltransferase activity.
  • the polyunsaturated fatty acids produced in the process are advantageously bound in membrane lipids and / or triacylglycerides, but may also be present as free fatty acids or bound in the form of other fatty acid esters in the organisms. As stated, they may be present as "pure products" or else advantageously in the form of mixtures of different fatty acids or mixtures of different glycerides.
  • the different fatty acids bound in the triacylglycerides can be derived from short-chain fatty acids having 4 to 6 C atoms, medium-chain fatty acids having 8 to 12 C atoms or long-chain fatty acids having 14 to 24 C atoms; preference is given to long-chain fatty acids the long-chain C 18 , C 20 and / or C22 fatty acids (LCPUFAs).
  • fatty acid esters with polyunsaturated C 16 , C 18 , C 20 and / or C 22 fatty acid molecules having at least two double bonds in the fatty acid ester.
  • the fatty acid ester with polyunsaturated C 16 -, C 18 -, C 2 0 - and / or C22-fatty acid molecules can be isolated from the organisms which have been used for the production of the fatty acid ester, in the form of an oil or lipid, for example in the form of compounds such as sphingolipids , Phosphoglycerides, lipids, glycolipids such as glycosphingolipid, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters containing the polyunsaturated fatty acids having at least two, preferably three , Containing double bonds, to be
  • Fatty acids in the organisms in an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 to 5% by weight of free fatty acids, 2 to 8 Wt .-% phospholipids, wherein the sum of the various compounds to 100 wt .-% complements.
  • the LCPUFAs produced are present in a content of at least 3% by weight, advantageously at least 5% by weight, preferably at least 8% by weight, more preferably at least 10% by weight, most preferably at least 15% by weight, based on the total fatty acids in the transgenic organisms, advantageously a transgenic plant.
  • a content of at least 3% by weight advantageously at least 5% by weight, preferably at least 8% by weight, more preferably at least 10% by weight, most preferably at least 15% by weight, based on the total fatty acids in the transgenic organisms, advantageously a transgenic plant.
  • Hexadecadienoic acid (Cl 6: 2), linoleic acid (Cl 8: 2) or linolenic acid (Cl 8: 3) are passed through several reaction steps, the end products of the process fall as For example, arachidonic acid (ARA) or eicosapentaenoic acid (EPA) not as absolute pure products, but there are always low levels of precursors in the final product. If both linoleic acid and linolenic acid are present in the starting organism or in the starting plant, for example, the end products such as ARA and EPA are present as mixtures.
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • the precursors should advantageously not more than 20 wt .-%, preferably not more than 15 wt .-%, more preferably not more than 10 wt .-%, most preferably not more than 5, 4, 3, 2, 1 wt .-%, based on the amount of the respective end product.
  • ARA or only EPA in the process according to the invention are advantageously bound as end products or prepared as free fatty acids. If both compounds (ARA + EPA) are prepared simultaneously, they are advantageously prepared in a ratio of at least 1: 2 (EPA: ARA), preferably of at least 1: 3, preferably of at least 1: 4, more preferably of at least 1: 5 ,
  • Fatty acid compositions can be prepared by the methods described above.
  • the fatty acids or fatty acid compositions from the organism such as the microorganisms or the plants or the culture medium in which or on which the organisms were grown, or from the organism and the culture medium in a known manner, for example via extraction,
  • organisms such as fungi such as Mortierella or Traustochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or.
  • Ceratodon non-human animals such as Caenorhabditis, algae such as Crypthecodinium or Phaeodactylum or plants such as dicotyledonous or monocotyledonous plants in question.
  • organisms are used which belong to the oil-producing organisms, that is, which are used for the production of oils, such as mushrooms such as Mortierella or
  • Traustochytrium algae such as Crypthecodinium or Phaeodactylum or plants, especially oil crops containing high levels of lipid compounds such as peanut, canola, canola, sunflower, safflower (safflower), poppy, mustard, hemp, castor, olive, sesame, calendula, punica, Evening Primrose, Mullein, Thistle, Wild Rose, Hazelnut, Almond, Macadamia, avocado, Bay Leaf, Pumpkin, Linseed, Soya, Pistachios, Borage, Trees (Oil Palm, Coconut or Walnut) or Crops such as Corn, Wheat, Rye, Oats, Triticale, Rice , Barley, cotton, cassava, pepper, tagetes, solanaceous plants such as potato, tobacco, aubergine and tomato, Vicia species, pea, alfalfa or bush plants (coffee, cocoa, tea), Salix species and perennial grasses and forage crops.
  • lipid compounds such as peanut
  • Preferred plants according to the invention are oil crop plants, such as peanut, oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, flax, soy, borage, trees (oil palm, coconut) , Particularly preferred are Cl 8: 2 and / or C18: 3 fatty acid-rich plants such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower. Most preferred are plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp.
  • genes of the fatty acid or lipid metabolism can advantageously be used in combination with the inventive acyl-CoA: lysophospholipid acyltransferase in the process for producing polyunsaturated fatty acids; genes of the fatty acid or lipid metabolism are advantageously selected from the group consisting of acyl-CoA Dehydrogenase (s), acyl-carrier protein-desaturase (s), acyl-ACP-thioesterase (s), fatty acid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase ( n), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s), fatty acid desaturase (s), fatty acid
  • genes selected from the group of ⁇ -4-desaturases, ⁇ -5-desaturases, ⁇ -6-desaturases, ⁇ -8-desatuases, ⁇ -9-desaturases, ⁇ -12-desaturases, ⁇ -5-elongases , ⁇ 6-elongases or ⁇ 9-elongases in combination with the inventive acyl-CoAiLysophospholipid acyltransferase used in the inventive method.
  • nucleic acids used in the method according to the invention which code for polypeptides having acyl-CoA: lysophospholipid acyltransferase activity, advantageously in combination with nucleic acid sequences which are suitable for polypeptides of fatty acid or lipid metabolism with ⁇ -4-, ⁇ -5-, ⁇ Encode ⁇ 6- ⁇ , ⁇ -8-desaturase or ⁇ -5, ⁇ -6 or ⁇ -9-Elongasecrettician, a variety of polyunsaturated fatty acids can be prepared in the process according to the invention.
  • the organisms used for the process according to the invention such as the advantageous plants, it is possible to use mixtures of the various polyunsaturated ones
  • fatty acids or individual polyunsaturated fatty acids such as EPA or ARA in free or bound form.
  • Fatty acids are produced as fatty acids derived from C18: 2 fatty acids, such as GLA, DGLA or ARA or those derived from C18: 3 fatty acids, such as SDA, ETA or EPA.
  • GLA, DGLA and ARA are produced as fatty acids derived from C18: 2 fatty acids, such as GLA, DGLA or ARA or those derived from C18: 3 fatty acids, such as SDA, ETA or EPA.
  • SDA, ETA and EPA can be produced as products of the process which, like may be present as free fatty acids or bound.
  • lysophospholipid acyltransferase advantageous in combination with the ⁇ -5 and ⁇ -6-desaturase and the ⁇ -6 elongase, or the ⁇ -5 and ⁇ -8 Desaturase and the ⁇ -9 elongase or in combination with only the first two genes of the synthesis chain, ⁇ -6-desaturase and ⁇ -6 elongase or ⁇ -8-desaturase and ⁇ -9 elongase, can be in the aforementioned Organisms, advantageously in the aforementioned plants, selectively produce only individual products.
  • ⁇ -6-desaturase and ⁇ -6 elongase Due to the activity of ⁇ -6-desaturase and ⁇ -6 elongase, depending on the starting plant and unsaturated fatty acid, for example, GLA and DGLA or SDA and ETA are formed. Preference is given to DGLA or ETA or mixtures thereof. If, in addition, ⁇ 5-desaturase is introduced into the organisms, advantageously into the plant, ARA or EPA are also produced. This also applies to organisms in which the ⁇ -8-desaturase and ⁇ -9 elongase were previously introduced.
  • the nucleic acids used in the method according to the invention are advantageously derived from plants such as algae such as Isochrysis or Crypthecodinium, algae / diatoms such as Phaeodactylum, mosses such as Physcomitrella or Ceratodon or higher plants such as Primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, microorganisms such as fungi Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, yeasts or animals such as nematodes such as Caenorhabditis, insects or humans.
  • the nucleic acids are derived from fungi, animals or from plants such as algae or mosses, preferably from nematodes such as Caenorhabditis.
  • Nucleic acid sequences or their derivatives or homologs which code for polypeptides which still possess the enzymatic activity of the proteins encoded by the wild-type nucleic acid sequences. These sequences are individually or in combination with the coding for the acyl-CoA: lysophospholipid acyltransferase nucleic acid sequence in expression constructs and used for introduction and expression in organisms. These expression constructs enable optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
  • the method further comprises the step of recovering a cell or a whole organism containing the nucleic acid sequences used in the method, wherein the cell and / or the organism with the nucleic acid sequence according to the invention, which for the acyl CoA: lysophospholipid acyltransferase, a gene construct or a vector as described below, alone or in combination with other nucleic acid sequences coding for proteins of the fatty acid or lipid metabolism is transformed.
