TRANSLATOR CERTIFICATION
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`Date: February 18, 2020
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`To whom it may concern:
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`I, Nadia Silva Castro, a translator fluent in the German and English languages, on behalf of Morningside
`Translations, do solemnly and sincerely declare that the following is, to the best of my knowledge and
`belief, a true and correct translation of the document(s) listed below in a form that best reflects the
`intention and meaning of the original text.
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`The document is designated as:
`• German Patent Application No: DE 10 2006 008 030.0
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`Signature
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`Nadia Silva Castro
`Print
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`www.morningsideIP.com
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`CSIRO Exhibit 1011
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`Method for producing polyunsaturated fatty acids
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`[0001] The present invention relates to a process for the production of eicosapentaenoic acid,
`docosapentaenoic acid and/or docosahexaenoic acid in transgenic plants, providing in the plant at least
`one nucleic acid sequence which codes for a polypeptide having a Δ6-desaturase activity; at least one
`nucleic acid sequence which codes for a polypeptide having a Δ6-elongase activity; at least one nucleic
`acid sequence which codes for a polypeptide having a Δ5-desaturase activity; and at least one nucleic
`acid sequence which codes for a polypeptide having a Δ5-elongase activity, where the nucleic acid
`sequence which codes for a polypeptide having a Δ5-elongase activity is modified by comparison with
`the nucleic acid sequence in the organism from which the sequence is derived in that it is adapted to the
`codon usage in one or more plant species.
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`[0002] In a preferred embodiment there is additionally provision of further nucleic acid sequences which
`code for a polypeptide having the activity of an ω3-desaturase and/or of a Δ4-desaturase in the plant.
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`[0003] In a further preferred embodiment there is provision of further nucleic acid sequences which
`code for acyl-CoA dehydrogenase(s), acyl-ACP (acyl carrier protein) desaturase(s), acyl-ACP
`thioesterase(s), fatty acid acyl transferase(s), acyl-CoA:lysophospholipid acyl transferase(s), fatty acid
`synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s),
`fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene oxide
`synthases, hydroperoxide lyases or fatty acid elongase(s) in the plant.
`
`[0004] The invention furthermore relates to recombinant nucleic acid molecules comprising at least one
`nucleic acid sequence which codes for a polypeptide having a Δ6-desaturase activity; at least one nucleic
`acid sequence which codes for a polypeptide having a Δ5-desaturase activity; at least one nucleic acid
`sequence which codes for a polypeptide having a Δ6-elongase activity; and at least one nucleic acid
`sequence which codes for a polypeptide having a Δ5-elongase activity and which is modified by
`comparison with the nucleic acid sequence in the organism from which the sequence originates in that it
`is adapted to the codon usage in one or more plant species.
`
`[0005] A further part of the invention relates to oils, lipids and/or fatty acids which have been produced
`by the process according to the invention, and to their use.
`
`[0006] Finally, the invention also relates to transgenic plants which have been produced by the process
`of the invention or which comprise a recombinant nucleic acid molecule of the invention, and to the use
`thereof as foodstuffs or feedstuffs.
`
`[0007] Lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to
`sn-glycerol-3-phosphate, and the addition or modification of a polar head group. Usual lipids which are
`used in membranes comprise phospholipids, glycolipids, sphingolipids and phosphoglycerides. Fatty acid
`synthesis starts with the conversion of acetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into
`acetyl-ACP by acetyl transacylase. After condensation reaction, these two product molecules together
`form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration
`reactions so that a saturated fatty acid molecule with the desired chain length is obtained. The
`production of the unsaturated fatty acids from these molecules is catalyzed by specific desaturases,
`either aerobically by means of molecular oxygen or anaerobically (regarding the fatty acid synthesis in
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`microorganisms, see F. C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., p.
`612-636 and references cited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme:
`Stuttgart, New York, and the references therein, and Magnuson, K., et al. (1993) Microbiological Reviews
`57:522-542 and the references therein). To undergo the further elongation steps, the resulting
`phospholipid-bound fatty acids must be returned to the fatty acid CoA ester pool. This is made possibly
`by acyl-CoA:lysophospholipid acyltransferases. Moreover, these enzymes are capable of transferring the
`elongated fatty acids from the CoA esters back to the phospholipids. If appropriate, this reaction
`sequence can be followed repeatedly.
