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`074017-0013-04-US-583539
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`Fisthamedmventor[PetaGIRPUSJPATENT APPLICATION inventor_|Petra CIRPUSust Named
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`rile.|OILS, LIPIDS AND FATTY ACIDS PRODUCEDIN
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`TRANSMITTAL
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`75
`150*
`330
`760
`380
`190
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`40
`160
`50
`100
`80
`600
`300
`150
`200
`Design
`105
`420
`50
`100
`210
`620
`310
`155
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`75
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`330
`2,200
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`117698130.1
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`3 of 667
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`074017-0013-04-US
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`Oils, Lipids And Fatty Acids Produced in Transgenic Brassica Plant
`
`RELATED APPLICATIONS
`
`This application is a continuation of patent application Serial No. 15/256,914, filed September6,
`
`2016, which is a continuation of patent application Serial No. 12/280,090, filed August 20, 2008,
`
`now U.S. 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
`
`10 2006 008 030.0,
`
`filed February 21, 2006 and European application 06120309.7,
`
`filed
`
`September 7, 2006.
`
`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 OF SEQUENCELISTING
`
`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_04583539ST25. Thesize ofthe 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.
`
`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
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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-elongaseactivity; at
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`20
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`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
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`the sequenceis derived in that it is adapted to the codon usage in one or moreplantspecies.
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`25
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`In a preferred embodiment there is additionally provision of further nucleic acid sequences
`
`which code for a polypeptide having the activity of an @3-desaturase and/or of a A4-desaturase
`
`in the plant.
`
`In a further preferred embodiment there is provision of further 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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`117649640.1
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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-
`
`074017-0013-04-US
`
`
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`coenzyme A_oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases,
`
`
`
`
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`triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase(s) in
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`the plant.
`
`The invention furthermore relates to recombinant nucleic acid molecules comprising 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 A5-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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`10
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`one nucleic acid sequence which codes for a polypeptide having a A5-celongase 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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`sequenceoriginates in that it is adapted to the codon usage in one or moreplant species.
`
`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, andto their use.
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`15
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`Finally, the invention also relates to transgenic plants which have been producedbythe process
`
`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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`20
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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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`25
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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, D.C., p. 612-636
`
`and references cited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme:
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`30
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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
`
`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.
`
`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
`
`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).
`
`10
`
`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 oftriacylglycerol, including the references is given by
`
`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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`15
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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
`
`(1995) Biochim. Biophys Acta 1256:181-186; Kunau et al. (1995) Prog. Lipid Res. 34:267-342;
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`Stymneetal. (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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`20
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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 w6
`
`and the 3 fatty acids, which differ with regard to their metabolism and their function, can be
`
`distinguished.
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`In the text which follows, polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA
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`25
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`or LCPUFAs (poly unsaturated fatty acids, PUFA,
`
`long chain poly unsaturated fatty acids,
`
`LCPUFA).
`
`The fatty acid linoleic acid (18:24'*) acts as starting material for the 6 metabolic pathway,
`while the 3 pathway proceeds via linolenic acid (18:34!”!5), Linolenic acid is formed from
`
`linoleic acid by the activity of an w3-desaturase (Tocher et al. (1998) Prog. Lipid Res. 37:
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`30
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`73-117; Domergueetal. (2002) Eur. J. Biochem. 269: 4105-4113).
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`Mammals, and thus also humans, have no corresponding desaturase activity (A12- and 3-
`
`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, 20:45*!1!4), an «w6-fatty acid and the two
`«93-fatty acids eicosapentaenoic acid (= EPA, 20:5°*!!.!417) and docosahexaenoic acid (DHA,
`22:6%4710,13,17,19) are synthesized via a sequence ofdesaturase and elongase reactions.
`
`The elongation of fatty acids, by elongases, by 2 or 4 C atomsis of crucial importance for the
`
`production of Cz0- 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
`
`10
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`(KCS, hereinbelow referred to as elongase). This is followed by a reduction step (ketoacyl-CoA
`
`reductase, KCR), a dehydratation 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).
`
`Fatty acids and triacylglycerides have a multiplicity of applications in the food industry,
`
`in
`
`15
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`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
`
`humannutrition. The polyunsaturated 3-fatty acids are supposed to have a positive effect on the
`
`20
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`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 w3-fatty acids to the
`
`food (Shimikawa (2001) World Rev. Nutr. Diet. 88: 100-108).
`
`w3-fatty acids also have a positive effect on inflammatory,
`
`specifically on chronically
`
`inflammatory, processes in association with immunological diseases such as rheumatoid arthritis
`
`25
`
`(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. w6-fatty acids such as arachidonic acid tend to have a negative effect
`
`in
`
`connection with these rheumatological diseases.
`
`w3- and w6-fatty acids are precursors of tissue hormones, known as eicosanoids, such as the
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`30
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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
`
`arachidonic acid and eicosapentaenoic acid. Eicosanoids (known as the PG? series) which are
`
`formed from the 6-fatty acids, generally promote inflammatory reactions, while eicosanoids
`
`(known as the PG3 series) from w3-fatty acids havelittle or no proinflammatory effect.
