EA037184B1 - Lipid comprising docosapentaenoic acid - Google Patents

Abstract

The present invention relates to extracted plant lipid or microbial lipid comprising docosapentaenoic acid, and processes for producing the extracted lipid.

Description

(54) LIPID CONTAINING DOCOSAPENTAENIC ACID (31) 2014902471; 20140104761; PCT / AU2014 / 050433; 14 / 575,756 (32) 2014.06.27; 2014.12.18; 2014.12.18;

2014.12.18 (33) AU; AR; AU; US (43) 2017.06.30 (86) PCT / AU2015 / 050340 (87) WO 2015/196250 2015.12.30 (71) (73) Applicant and patentee:

COMMONVELT SIGNIFIC

AND INDUSTRIAL RESEARCH ORGANIZATION; NUSID PTI LTD; GRANES RESERVE AND DIVELOPMENT CORPORATION (AU) (72) Inventor:

Petrie James Robertson, Singh Surinder Pal, Shresta Pushkar, McAllister Jason Timothy (AU), Devine Malcolm David (CA), De Feyter Robert Charles (AU) (74) Representative:

Rybina N.A., Rybin V.N. (RU) (56) US-A1-20100227924

WO-A1-2002092540

WO-A2-2007005727

WO-A1-2010147900

WO-A1-2010057246

WO-A2-2013185184

WO-A1-2005103253

037184 B1

037184 Bl (57) The invention relates to an extracted plant lipid or microbial lipid containing docosapentaenoic acid and methods for producing the extracted lipid.

Scope of invention

The present invention relates to a lipid containing docosapentaenoic acid obtained from plant cells or microbial cells, and methods for producing and using the lipid.

Background of the invention

Long-chain omega-3 polyunsaturated fatty acids (LC-PUFA) are widely recognized today as important compounds for human and animal health. These fatty acids can be obtained from food sources or by converting linoleic (LA, 18: 2ω6) or α-linolenic (ALA, 18: 3 ω3) fatty acids, both of which are considered essential fatty acids in the human diet. Although humans and many other vertebrates can convert plant-derived LA or ALA to C22, they do this at a very low rate. In addition, most modern societies have an unbalanced diet in which at least 90% of polyunsaturated fatty acids (PUFAs) are ω6 fatty acids, instead of a ratio of 4: 1 or less for ω6: ω3 fatty acids, which is considered ideal (Trautwein, 2001) ... The direct food source of DC-PUFA, for example, eicosapentaenoic acid (EPA, 20: 5ω3), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA, 22: 6ω3), for humans is mainly fish or fish oil. Thus, health professionals recommend regularly including fish containing significant levels of DC-PUFA in the human diet. More and more often, DC-PUFA obtained from fish oil are introduced, for example, into food products and baby food. However, by reducing global and national fishing, alternative sources of these health benefits are needed.

Flowering plants, in contrast to animals, do not have the ability to synthesize polyunsaturated fatty acids with a chain length of more than 18 carbon atoms. In particular, cultivated and garden plants, along with other angiosperms, do not contain the enzymes necessary for the synthesis of ω3 longer chain fatty acids, such as EPA, docosapentaenoic acid (DPC, 22: 5ω3) and DHA derived from ALA. An important goal in plant biotechnology, therefore, is to create cultivated plants that produce significant amounts of LCPUFA, thus providing an alternative source of these compounds.

Pathways of DC-PUFA biosynthesis.

The biosynthesis of DC-PUFA in organisms such as microalgae, mosses and fungi usually occurs as a series of reactions of oxygen-dependent desaturation and elongation (Fig. 1). The most common pathways for EPA production in these organisms include Δ6 desaturation, Δ6 elongation, and Δ5 desaturation (called the Δ6 desaturation pathway), while the less common pathway uses Δ9 elongation, Δ8 desaturation, and Δ5 desaturation (referred to as the Δ9 desaturation pathway). desamuration). These sequential desaturation and elongation reactions may start from the Δ6 fatty acid LC substrate, schematically illustrated in the upper left of FIG. 1 (ω6) or ω3 ALA substrate to EPA illustrated in the lower right portion of FIG. 1 (ω3). If the initial Δ6-desaturation is carried out on the substrate ω6 LA, the product of a series of three enzymes DC-PUFA will be ω6 fatty arachidonic acid (ARA). Organisms synthesizing DC-PUFA can convert ω6 fatty acids to ω3 fatty acids using ω3 desaturase, as illustrated as the Δ 17 desaturase step in FIG. 1, for the conversion of arachidonic acid (ARA, 20: 4ω6) to EPA. Some members of the ω3 desaturase family can act on a variety of substrates ranging from LA to ARA. Plant ω3 desaturases often specifically catalyze Δ15 desaturation of LA to ALA, whereas fungal and yeast ω3 desaturases may be specific for Δ 17 desaturation of ARA to EPA (Pereira et al., 2004a; Zank et al., 2005). Several reports suggest that there may be nonspecific ω3 desaturases capable of converting a wide range of ω6 substrates into corresponding ω3 products (Zhang et al., 2008).

