DocCode — SEQ.TXT
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`Form Revision Date: August 26, 2013
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`1 of 667
`1 of 667
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`CSIRO Exhibit 1010
`CSIRO Exhibit 1010
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`UTILITY
`074017-0013—04-US-583539
`
`__
`PATENT APPLICATION
`PetraC'RPUS
`”3
`ame ”V6”
`We
`OILS, LlPlDS AND FATTY ACIDS PRODUCED lN
`
`
`TRANSGENIC BRASSICA PLANT
`-_Lobe/No.
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`(Primnype) Bronwen M. Loeb, Ph.D.
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`1176981291
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`074017-0013—04-US—583539
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`Fees Paid (fl
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`-i'7" —
`Name (Print/Type) Bronwen M. Loeb, Ph.D.
`
`5393:3333?-
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`Telephone
`Date
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`April 1, 2019
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`1176981301
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`3 of 667
`3 of 667
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`CSIRO Exhibit 1010
`CSIRO Exhibit 1010
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`074017-0013-04-US
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`Oils, Lipids And Fat_ty Acids Produced in Transgenic Brassica Plant
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`RELATED APPLICATIONS
`
`This application is a continuation of patent application Serial No. 15/256,914, filed September 6,
`
`2016, which is a continuation of patent application Serial No. 12/280,090, filed August 20, 2008,
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`now US. Patent No. 10, 190,131, which is a national stage application (under 35 U.S.C. § 371)
`
`of PCT/EP2007/051675, filed February 21, 2007, which claims benefit of German application
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`10 2006 008 030.0,
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`filed February 21, 2006 and European application 06120309.7,
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`filed
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`September 7, 2006.
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`The entire content of each aforementioned application is hereby
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`10
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`incorporated by reference in its entirety.
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`SUBMISSION 0F SEQUENCE LISTING
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`The Sequence Listing associated with this application is filed in electronic format via EFS-Web
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`and hereby incorporated by reference into the specification in its entirety. The name of the text
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`file containing the Sequence Listing is 074017_0013_04_583539_ST25. The size of the text file
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`15
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`is 809,660 bytes, and the text file was created on March 27, 2019.
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`The present
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`invention relates to a process for the production of eicosapentaenoic acid,
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`docosapentaenoic acid and/or docosahexaenoic acid in transgenic plants, providing in the plant at
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`least one nucleic acid sequence which codes for a polypeptide having a A6-desaturase activity; at
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`least one nucleic acid sequence which codes for a polypeptide having a A6-elongase activity; at
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`least one nucleic acid sequence which codes for a polypeptide having a A5-desaturase activity;
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`and at least one nucleic acid sequence which codes for a polypeptide having a A5-elongase
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`activity, where the nucleic acid sequence which codes for a polypeptide having a A5-elongase
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`activity is modified by comparison with the nucleic acid sequence in the organism from which
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`the sequence is derived in that it is adapted to the codon usage in one or more plant species.
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`In a preferred embodiment there is additionally provision of fithher nucleic acid sequences
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`which code for a polypeptide having the activity of an (2)3-desaturase and/or of a A4-desaturase
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`in the plant.
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`In a fithher preferred embodiment there is provision of fithher nucleic acid sequences which
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`code for acyl-CoA dehydrogenase(s), acyl-ACP (acyl carrier protein) desaturase(s), acyl-ACP
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`074017-0013-04-US
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`thioesterase(s), fatty acid acyl transferase(s), acyl-CoA:lysophospholipid acyl transferase(s),
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`fatty acid synthase(s),
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`fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-
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`coenzyme A oxidase(s),
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`fatty acid desaturase(s),
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`fatty acid acetylenases,
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`lipoxygenases,
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`triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase(s) in
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`the plant.