  • this method further comprises the step of recovering the fine chemical from the culture.
  • the culture may be, for example, a fermentation culture, for example in the case of culturing microorganisms such as Mortierella, Saccharomyces or Traustochytrium, or a greenhouse or field crop of a plant.
  • the cell or organism thus produced is advantageously a cell of an oil-producing organism such as an oil crop such as peanut, canola, canola, flax, hemp, peanut, soybean, safflower, hemp, sunflower or borage.
  • cultivation is to be understood as cultivation on or in a nutrient medium or, in the case of the whole plant, cultivation on or in a substrate, for example in hydroponics, potting soil or on arable land.
  • a genetically linked to the nucleic acid sequence according to the invention genetic control sequence for example a promoter or c) (a) and (b)
  • modification may exemplarily be a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • Natural genetic environment means the natural genomic or chromosomal locus in the organ of origin or presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably at least partially conserved.
  • 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, more preferably at least 1000 bp, most preferably at least 5000 bp.
  • non-natural, synthetic ("artificial") methods such as For example, a mutagenization is changed.
  • a mutagenization is changed.
  • Corresponding methods are described, for example, in US Pat. No. 5,565,350 or WO 00/15815.
  • transgenic organism or transgenic plant is to be understood as meaning that the nucleic acids used in the method are not in their natural position in the genome of an organism, wherein the nucleic acids can be expressed homologously or heterologously.
  • transgene also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been altered. Is preferred Under transgenic to understand the expression of the nucleic acids according to the invention at unnatural site in the genome, that is, a homologous or preferably heterologous expression of the nucleic acids is present.
  • Preferred transgenic organisms are fungi such as Mortierella or plants such as oil crops.
  • all organisms which are able to synthesize fatty acids, especially unsaturated fatty acids, or which are suitable for the expression of recombinant genes are suitable in principle as organisms or host organisms for the nucleic acids, expression cassettes or vectors used in the method according to the invention.
  • plants such as Arabidopsis, Asteraceae such as calendula or crops such as soy, peanut, castor, sunflower, corn,
  • microorganisms such as fungi of the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae or protozoans such as Dinoflagellates called Crypthecodinium.
  • Organisms that can naturally synthesize oils in larger quantities such as fungi, e.g. Mortierella alpina, Pythium insidiosum or plants such as soybean, oilseed rape, coconut, oil palm, Klarbersafflor, flax, hemp, castor, calendula, peanut, cocoa or sunflower or yeasts such as Saccharomyces cerevisiae, particularly preferred are soy, flax, rape, Desirbersafflor, sunflower, Calendula, Morierella or Saccharomyces cerevisiae.
  • transgenic animals advantageously non-human animals such as C. elegans are suitable as host organisms in addition to the aforementioned transgenic organisms.
  • Transgenic plants which contain the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly, without the synthesized oils, lipids or fatty acids having to be isolated.
  • Plants in the method according to the invention include whole plants and all plant tissues, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, calli, kotelydons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures derived from the transgenic plant and / or used to produce the transgenic plant.
  • the seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue.
  • the compounds prepared in the process according to the invention can also be isolated from the organisms, advantageously plants, in the form of their oils, fats, lipids and / or free fatty acids. Because of this
  • Processed polyunsaturated fatty acids can be obtained by harvesting the organisms either from the culture in which they grow or from the field. This can be done by pressing or extraction of the plant parts, preferably the plant seeds.
  • the oils, fats, lipids and / or free fatty acids can be obtained by so-called cold striking or cold pressing without supplying heat by pressing.
  • the plant parts, especially the seeds, to be easier to digest they are first crushed, steamed or roasted. The pretreated seeds can then be pressed or extracted with solvents such as warm hexane. Subsequently, the solvent is removed again.
  • microorganisms these are, for example, extracted directly after the harvest, without further operations, or after digestion, extracted by various methods known to the person skilled in the art. In this way, more than 96% of the compounds produced in the process be isolated. Subsequently, the products thus obtained are further processed, that is refined. First of all, for example, the mucilages and turbid matter are removed. The so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid, such as phosphoric acid. Subsequently, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The product obtained is thoroughly washed 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 deodorized, for example, with steam.
  • the PUFAs or LCPUFAs produced by this process are C 18 , C 20 or C 22 fatty acid molecules having at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds.
  • These C 18 -, C 2 0 - or C22-fatty acid molecules can be prepared from the organism in the form of an oil, lipid or a free fatty acid isolate. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
  • One embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof prepared by the method described above, more preferably oils, lipids or a fatty acid composition comprising PUFAs derived from transgenic plants.
  • Another embodiment of the invention is the use of the oil, lipid, fatty acids and / or fatty acid composition in feed, food, cosmetics or pharmaceuticals.
  • oil is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s).
  • P refers in that the oil, lipid or fat contains a high proportion of polyunsaturated free or favorably esterified fatty acid (s), in particular linoleic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid.
  • the proportion of unsaturated esterified fatty acids is about 30%, more preferred is a proportion of 50%, most preferred is a proportion of 60%, 70%, 80% or more.
  • the proportion of a fatty acid can be determined by gas chromatography, for example, after conversion of the fatty acids into the methyl esters by transesterification.
  • the oil, lipid or fat may contain various other saturated or unsaturated fatty acids, eg calendulic acid, palmitic, palmitoleic, stearic, oleic acid, etc. In particular, depending on the starting organism, the proportion of the various fatty acids in the oil or fat may vary.
  • the polyunsaturated fatty acids having advantageously at least two double bonds produced in the process are, for example, sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
  • the polyunsaturated fatty acids having advantageously at least two double bonds prepared in the process according to the invention can be added, for example, via a
  • Alkali treatment for example, with aqueous KOH or NaOH, or acid hydrolysis advantageously in the presence of an alcohol such as methanol or ethanol or release via an enzymatic cleavage and isolate, for example, phase separation and subsequent acidification with eg H 2 SO 4 .
  • the release of the fatty acids can also be carried out directly without the workup described above.
  • the nucleic acids used in the method can either lie on a separate plasmid or be integrated into the genome of the host cell.
  • integration When integrated into the genome, integration may be at random or by such recombination as to replace the native gene with the incorporated copy, thereby modulating the production of the desired compound by the cell, or by using a gene in trans such that Gene having a functional expression unit, which contains at least one expression of a gene ensuring sequence and at least one polyadenylation of a functionally transcribed gene ensuring sequence is operably linked.
  • the nucleic acids are advantageously brought into the organisms via multi-expression cassettes or constructs for multiparallel expression, advantageously for multiparallel, seed-specific expression of genes in the plants.
  • Moose and algae are the only known plant systems that produce significant amounts of polyunsaturated fatty acids such as arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • Moose contain PUFAs in membrane lipids, while algae, algae-related organisms and some fungi also accumulate appreciable levels of PUFAs in the triacylglycerol fraction.
  • nucleic acid molecules isolated from strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process of the invention and thus for modification of the lipid and PUFA production system in a host, in particular plants such as oilseed crops, for example oilseed rape. Canola, flax, hemp, soy, sunflower, borage. They are therefore advantageous for use in the process according to the invention.
  • Suitable substrates of the acyl-CoA: lysophospholipid acyltransferases according to the invention are advantageously C 16 , C 18 , C 20 or C 22 fatty acids.
  • the polyunsaturated C 16 - or C 18 -fatty acids must first be desaturated by the enzymatic activity of a desaturase and then be extended by at least two carbon atoms via an elongase. After one round of elongation, this enzyme activity leads to C 18 or C 20 fatty acids, and after two or three elongation cycles to C 22 or C 2 4 fatty acids.
  • the activity of the desaturases and elongases used in the process of the invention preferably leads to C 18 -, C 2 0 - and / or C22-fatty acids, advantageously with at least two double bonds in the fatty acid molecule, preferably with three, four or five double bonds, especially preferably to C2 0 - and / or C22 fatty acids having at least two double bonds in the fatty acid molecule, preferably having three, four or five double bonds in the molecule.
  • Particularly preferred as products of the process according to the invention are dihomo- ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and / or docosahexaenoic acid.
  • the C 18 fatty acids having at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
  • the preferred biosynthesis site of fatty acids, oils, lipids or fats in the advantageously used plants is, for example, the seed in general or cell layers of the seed, so that a seed-specific expression of the nucleic acids used in the method makes sense.
  • biosynthesis of fatty acids, oils or lipids need not be limited to the seed tissue, but also in all other parts of the plant - For example, in epidermis cells or in the tubers - can be made tissue-specific.
  • the amount of polyunsaturated fatty acids produced in the process can be at least 10%, preferably at least 15%, particularly preferably at least 20%, very particularly preferably at least 50%, and most preferably at least 100%, of the wild type of organisms which do not recombinantly contain the nucleic acids.
  • the polyunsaturated fatty acids produced in the organisms used in the process can in principle be increased in two ways.
  • the pool of free polyunsaturated fatty acids and / or the proportion of esterified polyunsaturated fatty acids produced by the process can be increased.
  • the process according to the invention increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms.