The conversion of EPA to DHA in such organisms occurs by Δ5-elongation of EPA with the formation of duodenum, followed by Δ4-desaturation with the formation of DHA (Fig. 1). Conversely, mammals use the so-called Sprecher pathway, in which DNA is converted to DHA in three separate reactions independent of Δ4 desaturase (Sprecher et al., 1995).

Front-end desaturases, generally found in plants, mosses, microalgae and lower animals such as Caenorhabditis elegans, primarily accept fatty acid substrates esterified at the sn-2 position of the phosphatidylcholine (PC) substrate. These desaturases are thus known as lipid-associated front-end desaturase acyl-PCs (Domergue et al., 2003). Conversely, the front-end desaturases of higher animals generally accept acyl-CoA substrates, where the fatty acid substrate is bound to CoA instead of PC (Domergue et al., 2005). It is known that some desaturases of microalgae and one plant desaturase use fatty acid substrates esterified to CoA (Table 2).

Each polyunsaturated fatty acid elongation reaction consists of four stages catalyzed by a multicomponent protein complex: first, the condensation reaction leads to the addition of a 2C module from a malo- 1037184 nyl-CoA to a fatty acid, resulting in the formation of a β-ketoacyl intermediate. It is then reduced with NADPH, followed by dehydration and the formation of an enoyl intermediate. Finally, this intermediate is re-reduced to form an extended fatty acid. In general, the condensation step of these four reactions is considered to be substrate specific, while the other steps are not. In practice, this means that the natural plant elongation apparatus is capable of PUFA elongation, provided that a condensation enzyme (usually called elongase) specific for PUFA is introduced, although the efficiency of the natural plant elongation apparatus in terms of elongation of non-natural PUFA substrates may be low. In 2007, information was published regarding the identification and description of the yeast dehydratase elongation cycle (Denic and Weissman, 2007).

Desaturation of PUFA in plants, mosses and microalgae occurs naturally on fatty acid substrates, mainly in the acyl-PC pool, while elongation occurs on substrates in the acyl-CoA pool. The transfer of fatty acids from the acyl-PC molecules to the CoA carrier is performed by phospholipases (PLA), while the transfer of acyl-CoA fatty acids to the PC carrier is carried out by lysophosphatidylcholine acyltransferases (LPCAT) (Singh et al., 2005).

Designed production of DC-PUZhK.

Most of the metabolic construction of DC-PUFAs was carried out using the aerobic Δ6-desaturation / elongation pathway. The biosynthesis of γ-linolenic acid (GLA, 18: 3 ω6) in tobacco using Δ6 desaturase from the cyanobacterium Synechocystis was first reported in 1996 (Reddy and Thomas, 1996). Later, GLA was obtained in cultivated plants such as safflower (73% GLA in seed oil; WO 2006/127789) and soybeans (28% GLA; Sato et al., 2004). The production of DC-PUFAs, such as EPA and DHA, involves more sophisticated design by increasing the number of desaturation and elongation steps involved. EPA production in a terrestrial plant was first reported by Qi et al. (2004), who introduced genes encoding Δ9-elongase from Isochrysis galbana, Δ8-desaturase from Euglena gracilis, and Δ5-desamurase from Mortierella alpina into Arabidopsis, yielding up to 3% EPA. This work was completed by Abbadi et al. (2004), who reported the production of up to 0.8% EPA in flaxseed using genes encoding Δ6-desaturase and Δ6-elongase from Physcomitrella patens and Δ5-desaturase from Phaeodactylum tricornutum.

