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`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
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`7/2002
`11/2003
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`9/1997
`9/1991
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`5/1994
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`“Phaeodactylum tricornutum Elongase Encoding cDNA Seq ID No.
`9”,GeneSeq Database Accession No. ABV74261, Mar. 28, 2003.
`Bork, P., et al., “Go Hunting in Sequence Databases but Watch Out
`for the Traps”, Trends in Genet. 12:10 (1996), pp. 425-427.
`Broun, P., et al., “Catalytic Plasticity of Fatty Acid Modification
`Enzymes Underlying Chemical Diversity of Plant Lipids”, Science
`282:5392 (1998), pp. 1315-1317.
`Van de Loo, F. J., et al., “An Oleate 12-Hydroxylase from Ricinus
`communis L.
`is a Fatty Acyl Desaturase Homolog”, Proc. Natl.
`Acad. Sci. U S A 92:15 (1995), pp. 6743-6747.
`Doerks, T., et al., “Protein Annotation: Detective Work for Function
`Prediction”, Trends in Genet. 14:6 (1998), pp. 248-250.
`Brenner, S. E., “Errors in Genome Annotation”, Trends in Genet.
`15:4 (1999), pp. 132-133.
`Yu, 2., et al., “Study on Nutritional Function of Polyunsaturated
`Fatty Acid”, China Feed, 2003, Issue 24, pp. 21-23.
`Robert, S. 8., Production of Eicosapentaenoic and Docosahexaenoic
`Acid-Containing Oils in Transgenic Land Plants for Human and
`Aquaculture Nutrition, Marine Biotechnology, 2006, 8:103-109.
`“Codex Standard for Named Vegetable Oils%X-STAN 210-
`1999”, excerpt from Codex Alimentarius, 2001, vol. 8, pp. 11-25.
`“Danio rerio Polyunsaturated Fatty Acid Elongase mRNA, Com-
`plete cds”, Database GenBank, Accession No. AF532782, Feb. 15,
`2006.
`“Phaeodactylum tricornutum Delta 12 Fatty Acid Desaturase mRNA,
`Complete cds; Nuclear Gene for Microsomal Protein”, Database
`GenBank, Accession No. AY165023, Apr. 14, 2003.
`Girke, T., et al., “Identification of a Novel A6-Acyl-Group Desaturase
`by Targeted Gene Disruption in Physcomitrella patens”, The Plant
`Journal, 1998, vol. 15, No. 1, pp. 39-48.
`Michaelson, L. V., et al., “Functional Identification of a Fatty Acid
`A5 Desaturase Gene from Caenorhabditis elegans”, FEBS Letters,
`1998, vol. 439, No. 3, pp. 215-218.
`Michaelson, L. V., et al., “Isolation of a A5-Fatty Acid Desaturase
`Gene from Mortierella alpina”, The Journal of Biological Chemis-
`try, 1998, vol. 273, No. 30, pp. 19055-19059.
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`(56)
`
`References Cited
`OTHER PUBLICATIONS
`
`Moon, Y.-A., et al., “Identification of a Mammalian Long Chain
`Fatty Acyl Elongase Regulated by Sterol Regulatory Element-
`Binding Proteins”, The Journal of Biological Chemistry, 2001, vol.
`276, No. 48, pp. 45358-45366.
`Sayanova, et a1., “Expression of a Borage Desaturase cDNA Con-
`taining an N-Terminal Cytochrome b5 Domain Results in the
`Accumulation of High Levels of A6-Desaturated Fatty Acids in
`Transgenic Tobacco”, Proc. Natl. Acad. Sci USA, 1997, vol. 94, pp.
`421 1-4216.
`“Future Considerations”, p. 221 of “Bailey’s Industrial Oil and Fat
`Products”, Sixth Edition, vol. 6, Shahidi, F., Ed., John Wiley &
`Sons, Inc., 2005.
`Sperling, P., et al., “A Bifunctional A6-Fatty Acyl Acetylenase/
`Desaturase from the Moss Ceratodon purpureus”, European Journal
`of Biochemistry, 2000, vol. 267, No. 12, pp. 3801-3811.
`Tonon, T., et al., “Identification of a Very Long Chain Polyunsatu-
`rated Fatty Acid A4-Desaturase from the Microalga Pavlova lutheri”,
`FEBS letters, 2003, vol. 553, No. 3, pp. 440-444.
`
`Watts, J. L., et a1., “Isolation and Characterization of aA5 -Fatty Acid
`Desaturase from Caenorhabditis elegans”, Archives of Biochemis-
`try and Biophysics, 1999, vol. 362, No. 1, pp. 175-182.
`Kang, Z. B., et a1., “Adenoviral Gene Transfer of Caenorhabditis
`elegans n-3 Fatty Acid Desaturase Optimizes Fatty Acid Compo-
`sition in Mammalian Cells”, PNAS, 2001, vol. 98, No. 7, pp.
`4050-4054.
`Wagner, et a1., “Generation of glycerophospholipid molecular spe-
`cies in the yeast Saccharomyces cerevisiae. Fatty acid pattern of
`phospholipid classes and selective acyl turnover at sn-l and sn-2
`positions”, Yeast, vol. 10, 1994, pp. 1429-1437.
`Diedrich, et al., “The natural occurrence of unusual fatty acids. Part
`1. Odd numbered fatty acids”, Molecular Nutrition & Food Research,
`vol. 34, Issue 10, 1990, pp. 935-943.