  • microorganisms such as yeasts such as Saccharomyces or Schizosaccharomyces, fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Traustochytrium, algae such as Isochrysis, Phaeodactylum or Crypthecodinium are used in the process according to the invention, these organisms are advantageously cultivated by fermentation in a manner known to those skilled in the art or bred.
  • Microorganisms are usually found in a liquid medium that is 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
  • the pH of the nutrient fluid can be kept at a fixed value, that is, regulated during cultivation, or not.
  • the cultivation can be batchwise, semi-batchwise or continuous. Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.
  • the polyunsaturated fatty acids prepared can be isolated from the organisms by methods known to those skilled in the art, for example by extraction, distillation, crystallization, optionally salt precipitation and / or chromatography as described above. The organisms can be opened up for this purpose yet advantageous.
  • the method according to the invention when the host organisms are microorganisms, is advantageously at a temperature between 0 ° C to 95 ° C, preferably between 10 ° C to 85 ° C, more preferably between 15 ° C to 75 ° C, and most preferably carried out between 15 ° C to 45 ° C
  • the pH is advantageously maintained between pH 4 and 12, preferably between pH 6 and 9, more preferably between pH 7 and 8.
  • the process according to the invention can be operated batchwise, semi-batchwise or continuously.
  • a summary of known cultivation methods can be found in the textbook by Chmiel (Bioreatechnik 1.
  • the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various Microorganisms are included in the Manual of Method for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
  • these media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • sugars such as mono-, di- or polysaccharides.
  • very good sources of carbon are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar may also be added to the media via complex compounds, such as molasses or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
  • Other possible sources of carbon are oils and fats such. As soybean oil, sunflower oil, peanut oil and / or coconut oil, fatty acids such.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used singly or as a mixture.
  • Inorganic salt compounds which may be included in the media include the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • sulfur-containing fine chemicals in particular methionine
  • inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium salts can be used as the phosphorus source.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
  • the invention used for the cultivation of microorganisms
  • Fermentation media usually also contain other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
  • Growth factors and salts are often derived from complex media components such as yeast extract, molasses, corn steep liquor, and the like.
  • suitable precursors can be added to the culture medium.
  • the exact composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis. Information on the media optimization are available from the textbook "Applied Microbiol Physiology, A Practical Approach” (Ed PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).
  • Growth media can also be from commercial providers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
  • All media components are sterilized either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration.
  • the components can either be sterilized together or, if necessary, sterilized separately.
  • Media components may be present at the beginning of the culture or optionally added continuously or in batches.
  • the temperature of the culture is usually between 15 ° C and 45 ° C, preferably 25 ° C to 40 ° C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably 7.0.
  • the pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
  • B. fatty acid polyglycol esters are used.
  • the medium can be selected selectively acting substances such. As antibiotics, are added.
  • oxygen or oxygen-containing gas mixtures such. B. ambient air, registered in the culture. The culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
  • the fermentation broths thus obtained in particular containing polyunsaturated fatty acids, usually have a dry matter content of 7.5 to 25% by weight.
  • the fermentation broth can then be further processed.
  • the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or completely left in it.
  • the biomass is worked up after separation.
  • the fermentation broth can also without cell separation with known methods such.
  • production or productivity are known in the art and include the concentration of the fermentation product (compounds of formula I) formed in a given period of time and fermentation volume (e.g., kg of product per hour per liter).
  • concentration of the fermentation product compounds of formula I
  • fermentation volume e.g., kg of product per hour per liter
  • Production includes the time it takes to reach a given production quantity (eg how long the cell needs to set up a specific throughput rate of a fine chemical).
  • yield or product / carbon yield is known in the art and includes the efficiency of converting the carbon source into the product (ie, the fine chemical). This is usually expressed, for example, as kg of product per kg of carbon source.
  • biosynthesis or biosynthetic pathway are known in the art and involve the synthesis of a compound, preferably an organic compound, by a cell from intermediates, for example in a multi-step and highly regulated process.
  • Degradation or degradation pathways are known in the art and involve the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process.
  • metabolism is known in the art and includes the entirety of the biochemical reactions that take place in an organism.
  • the metabolism of a particular compound eg, the metabolism of a fatty acid
  • the fatty acids obtained in the process are also suitable as starting material for the chemical synthesis of further products of value.
  • they may be used in combination with each other or alone for the production of pharmaceuticals, foods, pet foods or cosmetics.
  • the invention furthermore relates to isolated nucleic acid sequences encoding polypeptides with acyl CoAiLysophospholipid acyltransferase activity which specifically C 16 -, C 18 -, C 20 - or C 22 fatty acids reacted with at least one double bond in the fatty acid molecule, wherein said nucleic acid sequences are selected from the group consisting of:
  • nucleic acid sequences which, as a result of the degenerate genetic code, can be deduced from the coding sequence contained in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5,
  • nucleic acid sequences originate from a eukaryotic organism, more preferably from Ostreococcus tauri or Mantoniella squamata.
  • the nucleic acids used in the method are advantageously subjected to amplification and ligation in a known manner.
  • one is based on the protocol of Pfu DNA polymerase or a
  • the primers are selected on the basis of the sequence to be amplified. Conveniently, the primers should be chosen so that the amplificate comprises the entire codogenic sequence from the start to the stop codon.
  • the amplificate is conveniently analyzed. For example, the analysis can be carried out after gel electrophoretic separation in terms of quality and quantity.
  • the amplificate can be purified according to a standard protocol (e.g., Qiagen). An aliquot of the purified amplificate is then available for subsequent cloning. Suitable cloning vectors are well known to those skilled in the art. These include in particular vectors that are in microbial infections, etc.
  • T-DNA-mediated transformation various suitable for the T-DNA-mediated transformation, binary and co-integrated vector systems.
  • Such vector systems are usually characterized in that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA-limiting sequences (T-DNA border).
  • these vector systems also include other cis-regulatory regions, such as promoters and terminators and / or selection markers, with which appropriately transformed organisms can be identified.
  • binary systems are based on at least two vectors, one of them vir genes, but no T-DNA and a second T-DNA, but no carries vir gene.
  • these binary vectors include vectors of the series pBIB-HYG, pPZP, pBecks, pGreen.
  • Preferably used according to the invention are pBin19, pBH01, pBinAR, pGPTV and pCAMBIA.
  • the vectors can first be linearized with restriction endonuclease (s) and then enzymatically modified in a suitable manner. The vector is then purified and an aliquot used for cloning. Upon cloning, the enzymatically cut and, if necessary, purified amplicon is cloned with similarly prepared vector fragments using ligase.
  • a particular nucleic acid construct or vector or plasmid construct can have one or more codogenic gene segments.
  • the codogenic gene segments in these constructs are functionally linked to regulatory sequences.
  • the regulatory sequences include, in particular, plant sequences such as the promoters and terminators described above.
  • the constructs can be advantageously in microorganisms, in particular Escherichia coli and Agrobacterium tumefaciens, stably propagate under selective conditions and allow transfer of heterologous DNA into plants or microorganisms.
  • nucleic acids used in the method can be introduced into organisms such as microorganisms or advantageously plants and thus used in plant transformation, such as those published in and cited herein: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, 15-38; Genes Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung and R.
  • nucleic acids used in the method, the inventive nucleic acids and nucleic acid constructs and / or vectors can thus be used for the genetic modification of a broad spectrum of organisms, advantageously on plants, so that they become better and / or more efficient producers of PUFAs.
  • acyl-CoA3 phospholipid acyltransferase protein of the invention can directly affect the yield, production and / or efficiency of production of a fine chemical in an oil crop or microorganism.
  • Acyltransferases, desaturases and / or elongases may be increased, so that larger amounts of the compounds produced are produced de novo because the Organisms lacked this activity and ability to biosynthesize prior to introduction of the corresponding gene (s).
  • the use of different divergent, ie different sequences on DNA sequence level may be advantageous or the use of promoters for gene expression, which allow a different time gene expression, for example, depending on the degree of ripeness of a seed or oil-storing tissue.
  • acyl-CoAlysophospholipid acyltransferase desaturase and / or elongase gene or several acyl-CoA: lysophospholipid acyltransferase, desaturase and / or elongase genes alone or in combination with other genes into an organism or Not only can a cell increase the biosynthetic flux to the final product, but also increase or de novo the corresponding triacylglycerol composition.
  • the number or activity of other genes required to import nutrients needed for the biosynthesis of one or more fine chemicals may be increased, such that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment, thereby further increasing the ability of the cells to produce PUFAs, as described below.
  • Fatty acids and lipids are desirable even as fine chemicals; by optimizing the activity or increasing the number of one or more acyl-CoA: lysophospholipid acyltransferases, desaturases and / or elongases involved in the biosynthesis of these compounds or by disrupting the activity of one or more desaturases involved in the degradation of these compounds It may be possible to increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from organisms and, advantageously, from plants.
  • the isolated nucleic acid molecules used in the method according to the invention encode proteins or parts thereof, wherein the proteins or the individual protein or parts thereof contain an amino acid sequence which is sufficiently homologous to an amino acid sequence of the sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, so that the protein or part thereof is an acyl
  • CoA maintains lysophospholipid acyltransferase activity.