The first report on the production of ODC-DHA is contained in WO 04/017467, which discloses the production of 3% DHA in soybean embryos, but not in the seed, through the introduction of genes encoding Saprolegnia diclina Δ6 desaturase, Mortierella alpina Δ6 desaturase, Mortierella alpina Δ5 desaturase , Δ4-desamurase Saprolegnia diclina, Δ 17-desaturase Saprolegnia diclina, Δ6-elongase Mortierella alpina, and Δ5-elongase Pavlova lutheri. The maximum level of EPA in embryos also producing DHA was 19.6%, indicating a low conversion efficiency of EPA to DHA (WO 2004/071467). This discovery was similar to that published by Robert et al. (2005), where the yield of conversion of EPA to DHA was low, with 3% EPA and 0.5% DHA production in Arabidopsis using Danio rerio Δ5 / 6-dsaturase, Caenorhabditis elegans Δ6-elongase, and Δ5-elongase and Δ4-desaturase Pavlova salina. Also in 2005, Wu et al. published a report on the production of 25% ARA, 15% EPA and 1.5% DHA in Brassica juncea using Δ6-desaturase Pythium irregulare, Δ5-desaturase Thraustochytrid, Δ6-desaturase Physcomitrella patens, Δ12-desaturase Calendula officianalis, Δ5-elongase, Thraustochytrid Phytophthora infestans Δ 17 desaturase, Oncorhyncus mykiss DC PUFA elongase, Thraustochytrid Δ4 desaturase, and Thraustochytrid LPKHAT (Wu et al., 2005). A summary of efforts to harvest oilseeds synthesized by ω3 DC-PUFA is given in Venegas-Caleron et al. (2010) and Ruiz-Lopez et al. (2012). As indicated by Ruiz-Lopez et al. (2012), at the date of publication, the results for DHA production in transgenic plants do not even remotely resemble the levels observed in fish oil. Later, Petrie et al. (2012) reported about 15% DHA production in Arabidopsis thaliana seeds, and WO2013 / 185184 reported the production of some oils from seeds containing 7 to 20% DHA. However, there are no reports of the production of vegetable oils containing more than 20% DHA.

There are no reports of the production of duodenum in recombinant cells at significant levels without concomitant DHA production. In fact, the present inventors are not aware of any published clue or idea of producing duodenum in recombinant cells without producing DHA.

Thus, there remains a need for a more efficient production of DC-PUFA in recombinant cells, in particular, duodenum in oil seeds.

The essence of the invention

Some organisms produce oil with more than 1–2% WPC and thus there is limited, if any, production of WPC on a large scale from natural sources. The present inventors have identified methods and plants for the production of lipid with much higher levels of Duodenum than in natural sources.

In a first aspect, the invention provides an extracted lipid, preferably an extracted plant lipid or an extracted microbial lipid containing fatty acids in

- 2,037184 esterified form, where fatty acids include oleic acid, palmitic acid, ω6 fatty acids including linoleic acid (LA), ω3 fatty acids including α-linolenic acid (ALA) and docosapentaenoic acid (DPA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), and the level of DPA in the total fatty acid content of the extracted lipid is from about 7 to 35%. In embodiments of this aspect, the level of DPC in the total fatty acid content of the extracted lipid is about 7, about 8, about 9, about 10, about 12, about 15, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 7 to about 28%, about 7 to about 25%, about 10 to 35%, about 10 to about 30%, about 10 to about 25%, about 10 to about 22 %, about 14 to 35%, about 16 to 35%, about 16 to about 30%, about 16 to about 25%, or about 16 to about 22%.

In an embodiment of the above aspect, DHA is present at less than 2% or less than 0.5% of the total fatty acid content of the extracted lipid, and more preferably absent in the total fatty acid content of the lipid.

In another aspect, the invention provides an extracted lipid, preferably an extracted plant lipid or an extracted microbial lipid, comprising fatty acids in an esterified form, the fatty acids comprising docosapentaenoic acid (DPA) and wherein at least 35% DPA esterified in the form of triacylglyceride (TAG) , esterified at the sn-2 position of TAG. In an embodiment, the extracted lipid further has one or more or all of the features of (i) it contains fatty acids including oleic acid, palmitic acid, ω6 fatty acids including linoleic acid (LA), ω 3 fatty acids including α-linolenic acid (ALA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), (ii) at least about 40, at least about 45, at least about 48% , from 35 to about 60% or from 35 to about 50% of the duodenum esterified in the form of triacylglyceride (TAG) are esterified at the sn-2 position of the TAG, and (iii) the level of duodenum in the total fatty acids of the extracted lipid is from about 1 to 35%, or about 7 to 35%, or about 20.1 to 35%. In embodiments of this aspect, the level of DPC in the total fatty acid content of the extracted lipid is about 7, about 8, about 9, about 10, about 12, about 15, about 18, about 20, about 22, about 24, about 26, about 28 about 30%, about 7 to about 28%, about 7 to about 25%, about 10 to 35%, about 10 to about 30%, about 10 to about 25%, about 10 to about 22 %, about 14 to 35%, about 16 to 35%, about 16 to about 30%, about 16 to about 25%, or about 16 to about 22%. In preferred embodiments, the extracted lipid has features (i) and (ii), (i) and (iii) or (ii) and (iii), more preferably all of (i), (ii) and (iii). Preferably, the extracted lipid is further characterized by the level of palmitic acid in the total fatty acid content of the extracted lipid, which is from about 2 to 16%, and, furthermore, the level of myristic acid (C14: 0) in the total fatty acid content of the extracted lipid, if present, is less than 1%.