`
`[0008] Furthermore, fatty acids must subsequently be transported to various modification sites and
`incorporated into the triacylglycerol storage lipid. A further important step during lipid synthesis is the
`transfer of fatty acids to the polar head groups, for example by glycerol fatty acid acyltransferase (see
`Frentzen, 1998, Lipid, 100(4-5):161-166).
`
`[0009] An overview of the biosynthesis of fatty acids in plants, desaturation, the lipid metabolism and
`the membrane transport of lipidic compounds, beta-oxidation, the modification of fatty acids, cofactors
`and the storage and assembly of triacylglycerol, including the references is given by the following
`papers: Kinney (1997) Genetic Engineering, Ed.: J K Setlow, 19:149-166; Ohlrogge and Browse (1995)
`Plant Cell 7:957-970; Shanklin and Cahoon (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 611–641;
`Voelker (1996) Genetic Engeneering, Ed.: J K Setlow, 18: 111–13; Gerhardt (1992) Prog. Lipid R. 31: 397–
`417; Guhnemann-Schafer & Kindl (1995) Biochim Biophys Acta 1256: 181–186; Kunau et al. (1995) Prog.
`Lipid Res. 34: 267–342; Stymne et al. (1993) in: Biochemistry and Molecular Biology of Membrane and
`Storage Lipids of Plants, Ed.: Murata and Somerville, Rockville, American Society of Plant Physiologists,
`150–158; Murphy & Ross (1998) Plant Journal. 13(1) :1–16.
`
`[0010] Depending on the desaturation pattern, two large classes of polyunsaturated fatty acids, the ω6
`and the ω3 fatty acids, which differ with regard to their metabolism and their function, can be
`distinguished.
`
`[0011] In the text which follows, polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or
`LCPUFAs (poly unsaturated fatty acids, PUFA, long chain poly unsaturated fatty acids, LCPUFA).
`
`[0012] The fatty acid linoleic acid (18:2Δ9,12) acts as starting material for the ω6 metabolic pathway, while
`the ω3 pathway proceeds via linolenic acid (18:3Δ9,12,15). Linolenic acid is formed from linoleic acid by the
`activity of an ω3-desaturase (Tocher et al. (1998) Prog. Lipid Res. 37: 73–117; Domergue et al. (2002)
`Eur. J. Biochem. 269: 4105–4113).
`
`[0013] Mammals, and thus also humans, have no corresponding desaturase activity (Δ12- and ω3-
`desaturase) for the formation of the starting materials and must therefore take up these fatty acids
`(essential fatty acids) via the food. Starting with these precursors, the physiologically important
`polyunsaturated fatty acids arachidonic acid (=ARA, 20:4Δ5,8,11,14), an ω6-fatty acid and the two ω3-fatty
`acids eicosapentaenoic acid (=EPA, 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,17,19) are
`synthesized via a sequence of desaturase and elongase reactions.
`
`[0014] The elongation of fatty acids, by elongases, by 2 or 4 C atoms is of crucial importance for the
`production of C20- and C22-PUFAs, respectively. This process proceeds via 4 steps. The first step is the
`condensation of malonyl-CoA onto the fatty acid acyl-CoA by ketoacyl-CoA synthase (KCS, hereinbelow
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`referred to as elongase). This is followed by a reduction step (ketoacyl-CoA reductase, KCR), a
`dehydration step (dehydratase) and a final reduction step (enoyl-CoA reductase). It has been postulated
`that the elongase activity affects the specificity and rate of the entire process (Millar and Kunst (1997)
`Plant Journal 12:121-131).
`
`[0015] Fatty acids and triacylglycerides have a multiplicity of applications in the food industry, in animal
`nutrition, in cosmetics and the pharmacological sector. Depending on whether they are free saturated
`or unsaturated fatty acids or else triacylglycerides with an elevated content of saturated or unsaturated
`fatty acids, they are suitable for very different applications. Thus, for example, lipids with unsaturated,
`specifically with polyunsaturated fatty acids, are preferred in human nutrition. The polyunsaturated ω3-
`fatty acids are supposed to have a positive effect on the cholesterol level in the blood and thus on the
`prevention of heart disease. The risk of heart disease, strokes or hypertension can be reduced markedly
`by adding these ω3-fatty acids to the food (Shimikawa (2001) World Rev. Nutr. Diet. 88: 100-108).
