(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2010/0227924 A1
`Cirpus et al.
`(43) Pub. Date:
`Sep. 9, 2010
`
`US 2010.0227924A1
`
`(54) METHOD FOR PRODUCING
`POLYUNSATURATED FATTY ACDS
`
`(75) Inventors:
`
`Petra Cirpus, Waldsee (DE); Jörg
`Bauer, Limburgerhof (DE); Xiao
`Qiu, Saskatoon (CA); Guohai Wu,
`Saskatoon (CA); Bifang Cheng,
`Saskatoon (CA); Martin Truksa,
`Edmonton (CA); Tom Wetjen,
`Mannheim (DE)
`Correspondence Address:
`CONNOLLY BOVE LODGE & HUTZ, LLP
`PO BOX 2207
`WILMINGTON, DE 19899 (US)
`
`(73) Assignee:
`
`BASF Plant Science GmbH,
`Ludwigshafen (DE)
`
`(21) Appl. No.:
`
`12/280,090
`
`(22) PCT Filed:
`
`Feb. 21, 2007
`
`(86). PCT No.:
`S371 (c)(1),
`(2), (4) Date:
`
`PCT/EP2007/051675
`
`Aug. 20, 2008
`
`(30)
`
`Foreign Application Priority Data
`
`Feb. 21, 2006 (DE) ......................... 102OO6008O3O.O
`
`Sep. 7, 2006 (EP) .................................. O612O309.7
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`A6II 3L/20
`(2006.01)
`CI2N 5/82
`(2006.01)
`AOIH 5/00
`(2006.01)
`C7H 2L/04
`(2006.01)
`A23D 9/00
`(52) U.S. Cl. ........ 514/558; 800/281:800/298; 536/23.6;
`426/601
`
`ABSTRACT
`(57)
`The invention relates to a method for producing eicosapen
`tanoic acid, docosapentanoic acid and/or docohexanoic acid
`in transgenic plants. According to said method, the plant is
`provided with at least one nucleic acid sequence coding for a
`polypeptide with a A6 desaturase activity, at least one nucleic
`acid sequence coding for a polypeptide with a A6 elongase
`activity, at least one nucleic acid sequence coding for a
`polypeptide with a A5 desaturase activity, and at least one
`nucleic acid sequence coding for a polypeptide with a A5
`elongase activity, the nucleic acid sequence coding for a
`polypeptide with a A5 elongase activity being modified in
`relation to the nucleic acid sequence in the organism from
`which the sequence originates, such that it is adapted to the
`codon use in at least one type of plant. For the production of
`docosahexanoic acid, at least one nucleic acid sequence cod
`ing for a polypeptide with a A4 desaturase activity is also
`introduced into the plant.
`
`CSIRO Exhibit 1019
`
`

`

`US 2010/0227924 A1
`
`Sep. 9, 2010
`
`METHOD FOR PRODUCING
`POLYUNSATURATED FATTY ACDS
`
`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 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 activ
`ity; 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 polypep
`tide 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 polypeptide having the activity of an ()3-de
`saturase and/or of a A4-desaturase in the plant.
`0002. 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:ly Sophospholipid 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, tria
`cylglycerol lipases, allene oxide synthases, hydroperoxide
`lyases or fatty acid elongase(s) in the plant.
`0003. The invention furthermore relates to recombinant
`nucleic acid molecules comprising at least one nucleic acid
`sequence which codes for a polypeptide having a A6-desatu
`rase 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 polypeptide 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.
`0004. 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.
`0005 Finally, the invention also relates to transgenic
`plants which have been produced by the process of the inven
`tion or which comprise a recombinant nucleic acid molecule
`of the invention, and to the use thereofas foodstuffs or feed
`stuffs.
`0006 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 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 aerobi
`cally by means of molecular oxygen oranaerobically (regard
`ing the fatty acid synthesis in 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 Magnu
`son, 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 pos
`sibly by acyl-CoA:lysophospholipid acyltransferases. More
`over, 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 repeat
`edly.
`0007 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 acyltrans
`ferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).
`0008. An overview of the biosynthesis of fatty acids in
`plants, desaturation, the lipid metabolism and the membrane
`transport of lipidic compounds, beta-oxidation, the modifica
`tion of fatty acids, cofactors and the storage and assembly of
`triacylglycerol, including the references is given by the fol
`lowing papers: Kinney (1997) Genetic Engineering, Ed.: JK
`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: Gühnemann-Schafer & Kindl
`(1995) Biochim. Biophys Acta 1256:181-186; Kunau et al.
`(1995) Prog. Lipid Res. 34:267-342; Stymine et al. (1993) in:
`Biochemistry and Molecular Biology of Membrane and Stor
`age Lipids of Plants, Ed.: Murata and Somerville, Rockville,
`American Society of Plant Physiologists, 150-158; Murphy
`& Ross (1998) Plant Journal. 13(1):1-16.
`0009. Depending on the desaturation pattern, two large
`classes of polyunsaturated fatty acids, the (D6 and the (p3 fatty
`acids; which differ with regard to their metabolism and their
`function, can be distinguished.
`0010. In the text which follows, polyunsaturated fatty
`acids are referred to as PUPA, PUFAs, LCPUFA or LCPUFAs
`(poly unsaturated fatty acids, PUFA, long chain poly unsat
`urated fatty acids, LCPUFA).
`0011. The fatty acid linoleic acid (18:2^*) acts as start
`ing material for the Co6 metabolic pathway, while the co3
`pathway proceeds via linolenic acid (18:3^''''). Linolenic
`acid is formed from linoleic acid by the activity of an (03-de
`saturase (Tocher et al. (1998) Prog. Lipid Res. 37: 73-117:
`Domergue et al. (2002) Eur. J. Biochem. 269: 4105-41 13).
`0012 Mammals, and thus also humans, have no corre
`sponding desaturase activity (A12- and (03-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 polyun
`saturated fatty acids arachidonic acid (ARA, 20:4:'''''),
`an (D-fatty acid and the two ()3-fatty acids eicosapentaenoic
`acid (=EPA, 20:5^'''''') and docosa-hexaenoic acid
`(DHA, 22:6''''''''') are synthesized via a sequence of
`desaturase and elongase reactions.
`
`CSIRO Exhibit 1019
`
`