Embodiments of each of the above aspects are disclosed in more detail below. As will be understood by the skilled person, any of the features of the described embodiment that are broader than the corresponding feature in the above aspect do not apply to this aspect.

In an embodiment of the invention, the extracted lipid has one or more of the following features:

i) the level of palmitic acid in the total fatty acid of the extracted lipid is about 2 to 18%, about 2 to 16%, about 2 to 15%, or about 3 to about 10%, ii) the level of myristic acid (C14: 0) in the total fatty acid content of the extracted lipid is less than 6, less than 3, less than 2, less than 1, or about 0.1%, iii) the level of oleic acid in the total fatty acid content of the extracted lipid is from about 1 to about 30%, from about 3 to about 30%, about 6 to about 30%, 1 to about 20%, about 30 to about 60%, about 45 to about 60%, about 30 or about 15 to about 30%, iv ) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is from about 4 to about 35%, from about 4 to about 20%, from about 4 to about 17%, or from about 5 to about 10%,

v) the level of α-linolenic acid (APA) in the total fatty acid content of the extracted lipid is from about 4 to about 40%, from about 7 to about 40%, from about 10 to about 35%, from about 20 to about 35%, about 4 to 16%, or about 2 to 16%, vi) the level of γ-linolenic acid (GLA) in the total fatty acids of the extracted lipid is less than 4, less than about 3, less than about 2, less than about 1 less than about 0.5%, 0.05 to about 7%, 0.05 to about 4%, 0.05 to about 3%, or 0.05 to about 2%, vii) stearidonic acid level (SDA) in the total fatty acid content of the extracted

3,037184 lipids are less than about 10, less than about 8, less than about 7, less than about 6, less than about 4, less than about 3%, from about 0.05 to about 7%, from about 0, 05 to about 6%, about 0.05 to about 4%, about 0.05 to about 3%, about 0.05 to about 10%, or 0.05 to about 2%, viii) eicosatetraenoic acid level (ETA) in the total fatty acid content of the extracted lipid is less than about 6, less than about 5, less than about 4, less than about 1, less than about 0.5%, from 0.05 to about 6%, from 0 , 05 to about 5%, 0.05 to about 4%, 0.05 to about 3%, or 0.05 to about 2%, ix) the level of eicosatrienoic acid (ETRA) in the total fatty acid content of the extracted lipid is less than 4, less than 2%, less than about 1%, from 0.05 to 4%, from 0.05 to 3%, or from 0.05 to about 2%, or from 0.05 to about 1%,

