EP1527182A2 - Verfahren zur herstellung von ungesättigten fettsäuren - Google Patents

Verfahren zur herstellung von ungesättigten fettsäuren

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Publication number
EP1527182A2
EP1527182A2 EP03716183A EP03716183A EP1527182A2 EP 1527182 A2 EP1527182 A2 EP 1527182A2 EP 03716183 A EP03716183 A EP 03716183A EP 03716183 A EP03716183 A EP 03716183A EP 1527182 A2 EP1527182 A2 EP 1527182A2
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EP
European Patent Office
Prior art keywords
desaturase
seq
fatty acid
plant
fatty acids
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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EP03716183A
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English (en)
French (fr)
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EP1527182A4 (de
Inventor
Andreas Renz
Martijn Gipmans
Ivo Feussner
Claire-Lise Rosenfield
Douglas C. Knipple
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BASF Plant Science GmbH
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BASF Plant Science GmbH
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Publication of EP1527182A2 publication Critical patent/EP1527182A2/de
Publication of EP1527182A4 publication Critical patent/EP1527182A4/de
Withdrawn legal-status Critical Current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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Definitions

  • the invention relates to processes for the production of unsaturated fatty acids, preferably of conjugated polyunsaturated fatty acids such as conjugated linoleic acid (CLA), by the recombinant expression of desaturases from insects of the order Lepidoptera.
  • Expression preferably takes place in an organism selected from the group of the plant organisms, yeasts, fungi and algae.
  • recombinant expression cassettes for the recombinant expression of desaturases from insects of the order Lepidoptera and the transgenic organisms transformed with these.
  • conjugated unsaturated fatty acids are used widely in the food industry, in animal nutrition, in cosmetics and in the pharmaceutical sector. Especially valuable and sought-after unsaturated fatty acids are what are known as conjugated unsaturated fatty acids. Conjugated polyunsaturated fatty acids are relatively rare in comparison with other polyunsaturated fatty acids. Examples of conjugated fatty acids are the conjugated linoleic acids (CLA; conjugated linoleic acid), ⁇ -parinaric acid (18:4 octadecatetraenoic acid), eleostearic acid (18:3 octadecatrienoic acid), the conjugated linolenic acids, dimorphecolic acid and calendulic acid (see scheme 1 ).
  • CLA conjugated linoleic acids
  • ⁇ -parinaric acid (18:4 octadecatetraenoic acid
  • eleostearic acid 18:3 octadecatrienoic acid
  • CLA is a collective term for positional and structural isomers of linoleic acid which are distinguished by a conjugated double bond system starting at carbon atom 8, 9, 10 or 11. Some examples are shown in scheme 2. Geometric isomers exist for each of these positional isomers, that is to say cis-cis, trans-cis, cis-trans, trans-trans.
  • the CLA isomers (9Z,11 E)-CLA and (10E,12Z)-CLA are known as the biologically active isomers.
  • CLA is found predominantly in foodstuffs of animal origin. High CLA concentrations are found in particular in the meat and in dairy products of ruminants: approx. 3 to 4 mg of CLA g fat in beef and lamb (Chin et al. (1992) J Food Comp Anal 5:185-197) and approx. 3 to 7 mg of CLA/g fat in dairy products (Dhiman et al. (1999) J Dairy Sci 82:2146-56), where (9Z,11 E)-CLA at a concentration of approximately 80% is in each case the predominant isomer. Higher plants only contain traces of CLA, with the two biologically active CLA isomers not having been found in plants to date.
  • CLA can be synthesized by alkaline isomerization of linoleic acid.
  • Vegetable oils with a high linoleic acid content are predominantly used on an industrial scale, for example sunflower oil, safflower oil. Heating to above 180°C under alkaline conditions catalyzes two reactions:
  • CLA oils contain a mixture of various CLA isomers and other saturated and unsaturated fatty acids. Owing to the presence of these biologically inactive and unnatural isomers, laborious purification of the biologically active isomers (9Z,1 1 E) CLA and (10E.12Z) CLA is required, or it must be demonstrated that the isomer mixture does not represent a health hazard for humans and animals. It has hitherto not been possible to produce individual CLA isomers by alkaline isomerization in an economically relevant process. Fractional crystallization makes it possible to concentrate the isomers (9Z,1 1 E)-CLA and (10E,12Z)-CLA, respectively.
  • reaction products are usually converted into methyl or ethyl esters so that the natural form of CLA, viz. the free fatty acids or the triacyl glyceride, are not available.
  • CLA isomerases convert free linoleic acid into CLA (Cepler and Tove (1967) J Biochem Chem 242:5686-5692).
  • linoleic acid predominantly exists in esterified form.
  • Lipids such as triacyl glycerides constitute the storage form, while thioesters such as acyl-CoA constitute the active form of the fatty acid.
  • Fatty acids with trans double bonds are extremely rare.
  • the seed oil of some plants contains fatty acids with double bonds in the trans position.
  • an E5-fatty acid has been detected in the seed oil of various Thalictrum species (Rankoff et al. (1971) J Amer Oil Chem Soc 48:700-701 ).
  • E5-desaturase activity has been described in Aquilegia vulgaris (Longman et al. (2000) Biochem Soc Trans 28:641- 643).
  • no plants have been described which contain either trans-vaccenic acid (E1 1 -octadecenoic acid) or E10-octadecenoic acid.
  • fatty acid ACP esters are desaturated by a soluble desaturase, predominantly at position 9, and
  • membrane lipids especially phosphatidyl cholins, are preferentially further desaturated at positions 6, 12 and 15 by membrane- bound desaturases.
  • conjugated fatty acids are produced by the activity of a conjugase (Crombie et al. (1984) J Chem Soc Chem Commun 15:953-955; Crombie et al. (1985) J Chem Soc Perkin Trans 1 :2425-2434; Fritsche et al. (1999) FEBS Letters 462: 249- 253; Cahoon et al. (2001 ) J Biol Chem 276:2637-2643; Qiu et al. (2001 ) Plant Physiol 125:847-855).
  • conjugated fatty acids such as calendulic acid, eleostearic acid or punicic acid proceeds via the desaturation of oleic acid to linoleic acid by a D12-desaturase and a further desaturation in conjunction with a rearrangement of the Z9- or Z12-double bond to the conjutrienic fatty acid by a specific conjutriene-forming desaturase (conjugase).
  • conjugated fatty acids such as calendulic acid, eleostearic acid or punicic acid proceeds via the desaturation of oleic acid to linoleic acid by a D12-desaturase and a further desaturation in conjunction with a rearrangement of the Z9- or Z12-double bond to the conjutrienic fatty acid by a specific conjutriene-forming desaturase (conjugase).
  • conjugated linoleic acid by the enzymatic activity of conjugase.
  • Liu et al. describe an E11- desaturase from the pheromone gland of a moth species ("light brown apple moth"), which is likely to play a role in pheromone biosynthesis (Liu WT et al. (2002) Proc Natl Acad Sci USA 99(2):620-624.
  • Knipple et al. compare various integral membrane desaturases from moths and flies which have been isolated from the pheromone glands of these insects.
  • a fragment of an acyl-CoA desaturase (PgosVASQ) from Pectinophora gossypiella is described.
  • the corresponding sequence has been deposited under the GeneBank Ace. No.: AF482921. Neither the complete sequence nor the specific activity of the desaturase are described. It was named merely on the basis of homologies with other desaturases.
  • a first subject matter of the invention relates to processes for the production of triglycerides comprising unsaturated fatty acids by the recombinant expression of at least one fatty acid desaturase from insects of the order Lepidoptera.
  • the process for the production of triglycerides comprising unsaturated fatty acids comprises the recombinant expression of at least one fatty acid desaturase from insects of the order Lepidoptera in an organism selected from the group of the plant organisms, yeasts, fungi and algae.
  • fatty acid desaturases are employed which are capable of generating a double bond at position C8, C9, C10, C1 1 or C12 in fatty acids, fatty acid CoA esters or other fatty acid derivatives.
  • fatty acid desaturases which are capable of specifically generating a cis or trans double bond in fatty acids, fatty acid CoA esters or other fatty acid derivatives with a fatty acid chain length of 16 or 18 C atoms.
  • Very especially preferred are fatty acid desaturases with at least 65% homology with one of the fatty acid desaturases described by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 22.
  • the process is employed for producing conjugated linoleic acid as the unsaturated fatty acid.
  • triglycerides prepared by the process according to the invention, and their use for preparing foodstuffs, feedstuffs, cosmetics or fine chemicals.
  • the advantage of the process according to the invention is, in particular, the possibility of the direct production of CLA-containing triglycerides, starting from organisms, preferably plants, yeasts, fungi or algae, with a high oil content, such as, for example, oilseed rape or sunflower.
  • organisms preferably plants, yeasts, fungi or algae
  • a high oil content such as, for example, oilseed rape or sunflower.
  • eukaryotic enzymes from insects of the order Lepidoptera makes possible good expression without the toxic effects frequently linked to prokaryotic proteins.
  • Fatty acid desaturases refers to enzymes which are capable of introducing a double bond into fatty acids or their derivatives, such as, for example, preferably fatty acid CoA esters. If appropriate, cofactors such as NADPH, NADH or else oxygen may additionally be necessary for this purpose. Preferred in this context are those desaturases which are capable of utilizing those acyl-CoA fatty acids as substrate whose fatty acid has a chain length of 14, 16, 18 or 20 C atoms, preferably 18 C atoms.
  • fatty acid desaturases encompasses those enzymes which are capable of generating a double bond at position C8, C9, C10, C1 1 or C12 in fatty acids or their derivatives, such as, for example, preferably fatty acid CoA esters.
  • those desaturases which lead to specific structural isomers, that is to say specifically to cis or trans double bonds. Specifically in this context is understood as meaning that the structural isomer in question is formed in an amount of at least 60%, preferably at least 80%, very especially preferably at least 90%, most preferably at least 95%.
  • desaturases which generate, in fatty acids or their derivatives, such as, for example, preferably fatty acid CoA esters, a double bond as is found in a CLA isomer.
  • Preferred essential characteristic of a fatty acid desaturase from lepidopterans refers to enzymes with at least one of the following characteristics:
  • substrate specificity refers to the characteristic of a fatty acid desaturase, of converting substrates of the stated chain length more rapidly than substrates of a different chain length.
  • the conversion rate of the preferred substrate is increased by at least 50%, preferably by at least 100%, very especially preferably by at least 200%, most preferably by at leat 500% in comparison with the substrates which are not preferred.
  • Preferred is at least in each case one of characteristics i) and ii).
  • fatty acid desaturases encompasses enzymes which are capable of introducing an isolated double bond, but also conjugases which, starting from one double bond in a substrate, are capable of generating a conjugated double bond system. In this context, it is preferably the first double bond which is moved.
  • these enzymes can be referred to as (E10,Z12)-conjugases.
  • These enzymes can also be referred to as E10-desaturases.