`

`US 2010/0227924 A1
`
`Sep. 9, 2010
`
`0013 The elongation of fatty acids, by elongases, by 2 or
`4C atoms is of crucial importance for the production of Co
`and C-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 referred to as elongase). This is followed by a
`reduction step (ketoacyl-CoA reductase, KCR), a dehydrata
`tion 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).
`0014 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, spe
`cifically with polyunsaturated fatty acids, are preferred in
`human nutrition. The polyunsaturated (D3-fatty acids are Sup
`posed 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 mark
`edly by adding these co3-fatty acids to the food (Shimikawa
`(2001) World Rev. Nutr. Diet. 88: 100-108).
`00.15
`(03-fatty acids also have a positive effect on inflam
`matory, specifically on chronically inflammatory, processes
`in association with immunological diseases such as rheuma
`toid 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. (D6-fatty acids
`Such as arachidonic acid tend to have a negative effect in
`connection with these rheumatological diseases.
`0016 (O3- and (06-fatty acids are precursors of tissue hor
`mones, known as eicosanoids, such as the prostaglandins,
`which are derived from dihomo-y-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 PG series)
`which are formed from the S.26-fatty acids, generally promote
`inflammatory reactions, while eicosanoids (known as the PG
`series) from S23-fatty acids have little or no proinflammatory
`effect.
`0017 Polyunsaturated long-chain S23-fatty acids such as
`eicosapentaenoic acid (=EPA, C20:5^''''''') or docosa
`hexaenoic acid (=DHA, C22:6-7'''''') 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, LA andYeoYK (1999) Pharmacol Res 40:211
`225).
`0018. Owing to the present-day composition of human
`food, an addition of polyunsaturated (D3-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, C22:6-7'''''')
`oreicosapentaenoic acid (=EPA, C20:5^*'''''') are added
`to infant formula to improve the nutritional value. There is
`therefore a demand for the production of polyunsaturated
`long-chain fatty acids.
`
`0019. The various fatty acids and triglycerides are mainly
`obtained from microorganisms such as Mortierella or
`Schizochytrium or from oil-producing plants such as Soy
`beans, 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 pre
`pared by hydrolyzing the triacylglycerides. Very long-chain
`polyunsaturated fatty acids such as DHA, EPA, arachidonic
`acid (ARA, C20:4^*'''), dihomo-y-linolenic acid
`(DHGL, C20:3''''') or docosapentaenoic acid (DPA, C22:
`5^''''''') are, however, not synthesized in oil crops such
`as oilseed rape, soybeans, Sunflowers and safflower. Conven
`tional natural Sources of these fatty acids are fish Such as
`herring, salmon, Sardine, redfish, eel, carp. trout, halibut,
`mackerel, Zander or tuna, or algae.
`0020. Owing to the positive characteristics of the polyun
`saturated fatty acids, there has been no lack of attempts in the
`past to make available genes which are involved in the Syn
`thesis of these fatty acids or triglycerides for the production of
`oils in various organisms with a modified content of unsatur
`ated fatty acids. Thus, WO 91/13972 and its US equivalent
`describea A9-desaturase. WO93/11245 claims a A15-desatu
`rase and WO94/11516 a A12-desaturase. Further desaturates
`are described, for example, in EPA-0550 162, WO94/18337.
`WO 97/30582, WO 97/21340, WO95/18222, EPA-0 794
`250, Stukey et al. (1990).J. Biol. Chem., 265: 20144-20149,
`Wada et al. (1990) Nature 347:200-203 or Huanget al. (1999)
`Lipids 34:649-659. However, the biochemical characteriza
`tion 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).
`0021. As a rule, membrane-bound desaturases are charac
`terized by being introduced into a Suitable organism which is
`Subsequently analyzed for enzyme activity by analyzing the
`starting materials and the products. A6-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 WO98/46763, 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 WO99/64616 or WO 98/46776. As regards the
`expression efficacy of desaturases and its effect on the forma
`tion 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.
`0022. 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 Syn
`thesis of monounsaturated long-chain fatty acids (C22:1) and
`for the synthesis of very long-chain fatty acids for the forma
`tion of waxes in plants (C-C). The synthesis of arachi
`donic 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 O2/4432O.
`
`CSIRO Exhibit 1019
`
`