x) The level of eicosapentaenoic acid (EPA) in the total fatty acids of the extracted lipid is 4 to 15%, less than 4%, less than about 3, less than about 2%, from 0.05 to 10%, from 0.05 to 5%, 0.05 to 3%, or from 0.05 to 2%, xi) the lipid comprises ω6-dokozanentaenovuyu acid (22: 5Δ ^{4,7,10,13,16)} including therein contained fatty acids, xii) the lipid comprises less than 0,1% ω6-docosapentaenoic acid (22: 5Δ ^{4,7,10,13,16)} include fatty acids contained in it, xiii) the lipid comprises less than 0.1% of one or more or all of the SDA, EPA and ETA in the number of fatty acids it contains, xiv) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is from about 4 to about 25%, from about 4 to about 20%, from about 6% to about 20% or about 6% to about 12%, xv) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 4 to about 40%, from about 4 to about 35%, from about 8 to about 25%, 8 to about 22%, about 15 to about 40%, or about 15 to about 35%, xvi) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 20 to about 75%, about 30 to about 75%, about 50 to about 75%, about 60, about 65, about 70, about 75%, or about 60 to about 75%, xvii) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is from about 35 to about 50%, from about 20 to about 35%, from about 6 to 20%, less than 20%, less than about 16, less than about 10%, from about 1 to about 16%, from about 2 to about 10%, or about 4 to about 10%, xviii) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10, less than about 8, less than about 6%, less than 4% , from about 1 to about 20%, from about 1 to about 10%, from 0.5 to about 8%, or from about 0.5 to 4%, xix) the level of total ω 3 fatty acids in the total content of fatty acids t of the extracted lipid is from 36 to about 65%, from 36 to about 70%, from about 40 to about 60%, from about 30 to about 60%, from about 35 to about 60%, from 40 to about 65%, from about 30 to about 65%, about 35 to about 65%, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or about 70%, xx) new ω 3 fatty acid levels in total the fatty acid content of the extracted lipid is 21 to about 45%, 21 to about 45%, about 23 to about 35%, about 25 to about 35%, about 27 to about 35%, about 23, about 25, about 27, about 30, about 34, about 40, or about 45%, xxi) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is from about 1.0 to about 3.0, from about 0, 1 to about 1, about 0.1 to about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15 , about 1.0, about 0.1, about 0.1 to about 0.4, or about 0.2, xxii) resp. wearing new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is from about 1.0 to about 3.0, from about 0.02 to about 0.1, from about 0.1 to about 1, from about 0.1 to about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.02, about 0.05, about 0.1, about 0.2, or about 1.0, xxiii) the fatty acid composition of the lipid is based on the efficiency of the conversion of oleic acid to LA by Δ12 desaturase at least about 60, at least about 70, at least about 80%, about 60 to about 98%, about 70 to about 95%, or about 75 to about 90%, xxiv) the fatty acid composition of the lipid is based on the efficiency of conversion of ALA to CDA by Δ6 desaturase by at least about 30, at least about 40, at least about 50, at least about 60, at least about 70%, about 30 to about 70%, about 35 to about 60%, or about 50 to about about 70%, xxv) the fatty acid composition of the lipid is based on the efficiency of converting SDA into acid

- 4 037184

ETA by Δ6-elongase at least about 60, at least about 70, at least about 75%, about 60 to about 95%, about 70 to about 88%, or about 75 to about 85%, xxvi ) the fatty acid composition of the lipid is based on the efficiency of conversion of ETA to EPA by Δ5-desaturase at least about 60, at least about 70, at least about

75%, about 60 to about 99%, about 70 to about 99%, or about 75 to about 98%, xxvii) the fatty acid composition of the lipid is based on the efficiency of conversion of EPA to DPC by Δ5-elongase at least about 80 at least about 85%, at least about 90%, about 50 to about 99%, about 85 to about 99%, about 50 to about 95%, or about 85 to about 95%, xxviii) composition fatty acids lipid based on the efficiency of converting oleic acid to DPC at least about 10, at least about 15, at least about 20, at least about 25%, about 20, about 25, about 30%, about 10 to about 50%, about 10 to about 30%, about 10 to about 25%, or about 20 to about 30%, xxix) the fatty acid composition of the lipid is based on the efficiency of converting LA to DPC at least about 15, at least about 20, at least about 22, at least about 25, at least about 30, at least about 40%, about 25, about 30, about 3 5, about 40, about 45, about 50%, about 15 to about 50%, about 20 to about 40%, or about 20 to about 30%, xxx) the fatty acid composition of the lipid is based on the efficiency of the conversion of ALA to DPC by at least about 17, at least about 22, at least about 24, at least about 30%, about 30, about 35, about 40, about 45, about 50, about 55, about 60%, about 22 to about 70%, about 17 to about 55%, about 22 to about 40%, or about 24 to about 40%, xxxi) the total fatty acids in the extracted lipid contain less than 1.5% C20: 1, less than 1 % C20: 1 or about 1% C20: 1, xxxii) the content of triacylglyceride (TAG) in the lipid is at least about 70, at least about 80, at least about 90, at least 95%, from about 70 to about 99% or about 90 to about 99%, xxxiii) the lipid contains diacylglyceride (DAG), and the DAG preferably contains DPC, xxxiv) the lipid contains less than about 10, less than about 5, less than about 1%, or about 0.001 to about 5% or substantially free of free (non-esterified) fatty acids and / or phospholipid, xxxv) at least 70, at least 72, or at least 80% of the esterified DPA in the form of TAG is in the sn- position 1 or sn-3 TAG, xxxvi), the most common DPC-containing TAG species in lipid are DPC / 18: 3/18: 3 (TAG 58:12), a lipid containing tri-DPC TAG (TAG 66:18), and xxxvii) the level of DPC in the total fatty acid content of the extracted lipid is about 7, about 8, about 9, about 10, about 12, about 15, about 18, about 20, about 22, about 24, about 26, about 28, about 31 %, about 7 to about 31%, about 7 to about 28%, about 10 to 35%, about 10 to about 30%, about 10 to about 25%, about 10 to about 22%, from about 14 to 35%, about 16 to 35%, about 16 to about 30%, about 16 to about 25%, or about 16 to about 22%, with the DHA level optionally being less than 0.5% of the total extractable fatty acid content bath lipid.