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`The invention furthermore relates to recombinant nucleic acid molecules comprising at least one
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`nucleic acid sequence which codes for a polypeptide having a A6-desaturase activity; at least one
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`nucleic acid sequence which codes for a polypeptide having a AS-desaturase activity; at least one
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`nucleic acid sequence which codes for a polypeptide having a A6-elongase activity; and at least
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`one nucleic acid sequence which codes for a polypeptide having a AS-elongase activity and
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`which is modified by comparison with the nucleic acid sequence in the organism from which the
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`sequence originates in that it is adapted to the codon usage in one or more plant species.
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`A further part of the invention relates to oils, lipids and/or fatty acids which have been produced
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`by the process according to the invention, and to their use.
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`Finally, the invention also relates to transgenic plants which have been produced by the process
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`of the invention or which comprise a recombinant nucleic acid molecule of the invention, and to
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`the use thereof as foodstuffs or feedstuffs.
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`Lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to
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`sn-glycerol-3-phosphate, and the addition or modification of a polar head group. Usual lipids
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`which are used in membranes comprise phospholipids, glycolipids,
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`sphingolipids and
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`phosphoglycerides. Fatty acid synthesis starts with the conversion of acetyl-CoA into malonyl-
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`CoA by acetyl-CoA carboxylase or into acetyl-ACP by acetyl transacylase. After condensation
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`reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a
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`series of condensation, reduction and dehydration reactions so that a saturated fatty acid
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`molecule with the desired chain length is obtained. The production of the unsaturated fatty acids
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`from these molecules is catalyzed by specific desaturases, either aerobically by means of
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`molecular oxygen or anaerobically (regarding the fatty acid synthesis in microorganisms, see
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`F.C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, DC, p. 612-636
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`and references cited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme:
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`Stuttgart, New York, and the references therein, and Magnuson, K., et al. (1993) Microbiological
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`Reviews 57:522-542 and the references therein). To undergo the further elongation steps, the
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`resulting phospholipid-bound fatty acids must be returned to the fatty acid CoA ester pool. This
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`is made possibly by acyl-CoA:lysophospholipid acyltransferases. Moreover, these enzymes are
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`capable of transferring the elongated fatty acids from the CoA esters back to the phospholipids.
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`If appropriate, this reaction sequence can be followed repeatedly.
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`Furthermore, fatty acids must subsequently be transported to various modification sites and
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`incorporated into the triacylglycerol storage lipid. A further important step during lipid synthesis
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`is the transfer of fatty acids to the polar head groups, for example by glycerol fatty acid
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`acyltransferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).
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`An overview of the biosynthesis of fatty acids in plants, desaturation, the lipid metabolism and
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`the membrane transport of lipidic compounds, beta-oxidation, the modification of fatty acids,
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`cofactors and the storage and assembly of triacylglycerol, including the references is given by
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`the following papers: Kinney (1997) Genetic Engineering, Ed.: JK Setlow, 19:149-166;
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`Ohlrogge and Browse (1995) Plant Cell 7:957-970; Shanklin and Cahoon (1998) Annu. Rev.
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`Plant Physiol. Plant Mol. Biol. 49:611-641; Voelker (1996) Genetic Engeneering, Ed.: JK
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`Setlow, 18:111-13; Gerhardt (1992) Prog. Lipid R. 31:397-417; Giihnemann-Schafer & Kindl
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`(1995) Biochim. Biophys Acta 1256:181-186; Kunau et al. (1995) Prog. Lipid Res. 34:267-342;
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`Stymne et al. (1993) in: Biochemistry and Molecular Biology of Membrane and Storage Lipids
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`of Plants, Ed.: Murata und Somerville, Rockville, American Society of Plant Physiologists, 150-
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`158; Murphy & Ross (1998) Plant Journal. 13(1):1-16.
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`Depending on the desaturation pattern, two large classes of polyunsaturated fatty acids, the 036
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`and the 033 fatty acids, which differ with regard to their metabolism and their function, can be
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`distinguished.
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`In the text which follows, polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA
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`or LCPUFAs (poly unsaturated fatty acids, PUFA,
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`long chain poly unsaturated fatty acids,
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`25
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`LCPUFA).