  • the essential characteristic of the very especially preferred E10-desaturates is the introduction of a trans double bond at position C-10 of a fatty acid or of a fatty acid CoA ester, where the fatty acid has a chain length of 18 C atoms.
  • conjugases are those which are identical to each other.
  • these enzymes can be referred to as (Z9,E1 1 )-conjugases.
  • These enzymes can also be referred to as E1 1 -desaturases.
  • the essential characteristic of the very especially preferred E1 1 -desaturases is the introduction of a trans double bond at position C1 1 of a fatty acid or of a fatty acid CoA ester, where the fatty acid has a chain length of 18 C atoms.
  • E10-, E1 1 -, Z10- and Z11 -desaturases and Z1 1 -(E10,Z12)- conjugase, E11 -(E10,Z12)-conjugase, Z10-(Z9,E11 )-conjugase and E10-(Z9,E11 )- conjugase with a substrate specificity for fatty acids or fatty acid derivative such as fatty acid CoA esters with a fatty acid chain length of 18 C atoms.
  • the fatty acid desaturases preferably originate from a Lepidoptera family selected from the group consisting of Acrolepiidae, Agaristidae, Arctiidae, Bombycidae, Carposinidae, Cochylidae, Cossidae, Eriocraniidae, Gelechiidae, Geometridae, Gracillariidae, Hepialidae, Ithomiidae, Lasiocampidae, Lycaenidae, Lymantriidae, Lyonetiidae, Nepticulidae, Noctuidae, Notodontidae, Nymphalidae, Oecophoridae, Papilionidae, Pieridae, Psychidae, Pterophoridae, Pyralidae, Saturniidae, Sesiidae, Sphingi
  • E1 1 -desaturases are preferably isolated from organisms of the species Diaphania hyalinata, Diaphania nitidalis, Leucinodes orbonalis, Ostrinia nubilalis, Sesamia grisescens, Brachmia macroscopa, Paraargyresthia japonica, Mnesictena flavidalis, Telorta edentata, Epiplema moza, Argyresthia chamaecypariae, Bradina sp., Dichrocrocis punctiferalis, Cryptoblabes gnidiella, Palpita unionalis, Sceliodes cordalis, Anisodes sp., Caloptilia theivora,
  • the E1 1 -desaturases, or the nucleic acid sequences encoding them, are especially preferably isolated from the pheromone glands of the above insects.
  • An example which may be mentioned is the E1 1 -desaturase from the pheromone glands of the light brown apple moth (Epiphyas postvittana) (SEQ ID NO: 2), from Ostrinia nubilalis (SEQ ID NO:4) and from Ostrinia furnicalis (SEQ ID NO: 6).
  • E11 -desaturase from Pectinophora gossypiella as shown in SEQ ID NO: 22.
  • the E1 1 -desaturases from the abovementioned organisms accept the plant acyl-CoA fatty acid derivatives as substrate.
  • desaturases are particularly advantageous in as far as it makes possible an E11 double bond.
  • Said desaturases are preferably optimized with a view to being able preferably to convert C16 and/or C18 acyl-CoA substrates, if they are not already capable of doing so (such as, for example, the desaturase from Pectinophora gossypiella as shown in SEQ ID NO: 22).
  • a further subject matter of the invention therefore relates to polypeptides with E11 -desaturase activity, the polypeptide preferentially converting C16 and/or C18 acyl-CoA fatty acids and encompassing at least one sequence selected from the group consisting of
  • amino acid sequences with at least 65% homology with one of the amino acid sequences as shown in SEQ ID NO: 2, 4, 6 or 22, and
  • amino acid sequences which encompass a fragment of at least 20 contiguous amino acid residues of a sequence as shown in SEQ ID NO: 2, 4, 6 or 22.
  • polypeptide is especially preferably described by an amino acid sequence as shown in SEQ ID NO: 22.
  • a further subject matter of the invention relates to nucleic acid molecules which encode said desaturases.
  • the sequence is especially preferably as shown in SEQ ID NO: 21.
  • E10-desaturases, or the nucleic acid sequences encoding them are preferably isolated from organisms of the species Dichrocrocis chlorophanta, Dichrocrocis punctiferalis, Bombyx mandarina, Bombyx mori, Coloradia velda, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nuttalli or Notarcha derogata.
  • the E10-desaturases, or the nucleic acid sequences encoding them are especially preferably isolated from the pheromone glands of the abovementioned insects.
  • E9-desaturases, or the nucleic acid sequences encoding them are preferably isolated from organisms of the species Epiplema plaqifera, Phyllonorycter coryli, Phyllonorycter harrisella, Phyllonorycter sylvella, Gelechiinae, Bryotropha sp., Bryotropha terrella, Gelechia betulae, Adoxophyes orana, Exartema appendiceum, Zeiraphera canadensis, Loxostege neobliteralis, Ostrinia nubilalis, Dioryctria clarioralis, Dioryctria merkeli, Dioryctria resinosella, Spodoptera exigua, Spodoptera triturata oder Polia grandis.
  • the E9-desaturases, or the nucleic acid sequences encoding them are especially preferably isolated from the pherom
  • E8-desaturases or the nucleic acid sequences encoding them, are preferably isolated from organisms of the species Phyllonorycter saportella, Phyllonorycter sp. or Dichrocrocis punctiferalis.
  • the E8-desaturases, or the nucleic acid sequences encoding them are especially preferably isolated from the pheromone glands of the abovementioned insects.
  • Z11 -desaturases as can be employed advantageously for producing Z1 1-octadecenoic acid have been described for Bombyx mori (Ando et al. (1988) Agric Biol Chem. 52:473-478), Trichoplusia ni and Helicoverpa zea (Knipple et al. (1998) Proc Nat Acad Sci USA 95:15287-15292).
  • Z11 -desaturases can preferably be isolated from Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. Examples which may be mentioned are the desaturases from Helicoverpa zea (SEQ ID NO: 8), Trichoplusia ni (SEQ ID NO: 10) and Argyrotaenia velutinana (SEQ ID NO: 12). Z11 -desaturases, or the nucleic acid sequences encoding them, are especially preferably isolated from pheromone glands of the abovementioned insects.
  • Z10-desaturases are preferably isolated from organisms of the species Ctenopseustis filicis, Pseudexentera spoliana, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nuttalli, Eurhodope advenella, Mamestra configurata or Dichrocrocis punctiferalis, especially preferably from the pheromone glands of the abovementioned insects.
  • An example which may be mentioned is the desaturase from Planototrix octo (SEQ ID NO: 14).
  • (E10,Z12)-conjugases, or the nucleic acid sequences encoding them are preferably isolated from Bombyx mori, Phyllonorycter crataegella, Amorbia cuneana, Notocelia incarnatana, Notocelia uddmanniana, Bombyx mandarina, Coloradia velda, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nuttalli, Notarcha derogata, Rondotia menciana, Amphion floridensis, Hyles gallii, Hyloicus pinastri, Manduca sexta, Sphinx drupiferarum, Earias insulana, Nola confusalis or Notarcha basipunctalis. (E10,Z12)-conjugases or the nucleic acid sequences encoding them, are especially preferably isolated from
  • (Z9.E1 1 )-conjugases are preferably isolated from Diatraea saccharalis , Xyrosaris lichneuta, Dioryctria abietella, Stenoma cecropia, Phalonidia manniana, Pselnophorus vilis, Dioryctria abietella; Dioryctria rubella, Myelopsis tetricella, Jodis lactearia, Scopula personata, Spodoptera descoinsi, Spodoptera eridania, Spodoptera latifascia, Spodoptera littoralis or Spodoptera litura.
  • (Z9.E11)-conjugases are especially preferably isolated from the pheromone glands of the abovementioned insects.
  • palmitelaidic acid (9E-hexadecenoic acid) is produced via trans-desaturation at position C9, for example by an E9-desaturase. Palmitelaidic acid can subsequently be elongated via the enzymatic activity of an elongase to give trans-vaccenic acid (scheme 4).
  • Trans-vaccenic acid can then be converted by a D9-desaturase to give the (9Z,1 1 E)-CLA isomer. It has been demonstrated for mammals and humans that trans-vaccenic acid is converted into CLA by the enzymatic activity of an endogenous D9-desaturase (WO 99/20123; Santora JE et al. (2000) J Nutr 130:208-215; Adlof RO et al. (2000) Lipids 35: 131-135). Likewise, it has been demonstrated that insect cells which express a E11 -desaturase are capable of converting the resulting E1 1 -fatty acid starting material into a Z9,E11 -fatty acid
  • plant D9- desaturases are capable of catalyzing the conversion of trans-vaccenic acid into the (Z9,E1 1 )-CLA isomer.
  • this conversion can be further increased by additional expression of a Z9-desaturase.
  • cytosolically active Z9-desaturases such as, for example, the yeast Z9-desaturase.
  • E10-octadecenoic acid is initially formed by trans-desaturation at position C10 (scheme 3).
  • E8- hexadecenoic acid can be produced starting from palmitic acid via trans- desaturation at position C8.
  • the E8-hexadecenoic acid can subsequently be illustrated via the enzymatic activity of an elongase to give E10-octadecenoic acid (scheme 4).
  • This fatty acid will be converted by the activity of a D 12-desaturase to give the (E10,Z12)-CLA isomer.
  • This reaction is also catalyzed by plant D12- desaturases. In a particularly preferred embodiment, this conversion rate can be increased further by additional expression of a Z12-desaturase.
  • Corresponding (E10,Z12)-conjugases from lepidopterans may also be employed. These conjugases convert for example Z11-octadecenoic acid (cis-vaccenic acid), E1 1-octadecenoic acid (trans-vaccenic acid) or Z10-otadecenoic acid into (E10,Z12)-CLA. To this end, stearic acid is first converted into Z1 1 -octadecenoic acid, as a result of the effect of a Z1 1 -desaturase, into trans-vaccenic acid as described above or into Z10-octadecenoic acid by a Z10-desaturase.
  • (Z9,E11 )-conjugases from lepidopterans can furthermore also be combined advantageously with (Z11 )-, (Z10)- or (E10)-desaturases.
  • these desaturases produce Z11 -octadecenoic acid (cis-vaccenic acid), Z10- octadecenoic acid or E10-octadecenoic acid, which are subsequently converted by a (Z9/E11 )-conjugase to give (Z9.E1 1 )-CLA.
  • the CLA fatty acids thus prepared, or their derivatives such as CoA fatty acid esters, are stored in the membrane lipids and triacyl glycerides.
  • the desirable enzyme activities have been localized in insects of the order lepidopterans, in particular in the abovementioned insect species.
  • the assay systems provided within the present invention were used for this purpose (Examples 2, 3 and 4).
  • the enzymes encoding these activities, or the nucleic acid sequences encoding them, can be isolated from the organisms in question in the manner with which the skilled worker is familiar or deduced by mutagenesis from corresponding known sequences.
  • Finding a protein sequence or a corresponding cDNA sequence to an enzymatic activity, functionality or phenotype is a traditional task in biochemistry and molecular biology.