In another embodiment, the extracted lipid has one or more of the following features:

i) the level of palmitic acid in the total fatty acid of the extracted plant lipid is between 2 and 15%, ii) the level of myristic acid (C14: 0) in the total fatty acid of the extracted plant lipid is about 0.1%, iii) the level of oleic acid in the total fatty acid content of the extracted plant lipid is 1 to 30%, iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted plant lipid is 4 to 20%,

v) the level of α-linolenic acid (ALA) in the total fatty acids of the extracted plant lipid is 4 to 40%, vi) the level of γ-linolenic acid (GLA) in the total fatty acids of the extracted plant lipid is 0.05 to 7 %, vii) the level of stearidonic acid (SDA) in the total fatty acids of the extracted plant lipid is from 0.05 to 10%, viii) the level of eicosatetraenoic acid (ETA) in the total fatty acids of the extracted plant lipid is less than 6%, ix ) the level of eicosatrienoic acid (EtrA) in the total fatty acid content of the extracted plant lipid is less than 4%,

- 5 037184

x) the extracted vegetable lipid comprises less than 0.1% ω6-docosapentaenoic acid (22: 5Δ ^{4,7,10,13,16)} include fatty acids contained therein, xi) the rate of new ω6 fatty acids in the total fatty acid content of the extracted plant lipid is less than 10%, xii) the ratio of the total ω6 fatty acids: the total ω3 fatty acids in the fatty acid content of the extracted plant lipid is 1.0 to 3.0 or 0.1 to 1, xiii) the ratio new ω6 fatty acids: new ω 3 fatty acids in the fatty acid content of the extracted plant lipid is 1.0 to 3.0, 0.02 to 0.1, or 0.1 to 1, xiv) the fatty acid composition of the extracted plant lipid lipid is based on a conversion efficiency of oleic acid to DPC of at least 10%, xv) the fatty acid composition of the extracted plant lipid is based on a conversion efficiency of LA to DPC of at least 15%, xvi) fatty acid composition of the extracted of this plant lipid is based on a conversion efficiency of ALA to DPC of at least 17%, xvii) the total fatty acids in the extracted plant lipid contain less than 1.5% C20: 1 and xviii) the triacylglyceride (TAG) content of the extracted plant lipid is at least 70% and may be characterized by one or more of the following:

xix) the extracted plant lipid contains diacylglyceride (DAG), which contains DPA, xx) the extracted plant lipid contains less than 10% free (non-esterified) fatty acids and / or phospholipid, or essentially does not contain them, xxi) at least 70% of DPA esterified in the form of TAG is in position sn-1 or sn-3 TAG, xxii) the most common DPC-containing TAG species in the extracted plant lipid are DPC / 18: 3/18: 3 (TAG 58:12) and xxiii ) the extracted plant lipid contains tri-DPA TAG (TAG 66:18).

In an embodiment, the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted plant lipid is between 0.05 and 10%.

In a further embodiment, the level of DHA in the total fatty acid content of the extracted plant lipid is less than 2%, preferably less than 1%, or from 0.1 to 2%, more preferably undetectable. Preferably, a plant or part thereof, such as a seed, or a microbial cell does not contain a polynucleotide encoding a Δ4 desaturase, or does not contain a Δ4 desaturase polypeptide. In another embodiment, the extracted lipid is in the form of an oil, the lipid comprising at least about 90%, at least about 95%, at least about 98%, or about 95 to about 98% by weight of the oil.

Preferably, the extracted lipid is a Brassica seed oil lipid or a Camelina sativa seed oil lipid.