`
`Polyunsaturated long-chain 3-fatty acids
`such
`as
`eicosapentaenoic
`acid
`(= EPA,
`C20:5458!114,17) or docosahexaenoic acid (= DHA, C22:6%47!913.16.19) 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
`
`10
`
`(1995) Lipids 30:1-14; Horrocks, LA and Yeo YK (1999) Pharmacol Res 40:211-225).
`
`Owing to the present-day composition of human food, an addition of polyunsaturated m3-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, C22:6%471013,16.19)
`or eicosapentaenoic acid (= EPA, C20:5%5*!114:17) are added to infant formula to improve the
`
`15
`
`nutritional value. There is therefore a demand for the production of polyunsaturated long-chain
`
`fatty acids.
`
`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
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`20
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`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, C20:45*!114), dihomo-y-linolenic acid (DHGL, C20:3°%!1"!4) or
`docosapentaenoic acid (DPA, C22:547!%13:16.19) are, however, not synthesized in oil crops such
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`25
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`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.
`
`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
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`30
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`acids ortriglycerides for the production of oils in various organisms with a modified content of
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`unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a A9-desaturase.
`
`WO 93/11245 claims a A15-desaturase and WO 94/11516 a A1l2-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, Stukeyet al. (1990) J. Biol. Chem., 265: 20144-20149, Wadaet
`
`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 (McKeonet al. (1981) Methods in Enzymol. 71: 12141-12147, Wang et al.
`
`(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
`
`organism which is subsequently analyzed for enzymeactivity by analyzing the starting materials
`
`and the products. A6-Desaturases are described in WO 93/06712, US 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 98/46765S.
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`15
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`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, 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
`
`(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
`
`(C2s-C32). The synthesis of arachidonic acid and EPA is described,
`
`for example,
`
`in WO
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`25
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`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
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`WO 02/44320.
`
`Especially suitable microorganisms for the production of PUFAs are microalgae such as
`
`Phaeodactylum tricornutum, Porphiridium species, Thraustochytrium species, Schizochytrium
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`30
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`species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as
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`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.
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`Oba (1987) Lipids 22: 1060-1062; M. Akimoto et al.
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`(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 producea 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-consuminganddifficult
`
`process. Moreover, 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,
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`they are generally obtained as fatty acid mixtures. This is why recombinant methods are
`
`preferred wheneverpossible.
`
`Higher plants comprise polyunsaturated fatty acids such as linoleic acid (C18:2) and linolenic
`
`acid (C18:3). ARA, EPA and DHAare foundnotatall in the seed oil of higher plants, or only in
`
`miniscule amounts (E. Ucciani: Nouveau Dictionnaire des Huiles Végétales [New Dictionary of
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`15
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`the Vegetable Oils]. Technique & Documentation — Lavoisier, 1995. ISBN: 2-7430-0009-0).
`
`However, the production of LCPUFAsin 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-
`
`quality LCPUFAsfor the food industry, animal nutrition and pharmaceutical purposes might be
`
`obtained economically. To this end, it is advantageous to introduce, into oilseeds, genes which
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`20
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`encode enzymes of the LCPUFA biosynthesis via recombinant methods and to express them
`
`therein. These genes encode for example A6-desaturases, A6-elongases, A5-desaturases or A4-
`
`desaturases. These genes can advantageously be isolated from microorganisms and lower plants
`
`which produce LCPUFAsandincorporate them in the membranesor 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.
`
`Transgenic plants which comprise and express genes encoding LCPUFAbiosynthesis enzymes
`
`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
`
`plants produce LCPUFAsin 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 onthe 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
`
`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.
`
`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 producedin large quantities and inexpensively in transgenic plants.
`
`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
`
`increased by expressing an optimized A5-elongase sequence in transgenic plants.
`
`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
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`are no longer able to produce themselves in sufficient amounts and thus must consumeadditional
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`amounts thereof, although they can easily be synthesized by other organismssuchasbacteria.
`
`Accordingly, the object of the invention is achieved by the processof the invention for producing
`
`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
`
`polypeptide having a A6-desaturase activity; at least one nucleic acid sequence which codesfor a
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`polypeptide having a A6-elongaseactivity; 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-elongaseactivity,
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`where the nucleic acid sequence which codesfor a polypeptide having a A5-elongaseactivity 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
`
`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.
`
`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 A5-desaturase activity and a
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`polypeptide having a A5-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
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`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.
`
`The nucleic acid sequence which codes for a polypeptide having a A5-elongase activity is
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`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
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`more plant species. This meansthat 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 beenaltered thereby.
`
`The genetic code is redundant because it uses 61 codons in order to specify 20 aminoacids.
`
`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,
`
`not used with the same frequency in a particular organism; on the contrary there are preferred
`
`codons whichare frequently used, and codons which are used morerarely. These differences in
`
`codon usage are attributed to selective evolutionary pressures and especially the efficiency of
`
`translation. One reason for the lowertranslation efficiency of rarely occurring codons might be
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`20
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`that the corresponding aminoacyl-tRNA pools are exhausted and thus no longer available for
`
`protein synthesis.
`
`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. coli) cells. It is therefore possible in some cases to increase
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`25
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`expression by replacing rarely used codons with frequently used codons. Without wishing to be
`
`boundto one theory, it is assumed that the codon-optimized DNA sequences make moreefficient
`
`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