`Gunstone FD, “Movements towards tailor-made fats”, Progress in
`Lipid Research, vol. 37, Issue 5, Nov. 1998, pp. 277-305.
`Thelen, et a1., “Metabolic Engineering of Fatty Acid Biosynthesis in
`Plants”, Metabolic Engineering, vol. 4, Issue 1, 2002, pp. 12-21.
`
`* cited by examiner
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`US 10,301,638 B2
`
`1
`OILS, LIPIDS AND FATTY ACIDS
`PRODUCED IN TRANSGENIC BRASSICA
`PLANT
`
`RELATED APPLICATIONS
`
`This application is a continuation of patent application
`Ser. No. 12/280,090 filed Aug. 20, 2008, which is a national
`stage application (under 35 U.S.C. § 371) of PCT/EP2007/
`051675, filed Feb. 21, 2007, which claims benefit of German
`application 10 2006 008 030.0, filed Feb. 21, 2006 and
`European application 061203097, filed Sep. 7, 2006. The
`entire content of each aforementioned application is hereby
`incorporated by reference in its entirety.
`
`SUBMISSION OF SEQUENCE LISTING
`
`The Sequence Listing associated with this application is
`filed in electronic format Via EFS-Web and hereby incorpo-
`rated by reference into the specification in its entirety. The
`name of the text file containing the Sequence Listing is
`Sequence_Listing_074017_0013_01. The size of the text
`file is 730 KB, and the text file was created on Sep. 2, 2016.
`The present invention relates to a process for the produc-
`tion 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 A6-desaturase activity; at least one
`nucleic acid sequence which codes for a polypeptide having
`a A6-elongase activity; at least one nucleic acid sequence
`which codes for a polypeptide having a A5-desaturase
`activity; and at least one nucleic acid sequence which codes
`for a polypeptide having a A5-elongase activity, where the
`nucleic acid sequence which codes for a polypeptide having
`a A5-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.
`In a preferred embodiment there is additionally provision
`of further nucleic acid sequences which code for a polypep-
`tide having the activity of an m3-desaturase and/or of a
`A4-desaturase in the plant.
`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) desatu-
`rase(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.
`The invention furthermore relates to recombinant nucleic
`
`least one nucleic acid
`acid molecules comprising at
`sequence which codes for a polypeptide having a A6-de-
`saturase activity; at least one nucleic acid sequence which
`codes for a polypeptide having a A5-desaturase activity; at
`least one nucleic acid sequence which codes for a polypep-
`tide having a A6-elongase activity; and at least one nucleic
`acid sequence which codes for a polypeptide having a
`A5-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.
`
`10
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`15
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`20
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`25
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`30
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`35
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`40
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`45
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`50
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`55
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`60
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`65
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`2
`
`A further part of the invention relates to oils, lipids and/or
`fatty acids which have been produced by the process accord-
`ing to the invention, and to their use.
`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 feedstulfs.
`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 phosphoglyc-
`erides. 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 conden-
`sation, reduction and dehydration reactions so that a satu-
`rated 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 microorganisms, see F.
`C. Neidhardt et al. (1996) E. coli and Salmonella. ASM
`Press: Washington, DC, 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 acyl-
`transferases. Moreover, these enzymes are capable of trans-
`ferring the elongated fatty acids from the CoA esters back to
`the phospholipids. If appropriate, this reaction sequence can
`be followed repeatedly.
`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).
`An overview of the biosynthesis of fatty acids in plants,
`desaturation, the lipid metabolism and the membrane trans-
`port 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.:
`I 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.: I K Setlow, 18:111-13; Gerhardt
`(1992) Prog. Lipid R. 31:397-417; Giihnemann-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 Mem-
`brane and Storage Lipids of Plants, Ed.: Murata and Somer-
`ville, Rockville, American Society of Plant Physiologists,
`150-158; Murphy & Ross (1998) Plant Journal. 13(1):1-16.
`Depending on the desaturation pattern, two large classes
`of polyunsaturated fatty acids, the 006 and the 003 fatty acids,
`which differ with regard to their metabolism and their
`function, can be distinguished.
`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).
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`US 10,301,638 B2
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`The fatty acid linoleic acid (18:2A9’12) acts as starting
`material for the 006 metabolic pathway, while the 003 path-
`way proceeds Via linolenic acid (18:3A9’12’15). Linolenic
`acid is formed from linoleic acid by the actiVity of an
`m3-desaturase (Tocher et al. (1998) Prog. Lipid Res. 37:
`73-117; Domergue et al.
`(2002) Eur. J. Biochem. 269:
`4105-4113).
`Mammals, and thus also humans, haVe no corresponding
`desaturase actiVity (A12- and m3-desaturase) for the forma-
`tion 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 polyunsatu-
`rated fatty acids arachidonic acid (:ARA, 20:4A5’8’11’14), an
`m6-fatty acid and the two m3-fatty acids eicosapentaenoic
`acid (:EPA, 20:5A5’8’11’14’17) and docosahexaenoic acid
`(DHA, 22:6A4’7’10’13’17’19) are synthesized Via a sequence of
`desaturase and elongase reactions.
`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, here-
`inbelow referred to as elongase). This is followed by a
`reduction step (ketoacyl-CoA reductase, KCR), a dehydra-
`tion step (dehydratase) and a final reduction step (enoyl-
`CoA reductase). It has been postulated that