`
`[0016] ω3-fatty acids also have a positive effect on inflammatory, specifically on chronically
`inflammatory, processes in association with immunological diseases such as rheumatoid arthritis (Calder
`(2002) Proc. Nutr. Soc. 61: 345-358; Cleland and James (2000) J. Rheumatol. 27: 2305-2307). They are
`therefore added to foodstuffs, specifically to dietetic foodstuffs, or are employed in medicaments. ω6-
`fatty acids such as arachidonic acid tend to have a negative effect in connection with these
`rheumatological diseases.
`
`[0017] ω3- and ω6-fatty acids are precursors of tissue hormones, known as eicosanoids, such as the
`prostaglandins, which are derived from dihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic
`acid, and of the thromboxanes and leukotrienes, which are derived from arachidonic acid and
`eicosapentaenoic acid. Eicosanoids (known as the PG2 series) which are formed from the ω6-fatty acids,
`generally promote inflammatory reactions, while eicosanoids (known as the PG3 series) from ω3-fatty
`acids have little or no proinflammatory effect.
`
`[0018] Polyunsaturated long-chain ω3-fatty acids such as eicosapentaenoic acid (=EPA, C20:5Δ5,8,11,14,17)
`or docosahexaenoic acid (=DHA, C22:6Δ4,7,10,13,16,19) are important components of human nutrition owing
`to their various roles in health aspects, including the development of the child brain, the functionality of
`the eyes, the synthesis of hormones and other signal substances, and the prevention of cardiovascular
`disorders, cancer and diabetes (Poulos, A (1995) Lipids 30:1-14; Horrocks, L A and Yeo Y K (1999)
`Pharmacol Res 40:211-225).
`
`[0019] Owing to the present-day composition of human food, an addition of polyunsaturated ω3-fatty
`acids, which are preferentially found in fish oils, to the food is particularly important. Thus, for example,
`polyunsaturated fatty acids such as docosahexaenoic acid (=DHA, C22:6Δ4,7,10,13,16,19) or eicosapentaenoic
`acid (=EPA, C20:5Δ5,8,11,14,17) are added to infant formula to improve the nutritional value. There is
`therefore a demand for the production of polyunsaturated long-chain fatty acids.
`
`[0020] The various fatty acids and triglycerides are mainly obtained from microorganisms such as
`Mortierella or Schizochytrium or from oil-producing plants such as soybeans, oilseed rape, and algae
`such as Crypthecodinium or Phaeodactylum and others, being obtained, as a rule, in the form of their
`triacylglycerides (=triglycerides=triglycerols). However, they can also be obtained from animals, for
`example, fish. The free fatty acids are advantageously prepared by hydrolyzing the triacylglycerides.
`Very long-chain polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (ARA, C20:4Δ5,8,11,14),
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`dihomo-γ-linolenic acid (DHGL, C20:3Δ8,11,14) or docosapentaenoic acid (DPA, C22:5Δ7,10,13,16,19) are,
`however, not synthesized in oil crops such as oilseed rape, soybeans, sunflowers and safflower.
`Conventional natural sources of these fatty acids are fish such as herring, salmon, sardine, redfish, eel,
`carp, trout, halibut, mackerel, zander or tuna, or algae.
`
`[0021] Owing to the positive characteristics of the polyunsaturated fatty acids, there has been no lack of
`attempts in the past to make available genes which are involved in the synthesis of these fatty acids or
`triglycerides for the production of oils in various organisms with a modified content of unsaturated fatty
`acids. Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. WO 93/11245 claims a Δ15-
`desaturase and WO 94/11516 a Δ12-desaturase. Further desaturates are described, for example, in EP-
`A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al.
`(1990) J. Biol. Chem., 265: 20144-20149, Wada et al. (1990) Nature 347: 200-203 or Huang et al. (1999)
`Lipids 34: 649-659. However, the biochemical characterization of the various desaturases has been
`insufficient to date since the enzymes, being membrane-bound proteins, present great difficulty in their
`isolation and characterization (McKeon et al. (1981) Methods in Enzymol. 71: 12141-12147, Wang et al.