In a preferred embodiment of the first aspect of the invention, the above is a lipid or oil, preferably a seed oil, more preferably a seed oil of the Brassica species or a seed oil of Camelina sativa, has the following characteristics: in the total fatty acid content of the lipid or oil, the DPA level is from about 7 to 30% or from 7 to 35%, palmitic acid level is about 2 to about 16%, myristic acid level is less than 1%, oleic acid level is 1 to about 30%, LA level is about 4 to about 35%, ALA is present , the level of total saturated fatty acids in the total fatty acids of the extracted lipid is about 4 to about 25%, the ratio of the total ω6 fatty acids: the total ω3 fatty acids in the fatty acid content of the extracted lipid is from 0.05 to about 3.0 , and the content of triacylglycerides (TAG) of the lipid is at least about 70%, and optionally the lipid is not substantially co holds cholesterol and / or lipid contains tri-DPA TAG (TAG 66:15). More preferably, the lipid or oil, preferably seed oil, further possesses one or more or all of the following characteristics: at least 70% of the DPA is esterified at the sn-1 or sn-3 position of the triacylglyceride (TAG), ALA is present at a level of 4 to 40 % of total fatty acids, GLA is present and / or GLA is less than 4% of total fatty acids, SDA is 0.05 to about 10%, ETA is less than about 4%, EPA is between 0.05 to about 10%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 4 to about 35%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 20 to about 75%, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is from about 0.03 to about 3.0, preferably less than about 0.50, the composition of fatty acids li-6 037184 pida is based on: the efficiency of conversion of oleic acid to LA under the action of Δ12 desaturase at least about 60%, the efficiency of conversion of SDA to ETA acid under the action of Δ6-elongase at least about 60%, the efficiency of conversion of EPA into DPC under the action of Δ5-elongase from about 50 to about 95%, and the efficiency of conversion of oleic acid to DPA at least about 10%. Most preferably, at least 81% of the DPA is esterified at the sn-1 or sn-3 position of the triacylglyceride (TAG).

In another preferred embodiment of the above second aspect of the invention, the lipid or oil, preferably seed oil, more preferably seed oil of the Brassica species or Camelina sativa seed oil containing WPC, has the following characteristics: in the total fatty acid content of the lipid or oil, the level of palmitic acid is from about 2 to about 16%, the level of myristic acid is less than 1%, the level of oleic acid is from about 1 to about 30%, the LA level is from about 4 to about 35%, ALA is present, the total level of saturated fatty acids in the total content fatty acids of the extracted lipid is from about 4 to about 25%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is from 0.05 to about 3.0, the content of triacylglycerides (TAG) of the lipid is at least about 70%, and optionally the lipid contains tri-DPA TAG (TAG 66:15), pr and wherein at least 35% of the duodenum esterified in the form of triacylglyceride (TAG) is esterified at the sn-2 position of the TAG. More preferably, the lipid or oil, preferably seed oil, further has one or more or all of the following: ALA is present at a level of 4 to 40% of the total fatty acids, GLA is present, and / or the GLA level is less than 4% of total fatty acids, SDA level is 0.05 to about 10%, ETA level is less than about 4%, EPA level is 0.05 to about 10%, total monounsaturated fatty acids level in total fatty acids of extracted lipid is from about 4 to about 35%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 20 to about 75%, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is from about 0 03 to about 3.0, preferably less than about 0.50, the fatty acid composition of the lipid is based on the conversion efficiency of oleic acid to LA under the action of Δ12-desaturase at least about 60%, the efficiency of conversion of SDA to acid ETA under the action of Δ6-elongase at least about 60%, the efficiency of conversion of EPA to DPC under the action of D5-elongase from about 50 to about 95%, the conversion efficiency oleic acid in WPC at least about 10%.

In the context of the extracted lipid or oil of the invention, in an embodiment of the invention, the level of DPA in the extracted lipid or oil is not increased or is substantially the same as the level of DPA in the lipid or oil of the plant or microbe part prior to extraction. In other words, after extraction, no procedures were performed to increase the level of duodenum in the lipid or oil relative to other fatty acids. As will be apparent, the lipid or oil can subsequently be treated by fractionation or other methods to modify the fatty acid composition.

In another preferred embodiment, the lipid or oil, preferably seed oil and more preferably Brassica seed oil, such as mustard oil or rapeseed oil or C. sativa seed oil, has the following characteristics: in the total fatty acid content of the lipid or oil, the DPA level is from about 7 to 35%, palmitic acid level is about 2 to about 16%, myristic acid level is less than about 6% and preferably less than 1%, oleic acid level is about 1 to about 30%, LA level is about 4 to about 35%, ALA is present, the SDA level is from about 0.05 to about 10%, the ETA level is less than about 6%, the EPA level is from about 0.05 to about 10%. DHA is detected or preferably not detected in lipid or oil. Preferably, DHA, if present, is present at a level of no more than 2 or no more than 0.5% of the total fatty acid content of the lipid or oil, and more preferably absent in the total fatty acid content of the lipid or oil. Optionally, the lipid is substantially free of cholesterol and / or the lipid contains triDPA TAG (TAG 66:15). More preferably, the lipid or oil, preferably seed oil, further possesses one or more or all of the following characteristics: at least 70% of the DPA is esterified at the sn-1 or sn-3 position of the triacylglyceride (TAG), ALA is present at a level of 4 to 40 % of total fatty acids, GLA is present and / or GLA is less than 4% of total fatty acids, SDA is 0.05 to about 10%, ETA is less than about 4%, EPA is between 0.05 to about 10%, the level of total unsaturated fatty acids in the total fatty acids of the extracted lipid is from about 4 to about 35%, the level of total polyunsaturated fatty acids in the total fatty acids of the extracted lipid is from about 20 to about 75%, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is from about 0.03 to about 3.0, preferably less than about 0.50, const av fatty acids lipi