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`The fatty acid linoleic acid (18:2A9’12) acts as starting material for the 036 metabolic pathway,
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`while the 033 pathway proceeds via linolenic acid (18:3A9’12’15). Linolenic acid is formed from
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`linoleic acid by the activity of an 033-desaturase (Tocher et al. (1998) Prog. Lipid Res. 37:
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`30
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`73-117; Domergue et al. (2002) Eur. J. Biochem. 269: 4105-4113).
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`Mammals, and thus also humans, have no corresponding desaturase activity (A12- and 033-
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`desaturase) for the formation of the starting materials and must therefore take up these fatty acids
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`(essential fatty acids) via the food. Starting with these precursors, the physiologically important
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`polyunsaturated fatty acids arachidonic acid (= ARA, 20:4A5’8’11’14), an oa6-fatty acid and the two
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`oa3-fatty acids eicosapentaenoic acid (= EPA, 20:5A5’8’11’14’17) and docosahexaenoic acid (DHA,
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`22:6A4’7’10’13’17’19) are synthesized via a sequence of desaturase and elongase reactions.
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`The elongation of fatty acids, by elongases, by 2 or 4 C atoms is of crucial importance for the
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`production of C20- and sz-PUFAs, respectively. This process proceeds via 4 steps. The first step
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`is the condensation of malonyl-CoA onto the fatty acid acyl-CoA by ketoacyl-CoA synthase
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`(KCS, hereinbelow referred to as elongase). This is followed by a reduction step (ketoacyl—CoA
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`reductase, KCR), a dehydratation step (dehydratase) and a final reduction step (enoyl-CoA
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`reductase). It has been postulated that the elongase activity affects the specificity and rate of the
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`entire process (Millar and Kunst (1997) Plant Journal 12: 121-131).
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`Fatty acids and triacylglycerides have a multiplicity of applications in the food industry,
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`in
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`animal nutrition, in cosmetics and the pharmacological sector. Depending on whether they are
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`free saturated or unsaturated fatty acids or else triacylglycerides with an elevated content of
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`saturated or unsaturated fatty acids, they are suitable for very different applications. Thus, for
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`example, lipids with unsaturated, specifically with polyunsaturated fatty acids, are preferred in
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`human nutrition. The polyunsaturated oa3-fatty acids are supposed to have a positive effect on the
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`cholesterol level in the blood and thus on the prevention of heart disease. The risk of heart
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`disease, strokes or hypertension can be reduced markedly by adding these oa3-fatty acids to the
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`food (Shimikawa (2001) World Rev. Nutr. Diet. 88: 100-108).
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`oa3-fatty acids also have a positive effect on inflammatory,
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`specifically on chronically
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`inflammatory, processes in association with immunological diseases such as rheumatoid arthritis
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`(Calder (2002) Proc. Nutr. Soc. 61: 345-358; Cleland and James (2000) J. Rheumatol. 27: 2305-
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`2307). They are therefore added to foodstuffs, specifically to dietetic foodstuffs, or are employed
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`in medicaments. oa6-fatty acids such as arachidonic acid tend to have a negative effect
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`in
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`connection with these rheumatological diseases.
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`033- and 036-fatty acids are precursors of tissue hormones, known as eicosanoids, such as the
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`prostaglandins, which are derived from dihomo-y-linolenic acid, arachidonic acid and
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`eicosapentaenoic acid, and of the thromboxanes and leukotrienes, which are derived from
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`arachidonic acid and eicosapentaenoic acid. Eicosanoids (known as the PG2 series) which are
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`formed from the oa6-fatty acids, generally promote inflammatory reactions, while eicosanoids
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`(known as the PG3 series) from oa3-fatty acids have little or no proinflammatory effect.
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`Polyunsaturated long-chain
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`oa3-fatty
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`acids
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`such
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`as
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`eicosapentaenoic
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`acid
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`(= EPA,
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`C2025A5’8’11’14’17) or docosahexaenoic acid (= DHA, C2226A4’7’10’13’16’19) are important components
`
`of human nutrition owing to their various roles in health aspects, including the development of
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`the child brain,
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`the functionality of the eyes,
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`the synthesis of hormones and other signal
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`substances, and the prevention of cardiovascular disorders, cancer and diabetes (Poulos, A
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`(1995) Lipids 3011-14; Horrocks, LA and Yeo YK (1999) Pharrnacol Res 40:211-225).