  • the protein or portion thereof encoded by the nucleic acid molecule still has its essential enzymatic activity and has the ability to interfere in the metabolism of compounds necessary for building cell membranes or lipid bodies in organisms, advantageously in plants, or in the transport of To participate in molecules via these membranes.
  • the protein encoded by the nucleic acid molecules is at least about 40%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and most preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to an amino acid sequence of the sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • SEQ ID NO: 2 amino acid sequence
  • lysophospholipid acyltransferases used is to be understood as meaning that they are different from the proteins / enzymes encoded by the sequence with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 and their derivatives in the Comparative still have an enzymatic activity of at least 10%, preferably at least 20%, more preferably at least 30% and most preferably at least 40% and thus the metabolism of the structure of fatty acids in an organism, preferably a plant cell, necessary compounds or transport of molecules via membranes, denatured C 16, C 18, C 20 or C 24 carbon chains having double bonds in at least two, preferably three, four or five positions.
  • Nucleic acids useful in the method are derived from fungi or plants such as algae or mosses such as the genera Physcomitrella, Thraustochytrium, Phytophthora, Ceratodon, Isochrysis, Aleurita, Muscarioides, Mortierella, Borago, Phaeodactylum, Crypthecodinium or from nematodes such as Caenorhabditis, especially from the genera and species Physcomitrella patens,
  • Phytophthora infestans Ceratodon purpureus, Isochrysis galbana, Aleurita farinosa, Muscarioides viallii, Mortierella alpina, Borago officinalis, Phaeodactylum tricornutum or particularly advantageous from Ostreococcus tauri or Mantoniella squamata.
  • CoA encode lysophospholipid acyltransferases which hybridize to a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, e.g. under stringent conditions.
  • the nucleic acid sequences used in the method are advantageously introduced into the respective organism in an expression cassette which enables expression of the nucleic acids in organisms such as microorganisms or plants.
  • nucleic acid sequences coding for the inventive acyl-CoA: lysophospholipid acyltransferases, the desaturases used and / or the elongases are advantageously linked functionally with one or more regulatory signals in order to increase gene expression.
  • These regulatory sequences are intended to allow the targeted expression of genes and proteins. Depending on the host organism, this may mean, for example, that the gene is expressed and / or overexpressed only after induction, or that it is expressed and / or overexpressed immediately.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences before the actual structural genes may still be present and possibly genetically altered, so that the natural regulation was switched off and the expression of genes was increased.
  • the gene construct may advantageously also contain one or more so-called "enhancer sequences” functionally linked to the promoter, which allow 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 acyl-CoAlysophospholipid acyltransferase genes as well as the advantageously used ⁇ -4-desaturase, ⁇ 5-desaturase, ⁇ -6-desaturase and / or ⁇ -8-desaturase genes and / or the ⁇ -5 elongase , ⁇ 6-elongase and / or ⁇ 9-
  • gene construct gene construct
  • This gene construct or gene constructs can be expressed together in the host organism.
  • the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else be inserted in the genome. It is advantageous for the insertion of further genes into the host genome when the genes to be expressed are present together in a gene construct.
  • the regulatory sequences or factors may, as described above, preferably positively influence the expression of the introduced genes and thereby increase them.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
  • a further embodiment of the invention are one or more gene constructs which contain one or more sequences which are defined by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or their derivatives and for polypeptides according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • the said acyl-CoA: lysophospholipid acyltransferases result in an exchange of the fatty acids between the mono-, di- and / or triglyceride pool of the cell and the CoA fatty acid ester pool, wherein the substrate advantageously one, two, three, four or five Double bonds and advantageously has 16, 18, 20, 22 or 24 carbon atoms in the fatty acid molecule.
  • Advantageous regulatory sequences for the novel process are, for example, in promoters such as cos, tac, trp, tet, trp tet, lpp, lac, lpp-lac, laclq, TJ, T5, T3 , gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and are advantageously used in Gram-negative bacteria.
  • promoters such as cos, tac, trp, tet, trp tet, lpp, lac, lpp-lac, laclq, TJ, T5, T3 , gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and 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, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S (Franck et (1980) Cell 21: 285-294), PRP1 (Ward et al (1993) Plant Mol. Biol. 22), SSU, OCS, Iib4, usp, STLS1, B33, nos or in ubiquitin or phaseolin Promoter.
  • inducible promoters such as those described in EP-AO 388 186 (benzylsulfonamide-inducible), Gatz et al. (1992) Plant J. 2: 397-404 (tetracycline-inducible), EP-A-0 335 528 (abscisic acid-inducible) or WO 93/21334 (ethanol- or cyclohexenol-inducible) described promoters.
  • suitable plant promoters are the cytosolic FBPase promoter or potato ST-LSI promoter (Stockhaus et al., (1989) EMBO J.
  • promoters which allow expression in tissues involved in fatty acid biosynthesis.
  • seed-specific promoters such as the USP promoter according to the embodiment, but also other promoters such as the LeB4, DC3, phaseolin or napin promoter.
  • Further particularly advantageous promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and in US Pat. No.
  • promoters are suitable for example for monocots: lpt-2 or lpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordein promoter from barley and other suitable promoters described in WO 99/16890.
  • PUFA biosynthesis genes should advantageously be seed-specifically expressed in oilseeds.
  • seed-specific promoters can be used or promoters that are active in the embryo and / or in the endosperm.
  • seed-specific promoters can be isolated from both dicotyledonous and monocotyledonous plants.
  • Lpt2 and lptl (barley) (WO 95/15389 and WO95 / 23230), seed-specific promoters from rice, corn u. Wheat (WO 99/16890), Amy32b, Amy 6-6 and Aleurain (US 5,677,474), Bce4 (rape) (US 5,530,149), glycinin (soybean) (EP 571 741), phosphoenol
  • Plant gene expression can also be achieved via a chemically inducible promoter (see review in Gatz (1997) Annu Rev. Plant Physiol Plant Mol. Biol. 48: 89-108).
  • Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2: 397-404) and an ethanol-inducible promoter.
  • each of the nucleic acids used in the process should be used for the acyl-CoA: lysophospholipid acyltransferase, the advantageous ⁇ -4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 8-desaturase and / or ⁇ 5-elongase, ⁇ 6-elongase and / or ⁇ -9 -Longase encode, under the control of its own, preferably a different promoter expressed as repetitive sequence motifs can lead to instability of the T-DNA or to recombination events.
  • Expression cassette is advantageously constructed so that a promoter is followed by a suitable interface for insertion of the nucleic acid to be expressed, which is advantageously in a polylinker and optionally followed by a terminator.
  • This sequence is repeated several times, preferably three, four or five times, so that up to five genes can be brought together in one construct and thus introduced into the transgenic plant for expression.
  • the sequence is repeated up to three times.
  • the nucleic acid sequences are inserted for expression via the appropriate interface, for example in the polylinker behind the promoter.
  • each nucleic acid sequence has its own promoter and optionally its own terminator. But it is also possible to have several
  • Nucleic acid sequences behind a promoter and possibly in front of a terminator to advertise are not of decisive importance, that is, a nucleic acid sequence may be inserted at the first or last position in the cassette, without this significantly affecting the expression.
  • different promoters such as the USP, LegB4 or DC3 promoter and different terminators may be used in the expression cassette. But it is also possible to use only one type of promoter in the cassette. However, this can lead to unwanted recombination events.
  • the transcription of the introduced genes should advantageously be stopped by suitable terminators at the 3 'end of the introduced biosynthetic genes (behind the stop codon).
  • suitable terminators at the 3 'end of the introduced biosynthetic genes can be used here, for example, the OCSl terminator.
  • the promoters different terminator sequences should be used for each gene.
  • the gene construct may, as described above, also include other genes to be introduced into the organisms. It is possible and advantageous to introduce into the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which intervene by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway, and to express therein. These genes may be of heterologous or homologous origin. Furthermore, further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct, or else these genes can be located on a further or several further nucleic acid constructs.
  • Fatty acid acyltransferase s
  • fatty acid synthase s
  • fatty acid hydroxylase s
  • acetyl coenzyme A carboxylase s
  • acyl coenzyme A oxidase s
  • fatty acid desaturase s
  • Fatty acid acetylenases lipoxygenases, triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase (s), or a combination of these genes.
  • nucleic acid sequences are biosynthesis genes of the fatty acid or lipid metabolism selected from the group of ⁇ -4-desaturase, ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -8-desatuase, ⁇ -9-desaturase, ⁇ -12-desaturase, ⁇ -5 elongase, ⁇ -6 elongase or ⁇ -9 elongase genes.
  • the abovementioned desaturases can be used in combination with other elongases and desaturases in expression cassettes according to the invention and used for the transformation of plants with the aid of Agrobacterium.
  • the regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
  • the expression cassettes can be used in principle directly for introduction into the plant or else be introduced into a vector.
  • These advantageous vectors contain the nucleic acids which code for acyl-CoA: lysophospholipid acyltransferases, or a nucleic acid construct which comprises the nucleic acid used alone or in combination with further biosynthesis genes of the fatty acid or lipid metabolism, such as ⁇ -4-.
  • vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached.
  • plasmid which is a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome.
  • Certain vectors may autonomously replicate in a host cell into which they have been introduced (eg bacterial vectors of bacterial origin of replication). Other vectors are advantageously integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome.
  • certain vectors may direct the expression of genes to which they are operably linked.
  • expression vectors used for Recombinant DNA recombinant techniques are the form of plasmids.
  • plasmid and “vector” can be used interchangeably because the plasmid is the most commonly used vector form.
  • the invention is intended to encompass other forms of expression vectors, such as viral vectors that perform similar functions.
  • vector is also intended to encompass other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.
  • Gene construct in a form suitable for expression of the nucleic acids used in a host cell which means that the recombinant expression vectors comprise one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed, include.
  • "operatively linked" means that the nucleotide sequence of interest is bound to the regulatory sequence (s) such that expression of the nucleotide sequence is possible and they are linked together such that both sequences fulfill the predicted function ascribed to the sequence (eg in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton , Florida, Ed .: Glick and Thompson, Chapter 7, 89-108, including references therein. regulatory sequences include those which direct the constitutive expression of a nucleotide sequence in many types of host cells and those which direct the direct expression of the nucleotide sequence only in certain host cells under certain conditions. One skilled in the art will appreciate that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the level of expression of the desired protein, etc.
  • acyl-CoAlysophospholipid acyltransferases desaturases and elongases in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construction are often carried out in microorganisms for the sake of simplicity.
  • acyl-CoA lysophospholipid acyltransferase, desaturase and / or elongase genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA, et al. (1992) "YEW 8: 423-488, van den Hondel, CAMJJ, et al.
  • Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector may alternatively be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerase.
  • Typical fusion expression vectors include i.a. pGEX (Pharmacia Biotech Inc., Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione-S Transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST glutathione-S Transferase
  • pTrc amann et al. (1988) Gene 69: 301-315
  • p ⁇ T 1 Id a coexpressed viral RNA polymerase
  • Polymerase is in the host strains BL21 (DE3) or HMS 174 (DE3) of a resident ⁇ prophage harbors a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • vectors useful in prokaryotic organisms are known to those of skill in the art, for example, in E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC series, such as pUC18 or pUC19, the Ml13mp series, pKC30, ⁇ Rep4 , pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 13-Bl, ⁇ gtl 1 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYeDesaturasecl (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 pYES2 (Invitrogen Co ⁇ oration, San Diego, CA).
  • Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J., & Punt, PJ.
  • yeast vectors are, for example, pAG-1, YEp6, YEp 13 or pEMBLYe23.
  • acyl-CoA lysophospholipid acyltransferases, desaturases and / or elongases can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • the above vectors provide only a brief overview of possible suitable vectors.
  • Other plasmids are known in the art and are described, for example, in: Cloning Vectors (Eds.
  • the acyl-CoA lysophospholipid acyltransferases, desaturases and / or elongases can be found in unicellular plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and cited therein, and plant cells from higher plants (eg spermatophytes such as crops) are expressed.
  • plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer to Higher Plauts; in: Transgenic Plauts, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, pp. 15-38.
  • a plant expression cassette preferably contains regulatory sequences that can direct gene expression in plant cells and are operably linked so that each sequence can perform its function, such as termination of transcription, for example, polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH5 known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, as well all other terminators functionally active in plants are suitable.
  • a plant expression cassette preferably contains other operably linked sequences, such as translation enhancers, for example the overdrive sequence containing the 5'-untranslated tobacco mosaic virus leader sequence, which has the protein / RNA ratio increases (Gallie et al., (1987) Nucl. Acids Research 15: 8693-8711).
  • translation enhancers for example the overdrive sequence containing the 5'-untranslated tobacco mosaic virus leader sequence, which has the protein / RNA ratio increases (Gallie et al., (1987) Nucl. Acids Research 15: 8693-8711).
  • the gene to be expressed must be operably linked to a suitable promoter that performs gene expression in an upright, cell or tissue-specific manner.
  • suitable promoters are consumptive promoters (Benfey et al (1989) EMBO J. 8: 2195-2202), such as those derived from plant viruses, such as 35S CAMV (Franck et al., (1980) Cell 21: 285-294), 19S CaMV (see also US 5,352,605 and WO 84/02913) or plant promoters, such as the Rubisco small subunit described in US 4,962,028.
  • sequences for use in the functional compound in plant gene expression cassettes are targeting sequences used to direct the gene product into its corresponding cell compartment, for example, the vacuole, nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, extracellular Space necessary for mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells (see review in Kermode (1996) Crit Rev. Plant Sci. 15: 4: 285-423 and references cited therein).
  • Plant gene expression can also be facilitated via a chemically inducible promoter as described above (see review in Gatz (1997) Annu Rev. Plant Physiol Plant Mol. Biol. 48: 89-108).
  • Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible Promoter (Gatz et al. (1992) Plant J. 2: 397-404) and an ethanol-inducible promoter.
  • Promoters which respond to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRPl gene promoter (Ward et al. (1993) Plant Mol. Biol. 22: 361-366), the heat-inducible hsp80 promoter Tomato (US 5,187,267), the potato inducible alpha-amylase promoter (WO 96/12814) or the wound-inducible pinII promoter (EP-A-0 375 091).
  • those promoters are preferred which bring about gene expression in tissues and organs in which the fatty acid, lipid and
  • Suitable promoters are the rapeseed napin promoter (US 5,608,152), the Viciafaba USP promoter (Baeumlein et al (1991) Mol Gen Genet 225 (3): 459-67), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the
  • Brassica Bce4 promoter (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al. (1992) Plant Journal 2 (2): 233-9) as well as promoters expressing seed-specific expression in monocotyledonous plants such as Corn, barley, wheat, rye, rice, etc. bring.
  • Suitable promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzine gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum casirin gene, the Rye-secalin gene).
  • promoters which are the plastid specific
  • plastids are the compartment in which the precursors and some end products of lipid biosynthesis are synthesized.
  • suitable Promoters are the viral RNA polymerase promoter described in
  • the multiparallel expression of the acyl-CoA: lysophospholipid acyltransferases used in the process may be desired alone or in combination with desaturases and / or elongases.
  • the introduction of such expression cassettes can be carried out via a simultaneous transformation of a plurality of individual expression constructs or preferably by combining a plurality of expression cassettes on a construct.
  • multiple vectors, each containing multiple expression cassettes can be transformed and transferred to the host cell.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection,” conjugation and transduction are intended to encompass a variety of methods known in the art for introducing foreign nucleic acids (eg, DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE- Dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment.
  • Suitable methods for transformation or transfection of host cells, including plant cells can be found in Sambrook and Russell (Molecular Cloning: A Laboratory Manual, 3rd ed., CoId Spring Harbor Laboratory, Col. Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 2001) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Eds: Gartland and Davey, Humana Press, Totowa, New Jersey.
  • Host cells which are suitable in principle for receiving the nucleic acid according to the invention, the gene product according to the invention or the vector according to the invention are all prokaryotic or eukaryotic organisms.
  • the host organisms which are advantageously used are microorganisms, such as fungi or yeasts or plant cells, preferably plants or parts thereof.
  • Fungi, yeasts or plants are preferably used, more preferably plants, most preferably plants such as oilseed crops containing high levels of lipid compounds such as rapeseed, evening primrose, hemp, thistle, peanut, canola, flax, soybean, safflower, sunflower, borage , or plants such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, tagetes, solanaceous plants such as potato, tobacco, eggplant and tomato, vicia species, pea, alfalfa, bush plants (coffee, Cocoa, tea), Salix species, trees (oil plan, coconut) and perennial grasses and forage crops.
  • Particularly preferred plants according to the invention are oil crop plants, such as soybean, peanut, rapeseed, canola, flax, hemp, evening primrose, sunflower, safflower, trees (oil palm, coconut).
  • nucleic acid sequences encoding acyl-CoA: lysophospholipid acyltransferase activity polypeptides as described above, wherein the acyl-CoA: lysophospholipid acyltransferases encoded by the nucleic acid sequences are C 16 , C 18 , C 20 or C 16 C 22 -fatty acids with at least one double bond in the fatty acid molecule implement.
  • nucleic acid sequence having the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, e) Nucleic acid sequences which can be derived as a result of the degenerate genetic code from the coding sequence contained in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5
  • SEQ ID NO: 2 SEQ ID NO: 4 or SEQ ID NO: 6 and have at least 40% homology at the amino acid level with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and a Acyl-CoA: have lysophospholipid acyltransferase activity.
  • nucleic acid sequences of the invention are derived from organisms such as animals, ciliates, fungi, plants such as algae or dinoflagellates which are capable of synthesizing PUFAs.
  • the nucleic acid sequences according to the invention are derived from Ostreococcus tauri or Mantonella squamata.
  • nucleic acid (molecule) as used here, in an advantageous embodiment, furthermore comprises the untranslated sequence located at the 3 'and at the 5' end of the coding gene region: at least 500, preferably 200, particularly preferably 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid preferably does not have sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (eg, sequences located at the 5 'and 3' ends of the nucleic acid).