  • the skilled worker is familiar with a variety of processes for solving this problem.
  • these methods are based on the assays provided within the present invention for determining the desaturase or conjugase activity (see, inter alia, Example 3).
  • the specific activity of a desaturase/conjugase in question is determined via analyzing the fatty acid pattern, for example by gas chromatography as described in Examples 2, 3 and 4.
  • expression cloning may be employed by way of method. This method has frequently been employed for isolating, starting from a particular enzymatic activity, a particular functionality or a particular phenotype, the gene responsible therefor or the cDNA corresponding to this gene (Dalboge H (1997) FEMS Microbiology Reviews 21 (1 ):29-42, Simonsen H and Lodish HF (1994) Trends Pharmacol Sci 15(12):437- 441 ). The traditional method of expression cloning has been described on a number of occasions, for example for membrane proteins, secreted factors and transmembrane channels (Masu Y et al.
  • an expression library can be generated starting from an organism, from cells or tissue which are capable of generating a desaturase or conjugase activity.
  • mRNA or, preferably, poly(A)-mRNA is first isolated from said organism, cell or tissue in a manner with which the skilled worker is familiar, and cDNA is prepared on the basis of what has been isolated (Gubler U, Hoffman BJ (1983) Gene 25:263-269).
  • the skilled worker is familiar with a variety of systems for isolating mRNA or poly(A)- mRNA and they are commercially available.
  • the synthesis can be carried out using the "Quick Prep Micro mRNA Purification Kit" (Amersham Pharmacia Biotech).
  • the first-strand cDNA synthesis is preferably carried out with an oligo(dT) primer using a reverse transcriptase (Borson ND et al. (1992) PCR Methods Appl 2:144-148; Chenchik A et al. (1994) CLONTECHniques 9(1 ):9-12).
  • a reverse transcriptase Bosset ND et al. (1992) PCR Methods Appl 2:144-148; Chenchik A et al. (1994) CLONTECHniques 9(1 ):9-12.
  • a variety of systems which make possible the generation of full-length cDNAs are known and commercially available. An example which may be mentioned is the "SMARTTM cDNA Library Construction Kit" (Clontech, Cat.#K1051 -1 ).
  • SMART Switch Mechanism At the 5' end of RNA Templates
  • cDNA libraries Herrler M (2000) J Mol Med 78(7):B23.
  • Specific oligonucleotides and the method which is known as long-distance PCR are employed. The method is described in Example 4.
  • Adaptors with single-stranded overhangs can be ligated to the double-stranded cDNAs; these adaptors make possible cloning for example in an expression vector which has been cleaved with restriction enzymes and has compatible cohesive ends.
  • cloning is directed so that the direction of reading is fixed and any promoter which may be present generates sense RNA starting from the cDNA insertion.
  • compatible Sfil (A) and Sfil (B) overhangs are used for directed cloning into an Sfil (recognition sequence GGCCNNNNINGGCC)- cleaved vector.
  • cDNA molecule is cloned into a vector molecule, so that eventually a multiplicity of vectors which are based on the same basic vector and which differ with regard to the integrated cDNA are present and form the expression libirary.
  • This expression library also contains vectors which are capable of expressing a desaturase or conjugase transgene. Suitable expression vectors are, in principle, all those which are capable of recombinantly expressing, in a transformed organism, desaturases or conjugases in active form.
  • Examples which may be mentioned are the lambda TriplEX2 vector (after excision pTriplEX2 vector; manufacturer: Clontech) for the recombinant expression in E.coli or the vector pYES2 (manufacturer: Invitrogen) for the recombinant expression in the yeast S.cerevisiae (see Example 4).
  • a normalization can be carried out in order to compensate for differences in the expression level of individual genes, so that the cDNA to each gene which is expressed is represented in the expression library with a similar copy number, independently of the actual expression level.
  • a variety of systems have been described for the generation of poly(A)-mRNA, cDNA and standardized cDNA (US 5,482,845) (Soares MB et al. (1994) Proc Natl Acad Sci USA 91 :9228-9232; Carninci P et al. (2000) Genome Res 10:1617-1630; Bonaldo MF et al. (1996) Genome Res 6:791 -806).
  • a subtractive expression library may also be established.
  • the cDNAs of an organism, of cells or of tissue with a desaturase or conjugase are compared with the cDNAs of an organism, of cells or of tissue without this activity, if possible of the same genus and species.
  • the skilled worker is familiar with methods for the targeted isolation of cDNAs which are only expressed in the biological material with desaturase or conjugase activity. Such methods and systems are described and commercially available.
  • the cDNAs in the expression library can be expressed recombinantly for example in prokaryotic or eukaryotic cells which are subsequently subjected to an assay for the desaturase or conjugase protein, for example an assay for a desaturase or conjugase activity.
  • the expression library is preferably transformed into an organism.
  • individual cells of the said organism are transformed in such a way that each cell, or each organism which is regenerated starting from this cell, is transformed with one type of vector molecule only.
  • all those organisms, or cells derived from them, which are capable of recombinantly expressing an active desaturase or conjugase are suitable for transformation. These organisms may be prokaryotic and eukaryotic. Preferred are all plants, cells derived from them, but also other photosynthetic organisms such as, for example, algae.
  • Preferred organisms are eukaryotic organisms, very especially preferably eukaryotic organisms which are capable of synthesizing fatty acids or acyl- CoA fatty acids.
  • the expression vectors used for the transformation contain a selection marker, for example a resistance antibiotic or an amino acid synthesis gene for selection for amino acid deficiency, which makes possible the selection of successfully transformed cells. Further selection methods are described hereinbelow.
  • expression cloning can be carried out in yeast.
  • yeast expression system from Invitrogen which is based on the vector pYES2.
  • pYES2 is a high-copy episomal vector for inducible expression of recombinant proteins in S. cerevisiae.
  • Transformed yeast strains are selected on the basis of uracil deficiency (pYES2 contains the ura3 gene).
  • the desaturase activity can be detected by culturing a transformed organism or cells derived therefrom, digesting it/them in a suitable buffer or solvent, bringing the digest into contact with fatty acids or acyl-CoA fatty acids and, if appropriate, with a cofactor such as NADH or NADPH or oxygen, and detecting the resulting desaturated fatty acids or acyl-CoA fatty acids.
  • the fatty acids or acyl-CoA fatty acids can preferably originate from the transformed organism itself if the organism or the cell derived from it is itself capable of synthesizing fatty acids or acyl-CoA fatty acids. If not, however, it is also possible to add fatty acids or acyl-CoA fatty acids.
  • the fatty acid or acyl-CoA fatty acid which has been modified by the desaturase or conjugase can be detected via customary methods with which the skilled work is familiar, if appropriate after extraction from the incubation mixture, for example with a solvent such as ethyl acetate.
  • Separation methods such as high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatogaphy (TLC) and detection methods such as mass spectroscopy (MS or MALDI), UV spectroscopy or autoradiography may be employed for this purpose.
  • the detection is carried out using gas chromatography as described in Examples 3, 4 and 5.
  • the transformed organisms, or cells derived from them, which are capable of recombinantly expressing a desaturase or conjugase can be isolated for example by dividing the total number of cells into subgroups, culturing these subgroups, if appropriate separately, and measuring the desaturase or conjugase activity of the individual subgroups.
  • the subgroup which contains a cell type which recombinantly expresses a desaturase or conjugase protein in functionally active form has an increased desaturase or conjugase activity.
  • This subgroup is subdivided further and the procedure is repeated until the result is a monoclonal culture, i.e. a culture which contains only one vector with a specific cDNA which encodes a desaturase or conjugase.
  • the vector which contains the nucleic acid encoding the desaturase or conjugase can be recovered from the transformed organism or cell. This can be done for example by means of polymerase chain reaction (PCR). To this end, oligonucleotide primers which are complementary to vector sequences which flank the desaturase or conjugase cDNA insert are used. A knowledge of the desaturase or conjugase nucleic acid sequence is not required in this context.
  • the vector if not integrated into the host genome, can be recovered from the cells, transformed into E.coli, propagated and sequenced in order to obtain the nucleic acid sequence encoding the desaturase or conjugase.
  • the desaturase or conjugase nucleic acid sequence can also be isolated from the isolated vector by means of PCR, without knowing its sequence order.
  • linkers which make possible directed cloning, may be added to the amplicon after the PCR reactions.
  • cloning can also be effected by means of blunt-end ligation or T-overhang ligation in a manner with which the skilled worker is familiar.
  • the desaturase or conjugase cDNA which has been amplified selectively by means of PCR may thus also be cloned directly into a vector suitable for generating a transgenic organism which expresses a desaturase or conjugase, without previous sequence elucidation.
  • This cloning procedure can be effected for example as a blunt-end cloning procedure using techniques with which the skilled worker is familiar. Customary recombination and cloning techniques as cited above are used for this purpose.
  • the above-described method of expression cloning can especially preferably be carried out in the yeast Saccharomyces cerevisiae. To this end, it is also possible to use systems in which the cDNAs to be expressed recombinantly are integrated, by means of homologous recombination, into an integration platform which has been generated (Lagarde D et al. (2000) Applied and Environmental Microbiology 66(1 ): 64- 72).
  • an oligo(dT)-primed cDNA library is constructed as described above and cloned into a high-copy expression plasmid with a T3, T7 or SP6 promoter.
  • This plasmid library is then transformed into E.coli.
  • approximately 50 to 100 independent tranformants are cultured on an agar plate with the corresponding selection antibiotic to a colony size of approx. 1 mm, collected and pooled. Plasmid DNA is isolated from part of this bacterial pool.
  • This plasmid DNA which is used as template, is transcribed directly in a reticulocyte system and translated.
  • the nucleic acid encoding the desaturase or conjugase can be amplified by PCR from the vectors present in this preparation and - if appropriate without knowledge and analysis of the sequence - used as described above for generating an organism which recombinantly expresses a desaturase or conjugase.
  • wheatgerm extract may also be used for the combined transcription/translation procedure (for example using the TNTTM Wheat Germ Extract System from Clontech).
  • the cDNAs can be cloned into a retroviral vector.
  • Retroviral expression vectors and expression systems are described (Kitamura T, International Journal of Hematology 1998, 67:351 -359) and commercially available (for example from Clontech; New Retroviral & ClonCapture Expression Libraries; CLONTECHniques October 1998, XIII(4):22-23).
  • Transfection is initially performed in a packaging cell line (for example EcoPackTM-293 or RetroPackTM PT67 Cell Line from Clontech). Using the viruses generated in this manner, the target cell line in question can be transfected and selected for the desired activity.
  • the inserts can be obtained and analyzed, for example by PCR. Furthermore, they can be cloned directly into a suitable expression vector, even without previously subjecting them to sequence analysis, and this expression vector can then be used for generating an organism which recombinantly expresses desaturase or conjugase.