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`Owing to the present-day composition of human food, an addition of polyunsaturated 033-fatty
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`acids, which are preferentially found in fish oils, to the food is particularly important. Thus, for
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`example, polyunsaturated fatty acids such as docosahexaenoic acid (= DHA, C2226A4’7’10’13’16’19)
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`or eicosapentaenoic acid (= EPA, C2025A5’8’11’14’17) are added to infant formula to improve the
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`nutritional value. There is therefore a demand for the production of polyunsaturated long-chain
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`fatty acids.
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`The various fatty acids and triglycerides are mainly obtained from microorganisms such as
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`Mortierella or Schizochytrium or from oil-producing plants such as soybeans, oilseed rape, and
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`algae such as Crypthecodinium or Phaeodaclylum and others, being obtained, as a rule, in the
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`form of their triacylglycerides (= triglycerides = triglycerols). However,
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`they can also be
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`obtained from animals, for example, fish. The free fatty acids are advantageously prepared by
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`hydrolyzing the triacylglycerides. Very long-chain polyunsaturated fatty acids such as DHA,
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`EPA, arachidonic acid (ARA, C2024A5’8’11’14), dihomo-y-linolenic acid (DHGL, C2023A8’11’14) or
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`docosapentaenoic acid (DPA, C2225A7’10’13’16’19) are, however, not synthesized in oil crops such
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`as oilseed rape, soybeans, sunflowers and safflower. Conventional natural sources of these fatty
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`acids are fish such as herring, salmon, sardine, redfish, eel, carp, trout, halibut, mackerel, zander
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`or tuna, or algae.
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`Owing to the positive characteristics of the polyunsaturated fatty acids, there has been no lack of
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`attempts in the past to make available genes which are involved in the synthesis of these fatty
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`acids or triglycerides for the production of oils in various organisms with a modified content of
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`unsaturated fatty acids. Thus, W0 91/ 13972 and its US equivalent describe a A9-desaturase.
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`W0 93/11245 claims a A15-desaturase and W0 94/11516 a A12—desaturase. Further desaturates
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`are described, for example, in EP-A-0 550 162, W0 94/18337, W0 97/30582, W0 97/21340,
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`W0 95/ 18222, EP-A-0 794 250, Stukey et al. (1990) J. Biol. Chem., 265: 20144-20149, Wada et
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`al. (1990) Nature 347: 200-203 or Huang et al. (1999) Lipids 34: 649-659. However,
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`the
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`biochemical characterization of the various desaturases has been insufficient to date since the
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`enzymes, being membrane-bound proteins, present great difficulty in their
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`isolation and
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`characterization (McKeon et al. (1981) Methods in Enzymol. 71: 12141-12147, Wang et al.
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`(1988) Plant Physiol. Biochem., 26: 777-792).
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`As a rule, membrane-bound desaturases are characterized by being introduced into a suitable
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`organism which is subsequently analyzed for enzyme activity by analyzing the starting materials
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`and the products. A6-Desaturases are described in W0 93/06712, US 5,614,393, W0 96/21022,
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`W0 00/21557 and W0 99/27111. The application of this enzyme for the production of fatty
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`acids in transgenic organisms is described in W0 98/46763, W0 98/46764 and W0 98/46765.
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`The expression of various desaturases and the formation of polyunsaturated fatty acids is also
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`described and claimed in W0 99/64616 or W0 98/46776. As regards the expression efficacy of
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`desaturases and its effect on the formation of polyunsaturated fatty acids, it must be noted that
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`the expression of a single desaturase as described to date has only resulted in low contents of
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`unsaturated fatty acids/lipids such as, for example, y-linolenic acid and stearidonic acid.