  • the isolated acyl-CoA: lysophospholipid acyltransferase molecule may be less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences naturally flanking the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.
  • nucleic acid molecules used in the method for example a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or a part thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. It is also possible with the aid of comparative algorithms to identify, for example, a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level.
  • nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or a part thereof can be isolated by polymerase chain reaction using oligonucleotide primers based on this sequence or portions thereof (For example, a nucleic acid molecule comprising the complete sequence or a portion thereof can be isolated by polymerase chain reaction using oligonucleotide primers prepared on the basis of this same sequence).
  • mRNA can be isolated from cells (eg, by the guanidinium thiocyanate extraction method of Chirgwin et al., (1979) Biochemistry 18: 5294-5299) and cDNA by reverse transcriptase (eg, Moloney MLV reverse transcriptase, available from Gibco / BRL , Bethesda, MD, or AMV Reverse Transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase eg, Moloney MLV reverse transcriptase, available from Gibco / BRL , Bethesda, MD, or AMV Reverse Transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for polymerase chain reaction amplification can be synthesized on the basis of one of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or with the aid of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • a nucleic acid of the invention may be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • homologs of the acyl-CoA3 phospholipid acyltransferase nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 used are allelic variants of at least about 40 to 60%, preferably at least about 60 to 70 %, more preferably at least about 70 to 80%, 80 to 90% or 90 to 95%, and most preferably at least about 95%, 96%, 97%, 98%, 99% or more homology to any of the amino acid sequences set forth in SEQ ID NO : 1, SEQ ID NO: 3 or SEQ ID NO: 5 shown nucleotide sequences or their homologs, derivatives or analogs or parts thereof.
  • allelic variants comprise functional variants which can be obtained by deletion, insertion or substitution of nucleotides from / in the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, but the intention is that the enzyme activity of the resulting proteins is substantially retained.
  • Homologs of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 also mean, for example, bacterial, fungal and plant homologs, truncated sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence.
  • Homologs of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 also mean derivatives such as promoter variants.
  • the promoters upstream of the indicated nucleotide sequences may be modified by one or more nucleotide exchanges, insertion (s) and / or deletion (s), without, however, interfering with the functionality or activity of the promoters. It is also possible that the activity of the promoters is increased by modification of their sequence or that they are completely replaced by more active promoters, even from heterologous organisms.
  • nucleic acid molecules which code for proteins having acyl-CoA: lysophospholipid acyltransferase activity, which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds via membranes, are used in the process according to the invention for modulating the production of PUFAs in transgenic organisms advantageous in plants such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, linum species such as oil or fiber kidney, Brassica species such as rapeseed, canola and turnip rape, pepper, sunflower, Borage, evening primrose and Tagetes, Solanacaen plants such as potato, tobacco, aubergine and tomato, Vicia species, pea, cassava, alfalfa, bush plants (coffee, cocoa, tea), Salix species, trees (oil palm, coconut) and perennials Grasses and forage crops, either directly (
  • PUFAs polyunsaturated fatty acids
  • the lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate as well as the addition or modification of a polar head group.
  • Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides.
  • Fatty acid synthesis begins with the conversion of acetyl-CoA into malonyl-CoA by the acetyl-CoA carboxylase or into acetyl-ACP by the acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which through a series of
  • the phospholipid-bound fatty acids thus produced must then be converted again for further elongations from the phospholipids into the fatty acid CoA ester pool.
  • these enzymes can transfer the elongated fatty acids again from the CoA esters to the phospholipids. This reaction sequence can optionally be repeated several times (see Fig. 4).
  • Precursors for the PUFA biosynthesis are, for example, oleic acid, linoleic acid and
  • Linolenic acid These C 18 -carbon fatty acids must be elongated 20 and C 22 to obtain fatty acids of eicosapentaenoic and docosa chain type on C.
  • desaturases such as the ⁇ -4-, ⁇ -5, ⁇ -6 and ⁇ -8-desaturases and / or the ⁇ -5, ⁇ - 6-, ⁇ -9 elongases may be obtained, extracted and used for various purposes in food, feed, cosmetic or pharmaceutical applications, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid, as well as various other long-chain PUFAs.
  • C 18 , C 20 , and / or C 22 fatty acids having at least two, advantageously at least three, four, five or six double bonds in the fatty acid molecule, preferably to C 20 -, and / or C 22 -fatty acids, may preferably be used with the abovementioned enzymes be prepared with advantageous three, four or five double bonds in the fatty acid molecule.
  • the desaturation can be carried out before or after elongation of the corresponding fatty acid.
  • the products of desaturase activities and possible further desaturation and elongation result in preferred PUFAs having a higher degree of desaturation, including a further elongation of C 20 to C 22 fatty acids, to fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, stearidonic acid, Eicosatetraenoic acid or eicosapentaenoic acid.
  • Substrates of the acyl-CoA are C 16 , C 18 , C 20 or C 22 fatty acids, for example palmitic acid, palmitoleic acid, linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, eicosatetraenoic acid or stearidonic acid.
  • Preferred substrates are linoleic acid, ⁇ -linolenic acid and / or ⁇ - Linolenic acid, dihomo- ⁇ -linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
  • the C 18 , C 20 or C 22 fatty acids having at least two double bonds in the fatty acid are obtained in the novel process in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.
  • glycolide is understood as meaning a glycerol (mono-, di- or triglyceride) esterified with one, two or three carboxylic acid residues.
  • glycolide is also meant a mixture of different glycerides.
  • the glyceride or glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.
  • a "glyceride” in the sense of the process according to the invention is also understood as meaning derivatives derived from glycerol.
  • these also include glycerophospholipids and glyceroglycolipids. Examples include the glycerophospholipids lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and Alkylacylglycerophospholipide called.
  • fatty acids must then be transported to various modification sites and incorporated into the triacylglycerol storage lipid.
  • Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol-fatty acid acyltransferase (see Frentzen (1998) Lipid 100 (4-5): 161-166).
  • the PUFAs produced in the process comprise a group of molecules that are no longer able to (sufficiently) synthesize higher animals, and thus must (additionally) take up, although they are readily synthesized by other organisms such as bacteria.
  • acyl-CoA lysophospholipid acyltransferases
  • phospholipids are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and / or phosphatidylinositol, advantageously phosphatidylcholine.
  • acyl-CoA ⁇ ssophospholipid acyltransferase nucleic acid sequence (s) in the sense of the invention comprises nucleic acid sequences which encode an acyl-CoA: lysophospholipid acyltransferase, in particular an acyl-CoA: lysophosphatidylcholine acyltransferase, and a coding region and optionally corresponding ones 5'- and 3'-untranslated sequence regions.
  • the invention also comprises nucleic acid molecules which differ from one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 (and parts thereof) due to the degenerate genetic code and thus the same acyl CoA: Lysophospholipid acyltransferase encode such as that encoded by the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
  • acyl-CoA3sophospholipid acyltransferase nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 those skilled in the art will recognize that within a population, DNA sequence polymorphisms may exist that result in changes in the DNA sequence Amino acid sequences of the acyl-CoA: lysophospholipid acyltransferases can lead. These genetic polymorphisms in the acyl-CoA3sophospholipid acyltransferase gene may exist due to natural variation between individuals within a population.
  • Nucleic acid molecules useful in the method of the present invention can be isolated based on their homology to the acyl-CoA: lysophospholipid acyltransferase nucleic acids disclosed herein using the sequences or a portion thereof as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • isolated nucleic acid molecules can be used which are at least 15 nucleotides long and which hybridize under stringent conditions with the nucleic acid molecules which comprise a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
  • Nucleic acids of at least 25, 50, 100, 250 or more nucleotides may also be used.
  • hybridized under stringent conditions is intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 60% homologous to one another usually remain hybridized to one another.
  • the conditions are preferably such that sequences which are at least about 65%, preferably at least about 70%, and more preferably at least about 75% or more homologous, are usually hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridizations in 6 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 50 to 65 ° C. It is known to the person skilled in the art that these hybridization conditions may differ with regard to the temperature and the concentration of the buffer, depending on the type of nucleic acid and, for example, whether organic solvents are present. The temperature is for example under "standard hybridization conditions" depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2).
  • the temperature is about 42 ° C under standard conditions.
  • the hybridization conditions for DNA: DNA hybrids are, for example, 0.1 x SSC and 20 ° C to 45 ° C, preferably between 30 ° C and 45 ° C.
  • the hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 ° C to 55 ° C, preferably between 45 ° C and 55 ° C.
  • the sequences are written among each other for optimal comparison (eg, gaps in the sequence of a protein or a nucleic acid inserted to create an optimal alignment with the other protein or nucleic acid).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared.
  • An isolated nucleic acid molecule encoding an acyl-CoA: lysophospholipid acyltransferase that is homologous to a protein sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 may be prepared by introducing one or more nucleotide substitutions , additions or deletions into a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into any of the sequences of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made on one or more of the predicted nonessential amino acid residues.
  • an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • families of amino acid residues have been defined with similar side chains.