`(1988) Plant Physiol. Biochem., 26: 777-792).
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`[0022] As a rule, membrane-bound desaturases are characterized by being introduced into a suitable
`organism which is subsequently analyzed for enzyme activity by analyzing the starting materials and the
`products. Δ6-Desaturases are described in WO 93/06712, U.S. Pat. No. 5,614,393, WO 96/21022, WO
`00/21557 and WO 99/27111. The application of this enzyme for the production of fatty acids in
`transgenic organisms is described in WO 98/46763, WO 98/46764 and WO 98/46765. The expression of
`various desaturases and the formation of polyunsaturated fatty acids is also described and claimed in
`WO 99/64616 or WO 98/46776. As regards the expression efficacy of desaturases and its effect on the
`formation of polyunsaturated fatty acids, it must be noted that the expression of a single desaturase as
`described to date has only resulted in low contents of unsaturated fatty acids/lipids such as, for
`example, γ-linolenic acid and stearidonic acid.
`
`[0023] There have been a number of attempts in the past to obtain elongase genes. Millar and Kunst
`(1997) Plant Journal 12: 121-131 and Millar et al. (1999) Plant Cell 11:825-838 describe the
`characterization of plant elongases for the synthesis of monounsaturated long-chain fatty acids (C22:1)
`and for the synthesis of very long-chain fatty acids for the formation of waxes in plants (C28-C32). The
`synthesis of arachidonic acid and EPA is described, for example, in WO 01/59128, WO 00/12720, WO
`02/077213 and WO 02/08401. The synthesis of polyunsaturated C24-fatty acids is described, for
`example, in Tvrdik et al. (2000) J. Cell Biol. 149: 707–718 or in WO 02/44320.
`
`[0024] Especially suitable microorganisms for the production of PUFAs are microalgae such as
`Phaeodactylum tricornutum, Porphiridium species, Thraustochytrium species, Schizochytrium species or
`Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella,
`Entomophthora or Mucor and/or mosses such as Physcomitrella, Ceratodon and Marchantia (R.
`Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22: 1060-
`1062; M. Akimoto et al. (1998) Appl. Biochemistry and
`
` Biotechnology 73: 269-278). Strain selection has resulted in the development of a number of mutant
`strains of the microorganisms in question which produce a series of desirable compounds including
`PUFAs. However, the mutation and selection of strains with an improved production of a particular
`molecule such as the polyunsaturated fatty acids is a time-consuming and difficult process. Moreover,
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`only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or ARA can be
`produced with the aid of the abovementioned microorganisms; in addition, they are generally obtained
`as fatty acid mixtures. This is why recombinant methods are preferred whenever possible.
`
`[0025] Higher plants comprise polyunsaturated fatty acids such as linoleic acid (C18:2) and linolenic acid
`(C18:3). ARA, EPA and DHA are found not at all in the seed oil of higher plants, or only in miniscule
`amounts (E. Ucciani: Nouveau Dictionnaire des Huiles Vegetales [New Dictionary of the Vegetable Oils].
`Technique & Documentation--Lavoisier, 1995. ISBN: 2-7430-0009-0). However, the production of
`LCPUFAs in higher plants, preferably in oil crops such as oilseed rape, linseed, sunflowers and soybeans,
`would be advantageous since large amounts of high-quality LCPUFAs for the food industry, animal
`nutrition and pharmaceutical purposes might be obtained economically. To this end, it is advantageous
`to introduce, into oilseeds, genes which encode enzymes of the LCPUFA biosynthesis via recombinant
`methods and to express them therein. These genes encode for example Δ6-desaturases, Δ6-elongases,
`Δ5-desaturases or Δ4-desaturases. These genes can advantageously be isolated from microorganisms
`and lower plants which produce LCPUFAs and incorporate them in the membranes or triacylglycerides.
`Thus, it has already been possible to isolate Δ6-desaturase genes from the moss Physcomitrella patens
`and Δ6-elongase genes from P. patens and from the nematode C. elegans.
`
`[0026] Transgenic plants which comprise and express genes encoding LCPUFA biosynthesis enzymes and
`which, as a consequence, produce LCPUFAs have been described, for example, in DE-A-102 19 203
`(process for the production of polyunsaturated fatty acids in plants). However, these plants produce
`LCPUFAs in amounts which require further optimization for processing the oils which are present in the
`plants. Thus, the ARA content in the plants described in DE-A-102 19 203 is only 0.4 to 2% and the EPA
`content only 0.5 to 1%, in each case based on the total lipid content of the plant.