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`20
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`There have been a number of attempts in the past to obtain elongase genes. Millar and Kunst
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`(1997) Plant Journal 12:121-131 and Millar et al. (1999) Plant Cell 11:825-838 describe the
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`characterization of plant elongases for the synthesis of monounsaturated long-chain fatty acids
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`(C22:1) and for the synthesis of very long-chain fatty acids for the formation of waxes in plants
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`(Czs-C32). The synthesis of arachidonic acid and EPA is described,
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`for example,
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`in W0
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`01/59128, W0 00/ 12720, W0 02/077213 and W0 02/08401. The synthesis of polyunsaturated
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`C24-fatty acids is described, for example, in Tvrdik et al. (2000) J. Cell Biol. 149:707-718 or in
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`W0 02/44320.
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`Especially suitable microorganisms for the production of PUFAs are microalgae such as
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`Phaeodaclylum tricornutum, Porphiridium species, Thraustochytrium species, Schizochytrium
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`species or Crypthecodinium species, ciliates such as Siylonychia or Colpidium, fiJngi such as
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`Mortierella, Entomophthora or Mucor and/or mosses such as Physcomitrella, Ceratodon and
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`Marchantia (R. Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558; K. Totani & K.
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`Oba (1987) Lipids 22: 1060-1062; M. Akimoto et al.
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`(1998) Appl. Biochemistry and
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`Biotechnology 73: 269-278). Strain selection has resulted in the development of a number of
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`mutant strains of the microorganisms in question which produce a series of desirable compounds
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`including PUFAs. However, the mutation and selection of strains with an improved production
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`of a particular molecule such as the polyunsaturated fatty acids is a time-consuming and difficult
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`process. Moreover, only limited amounts of the desired polyunsaturated fatty acids such as DPA,
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`EPA or ARA can be produced with the aid of the abovementioned microorganisms; in addition,
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`they are generally obtained as fatty acid mixtures. This is why recombinant methods are
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`preferred whenever possible.
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`Higher plants comprise polyunsaturated fatty acids such as linoleic acid (C18:2) and linolenic
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`acid (C18:3). ARA, EPA and DHA are found not at all in the seed oil of higher plants, or only in
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`miniscule amounts (E. Ucciani: Nouveau Dictionnaire des Huiles Végétales [New Dictionary of
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`the Vegetable Oils]. Technique & Documentation — Lavoisier, 1995. ISBN: 2-7430-0009—0).
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`However, the production of LCPUFAs in higher plants, preferably in oil crops such as oilseed
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`rape, linseed, sunflowers and soybeans, would be advantageous since large amounts of high-
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`quality LCPUFAs for the food industry, animal nutrition and pharmaceutical purposes might be
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`obtained economically. To this end, it is advantageous to introduce, into oilseeds, genes which
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`encode enzymes of the LCPUFA biosynthesis via recombinant methods and to express them
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`therein. These genes encode for example A6-desaturases, A6-elongases, A5-desaturases or A4-
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`desaturases. These genes can advantageously be isolated from microorganisms and lower plants
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`which produce LCPUFAs and incorporate them in the membranes or triacylglycerides. Thus, it
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`has already been possible to isolate A6-desaturase genes from the moss Physcomitrella patens
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`and A6-elongase genes from P. patens and from the nematode C. elegans.
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`Transgenic plants which comprise and express genes encoding LCPUFA biosynthesis enzymes
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`and which, as a consequence, produce LCPUFAs have been described, for example, in DE-A-
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`102 19 203 (process for the production of polyunsaturated fatty acids in plants). However, these
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`plants produce LCPUFAs in amounts which require further optimization for processing the oils
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`which are present in the plants. Thus, the ARA content in the plants described in DE-A-102 19
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`203 is only 0.4 to 2% and the EPA content only 0.5 to 1%, in each case based on the total lipid
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`content of the plant.
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`To make possible the fortification of food and of feed with polyunsaturated, long-chain fatty
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`acids, there is therefore a great need for a simple, inexpensive process for the production of
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`polyunsaturated, long-chain fatty acids, specifically in plant systems.