  • E10- or E11 -desaturases can be obtained by modifying known cis-desaturases via mutagenesis in such a way that they are specific for desaturation in the trans-position.
  • mutagenesis for example the following nucleic acid sequences encoding cis- desaturases can act as starting sequences and be subjected to mutagenesis:
  • E1 1 -desaturases can be modified by mutagenesis in such a way that longer- chain fatty acids can be converted more efficiently, or at all.
  • Nucleic acid sequences encoding desaturases/conjugases for use in the method according to the invention can also be isolated by means of polymerase chain reaction using suitable degenerate oligonucleotide primers from cDNA preparations or libraries of the respective abovementioned Lepidoptera species. Those which are preferably employed are the oligonucleotide primer pair which is described by SEQ ID NO: 15 and 16 or the oligonucleotide primer pair which is described by SEQ ID NO: 17 and 18.
  • nucleic acid sequence of the fatty acid desaturase from lepidopterans which is employed in the process according to the invention preferably comprises
  • the protein sequence of the fatty acid desaturase from lepidopterans which is used in the process according to the invention, preferably has at least 65%, preferably at least 70%, especially preferably at least 80%, very especially preferably at least 90%, homology with one of the fatty acid desaturases described by SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 and - optionally and preferably - has at least one of the preferred essential characteristics of a desaturase or conjugase.
  • These proteins can also encompass natural or artificial mutations of one of the abovementioned desaturase nucleic acid sequences and their homologs from other animal or plant genera and species.
  • Mutations encompass substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. Where insertions, deletions or substitutions, such as, for example, transitions and transversions, are suitable, techniques which are known per se, such as in vitro mutagenesis, primer repair, restriction or ligation can be used. Complementary ends of the fragments can be provided for ligation by manipulations such as, for example restriction, chewing-back or filling up overhangs for blunt ends. Analogous results can also be obtained using the polymerase chain reaction (PCR) using specific oligonucleotide primers.
  • PCR polymerase chain reaction
  • the nucleic acid sequence encoding the fatty acid desaturase from lepidoterans which is employed in the process according to the invention preferably has at least 65%, preferably at least 70%, especially preferably at least 80%, very especially preferably at least 90% homology with one of the nucleic acid sequences described by SEQ ID NO: 1 , 3, 5, 7, 9, 11 or 13 and which - optionally and preferably - encodes a protein which has at least one of the preferred essential characteristics of a desaturase or conjugase.
  • Gap Weight 12 Length Weight: 4
  • nucleic acid sequence encoding the fatty acid desaturase from lepidopterans which is employed in the method according to the invention preferably hybridizes under standard conditions with one of the abovementioned nucleic acid sequences encoding desaturases, preferably with the sequence as shown in SEQ ID NO: 1 , 3, 5, 7, 9, 11 or 13.
  • Standard hybridization conditions is to be understood in the broad sense and refers to stringent or else less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning (A Laboratory Manual), 2 nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31 -9.57) or in Current Protocols in Molecular Biology, John Wiley
  • the conditions during the wash step can be selected from the range of conditions delimited by those with low stringency
  • the temperature during the wash step can be raised from low-stringency conditions at room temperature, approximately 22°C, to higher-stringency conditions at approximately 65°C.
  • the two parameters, salt concentration and temperature can be varied simultaneously or else one of the two parameters can be kept constant while only the other one is varied.
  • Denaturing agents such as, for example, formamide or
  • SDS can also be employed during the hybridization reaction.
  • the hybridization reaction is preferably carried out at 42°C.
  • nucleic acid sequence refers to, for example, a genomic or a complementary DNA (cDNA) sequence or an RNA sequence, and semi-synthetic or fully synthetic analogs thereof. These sequences can exist in linear or circular form, extrachromosomally or integrated into the genome.
  • the nucleotide sequences of the expression cassettes or nucleic acids according to the invention can be generated synthetically or obtained naturally or comprise a mixture of synthetic and natural DNA constituents, and consist of various heterologous gene segments of various organisms.
  • artificial nucleic acid sequences are suitable as long as they have the desired essential characteristics. For example, synthetic nucleotide sequences with codons which are preferred by plants to be transformed can be generated.
  • codons which are preferred by plants can be determined in the customary manner on the basis of the codon usage from codons with the highest protein frequency.
  • coding nucleotide sequences which have been obtained by backtranslation of a polypeptide sequence in accordance with the host-plant-specific codon usage.
  • nucleotide sequences can be synthesized chemically in the manner known per se from the nucleotide units, such as, for example, by fragment condensation of individual, overlapping complementary nucleic acid units of the double helix.
  • Oligonucleotides can be synthesized chemically for example in a manner known per se by the phosphoamidite method (Voet, Voet, 2 nd edition, Wiley Press New York, pages 896-897).
  • various DNA fragments can be manipulated in such a way that a nucleotide sequence with the correct direction of reading and the correct reading frame is obtained.
  • Adapters or linkers can be added to the fragments in order to link the nucleic acid fragments with each other.
  • the addition of synthetic oligonucleotides and the filling up of gaps with the aid of the Klenow fragment of the DNA polymerase, ligation reactions and general cloning methods are described by Sambrook et al. (1989). Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
  • transgenicTrecombinant refers to all those constructions which have come about by recombinant methods, or to their use, in which either
  • a genetic control sequence for example a promoter, linked operably to the nucleic acid sequence encoding a desaturase, or
  • the term natural genetic environment refers to the natural chromosomal locus in the organism of origin or to the presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably retained at least in part.
  • the environment flanks the nucleic acid sequence at least unilaterally and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp.
  • non-natural, synthetic processes such as, for example, mutagenization.
  • Recombinant expression refers to the use of a recombinant expression cassette for expressing a nucleic acid sequence.
  • the invention furthermore relates to recombinant expression cassettes which comprise a desaturase-encoding nucleic acid sequence, and vectors encompassing these expression cassettes.
  • a desaturase-encoding nucleic acid molecule is preferably in operable linkage with at least one genetic control element (for example a promoter) which ensures the recombinant expression (transcription and/or translation) of said desaturase in an organism, preferably in plants, plant organisms, algae, yeasts or fungi.
  • a genetic control element for example a promoter
  • plant-specific genetic control elements for example promoters
  • the desaturase can also be expressed in other organisms or in vitro.
  • preferred prokaryotic or eukaryotic genetic control elements are all those which allow for expression in the organism chosen in each case for the production.
  • the invention furthermore therefore relates to recombinant expression cassettes comprising at least one nucleic acid sequence encoding a fatty acid desaturase from insects of the order Lepidoptera under the control of a promoter which is functional in plants, plant organisms, algae, yeasts or fungi.
  • the nucleic acid sequence which is present in the expression cassettes encodes a polypeptide which encompasses at least one sequence selected from the group consisting of
  • amino acid sequences as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 22 and b) amino acid sequences with at least 65% homology with one of the amino acid sequences as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 22, and
  • amino acid sequences which encopass a fragment of at least 20 contiguous amino acid sequences of a sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12,
  • the fatty acid desaturase is especially preferably described in this context by an amino acid sequence as shown in SEQ ID NO: 22.
  • the invention furthermore relates to recombinant expression vectors which comprise at least one of the expression cassettes according to the invention and to transgenic organisms which comprise at least one of the recombinant expression cassettes according to the invention or one of the recombinant expression vectors according to the invention.
  • the transgenic organism is a plant organism, especially preferably selected from the plants used for oil production such as, for example, sunflower, sesame, safflower, olive tree, soya, linseed, peanut, castor-oil plant, oil palm, maize, wheat, cocoa bush and nut species.
  • Operable linkage is understood as meaning, for example, the sequential arrangement of a promoter with the desaturase nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that each of the regulatory elements can fulfil its function when the nucleic acid sequence is expressed recombinantly.
  • Direct linkage in the chemical sense is not necessarily required for this purpose.
  • Genetic control sequences such as, for example, enhancer sequences can also exert their function on the target sequence from positions which are further removed or indeed from other DNA molecules.
  • Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter so that the two sequences are linked covalently to each other.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.
  • Operable linkage and a transgenic expression cassette can both be produced by means of conventional recombination and cloning techniques as they are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML und Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley
  • the recombinant expression cassette composed of a promoter linked to a nucleic acid sequence to be expressed can be in a vector-integrated form and can be inserted into a plant genome, for example by transformation.
  • transgenic expression cassette is also understood as meaning those constructs where the nucleic acid sequence encoding a desaturase is placed behind an endogenous promoter in such a way that the latter controls the expression of the desaturase.
  • the fusion of endogenous promoter and desaturase nucleic acid sequence, which is brought about by the insertion, is a recombinant expression cassette for the purposes of the invention.
  • Plant-specific promoters are understood as meaning any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues or plant cultures.
  • expression may be, for example, constitutive, inducible or development-dependent. The following are preferred:
  • Preferred vectors are those which make possible a constitutive expression in plants (Benfey et al. (1989) EMBO J.8, 2195-2002).
  • “Constitutive” promoter refers to those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of plant development, preferably at all times during plant development.
  • a plant promoter or promoter originating from a plant virus is especially preferably used.
  • the promoter of the CaMV (cauliflower mosaic virus) 35S transcript (Franck et al. (1980) Cell 21 :285-294; Odell et al. (1985) Nature 313:810-812; Shewmaker et al.
  • X03677 the promoter of the nopalin synthase from Agrobacterium, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), the ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18:675-689; Bruce et al.
  • promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds.
  • Seed-specific promoters such as, for example, the phaseolin promoter (US 5,504,200; Bustos MM et al.
  • seed-specific promoters are those of the gene encoding high-molecular weight glutenin (HMWG), gliadin, branching enyzme, ADP glucose pyrophosphatase
  • Promoters which are furthermore preferred are those which permit a seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like.
  • the promoter of the Ipt2 or Ipt1 gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzin gene, the prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the casirin gene or the secalin gene) can advantageously be employed.
  • Tuber-, storage-root- or root-specific promoters such as, for example, the class I patatin promoter (B33) and the promoter of the cathepsin D inhibitor from potato.
  • Leaf-specific promoters such as the promoter of the potato cytosolic FBPase (WO 97/05900), the SSU promoter (small subunit) of Rubisco (ribulose-1 ,5-bisphosphate carboxylase) or the potato ST-LSI promoter (Stockhaus et al. (1989) EMBO J 8:2445-2451 ).
  • Flower-specific promoters such as, for example, the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
  • Anther-specific promoters such as the 5126 promoter (US 5,689,049, US 5,689,051 ), the glob-l promoter and the g-zein promoter.
  • the recombinant expression cassettes may also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a particular point in time.
  • a chemically inducible promoter such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361 -366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide- inducible promoter (EP 0 388 186), a tetracyclin-inducible promoter (Gatz et al.
  • promoters which are preferred are those which are induced by biotic or abiotic stress, such as, for example, the pathogen-inducible promoter of the PRP1 gene (Ward et al. (1993) Plant Mol Biol 22:361 -366), the heat-inducible hsp70 or hsp80 promoter from tomato (US 5,187,267), the low-temperature- inducible alpha-amylase promoter from potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinll promoter (EP375091 ).