`
`[0027] To make possible the fortification of food and of feed with polyunsaturated, long-chain fatty
`acids, there is therefore a great need for a simple, inexpensive process for the production of
`polyunsaturated, long-chain fatty acids, specifically in plant systems.
`
`[0028] One object of the invention is therefore to provide a process with which long-chain
`polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and/or
`docosahexaenoic acid can be produced in large quantities and inexpensively in transgenic plants.
`
`[0029] It has now surprisingly been found that the yield of long-chain polyunsaturated fatty acids,
`especially eicosapentaenoic, docosapentaenoic acid and/or docosahexaenoic acid, can be increased by
`expressing an optimized Δ5-elongase sequence in transgenic plants.
`
`[0030] The PUFAs produced by the process of the invention comprise a group of molecules which higher
`animals are no longer able to synthesize and thus must consume, or which higher animals are no longer
`able to produce themselves in sufficient amounts and thus must consume additional amounts thereof,
`although they can easily be synthesized by other organisms such as bacteria.
`
`[0031] Accordingly, the object of the invention is achieved by the process of the invention for producing
`eicosapentaenoic acid, docosapentaenoic acid and/or docosahexaenoic acid in a transgenic plant,
`comprising the provision in the plant of at least one nucleic acid sequence which codes for a polypeptide
`having a Δ6-desaturase activity; at least one nucleic acid sequence which codes for a polypeptide having
`a Δ6-elongase activity; at least one nucleic acid sequence which codes for a polypeptide having a Δ5-
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`desaturase activity; and at least one nucleic acid sequence which codes for a polypeptide having a Δ5-
`elongase activity, where the nucleic acid sequence which codes for a polypeptide having a Δ5-elongase
`activity is modified by comparison with the nucleic acid sequence in the organism from which the
`sequence is derived in that it is adapted to the codon usage in one or more plant species. To produce
`DHA it is additionally necessary to provide at least one nucleic acid sequence which codes for a
`polypeptide having a Δ4-desaturase activity in the plant.
`
`[0032] The "provision in the plant" means in the context of the present invention that measures are
`taken so that the nucleic acid sequences coding for a polypeptide having a Δ6-desaturase activity, a
`polypeptide having a Δ6-elongase activity, a polypeptide having a Δ5-desaturase activity and a
`polypeptide having a Δ5-elongase activity are present together in one plant. The "provision in the plant"
`thus comprises the introduction of the nucleic acid sequences into the plant both by transformation of a
`plant with one or more recombinant nucleic acid molecules which comprise said nucleic acid sequences,
`and by crossing suitable parent plants which comprise one or more of said nucleic acid sequences.
`
`[0033] The nucleic acid sequence which codes for a polypeptide having a Δ5-elongase activity is
`modified according to the invention by comparison with the nucleic acid sequence in the organism from
`which the sequence originates in that it is adapted to the codon usage in one or more plant species. This
`means that the nucleic acid sequence has been specifically optimized for the purpose of the invention
`without the amino acid sequence encoded by the nucleic acid sequence having been altered thereby.
`
`[0034] The genetic code is redundant because it uses 61 codons in order to specify 20 amino acids.
`Therefore, most of the 20 proteinogenic amino acids are therefore encoded by a plurality of triplets
`(codons). The synonymous codons which specify an individual amino acid are, however, not used with
`the same frequency in a particular organism; on the contrary there are preferred codons which are
`frequently used, and codons which are used more rarely. These differences in codon usage are
`attributed to selective evolutionary pressures and especially the efficiency of translation. One reason for
`the lower translation efficiency of rarely occurring codons might be that the corresponding aminoacyl-
`tRNA pools are exhausted and thus no longer available for protein synthesis.