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`One object of the invention is
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`therefore to provide a process with which long-chain
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`polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and/or
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`docosahexaenoic acid can be produced in large quantities and inexpensively in transgenic plants.
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`It has now surprisingly been found that the yield of long-chain polyunsaturated fatty acids,
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`especially eicosapentaenoic, docosapentaenoic acid and/or docosahexaenoic acid, can be
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`increased by expressing an optimized A5-elongase sequence in transgenic plants.
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`The PUFAs produced by the process of the invention comprise a group of molecules which
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`higher animals are no longer able to synthesize and thus must consume, or which higher animals
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`are no longer able to produce themselves in sufficient amounts and thus must consume additional
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`amounts thereof, although they can easily be synthesized by other organisms such as bacteria.
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`Accordingly, the object of the invention is achieved by the process of the invention for producing
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`eicosapentaenoic acid, docosapentaenoic acid and/or docosahexaenoic acid in a transgenic plant,
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`comprising the provision in the plant of at least one nucleic acid sequence which codes for a
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`polypeptide having a A6-desaturase activity; at least one nucleic acid sequence which codes for a
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`polypeptide having a A6-elongase activity; at least one nucleic acid sequence which codes for a
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`polypeptide having a A5-desaturase activity; and at least one nucleic acid sequence which codes
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`for a polypeptide having a A5-elongase activity,
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`where the nucleic acid sequence which codes for a polypeptide having a A5-elongase activity is
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`modified by comparison with the nucleic acid sequence in the organism from which the sequence
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`is derived in that it is adapted to the codon usage in one or more plant species. To produce DHA
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`it
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`is additionally necessary to provide at least one nucleic acid sequence which codes for a
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`polypeptide having a A4-desaturase activity in the plant.
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`The “provision in the plant” means in the context of the present invention that measures are
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`taken so that the nucleic acid sequences coding for a polypeptide having a A6-desaturase activity,
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`a polypeptide having a A6-elongase activity, a polypeptide having a AS-desaturase activity and a
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`polypeptide having a AS-elongase activity are present together in one plant. The “provision in the
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`plant” thus comprises the introduction of the nucleic acid sequences into the plant both by
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`transformation of a plant with one or more recombinant nucleic acid molecules which comprise
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`said nucleic acid sequences, and by crossing suitable parent plants which comprise one or more
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`of said nucleic acid sequences.
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`The nucleic acid sequence which codes for a polypeptide having a AS-elongase activity is
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`modified according to the invention by comparison with the nucleic acid sequence in the
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`organism from which the sequence originates in that it is adapted to the codon usage in one or
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`more plant species. This means that the nucleic acid sequence has been specifically optimized for
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`the purpose of the invention without the amino acid sequence encoded by the nucleic acid
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`sequence having been altered thereby.
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`The genetic code is redundant because it uses 61 codons in order to specify 20 amino acids.
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`Therefore, most of the 20 proteinogenic amino acids are therefore encoded by a plurality of
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`triplets (codons). The synonymous codons which specify an individual amino acid are, however,
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`not used with the same frequency in a particular organism; on the contrary there are preferred
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`codons which are frequently used, and codons which are used more rarely. These differences in
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`codon usage are attributed to selective evolutionary pressures and especially the efficiency of
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`translation. One reason for the lower translation efficiency of rarely occurring codons might be
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`that the corresponding aminoacyl-tRNA pools are exhausted and thus no longer available for
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`protein synthesis.
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`In addition, different organisms prefer different codons. For this reason, for example,
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`the
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`expression of a recombinant DNA derived from a mammalian cell frequently proceeds only
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`suboptimally in Escherichia coli (E. 0011) cells. It is therefore possible in some cases to increase
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`expression by replacing rarely used codons with frequently used codons. Without wishing to be
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`bound to one theory, it is assumed that the codon-optimized DNA sequences make more efficient
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`translation possible, and the mRNAs formed therefrom possibly have a greater half-life in the
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`cell and therefore are available more frequently for translation. From what has been said above,
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`it follows that codon optimization is necessary only if the organism in which the nucleic acid
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`sequence is to be