  • the pathogen-inducible promoter of the PRP1 gene Ward et al. (1993) Plant Mol Biol 22:361 -366
  • the heat-inducible hsp70 or hsp80 promoter from tomato US 5,187,267
  • the low-temperature- inducible alpha-amylase promoter from potato
  • Pathogen-inducible promoters encompass the promoters of genes which are induced as the consequence of a pathogen attack, such as, for example, genes of PR proteins, SAR proteins, b-1 ,3-glucanase, chitinase and the like (for example Redolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al.
  • wound-inducible promoters such as the promoter of the pinll gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) Nat
  • suitable promoters are, for example, fruit-maturation-specific promoters, such as, for example, the fruit-maturation-specific promoter from tomato (WO
  • Development-dependent promoters include some of the tissue-specific promoters since some tissues are formed naturally as the function of development.
  • promoters which make possible expression in further plant tissues or in other organisms such as, for example, E.coli bacteria, may be linked operably with the nucleic acid sequence to be expressed.
  • Suitable plant promoters are, in principle, all of the above-described promoters.
  • nucleic acid sequences present in the recombinant expression cassettes or vectors according to the invention can be linked operably with further genetic control sequences besides a promoter.
  • genetic control sequences is to be understood in the broad sense and refers to all those sequences which have an effect on the establishment or the function of the expression cassette according to the invention. Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms.
  • the recombinant expression cassettes according to the invention preferably encompass the promoter with specificity for the embryonal epidermis and/or the flower 5'-upstream of the nucleic acid sequence to be expressed recombinantly in each case and, as additional genetic control sequence, a terminator sequence 3'-downstream, and, if appropriate, further customary regulatory elements, in each case operably linked with the nucleic acid sequence to be expressed recombinantly.
  • Genetic control sequences also encompass further promoters, promoter elements or minimal promoters capable of modifying the expression-controlling properties.
  • genetic control sequences can, for example, bring about tissue-specific expression which is additionally dependent on certain stress factors.
  • Such elements are, for example, described for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991 ; 266(26): 17131 -17135) and thermal stress (Schoffl F et al. (1989) Mol Gen Genetics 217(2-3):246-53).
  • control sequences are, for example, in the Gram-positive promoters amy and SP02, and in the yeast or fungal promoters ADC1 , MFa, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH.
  • Genetic control sequences also further encompass the 5'-untranslated regions, introns or nonencoding 3'-region of genes, such as, for example, the actin-1 intron, or the Adh1 -S introns 1 , 2 and 6 (for general reference, see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been demonstrated that these may play a significant role in regulating gene expression. Thus, it has been demonstrated that 5'-untranslated sequences can enhance the transient expression of heterologous genes. Translation enhancers which may be mentioned by way of example are the tobacco mosaic virus 5' leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-871 1 ) and the like. They may furthermore promote tissue specificity (Rouster J et al. (1998) Plant J 15:435-440).
  • the recombinant expression cassette can advantageously contain one or more of what are known as enhancer sequences in operable linkage with the promoter, and these make possible an increased recombinant expression of the nucleic acid sequence. Additional advantageous sequences such as further regulatory elements or terminators may also be inserted at the 3' end of the nucleic acid sequences to be expressed recombinantly. One or more copies of the nucleic acid sequences to be expressed recombinanly may be present in the gene construct.
  • Polyadenylation signals which are suitable as control sequences are plant polyadenylation signals, preferably those which correspond essentially to Agrobacterium tumefaciens T-DNA polyadenylation signals, in particular those of gene
  • EMBO J 3:835 et seq. or functional equivalents thereof.
  • particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopaline synthase) terminator.
  • Control sequences are furthermore understood as those which make possible homologous recombination or insertion into the genome of a host organism, or removal from the genome.
  • Methods such as the cre/lox technology permit the tissue-specific, possibly inducible, removal of the recombinant expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14(4):381 -92).
  • certain flanking sequences are added to the target gene (lox sequences), and these make possible removal by means of ere recombinase at a later point in time.
  • a recombinant expression cassette and the recombinant vectors derived from it may comprise further functional elements.
  • the term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, replication or function of the recombinant expression cassettes, vectors or transgenic organisms according to the invention. Examples which may be mentioned, but not by way of limitation, are:
  • Selection markers which confer resistance to a metabolism inhibitor such as 2- deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinothricin and the like. Particularly preferred selection markers are those which confer resistance to herbicides.
  • Reporter genes which encode readily quantifiable proteins and which allow the transformation efficiency or the expression site or time to be assessed via their color or enzyme activity.
  • reporter proteins such as the "green fluorescent protein” (GFP) (Sheen et al.(1995) Plant Journal 8(5):777-784); Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et al.
  • Replication origins which allow replication of the recombinant expression cassettes or vectors according to the invention in, for example, E.coli.
  • ORI oil of DNA replication
  • the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a selectable marker which confers resistance to a biocide for example a herbicide
  • a metabolism inhibitor such as 2-deoxyglucose-6-phosphate
  • the recombinant expression cassette according to the invention can have the following structure:
  • the recombinant expression cassette according to the invention preferably has the following structure:
  • Cotransformation with more than one of the abovementioned examples a) or b) may be advantageous for the advantageous CLA biosynthesis processes according to the invention.
  • transformation with one or more vectors, each of which contains a combination of the abovementioned recombinant expression cassettes, may be advantageous.
  • said recombinant expression cassette may comprise a nucleic acid sequence whose recombinant expression brings about an increase in fatty acid biosynthesis (hereinbelow proOIL).
  • This proOIL nucleic acid sequence which is additionally expressed recombinantly can be selected by way of example but not by limitation from among nucleic acids encoding acetyl-CoA carboxylase (ACCase), glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT), diacylglycerol acyltransferase (DAGAT) and phospholipid:diacylglycerol- acyltransf erase (PDAT).
  • ACCase acetyl-CoA carboxylase
  • GPAT glycerol-3-phosphate acyltransferase
  • LPAT lysophosphatidate acyltransferase
  • DGAT diacyl
  • the proOIL nucleic acid sequences also encompass those nucleic acids whose recombinant expression generates an antisense-RNA or a duplex RNA which also brings about an increase in the fatty acid production.
  • Preferred examples encompass vectors comprising the following recombinant expression cassettes:
  • the desaturase or proOIL nucleic acids or recombinant expression cassettes according to the invention can be cloned into suitable vectors which allow their propagation in, for example, E. coli.
  • suitable cloning vectors are pBR332, pUC series, M13mp series and pACYC184, inter alia.
  • binary vectors which are capable of replication both in E. coli and in agrobacteria.
  • the desaturase or proOIL nucleic acids or recombinant expression cassettes according to the invention are preferably inserted into suitable transformation vectors.
  • suitable vectors are described, inter alia, in "Methods in Plant Molecular Biology and Biotechnology” (CRC Press), chapter 6/7, p. 71-119 (1993). Transformation examples and transformation processes are described hereinbelow.
  • the invention furthermore relates to transgenic organisms transformed with at least one recombinant expression cassette according to the invention or with a vector according to the invention, and to cells, cell cultures, tissues, parts - such as, for example, leaves, roots and the like in the case of plant organisms - or propagation material derived from such organisms.
  • organism starting organisms or host organisms are understood as meaning prokaryotic or eukaryotic organisms such as, for example, microorganisms or plant organisms.
  • Preferred microorganisms are bacteria, yeasts, algae or fungi.
  • Preferred bacteria are bacteria from the genus Escherichia, Corynebacterium, Bacillus, Clostrridium, Proionibacterium, Butyrivibrio, Eubacterium, Lactobacillus, Erwinia,
  • Agrobacterium Flavobacterium, Alcaligenes, Phaeodactylum, Colpidium, Mortierella, Entomophthora, Mucor, Crypthecodinium or cyanobacteria, for example of the genus Synechocystis.
  • microorganisms which are capable of infecting plants and thus of transferring the constructs according to the invention.
  • Preferred microorganisms are those of the genus Agrobacterium, in particular of the species Agrobacterium tumefaciens.
  • Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.
  • Particularly preferred yeasts are those in which fatty acids/fatty acid derivatives amount to at least 20%, preferably 40%, especially preferably 60% of the cell dry matter, in particular yeasts such as Cryptococcus curvatus (Ratledge (1989) Biotechnology of oils and fats. In: Microbiol lipids Vol.2 Academic Press, London, pp. 567-668).
  • Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria, Phytophthora infestans or further fungi described in Indian Chem Engr. Section B. Vol 37, No 1 ,2 (1995), page 15, Table 6.
  • fungi in which fatty acids/fatty acid derivatives amount to at least 10%, preferably 20 % of the cell dry matter, in particular Mucor circinelloides and Mortierella alpina (Wynn et al. (2001 ) Microbiology 147: 2857-2864).
  • Preferred transgenic organisms are, in particular, plant organisms.
  • plant organism encompasses any organism which is capable of photosynthesis and cells, tissues, parts or propagation material (such as seeds or fruit) derived therefrom. Included for the purpose of the invention are all genera and species of higher and lower plants of the plant kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants and gymnosperms are preferred. Included are the mature plant, seeds, shoots and seedlings, and parts derived therefrom, propagation material (for example tubers, seeds or fruit) plant organs, tissues, protoplasts, callus cultures and other cultures, for example cell cultures.
  • aliveMature plants refers to plants at any developmental stage beyond the seedling stage. The term seedling refers to a young immature plant at an early developmental stage.
  • Plant encompasses all annual and perennial monocotyldedonous or dicotyledonous plants and includes by way of example, but not by limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemericallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus,
  • Preferred plants are those from the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceae, Umbelliferae.
  • Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants such as, for example, the Gramineae family, such as alfalfa, rice, maize, wheat or other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
  • the Gramineae family such as alfalfa, rice, maize, wheat or other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
  • the invention is applied very particularly preferably to dicotyledonous plant organisms.
  • Preferred dicotyledonous plants are selected in particular from the dicotyledonous crop plants such as, for example,
  • Asteraceae such as sunflower, tagetes or calendula and others,
  • Compositae especially the genus Lactuca, very particularly the species sativa (lettuce) and others,
  • Cruciferae particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other cabbages; and the genus Arabidopsis, very particularly the species thaliana, and cress or canola and others,
  • Cucurbitaceae such as melon, pumpkin/squash or zucchini and others
  • Leguminosae particularly the genus Glycine, very particularly the species max (soybean), soya, and alfalfa, pea, bean or peanut and others,
  • Rubiaceae preferably the subclass Lamiidae such as, for example Coffea arabica or Coffea liberica (coffee bush) and others,
  • Sterculiaceae preferably the subclass Dilleniidae such as, for example, Theobroma cacao (cacao bush) and others,
  • Theaceae preferably the subclass Dilleniidae such as, for example, Camellia sinensis or Thea sinensis (tea shrub) and others, Umbelliferae, particularly the genus Daucus (very particularly the species carota
  • angiosperms such as, for example, Hepaticae (liverworts) and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae, the families of the Rosaceae such as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, freesi
  • plant organisms for the purposes of the invention are further organisms capable of being photosynthetically active such as, for example, algae, cyanobacteria and mosses.