`

`US 2010/0227924 A1
`
`Sep. 9, 2010
`
`0023 Especially suitable microorganisms for the produc
`tion of PUFAs are microalgae such as Phaeodactylum tricor
`nutum, 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 Physcomi
`trella, 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 selec
`tion 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 pro
`duction of a particular molecule Such as the polyunsaturated
`fatty acids is a time-consuming and difficult process. More
`over, 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.
`0024 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 Végétales New Dictionary
`of the Vegetable Oils. Technique & Documentation—
`Lavoisier, 1995. ISBN: 2-7430-0009-0). However, the pro
`duction 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 LCPU
`FAS for the food industry, animal nutrition and pharmaceuti
`cal 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 meth
`ods 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
`LCPUFAs and incorporate them in the membranes or tria
`cylglycerides. Thus, it has already been possible to isolate
`A6-desaturase genes from the moss Physcomitrella patens
`and A6-elongase genes from P patens and from the nematode
`C. elegans.
`0025 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 19203 (process for the production
`of polyunsaturated fatty acids in plants).
`0026. 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 19203 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 eicosapen
`taenoic, docosapentaenoic acid and/or docosahexaenoic acid,
`can be increased by expressing an optimized A5-elongase
`sequence in transgenic plants.
`0030. The PUFAs produced by the process of the inven
`tion 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 eicosapen
`taenoic 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 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 activ
`ity; 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. To produce DHA it is additionally
`necessary to provide at least one nucleic acid sequence which
`codes for a polypeptide having a A4-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
`A6-desaturase activity, a polypeptide having a A6-elongase
`activity, a polypeptide having a A5-desaturase activity and a
`polypeptide having a A5-elongase activity are present
`together in one plant. The “provision in the plant' thus com
`prises 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 A5-elongase activity is modified
`according to the invention by comparison with the nucleic
`acid sequence in the organism from which the sequence origi
`nates 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
`
`CSIRO Exhibit 1019
`
`