  • amino acids with basic side chains eg, lysine, Arginine, histidine
  • acidic side chains eg aspartic acid, glutamic acid
  • uncharged polar side chains eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains eg alanine, valine, leucine, isoleucine, proline, phenylalanine, Methionine, tryptophan
  • beta-branched side chains eg threonine, valine, isoleucine
  • aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in an acyl-CoA: lysophospholipid acyltransferase is thus preferably replaced by another amino acid residue from the same side chain family.
  • the mutations may be randomly introduced over all or part of the acyl-CoAi lysophospholipid acyltransferase coding sequence, eg, by saturation mutagenesis, and the resulting mutants screened by the acyl-CoAlysophospholipid acyltransferase activity described herein. to identify mutants that have retained the acyl-CoA: lysophospholipid acyltransferase activity.
  • the encoded protein can be recombinantly expressed and the activity of the protein can be determined, for example, using the assays described herein.
  • DNA modifying enzymes and molecular biology kits were purchased from AGS (Heidelberg), Amersham (Brunswick), Biometra (Göttingen), Roche (Mannheim), Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach / Taunus), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Amsterdam, The Netherlands). Unless otherwise stated, they were used according to the manufacturer's instructions.
  • T-120-Ot-LPCAT-XhoI ACT CGA GTC ACG AGT TGT TCA CGA GGC The positions of the primers used are shown in FIG.
  • the amplification of the OtLPCAT DNA was carried out in each case with 1 .mu.l thawed cells, 200 .mu.M dNTPs, 2.5 U T ⁇ ⁇ polymerase and 100 pmol of each primer in a total volume of 50 ul.
  • the conditions for the PCR were as follows: first denaturation at 95 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes and a final extension step at 72 ° C for 10 minutes.
  • Example 3 Cloning of Expression Plasmids for the Heterologous Expression of OtLPCAT in Yeasts
  • the corresponding primer pairs were selected to carry the yeast consensus high-efficiency translation sequence (Kozak (1986) Cell 44: 283-292) adjacent to the start codon.
  • the amplification of OtLPCAT was carried out in each case with 1 .mu.l cDNA, 200 .mu.M dNTPs, 2.5 U Advantage-Volymerase and 100 pmol of each primer in a total volume of 50 ul.
  • the conditions for the PCR were as follows: first denaturation at 95 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes and a final extension step at 72 ° C for 10 minutes.
  • the PCR products were incubated for 30 min at 21 0 C with the yeast expression vector pYES2.1 -TOPO (Invitrogen) according to manufacturer's instructions.
  • the PCR product is ligated by a T-overhang and activity of a topoisomerase (Invitrogen) in the vector.
  • transformation of E. coli DH5 ⁇ cells was then carried out.
  • Corresponding clones were identified by PCR, the plasmid DNA isolated using Qiagen DNAeasy kit and verified by sequencing. The sequence of the resulting plasmid pYES2.1 -OtLPCAT is shown in SEQ ID no. 7 indicated.
  • the correct sequence was then inserted into the S ⁇ cch ⁇ romyces Strain INVScI (Invitrogen) transformed by electroporation (1500V).
  • the empty vector pYES2.1 was transformed in parallel.
  • the yeasts were plated on complete minimal medium without uracil with 2% glucose. Cells which were capable of growth in the medium without uracil contained the corresponding plasmids pYES2.1 or pYES2.1-OtLPCAT. After selection, two transformants were selected for further functional expression.
  • pSUN300 is a derivative of the plasmid pPZP (Hajdukiewicz, P. et al. (1994) Plant Mol. Biol. 25: 989-994).
  • pSUN-USP was generated from pSUN300 by inserting into pSUN300 a USP promoter as an EcoRI fragment.
  • the USP promoter corresponds to nucleotides 1-684 (Genbank Accession X56240), with part of the non-coding region of the USP gene contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial 17 standard primer (Stratagene) and a synthesized primer
  • the PCR fragment was rescored with EcoRI / SalI and inserted into the vector pSUN300 with OCS terminator.
  • the polyadenylation signal is that of the octopine synthase gene from the A. tumefaciens Ti plasmid (ocs terminator, Genbank Accession V00088) (De Greve, H. et al., (1982) J. Mol. Appl. Genet. 1 (6): 499-511).
  • the result was the plasmid with the name pSUN-USP.
  • the construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • Notl cleavage sites were inserted at the 5 'and 3' end of the coding sequence with the following primer pair:
  • composition of the PCR mixture (50 ⁇ l):
  • Annealing temperature 1 min 55 ° C denaturation temperature: 1 min 94 ° C elongation temperature: 2 min 72 ° C number of cycles: 35
  • the PCR products were incubated for 16 h at 37 0 C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was incubated in the same way. Subsequently, the PCR products and the 7624 bp vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The purification of the DNA fragments was carried out using Qiagen Gel Purification Kit according to the manufacturer. Subsequently, vector and PCR products ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmid pSUN-OtLPCAT was verified by sequencing.
  • OtLPCAT The activity of OtLPCAT was determined after expression of OtLPCAT in yeasts and feeding of various fatty acids (Figure 2).
  • the double construct pESCLeu-PpD6-PSE1 was furthermore prepared which has the open reading frames of a ⁇ 6-desaturase (PpD6) and a ⁇ 6 elongase (PSE1) from Physcomitrella patens ( see DE 102 19 203), and together with either the empty vector pYES2.1 or the vector pYES2.1 -
  • OtLPCAT transformed OtLPCAT transformed.
  • the cloning of the construct pESCLeu-PpD6-PSE1 can be found in WO 2004/076617, the content of which is hereby incorporated by reference.
  • yeast cells from the major cultures were harvested by centrifugation (100 xg, 10 min, 20 ° C) and washed with 100 mM NaHCO 3 , pH 8.0 to remove residual medium and fatty acids.
  • fatty acid methyl esters FAMEs
  • FAMEs fatty acid methyl esters
  • FIG. 2A shows the conversion of the nutritious fatty acid 18: 2 A9 '12 to 20: 3 A8>11> 14 by yeasts which had been transformed with the plasmids pESCLeu-PpD6-PSE1 and pYES2.1.
  • FIG. 2B shows the conversion into yeasts which, in addition to the plasmid pESCLeu-PpD6-PSE1, additionally contain the plasmid pYES2.1-OtLPCAT.
  • the fed substrate was abundant in all transgenic yeasts. Both transgenic yeasts showed a synthesis of 18: 3 ⁇ 6 ' 9 ' 12 and
  • Example 6 Cloning of acyltransferase genes from Mantoniella squamata By searching for conserved regions in the protein sequences of the previously isolated LPCAT from C. elegans (WO 2004/76617), it was possible to identify sequences with corresponding motifs in a Mantoniella sequence database (see FIG. 1). These are the following sequences:
  • a 2 1 culture of M ⁇ ntoniell ⁇ squ ⁇ m ⁇ t ⁇ was prepared in f / 2 medium (Guillard, RRL (1975) Culture of phytoplankton for feeding marine invertebrates.) In Culture of Marine Invertebrate Animals (Eds. Smith, WL and Chanley, MH), Plenum Press, New York, pp 29-60) for 14 days at a luminous intensity of 80 U / cm 2 .
  • RACE rapid amplification of cDNA ends
  • the associated genomic DNAs were amplified by PCR.
  • the corresponding primer pairs were selected such that they contain the first 20 nucleotides at the 5 'end and the last 20 nucleotides at the 3' end (including stop codon) and at the 5 'end additionally the yeast consensus sequence for highly efficient translation ( Kozak (1986) Cell 44: 283-292).
  • the following primers were used:
  • MA LPCAT BamHI AGG ATC CAT GTC TTT TTA CCT CGT CAC CTT CAC C 3 ⁇ -113
  • MA LPCAT_XhoI ACT CGA GTC ACG AGT ACT TGA CAA GGC for the shorter form of LPCAT from Mantoniella squamata and
  • the amplification of the MsLPC AT-DNAs was carried out in each case with 1 .mu.l thawed cells, 200 .mu.M dNTPs, 2.5 U T ⁇ ⁇ -Polymerase and 100 pmol of each primer in a total volume of 50 ul.
  • the conditions for the PCR were as follows: first denaturation at 95 ° C for 5 minutes followed by 30 cycles at 94 ° C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes and a final extension step at 72 ° C for 10 minutes.
  • the corresponding primer pairs were selected to carry the yeast consensus high-efficiency translation sequence (Kozak (1986) Cell 44: 283-292) adjacent to the start codon.
  • the amplification of the MsLPCATs was carried out in each case with 1 .mu.l cDNA, 200 .mu.M dNTPs, 2.5 U Advantage-Volymerase and 100 pmol of each primer in a total volume of 50 ul.
  • the conditions for the PCR were as follows: first denaturation at 95 ° C for 5 minutes followed by 30 cycles at 94 ° C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes and a final extension step at 72 ° C for 10 minutes.
  • the following oligonucleotides were used for the PCR reaction:
  • PCR products were incubated for 30 min at 21 0 C with the yeast expression vector pYES2.1 -TOPO (Invitrogen) according to manufacturer's instructions.