  • Preferred algae are green algae such as, for example, algae from the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.
  • plants which are suitable for oil production such as, for example, oilseed rape, sunflower, sesame, safflower (Carthamus tinctorius), olive tree, soya, linseed, peanut, castor-oil plant, oil palm, maize, wheat, cocoa bush or various nut species such as, for example, walnut, coconut or almond.
  • oilseed rape sunflower, sesame, safflower (Carthamus tinctorius), olive tree, soya, linseed, peanut, castor-oil plant, oil palm, maize, wheat, cocoa bush or various nut species such as, for example, walnut, coconut or almond.
  • oilseed rape sunflower
  • sesame sesame
  • safflower Carthamus tinctorius
  • olive tree soya
  • linseed peanut
  • castor-oil plant oil palm
  • maize wheat, cocoa bush
  • various nut species such as, for example, walnut, coconut or almond.
  • Preferred algae are green algae such as, for example, algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Others which may be mentioned as being preferred are protozoa such as dinoflagellates.
  • Preferred among abovementioned organisms are, in particular, those which are naturally capable of synthesizing oils in substantial amounts, such as fungi, e.g. Mucor circinelloides, Mortierella alpina, Pythium insidiosum, yeasts such as Saccharomyces cerevisiae or Cryptococcus curvatus, or plants such as soya, linseed, oilseed rape, coconut, oil palm, safflower, castor-oil plant, peanut, cacao tree or sunflower, especially preferably soya, oilseed rape, sunflower, Mucor circinelloides, Mortierella alpina, Pythium insidiosum, Cryptococcus curvatus or Saccharomyces cerevisiae.
  • fungi e.g. Mucor circinelloides, Mortierella alpina, Pythium insidiosum, yeasts such as Saccharomyces cerevisiae or Cryptococcus curvatus
  • yeasts such as Sac
  • microorganisms are grown in a liquid medium which contains a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron salts, manganese salts, magnesium salts and, if appropriate, vitamins, at temperatures between 0°C and 100°C, preferably between 10°C and 60°C, while passing in oxygen.
  • the pH value of the liquid nutrient medium may be kept constant, i.e. can be regulated during culturing or not. Culturing can be effected batchwise, semibatchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semicontinuously or continuously.
  • a recombinant expression cassette according to the invention can advantageously be introduced into an organism or into cells, tissues, organs, parts or seeds thereof (preferably into plants or plant cells, tissues, organs, parts or seeds) by using vectors in which the recombinant expression cassettes are present.
  • the recombinant expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site.
  • the resulting plasmid is first introduced into E.coli. Correctly transformed E.coli are selected, grown, and the recombinant plasmid is obtained by methods with which the skilled worker is familiar. Restriction analysis and sequencing may be used for verifying the cloning step.
  • An expression cassette according to the invention can advantageously be introduced into cells, preferably plant cells, using vectors.
  • vectors are plasmids, cosmids, phages, viruses or else agrobacteria.
  • the expression cassette is introduced by means of plasmid vectors.
  • Preferred vectors are those which make possible stable integration of the expression cassette into the host genome.
  • Generating a transformed organism requires introducing the DNA, RNA or protein in question into the host cell in question.
  • a multiplicity of methods are available for this procedure, which is referred to as transformation (or transduction or transfection) (Keown et al. (1990) Methods in
  • the DNA or RNA can be introduced directly by means of microinjection or by bombardment with DNA-coated microparticles.
  • the cell can be permeabilized chemically, for example using polyethylene glycol, so that the DNA can enter the cell by diffusion.
  • the DNA can also be introduced by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes.
  • Another suitable method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an electrical pulse. Such methods are described (Bilang et al. (1991 ) Gene 100:247-250; Scheid et al.
  • the desaturases or conjugases can be used for the recombinant modification of a wide range of organisms, preferably of plants, so that these become a de-novo producer of one or more products derived from lipids, such as the two CLA isomers. Plants are initially regenerated after the transformation step and subsequently cultured or grown as usual.
  • the plasmid used need not meet any particular requirements. It is possible to use simple plasmids such as those from the pUC series, pBR322, M13mp series, pACYCI 84 and the like. If intact plants are to be regenerated from the transformed cells, the plasmid must contain an additional selectable marker gene.
  • transformation may also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid (Ti and ⁇ i plasmid, respectively) which is transferred to the plant following infection with Agrobaterium. Part of this plasmid, referred to as T-DNA (transferred DNA), is integrated into the genome of the plant cell.
  • agrobacterium is also capable of transferring binary vectors (mini-Ti plasmids) to plants, and these vectors are integrated into the plants' genome.
  • Agrobacterium-mediated transformation is best suited to dicotyledonous, diploid plant cells, while the direct transformation techniques are suitable for any cell type. Methods for the agrobacterium-mediated transformation are described, for example, by Horsch RB et al. (1985) Science 225:1229f. If agrobacteria are used, the expression cassette is integrated into specific plasmids, namely either into a shuttle vector (intermediate vector) or into a binary vector. If a Ti or Ri plasmid is used for the transformation, at least the right border, but in this case the right and left border, of the Ti or Ri plasmid T-DNA is linked to the expression cassette to be inserted as flanking region.
  • Binary vectors are preferably used for transformation with Agrobacterium.
  • Binary vectors are capable of replication both in E.coli and in Agrobacterium.
  • they contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181 -187).
  • the selection marker gene which is, for example, the nptll gene, which confers resistance to kanamycin, permits a selection of transformed agrobacteria.
  • the agrobacterium which acts as host organism in this case should already contain a plasmid with the vir region. The latter is required for transferring the T-DNA to the plant cells.
  • An agrobacterium transformed in this way can be used for transforming plant cells.
  • the use of T-DNA for the transformation of plant cells has been studied intensively and described (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287).
  • Various binary vectors are known, some of which are commercially available, such as, for example, pBH 01.2 or pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al.(1984) Nucl Acids Res 12:8711 ), pBinAr, pPZP200 or pPTV.
  • the agrobacteria which have been transformed with such a vector can then be used in the known manner for transforming plants, in particular crop plants, such as, for example, oilseed rape, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently culturing them in suitable media.
  • the transformation of plants with agrobacteria is described (White FF, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15 - 38; Jenes B et al.(1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, edited by S.D. Kung and R.
  • Transgenic plants which contain the above-described desaturase or conjugase, pro-desaturase or conjugase or proOIL nucleic acids or recombinant expression cassettes or vectors according to the invention which are integrated can be regenerated in the known manner from the transformed cells of the scarified leaves or leaf segments.
  • Stably transformed cells i.e. those which contain the inserted DNA integrated into the DNA of the host cell, can be selected from untransformed cells when a selectable marker is part of the inserted DNA.
  • a selectable marker is part of the inserted DNA.
  • any gene which is capable of conferring resistance to antibiotics or herbicides such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin and the like
  • Transformed cells which express such a marker gene are capable of surviving in the presence of concentrations of such an antibiotic or herbicide which kill an untransformed wild type. Examples are mentioned above and preferably comprise the bar gene, which confers resistance to the herbicide phosphinothricin (Rathore KS et al.
  • the selection marker permits selection of transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81 -84).
  • the plants obtained can be bred and hybridized in the customary manner. Two or more generations should preferably be grown in order to ensure that the genomic integration is stable and hereditary.
  • CLA production means for the purposes of the present invention for example the artificially acquired ability of an increased biosynthesis rate of at least one compound from the group of CLA, its esters, such as, for example, CoA esters or glyceride esters, in the transgenic organism in comparison with the non-genetically-modified starting organism.
  • CLA production in the transgenic organism is preferably increased by 10%, especially preferably by 50%, very especially preferably by 100% in comparison with the non-genetically-modified organism.
  • Improved may also refer to an advantageously modified qualitative composition of the CLA mixture, i.e. an increased content in (9Z,1 1 E)-CLA and/or (10E,12Z)-CLA in comparison with the starting organism.
  • transgenic plant organisms Also in accordance with the invention are cells, cell cultures, parts - such as, for example, roots, leaves and the like in the case of transgenic plant organisms - and transgenic propagation material such as seeds or fruits which are derived from the above-described transgenic organisms.
  • the invention furthermore relates to the use of the above-described transgenic organisms according to the invention and to the cells, cell cultures, parts - such as, for example, roots, leaves and the like in the case of transgenic plant organisms - and transgenic propagation material such as seeds or fruits which are derived from them for the production of foodstuffs or feedstuffs, cosmetics or fine chemicals, such as free fatty acids, in particular CLA.
  • transgenic plant organisms - and transgenic propagation material such as seeds or fruits which are derived from them for the production of foodstuffs or feedstuffs, cosmetics or fine chemicals, such as free fatty acids, in particular CLA.
  • Particularly preferred is the use for the production of CLA- containing lipids, preferably triglycerides.
  • Genetically modified plants according to the invention which can be consumed by humans and animals may also be used as foodstuffs or feedstuffs, for example directly or following processing known per se.
  • the lipids are obtained in the customary manner.
  • the organisms can first be digested post-harvest or else used directly.
  • the lipids are advantageously extracted with suitable solvents such as apolar solvents, such as hexane or ethanol, isopropanol or mixtures such as hexane/isopropanol, phenol/chloroform/isoamyl alcohol, at temperatures between 0°C to 80°C, preferably between 20°C and 50°C.
  • suitable solvents such as apolar solvents, such as hexane or ethanol, isopropanol or mixtures such as hexane/isopropanol, phenol/chloroform/isoamyl alcohol, at temperatures between 0°C to 80°C, preferably between 20°C and 50°C.
  • the biomass is extracted with an excess of solvent, for example with a 1 :4 excess of solvent to biomass.
  • the solvent is subsequently removed, for example via distillation
  • the crude oil obtained in this manner can subsequently be purified further, for example by removing cloudiness by treatment with polar solvents such as acetone or chloroform, followed by filtration or centrifugation. Further purification over columns is also possible.
  • polar solvents such as acetone or chloroform
  • the invention furthermore relates to vegetable oils, fatty acid mixtures and/or triglyceride mixtures with an increased content of unsaturated fatty acids, preferably CLA, and which has been produced by the abovementioned processes according to the invention, and to their use for the production of foodstuffs, animal feed, cosmetics or pharmaceuticals. To this end, they are added in customary amounts to the foodstuffs, the animal feed, the cosmetics or pharmaceuticals.
  • SEQ ID NO: 1 Nucleic acid sequence encoding an acyl-CoA E1 1 -desaturase from Epiphyas postvittana.