`

`US 2010/0227924 A1
`
`Sep. 9, 2010
`
`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 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 trans
`lation 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 interalia at the following Internet address: http://www.
`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 GeneOpti
`mizer).
`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 par
`ticular base triplet is used can take place at random in this
`case. The DNA sequence was adapted according to the inven
`tion 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 A5-elongase activity, Such as, for
`example, the polypeptide depicted in SEQID No. 110, 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
`A5-elongase is adapted in at least 20% of the positions, pref
`erably 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 SEQID No. 64.
`0040. It will be appreciated that the invention also encom
`passes those codon-optimized DNA sequences which code
`for a polypeptide having the activity of a A5-elongase and
`whose amino acid sequence is modified in one or more posi
`
`tions by comparison with the wild-type sequence but which
`still has substantially the same activity as the wild-type pro
`tein.
`0041. The nucleic acid sequence which codes for a
`polypeptide having a A6-desaturase activity is preferably
`selected from the group consisting of:
`a) nucleic acid sequences having the sequence depicted in
`SEQID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
`31, 33, 35, 37, 39 or 41, preferably having the sequence
`depicted in SEQID No. 1,
`b) nucleic acid sequences which code for the amino acid
`sequence indicated in SEQID No. 2, 4, 6, 8, 10, 12, 14, 16, 18,
`20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42, preferably in
`SEQID No. 2,
`c) nucleic acid sequences which hybridize with the comple
`mentary Strand of the nucleic acid sequences indicateda) orb)
`above, in particular of the nucleic acid sequence indicated in
`SEQID No. 1, under stringent conditions,
`d) nucleic acid sequences which are at least 60%. 65%, 70%,
`75% or 80%, preferably at least 81%, 82%, 83%, 84%, 85%,
`86%, 87%, 88%, 89% or 90%, particularly preferably at least
`91%, 92%, 93%, 94% or 95% and especially at least 96%,
`97%, 98% or 99%, identical to the nucleic acid sequences
`indicated in a) or b) above, especially to the sequence indi
`cated in SEQID No. 1, and
`e) nucleic acid sequences which code for an amino acid
`sequence and which have at least one, for example 2, 3, 4, 5,
`6, 7 or 8, preferably all of the amino acid pattern indicated in
`SEQID No. 43,44, 45, 46, 47, 48, 49 or 50.
`0042 Amino acid pattern means short amino acid
`sequences which preferably comprise less than 50, particu
`larly preferably less than 40 and especially from 10 to 40 and
`even more preferably from 10 to 30 amino acids.
`0043. For the present invention, the identity is ascertained
`preferably over the full length of the nucleotide or amino acid
`sequences of the invention, for example for the nucleic acid
`sequence indicated in SEQID NO: 64 over the full length of
`903 nucleotides.
`0044) The nucleic acid sequence which codes for a
`polypeptide having a A6-elongase activity is preferably
`selected from the group consisting of:
`a) nucleic acid sequences having the sequence depicted in
`SEQID No. 171, 173, 175, 177, 179, 181 or 183, especially
`having the sequence depicted in SEQID No. 171,
`b) nucleic acid sequences which code for the amino acid
`sequence indicated in SEQID No. 172, 174, 176, 178, 180,
`182 or 184, especially for the amino acid sequence indicated
`in SEQID No. 172,
`c) nucleic acid sequences which hybridize with the comple
`mentary Strand of the nucleic acid sequences indicateda) orb)
`above, especially of the nucleic acid sequence indicated in
`SEQID No. 1, under stringent conditions,
`d) nucleic acid sequences which are at least 60%. 65%, 70%,
`75% or 80%, preferably at least 81%, 82%, 83%, 84%, 85%,
`86%, 87%, 88%, 89% or 90%, particularly preferably at least
`91%, 92%, 93%, 94% or 95% and especially at least 96%,
`97%, 98% or 99%, identical to the nucleic acid sequences
`indicated in a) or b) above, especially to the sequence indi
`cated in SEQID No. 171, and
`e) nucleic acid sequences which code for an amino acid
`sequence and which have at least one, for example 2, 3, 4, 5,
`6, 7 or 8, preferably all of the amino acid pattern indicated in
`SEQID No. 185, 186, 187, 188, 189, 190, 191 or 192.
`
`CSIRO Exhibit 1019
`
`

`

`US 2010/0227924 A1
`
`Sep. 9, 2010
`
`0045. The nucleic acid sequence which codes for a
`polypeptide having a A6-elongase activity is in particular
`likewise a codon-optimized sequence according to the
`present invention, preferably the nucleic acid sequence
`depicted in SEQID NO: 122.
`0046. The nucleic acid sequence which codes for a
`polypeptide having a A5-desaturase activity is preferably
`selected from the group consisting of:
`a) nucleic acid sequences having the sequence depicted in
`SEQ ID No. 51, 53 or 55, preferably having the sequence
`depicted in SEQID No. 51,
`b) nucleic acid sequences which code for the amino acid
`sequence indicated in SEQID No. 52, 54 or 56, preferably for
`the amino acid sequence indicated in SEQID No. 52,
`c) nucleic acid sequences which hybridize with the comple
`mentary Strand of the nucleic acid sequences indicated ina) or
`b) above, especially of the nucleic acid sequence indicated in
`SEQID No. 51, under stringent conditions,
`d) nucleic acid sequences which are at least 60%. 65%, 70%,
`75% or 80%, preferably at least 81%, 82%, 83%, 84%, 85%,
`86%, 87%, 88%, 89% or 90%, particularly preferably at least
`91%, 92%, 93%, 94% or 95% and especially at least 96%,
`97%, 98% or 99%, identical to the nucleic acid sequences
`indicated in a) or b) above, especially to the nucleic acid
`indicated under SEQID No. 51, and
`e) nucleic acid sequences which code for an amino acid
`sequence which have at lea