  • the PCR product is ligated by a T-overhang and activity of a topoisomerase (Invitrogen) in the vector.
  • transformation of E. coli DH5 ⁇ cells was then carried out.
  • Corresponding clones were identified by PCR, the plasmid DNA isolated using Qiagen DNAeasy kit and verified by sequencing.
  • the sequences of the resulting plasmids pYES2.1 -MsLPCATl 12 and pYES2.1 -MsLPCATl 18 are shown in SEQ ID Nos. 8 or 9 indicated.
  • composition of the PCR mixture (50 ⁇ l):
  • Annealing temperature 1 min 55 ° C denaturation temperature: 1 min 94 ° C elongation temperature: 2 min 72 ° C number of cycles: 35
  • the PCR products were incubated for 16 h at 37 0 C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was used in the same way incubated. Subsequently, the PCR products and the 7624 bp vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The purification of the DNA fragments was carried out using Qiagen Gel Purification Kit according to the manufacturer. Subsequently, vector and PCR products were ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmids pSUN-MsLPCATl 12 and pSUN-MsLPCATl 18 were verified by sequencing.
  • the activity of the MsLPCATs could be determined after expression of the MsLPCATs in yeasts and feeding of various fatty acids ( Figures 3 A, B and C).
  • the construct pESCLeu-PpD6-PSE1 was introduced into the yeast together with either the empty vector pYES2.1 or the plasmid pYES2.1 -MsLPCATl 12 or pYES2.1 -MsLPCATl 18 here.
  • FIG. 3A shows the conversion of the fed fatty acid 18: 2 A9 '12 to 20: 3 A8> 11 ' 14 by yeasts which had been transformed with the plasmid pESCLeu-PpD6-PSE1 and pYES2.1.
  • FIG. 3B shows the reaction in yeasts which, in addition to the plasmid pESCLeu-PpD6-PSE1, additionally contain the plasmid pYES2.1-MsLPCAT112.
  • FIG. 3C describes the fatty acid spectrum of yeasts which are associated with the
  • LCPUF A-specific acyltransferases in transgenic plants is advantageous in order to increase the LCPUF A content in these plants.
  • the acyltransferase cDNAs according to the invention were cloned into binary vectors (see Examples 4 and 8) and via Agrobacterium-mediated DNA transfer in
  • transgenic plants which already express the desaturases and elongases necessary for the synthesis of LCPUFAs and produce small amounts of these LCPUFAs.
  • Such plants are for example those in DE 102 19 203 which contain functional ⁇ 6-desaturase genes, ⁇ 6-elongase and ⁇ 5-desaturase and produce small amounts of ARA and EPA.
  • the resulting binary vectors with acyltransferase genes were transformed into Agrobacterium tumefaciens (Hofgen and Willmitzer (1988) Nucl. Acids Res. 16: 9877).
  • the transformation of A thaliana was carried out by means of "floral dip" (Clough and Bent (1998) Plant Journal 16: 735-743), by N. tabacum on cocultivation of tobacco leaf pieces with transformed A. tumefaciens cells, of linseed and oilseed rape Cocultivation of hypocotyl pieces with transformed A. tumefaciens' cells. Corresponding methods are known to the person skilled in the art.
  • acyltransferase genes in transgenic Arabidopsis, tobacco, rapeseed and linseed plants was examined by Northern blot analysis. Selected plants were examined for their content of PUFAs in seed oil.
  • Agrobacterium-mediated plant transformation can be carried out using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Central Signature: BTI 1 -P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, B. Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).
  • rape can be transformed by cotyledon or hypocotyl transformation (Moloney et al., (1989) Plant Cell Report 8: 238-242; De Block et al. (1989) Plant Physiol. 91: 694-701).
  • the use of antibiotics for Agrobacterium and plant selection depends on the binary vector and Agrobacterium strain used for the transformation.
  • the rape selection will usually performed using kanamycin as a selectable plant marker.
  • the Agrobacterium-mediated gene transfer in flax (Linum usitatissimum) can be determined, for example, using a method described by Mlynarova et al. (1994) Plant Cell Report 13: 282-285.
  • soy may be carried out using, for example, a technique described in EP-A-0 0424047 (Pioneer Hi-Bred International) or in EP-A-0 039 7687, US 5,376,543, US 5,169,770 (University Toledo). Plant transformation using particle bombardment, polyethylene glycol mediated DNA uptake or via the silicon carbonate fiber technique is described, for example, by Freeling and Walbot "The Maize Handbook” (1993) ISBN 3-540-97826-7, Springer, New York.
  • TAG fatty acids or triacylglycerol
  • FAME fatty acid methyl ester
  • GC-MS gas-liquid chromatography-mass spectrometry
  • TLC thin-layer chromatography
  • the unambiguous evidence for the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods such as GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition: Christie , OiIy Press, Dundee, 119-169; 1998, Gas Chromatography Mass Spectrometry Method, Lipids 33: 343-353).
  • the plant material to be analyzed may be broken up either by sonication, milling in the glass mill, liquid nitrogen and milling or by other applicable methods. The material is then centrifuged after rupture. The sediment is then distilled in aqua. re-suspended, heated at 100 ° C for 10 min, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 h at 90 ° C resulting in hydrolyzed oil and lipid compounds. which give transmethylated lipids.
  • fatty acid methyl esters can then be extracted into petroleum ether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ° C and 240 ° C for 20 min and 5 min at 240 ° C are subjected.
  • the identity of the resulting fatty acid methyl esters can be defined using standards available from commercial sources (i.e., Sigma).
  • the seeds were taken up with 1% sodium methanolate in methanol and incubated for 20 min at RT (about 22 ° C). It was then washed with NaCl solution and taken up the FAME in 0.3 ml of heptane.
  • the samples were separated on a ZEBRON ZB Wax capillary column (30 m, 0.32 mm, 0.25 micron, Phenomenex) in a Hewlett Packard 6850 gas chromatograph with a flame ionization detector.
  • the furnace temperature was (hold 1 min) of 7O 0 C to 200 0 C at a rate of 20 ° C / min, then to 25O 0 C (5 hold min) at a rate of 5 ° C / min and finally to 26O 0 C programmed at a rate of 5 ° C / min.
  • the carrier gas was nitrogen employed (4.5 ml / min at 7O 0 C).
  • the fatty acids were identified by comparison with retention times of FAME standards (SIGMA).
  • acyl-CoA lysophopholipid acyltranserase
  • the biosynthetic pathway of the LCPUFAS is thus as follows:
  • Desaturases catalyze the introduction of double bonds into lipid-coupled fatty acids (s ⁇ -acyl-phosphatidylcholine), while the elongases exclusively catalyze the elongation of coenzyme A-esterified fatty acids (acyl-CoAs). According to this mechanism requires the alternating action of desaturases and
  • Elongases a constant exchange of acyl substrates between phospholipids and the acyl-CoA-pool and thus the existence of an additional activity, the acyl substrates in the respectively necessary substrate form, ie lipids (for desaturases) or CoA thioesters (for elongases) , convicted.
  • This exchange between acyl-CoA pool and phospholipids is facilitated by LCPUF
  • Fig. 1 Amino acid sequence comparison of OtLPCAT, MsLPCAT and CeLPCAT
  • Fig. 2 Fatty acid analysis of yeasts which had been transformed with the plasmids pESCLeu-PpD6-PSE1 and pYES2.1 (A) or pLEU-PSE1 (Pp) _d6Des (Pp) and pYES2.1-OtLPCAT (B) Feeding with 18: 2 A9 '12 .
  • Fig. 3 Fatty acid analysis of yeasts infected with the plasmids pESCLeu-PpD6-PSE1 and pYES2.1 (A), pESCLeu-PpD6-PSE1 and pYES2.1 -MsLPCAT12 (B) and pESCLeu-PpD6-PSE1 and pYES2, respectively.
  • Fig. 4 Biosynthetic pathway of LCPUFAs

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Abstract

L'invention concerne un procédé de production d'acides gras multi-insaturés dans un organisme, procédé consistant à introduire dans l'organisme, des acides nucléiques codant pour un polypeptide à activité acyl-CoA : lysophospholipidacyltransférase. Avantageusement, ces séquences d'acides nucléiques peuvent être exprimées dans l'organisme transgénique, éventuellement, avec d'autres séquences d'acides nucléiques codant pour un polypeptide du métabolisme acide gras ou lipidique. L'invention concerne en outre les séquences d'acides nucléiques conformes à l'invention, des constructions d'acides nucléiques renfermant lesdites séquences d'acides nucléiques selon l'invention, des vecteurs renfermant les séquences d'acides nucléiques selon l'invention et/ou les constructions d'acides nucléiques, ainsi que les organismes transgéniques renfermant les séquences d'acides nucléiques précitées, les constructions d'acides nucléiques et/ou les vecteurs précités. L'invention concerne en outre des huiles, des lipides et/ou des acides gras fabriqués conformément au procédé de l'invention, ainsi que l'utilisation de ces composés.
EP05821795A 2004-12-23 2005-12-20 Procede de production d'acides gras a longues chaines multi-insatures dans des organismes transgeniques Withdrawn EP1831358A2 (fr)

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