  • SEQ ID NO: 2 Protein sequence encoding an acyl-CoA E11 -desaturase from Epiphyas postvittana.
  • SEQ ID NO: 3 Nucleic acid sequence encoding an acyl-CoA Z/E1 1 -desaturase from Ostrinia nubilalis.
  • SEQ ID NO: 4 Protein sequence encoding an acyl-CoA Z/E1 1 -desaturase from Ostrinia nubilalis.
  • SEQ ID NO: 5 Nucleic acid sequence encoding an acyl-CoA Z/E1 1 -desaturase from Ostrinia fumacalis. 6.
  • SEQ ID NO: 6 Protein sequence encoding an acyl-CoA Z/E11 -desaturase from Ostrinia furnacalis.
  • SEQ ID NO: 7 Nucleic acid sequence encoding an acyl-CoA ⁇ 11 -desaturase from Helicoverpa zea.
  • SEQ ID NO: 8 Protein sequence encoding an acyl-CoA ⁇ 11 -desaturase from Helicoverpa zea.
  • SEQ ID NO: 9 Nucleic acid sequence encoding an acyl-CoA ⁇ 11 -desaturase from Trichoplusia ni.
  • SEQ ID NO: 10 Protein sequence encoding an acyl-CoA ⁇ 11 -desaturase from Trichoplusia ni.
  • SEQ ID NO: 11 Nucleic acid sequence encoding an acyl-CoA ⁇ 11 -desaturase from Argyrotaenia velutinana.
  • SEQ ID NO: 12 Protein sequence encoding an acyl-CoA ⁇ 11 -desaturase from Argyrotaenia velutinana.
  • SEQ ID NO: 13 Nucleic acid sequence encoding an acyl-CoA Z10-desaturase from Planotortrix octo.
  • SEQ ID NO: 14 Protein sequence encoding an acyl-CoA Z10-desaturase from Planotortrix octo.
  • SEQ ID NO: 15 5'-ATYACHGCCGGKKMYCAYMG-3'
  • SEQ ID NO: 16 5'-GGRAABDYGTGRTGGWAGTT-3'
  • SEQ ID NO: 17 5'-CCCCAYCRNCTSTGGWCNCA-3'
  • SEQ ID NO: 18 5'-CCCTCTAGARTGRRWARTTRTGRWA-3'
  • SEQ ID NO: 19 5'-TAATACGACTCACTATAG-3'
  • SEQ ID NO: 20 5'-ACATAACTAATTACATGAT-3' 21.
  • SEQ ID NO: 21 Nucleic acid sequence encoding an acyl-CoA Z/E1 1- desaturase from Pectinophora gossypiella
  • SEQ ID NO: 22 Amino acid sequence encoding an acyl-CoA Z/E11 -desaturase from Pectinophora gossypiella
  • cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoreses, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, bacterial culture and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Oligonucleotides can be synthesized chemically for example in the known manner using the phosphoamidite method (Voet, Voet, 2 nd edition, Wiley Press New York, pp.896-897).
  • the cloning steps carried out within the present invention such as, for example, restriction cleavages, agarose gel electrophosesis, purification of DNA fragments, transfer of nucleic acids to vitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, propagation of phages and sequence analysis of recombinant DNA were carried out as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules were sequenced using a laser fluorescence DNA sequencer from Licor (sold by MWG Biotech, Ebersbach) following the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463-5467).
  • Example 1 Breeding the Lepidoptera insects
  • insects are kept on suitable host plants in terrarria.
  • the growth conditions are: 27°C, day-night rhythm: 14 h light, 10 hours darkness.
  • Insects and larvae are transferred to fresh plants twice per week.
  • Puppae are collected, and males and females are kept separately in terrarria until the adult insects hatch. Approximately 1 to 2 days after the insects have hatched, the pheromone glands can be isolated.
  • Example 2 Isolation of desaturase or conjugase cDNAs by means of degenerate primers
  • RNA is isolated from the abdomen of adult moths and frozen in liquid N 2 until total RNA is isolated, for example by means of TRIzol (Gibco/BRL) following the manufacturer's instructions.
  • TRIzol Gibco/BRL
  • RNA is employed to produce a first-strand cDNA with an oligo(dT) primer. This may be done for example using the SMART RACE cDNA amplification kit (Clontech) following the manufacturer's instructions.
  • This single-strand cDNA acts as template for a PCR in which the central region of the desaturase/ conjugase cDNA is amplified.
  • Two degenerate primers are designed in such a way that they are capable of amplifying the conserved central region of the desaturases/conjugases from lepidopterans and employed in the following PCR protocol:
  • the degenerate primers with SEQ ID NO: 10 and SEQ ID NO: 11 are used as primer pair for the amplification of the central desaturase region.
  • the PCR was carried out under the following cycle conditions:
  • the PCR product is ligated directly, for example into the linearized TOPO TA PCR 2.1 vector (Invitrogen) and subsequently transformed into competent E.coli TOP 10 cells (Invitrogen). Positive colonies are reamplified, the plasmid DNA is purified (Qiagen Plasmid Mini Kit) and subsequently sequenced. Owing to the sequence information obtained, gene-specific primers can be derived and used for amplifying 5' and 3' regions (SMART RACE cDNA Amplification Kit (Clontech)). Again, these PCR products are ligated into the TOPO TA PCR 2.1 vector (Invitrogen) and subsequently transformed into competent TOP 10 cells (Invitrogen). Positive colonies are reamplified, and the plasmid DNA is purified (Qiagen Plasmid Mini Kit) and subsequently sequenced.
  • the complete sequence of a desaturase/conjugase can be put together using standard cloning techniques and transferred for characterization purposes for example into the pYES2 vector (Invitrogen) for expression in yeast.
  • Saccharomyces INVSd (Invitrogen) are transformed with the corresponding pYES2 expression vectors by means of a modified PEG/lithium acetate protocol (Ausubel et al. (1996) Current Protocols in Molecular Biology. John Wiley and Sons, New York).
  • pYES2DESAT transformants pYES2DESATa-d
  • one pYES2 transformant are selected for further cultivation and functional expression.
  • Preculture 20 ml of CMdum liquid medium supplemented with 2% (w/v) raffinose were inoculated with the transgenic yeast clones (pYES2DESATa-d, pYES2) and cultured for 3 days at 30°C, 200 rpm, until an optical density at 600 nm (OD 60 o) of 1.5-2 had been reached.
  • CMdum liquid medium supplemented with 2% of raffinose and 1% (v/v) Tergitol NP-40 were concentrated with the corresponding substrates such as stearic acid, palmitic acid or myristic acid, to a final concentration of 0.003% (w/v).
  • the media were inoculated with the precultures to an OD 6 oo of 0.05.
  • Expression was induced for 16 hours at an OD 600 of 0.2 using 2% (w/v) galactose, whereafter the cultures had reached an OD 600 of 0.8 to 1.2.
  • Fatty acid analysis The total fatty acids were extracted from yeast cultures and analyzed by means of gas chromatography. To this end, cells of 5 ml of culture were harvested by centrifugation (1000 x g, 10 min, 4°C) and washed once with 100 mM NaHC03, pH 8.0 in order to remove residues of medium and fatty acids. To prepare the fatty acid methyl esters (FAMES), the cell sediments were shock-frozen in liquid N 2 and lyophilized at 30°C under N 2 gas. The pellet is homogenized in 1 % sodium methoxide in methanol and the homogenate is incubated for 20 minutes at room temperature.
  • Equal volumes of 1 M NaCI and n-heptane are subsequently added, the sample is mixed and the mixture is transferred into a GC tube.
  • the samples are separated from a DB-wax capillary column (30 m, 0.25 mm, 0.25 ⁇ m; Agilent J & W) in a Hewlett Packard 6890 gas chromatograph equipped with a flame ionization detector.
  • the oven temperature was programmed from 60°C (hold 5 min) to 200°C at a rate of 20°C/min (hold 20 min) and finally to 250°C (hold 30 min) at a rate of 20°C/min.
  • the carrier gas used was nitrogen (1.6 ml/min).
  • the fatty acids were identified by comparison with retention times of FAME standards (SIGMA).
  • the following fatty acids are preferably used as standard for this purpose: c9-16:1 , t9-16:1 , 18:0, c9-18:1 , t9- 18:1 , d 1 -18:1 , t11 -18:1 , c9,d 2-18:2, t9,t12-18:2, c9,t1 1 -18:2, t10,c12-18:2.
  • Example 4 Expression cloning of desaturase/conjugase from Lepidoptera in Saccharomyces cerevisiae
  • RNA The pheromone glands are isolated from the abdomen of adult moths and frozen in liquid N 2 until total RNA is isolated, for example by means of TRIzol (Gibco/BRL) following the manufacturer's instructions. Experience has shown that approximately 60 to 80 ⁇ g of total RNA can be isolated from approximately 30 mg fresh tissue. Approximately 5 ⁇ g of total RNA is employed for generating a cDNA library. This may be done for example using the SMART cDNA Library Construction Kit (Clontech). The isolated double-stranded cDNA is finally ligated into the linearized pYES2 (Invitrogen) yeast expression vector.
  • TRIzol Gibco/BRL
  • the multiple cloning site of the pTriplEx2 vector (Clontech) is first inserted into pYES2.
  • the double-stranded cDNA is digested with SfilA and SfilB and then cloned in a directed fashion into the vector which has been modified.
  • plasmid DNA is transformed into INVSd (Invitrogen) using the Saccharomyces cerevisiae EASY COMP Transformation kit (Invitrogen) following the manufacturer's instructions.
  • INVSd Invitrogen
  • Saccharomyces cerevisiae EASY COMP Transformation kit Invitrogen
  • Two 50 ⁇ l-batches are plated onto selection medium in a large square Petri dish (245x245 mm) and incubated for 3 days at 30°C.
  • MTP microtiter plates
  • the preculture is carried out in 200 ⁇ of medium [1 x CSM-Ura; 1 x YNB without amino acids and sugar; 0.5% raffinose; 5% glycerol; 40 mg / 1 adenin sulfate; 0.5% ammonium sulfate] for 72 hours at 30°C and 250 revolutions per minute.
  • An OD 6 oo of 0.2 is adjusted in the main culture by addition of an average volume from the preculture.
  • the main culture is carried out in 1 ml of medium [1 x CSM-Ura; 1 x YNB without amino acids and sugar; 0.5% raffinose; 2% galactose; 0.2% Tergitol NP-40; 40 mg/l adenin sulfate; 0.5% ammonium sulfate; 0.3 mM fatty acid substrate] for 2 to 3 weeks at 16°C and 250 revolutions per minute until an OD 600 of 3 to 4 has been reached.
  • medium [1 x CSM-Ura; 1 x YNB without amino acids and sugar; 0.5% raffinose; 2% galactose; 0.2% Tergitol NP-40; 40 mg/l adenin sulfate; 0.5% ammonium sulfate; 0.3 mM fatty acid substrate] for 2 to 3 weeks at 16°C and 250 revolutions per minute until an OD 600 of 3 to 4 has been reached.