`
`[0035] In addition, different organisms prefer different codons. For this reason, for example, the
`expression of a recombinant DNA derived from a mammalian cell frequently proceeds only suboptimally
`in Escherichia coli (E. coli) cells. It is therefore possible in some cases to increase expression by replacing
`rarely used codons with frequently used codons. Without wishing to be bound to one theory, it is
`assumed that the codon-optimized DNA sequences make more efficient translation possible, and the
`mRNAs formed therefrom possibly have a greater half-life in the cell and therefore are available more
`frequently for translation. From what has been said above, it follows that codon optimization is
`necessary only if the organism in which the nucleic acid sequence is to be expressed differs from the
`organism from which the nucleic acid sequence is originally derived.
`
`[0036] For many organisms of which the DNA sequence of a relatively large number of genes is known
`there are tables from which the frequency of use of particular codons in the respective organism can be
`taken. It is possible with the aid of these tables to translate protein sequences with relatively high
`accuracy back into a DNA sequence which comprises the codons preferred in the respective organism
`for the various amino acids of the protein. Tables on codon usage can be found inter alia at the following
`Internet address: kazusa.or.ip/Kodon/E.html. In addition, several companies provide software for gene
`optimization, such as, for example, Entelechon (Software Leto) or Geneart (Software GeneOptimizer).
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`[0037] Adaptation of the sequences to the codon usage in a particular organism can take place with the
`aid of various criteria. On the one hand, it is possible to use for a particular amino acid always the codon
`which occurs most frequently in the selected organism but, on the other hand, the natural frequency of
`the various codons can also be taken into account, so that all the codons for a particular amino acid are
`incorporated into the optimized sequence according to their natural frequency. Selection of the position
`at which a particular base triplet is used can take place at random in this case. The DNA sequence was
`adapted according to the invention taking account of the natural frequency of individual codons, it also
`being suitable to use the codons occurring most frequently in the selected organism.
`
`[0038] It is particularly preferred for a nucleic acid sequence from Ostreococcus tauri which codes for a
`polypeptide having a Δ5-elongase activity to be adapted at least to the codon usage in oilseed rape,
`soybean and/or flax. The nucleic acid sequence originally derived from Ostreococcus tauri is preferably
`the sequence depicted in SEQ ID NO: 109. The DNA sequence coding for the Δ5-elongase is adapted in at
`least 20% of the positions, preferably in at least 30% of the positions, particularly preferably in at least
`40% of the positions and most preferably in at least 50% of the positions to the codon usage in oilseed
`rape, soybean and/or flax.
`
`[0039] The nucleic acid sequence used is most preferably the sequence indicated in SEQ ID NO: 1.
`
`[0040] It will be appreciated that the invention also encompasses those codon-optimized DNA
`sequences which code for a polypeptide having the activity of a Δ5-elongase and whose amino acid
`sequence is modified in one or more positions by comparison with the wild-type sequence but which
`still has substantially the same activity as the wild-type protein.
`
`[0041] The nucleic acid sequence which codes for a polypeptide having a Δ6-desaturase activity is
`preferably selected from the group consisting of:
`
`a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 3,
`
`b) nucleic acid sequences which code for the amino acid sequence indicated in SEQ ID NO:
`
`4,
`
`c) nucleic acid sequences which hybridize with the complementary strand of the nucleic acid sequence
`indicated in SEQ ID NO: 3, under stringent conditions, and
`
`d) nucleic acid sequences which are at least 60% identical to the sequence indicated in SEQ ID NO: 3.
`
`[0042] The nucleic acid sequence which codes for a polypeptide having a Δ6-elongase activity is
`preferably selected from the group consisting of:
`
`a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 5,
`
`b) nucleic acid sequences which code for the amino acid sequence indicated in SEQ ID NO: 6,
`
`c) nucleic acid sequences which hybridize with the complementary strand of the nucleic acid sequence
`indicated in SEQ ID NO: 5, under stringent conditions, and
`
`d) nucleic acid sequences which are at least 60% to the sequence indicated in SEQ ID NO: 5.
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` [0043] The nucleic acid sequence which codes for a polypeptide having a Δ5-desaturase activity is
`preferably selected from the group consisting of:
`
`a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 7,
`
`b) nucleic acid sequences which code for the amino acid sequence indicated in SEQ ID NO: 8,
`
`c) nucleic acid sequences which hybridize with the complementary strand of the nucleic acid sequence
`indicated in SEQ ID NO: 7, under stringent conditions,
`
`d) nucleic acid sequences which are at least 60% identical to the nucleic acid indicated under SEQ ID NO:
`7.