  • the yeast cells are sedimented (1000 x g, 10 min, 4°C) and stored at -80°C until further processing.
  • FMES fatty acid methyl esters
  • the cell sediments are shock-frozen in liquid N 2 and lyophilized under N 2 gas at 30°C.
  • the pellet is homogenized in 1 % sodium methoxide in methanol and incubated for 20 minutes at room temperature. Equal volumes of 1 M NaCI and n-heptane are subsequently added, the samples are mixed and the supernatant is transferred into a GC tube.
  • the samples are separated on a DB-wax capillary column (30 m, 0.25 mm, 0.25 ⁇ m; Agilent J & W) in a Hewlett Packard 6890 gas chromatograph equipped with a flame ionization detector.
  • the oven temperature was programmed from 60°C (hold 5 min) to 200°C at a rate of 20°C/min (hold 20 min) and finally to 250°C (hold 30 min) at a rate of 20°C/min.
  • the carrier gas used was nitrogen (1.6 ml/min).
  • the fatty acids were identified by comparison with retention times of FAME standards (SIGMA). The same standards as listed in Example 3 are used.
  • STE buffer [2% SDS; 50 mM Tris/HCI, pH 8.0; 10 mM EDTA] are added to the cell lysate and the mixture is incubated for 10 minutes at room temperature.
  • 200 ⁇ l of 5 M sodium acetate are added and the mixture is placed on ice for 30 minutes. After centrifugation, the supernatant is transferred and precipitated with 2.5 volumes of ethanol. The pellet which has sedimented is washed with 70% ethanol and finally taken up in sterile water.
  • sequence of the desaturase/conjugase can be determined by sequencing using vector-specific primers (SEQ ID NO: 12 and 13).
  • Example 5 Manipulation of the plant fatty acid biosynthesis
  • transgenic A.thaliana or B.napus plants which express the desaturases/conjugases from lepidopterans
  • pBinAR H ⁇ fgen and Willmitzer (1990) Plant Sci 66:221 -230.
  • This vector contains the CaMV (cauliflower mosaic virus) 35S promoter (Franck et al. (1980) Cell 21 (1 ):285-294) and the termination signal of the octopine synthase gene (Gielen et al. (1984) EMBO J 3:835-846).
  • Agroabacterium tumefaciens strain (GV3101 [pMP90]) on the basis of a modified vacuum infiltration method (Clough S and Bent A (1998) Plant J 16(6):735-43; Bechtold N et al. (1993) in: Planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CRAcad Sci Paris 1 144(2):204-212).
  • the Agrobacterium tumefaciens cells which were used had previously been transformed with the plasmids.
  • Seeds of the primary transformants are selected on the basis of their resistance to antibiotics. Seedlings with resistance to antibiotics are planted into soil and subjected to biochemical analysis as fully-developed plants.
  • Transgenic oilseed rape plants are generated following a protocol of Bade JB and Damm B (in Gene Transfer to Plants, Potrykus I and Spangenberg G (eds.) Springer Lab Manual, Springer Verlag, 1995, 30-38), which also states the composition of the media and buffers used.
  • the transformations are carried out with Agrobacterium tumefaciens strain GV3101 [pMP90].
  • the plasmids pBinAR-TkTP/Vit E-AT are used for the transformation.
  • Seeds of Brassica napus var. Westar are surface-sterilized with 70% ethanol (v/v), washed for 10 minutes in water at 55°C, incubated for 20 minutes in 1 % strength hypochlorite solution (25% v/v Teepol, 0.1 % v/v Tween 20) and washed six times with sterile water for in each case 20 minutes.
  • the seeds are dried for three days on filter paper, and 10 to 15 seeds are germinated in a glass flask containing 15 ml of germination medium.
  • the roots and apices are removed from several seedlings (approximate size 10 cm), and the hypocotyls which remain are cut into pieces approximately 6 mm in length.
  • the approx. 600 explants thus obtained are washed for 30 minutes with 50 ml of basal medium and transferred into a 300 ml flask. After addition of 100 ml of callus induction medium, the cultures are incubated for 24 hours at 100 rpm.
  • An overnight culture of the Agrobacterium strain is established at 29°C in Luria broth medium supplemented with kanamycin (20 mg/l), and 2 ml of this culture are incubated in 50 ml of Luria broth medium without kanamycin for 4 hours at 29°C until an OD 6 oo of 0.4 to 0.5 has been reached. After the culture has been pelleted for 25 minutes at 2000 rpm, the cell pellet is resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution is brought to an OD 60 o of 0.3 by addition of further basal medium.
  • the callus induction medium is removed from the oilseed rape explants using sterile pipettes, 50 ml of Agrobacterium solution are added, and the culture is mixed carefully and incubated for 20 minutes.
  • the agrobacterial suspension is removed, the oilseed rape explants are washed for 1 minute using 50 ml of callus induction medium, and 100 ml of callus induction medium are subsequently added.
  • Coculturing is performed for 24 hours on a orbital shaker at 100 rpm. Coculturing is stopped by withdrawing the callus induction medium, and the explants are washed twice for in each case 1 minute with 25 ml and twice for 60 minutes with in each case 100 ml of wash medium at 100 rpm.
  • the wash medium together with the explants is transferred to 15 cm Petri dishes, and the medium is removed using sterile pipettes.
  • batches of 20 to 30 explants are transferred into 90 mm Petri dishes which contain 25 ml of shoot induction medium supplemented with kanamycin.
  • the Petri dishes are filled with 2 layers of Leukopor and incubated at 25°C and 2000 lux at a 16-hour light/8-hour darkness photo period. Every 12 days, the developing calli are transferred to fresh Petri dishes containing shoot induction medium. All further steps for regenerating intact plants are carried out as described by Bade, J.B and Damm, B. (in: Gene Transfer to Plants, Potrykus I and Spangenberg G (eds.) Springer Lab Manual, Springer Verlag, 1995, 30-38).
  • the seeds of transgenic plants are homogenized directly in 1 % sodium methoxide in methanol and the homogenate is incubated for 20 minutes at room temperature. Equal volumes of 1 M NaCI and n-heptane are subsequently added, the sample is mixed and the supernatant is transferred into a GC tube. The samples are separated on a DB-wax capillary column (30 m, 0.25 mm, 0.25 ⁇ m; Agilent J & W) in a Hewlett Packard 6890 gas chromatograph equipped with a flame ionization detector.
  • the oven temperature was programmed from 60°C (hold 5 min) to 200°C at a rate of 20°C/min (hold 20 min) and finally to 250°C (hold 30 min) at a rate of 20°C/min.
  • the carrier gas used was nitrogen (1.6 ml/min).
  • the fatty acids were identified by comparison with retention times of FAME standards (SIGMA). The same standards as stated in Example 3 are used.
  • Example 6 Functional expression in yeast of a desaturase from the Lepidoptera Pectinophora gossypiella
  • a desaturase isolated from gland material of the moth Pectinophora gossypiella was cloned into the yeast expression vector pYES2 (Invitrogen) by means of BamHI and EcoRI.
  • the resulting construct was named pYES2::DesPgos.
  • Both pYES2::DesPgos and the control pYES2 were transformed into yeast cells (Saccharomyces cerevisiae INVSd [MAT, his3-1 , Ieu2, trp1 -289, ura3-52]; Invitrogen) using the S.c. Easy Comp Transformation Kit (Invitrogen).
  • yeast cells contained additionally the construct pESC::ACS for expressing a Brassica napus acetyl-CoA synthase.
  • pESC :ACS for expressing a Brassica napus acetyl-CoA synthase.
  • a single colony of both pYES2::DesPgos and of the control pYES2 was isolated on selection medium and grown for 2 days at 30°C and 200 revolutions per minute in 50 ml of preculture medium (1 * CSM -Leu ⁇ Ura; 1 * YNB w/o AA; 0.5% raffinose; 5% glycerol and 0.5% ammonium sulfate) to give a preculture.
  • the main cultures were adjusted to OD ⁇ 2 , and 10 ml were centrifuged for 30 minutes at RT and 17500 g.
  • the pellet was resuspended in H 2 0, heated for 10 minutes at 90°C and again sedimented under the same conditions.
  • the pellet was washed with H 2 0, sedimented, shock-frozen in liquid nitrogen (N 2 ) and subsequently lyophilized under N 2 .
  • N 2 liquid nitrogen
  • FAME fatty acid methyl esters
  • Table 1 shows the relative distribution of the FAME of lipids from pYES2::DesPgos in comparison with the control pYES2 at 16°C. It can be seen that the expression of the Pectinophora gossypiella desaturase results in an accumulation of the following monounsaturated fatty acids: C16:1 D11 , C18:1 D11 and C18:1 D13. Moreover, the heterologous expression of the P. gossypiella desaturase leads to the formation of (9Z,1 1 E)-CLA.
  • Table 2 shows the relative distribution of the FAME of lipids of pYES2::DesPgos in comparison with the control pYES2 at 30°C.
  • the expression results at 30°C correspond to those at 16°C: expression of the Pectinophora gossypiella desaturase in the yeast leads to an accumulation of the monounsaturated fatty acids C16:1 D1 1 , C18:1 D1 1 and C18:1 D13.
  • the heterologous expression of the P. gossypiella desaturase leads to the formation of (9Z,11 E)-CLA.
  • Table 1 Heterologous expression of the Pectinophora gossypiella desaturase in S. cerevisiae at 16°C.
  • the values represent the relative amount of the individual fatty acids in the total fatty acid spectrum.
  • Table 2 Heterologous expression of the Pectinophora gossypiella desaturase in S. cerevisiae at 30°C.
  • the values represent the relative amount of the individual fatty acids in the total fatty acid spectrum.

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KR101540746B1 (ko) 2011-12-28 2015-08-03 대한민국 공액리놀레산(Ｃｏｎｊｕｇａｔｅｄ Ｌｉｎｏｌｅｉｃ Ａｃｉｄ, ＣＬＡ)의 생산방법
CN108138202B (zh) 2015-06-26 2023-05-16 丹麦科技大学 用于在酵母中产生蛾信息素的方法
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US11214818B2 (en) 2016-06-06 2022-01-04 Provivi, Inc. Semi-biosynthetic production of fatty alcohols and fatty aldehydes
ES2930358T3 (es) 2016-12-16 2022-12-09 Univ Danmarks Tekniske Producción de alcoholes grasos desaturados y acetatos de acilo grasos desaturados en levaduras
US11345922B2 (en) 2016-12-16 2022-05-31 Danmarks Tekniske Universitet Methods for producing fatty alcohols and derivatives thereof in yeast
BR112019024258A2 (pt) 2017-05-17 2020-08-18 Provivi, Inc. Microorganismos yarrowia lipolytica recombinante e método para produzir um c6-c24 álcool graxo mono- ou poliinsaturado a partir de uma fonte endógena ou exógena de c6-c24 ácidograxo saturado
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