`
`[0044] Further suitable nucleic acid sequences can be found by the skilled worker from the literature or
`the well-known gene libraries such as, for example, ncbi.nlm.nih.gov.
`
`[0045] In a further preferred embodiment of the process, additionally one or more nucleic acid
`sequences which code for a polypeptide having the activity of an ω-3-desaturase and/or of a Δ4-
`desaturase are introduced into the plant.
`
`[0046] The nucleic acid sequence which codes for a polypeptide having an ω-3-desaturase activity is
`preferably selected from the group consisting of:
`
`a) nucleic acid sequences having the sequence depicted in SEQ ID NO:9,
`
`b) nucleic acid sequences which code for the amino acid sequence indicated in SEQ ID NO: 10,
`
`c) nucleic acid sequences which hybridize with the complementary strand of the nucleic acid sequence
`indicated in SEQ ID NO: 9 under stringent conditions, and
`
`d) nucleic acid sequences which are at least 60% identical to the sequence indicated in SEQ ID NO: 9.
`
`[0047] The ω-3-desaturase advantageously used in the process of the invention makes it possible to shift
`from the ω-6 biosynthetic pathway to the ω-3 biosynthetic pathway, leading to a shift from C18:2 to C18:3
`fatty acids. It is further advantageous for the ω-3-desaturase to convert a wide range of phospholipids
`such as phosphatidylcholine (=PC), phosphatidylinositol (=PIS) or phosphatidylethanolamine (=PE).
`Finally, desaturation products can also be found in the neutral lipids (=NL), that is to say in the
`triglycerides.
`
`[0048] The nucleic acid sequence which codes for a polypeptide having a Δ4-desaturase activity is
`preferably selected from the group consisting of:
`
`a) nucleic acid sequences having the sequence depicted in SEQ ID NO: 11,
`
`b) nucleic acid sequences which code for the amino acid sequence indicated in SEQ ID NO: 12,
`
`c) nucleic acid sequences which hybridize with the complementary strand of the nucleic acid sequence
`indicated in SEQ ID NO: 11, under stringent conditions,
`
`d) nucleic acid sequences which are at least 60% identical to the sequence indicated in SEQ ID NO: 11.
`[0049] The Δ4-desaturase which is advantageously used in the process of the invention catalyzes the
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`CSIRO Exhibit 1011
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`introduction of a double bond into the fatty acid docosapentaenoic acid, leading to formation of
`docosahexaenoic acid.
`
`[0050] It is advantageous for the described process of the invention additionally to introduce further
`nucleic acids which code for enzymes of fatty acid or lipid metabolism into the plants in addition to the
`nucleic acid sequences which code for polypeptides having a Δ6-desaturase activity, a Δ6-elongase
`activity, a Δ5-desaturase activity and a Δ5-elongase activity, and to the nucleic acid sequences which are
`introduced if appropriate and which code for a polypeptide having an ω-3-desaturase activity and/or a
`Δ4-desaturase activity.
`
`[0051] It is possible in principle to use all genes of fatty acid or lipid metabolism in combination with the
`nucleic acid sequences used in the process of the invention; genes of fatty acid or lipid metabolism
`selected from the group of acyl-CoA dehydrogenase(s), acyl-ACP (acyl carrier protein) desaturase(s),
`acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:lysophospholipid acyltransferases, fatty
`acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A
`oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene
`oxide synthases, hydroperoxide lyases or fatty acid elongase(s) are preferably used in combination with
`the Δ6-elongase, Δ6-desaturase, Δ5-desaturase and the Δ5-elongase, and if appropriate the ω3-
`desaturase and/or the Δ4-desaturase, it being possible to use individual genes or a plurality of genes in
`combination.
`
`[0052] The nucleic acids used in the process of the invention are advantageously expressed in vegetative
`tissues (somatic tissue). Vegetative tissue means in the context of this invention a tissue which is
`propagated through mitotic divisions. Tissue of this type also arises through asexual reproduction
`(apomixis) and propagation. Propagation is the term used when the number of individuals increases in
`consecutive generations. These individuals arising through asexual propagation are very substantially
`identical to their parents. Examples of such tissues are leaf, flower, root, stalk, runners above or below
`ground (side s