- 7 037184 yes is based on: the efficiency of conversion of oleic acid to LA under the action of Δ12desaturase at least about 60%, the efficiency of conversion of SDA to ETA acid under the action of Δ6-elongase at least about 60%, the efficiency of conversion of EPA into DPA under the action of Δ5- elongase from about 50 to about 95% and the efficiency of conversion of oleic acid to WPC at least about 10%. In an embodiment, at least 81% of the DPA is esterified at the sn-1 or sn-3 position of the triacylglyceride (TAG). Alternatively, at least 35% of the TAG esterified DPA is esterified at the sn-2 position of the TAG.

In a further embodiment, the extracted lipid of the invention further comprises one or more sterols, preferably plant sterols.

In another embodiment, the extracted lipid is in the form of an oil and contains less than about 10, less than about 7 mg sterols / g oil, about 1.5 to about 10 mg sterols / g oil, or about 1.5 to about 7 mg sterols / g oil.

Examples of sterols that may be present in the extracted lipid include, but are not necessarily limited to, one or more or all of the following: campestrol / 24-methylcholesterol, Δ5stigmasterol, eburicol, β-sitosterol / 24-ethylcholesterol, Δ5-avenasterol / isofucosterol, Δ7 / stigmast-7-en-3v-ol and Δ7-avenasterol.

In an embodiment, the plant species is one of those listed in table. 11, for example rapeseed, and the level of sterols is about the same as shown in table. 11 for this particular plant species. The plant species may be B. napus, mustard (B. juncea) or C. sativa, and contain sterol levels close to that found in the extracted wild-type mustard oil B. napus, mustard or C. sativa, respectively.

In an embodiment, the extracted plant lipid comprises one or more or all of campesterol / 24-methylcholesterol, Δ5-stigmasterol, eburicol, β-sitosterol / 24-ethylcholesterol, Δ5-avenasterol / isofucosterol, Δ7-stigmasterol / stigmast-3β-7-ene- α-ol and Δ7-avenasterol, or contains sterol levels substantially the same as in wild-type rapeseed oil.

In an embodiment, the extracted lipid contains a sterol level substantially the same as in rapeseed oil, mustard oil, or wild type C. sativa oil.

In an embodiment, the extracted lipid contains less than about 0.5, less than about 0.25 mg cholesterol / g oil, about 0 to about 0.5 mg cholesterol / g oil, or about 0 to about 0.25 mg cholesterol / g oil, or essentially no cholesterol.

In a further embodiment, the lipid is an oil, preferably an oilseed oil. Examples of such oils include, but are not limited to, Brassica oil such as rapeseed or mustard oil, Gossypium hirsutum oil, Linum usitatissimum oil, Helianthus spp. Oil, Carthamus tinctorius oil, Glycine max oil, Zea mays oil, Arabidopsis thaliana oil, Sorghum bicolor oil , Sorghum vulgare oil, Avena sativa oil, Trifolium oil, Elaesis guineenis oil, Nicotiana benthamiana oil, Hordeum vulgare oil, Lupinus angustifolius oil, Oryza sativa oil, Oryza glaberrima oil, Camelina sativa oil, Crambigan abythus oil, Miscellaneous oil, Misth. or Miscanthus sinensis oil. More preferably the oil is Brassica species oil, Camelina sativa oil or Glycine max (soybean) oil. In an embodiment, the lipid comprises or is a Brassica spp. Oil such as Brassica napus oil or Brassica juncea oil, Gossypium hirsutum oil, Linum usitatissimum oil, Helianthus spp. Oil, Carthamus tinctorius oil, Glycine max oil, Zea mays oil, Elaesis guineenis oil, Nicotiana benthamiana oil, Lupinus angustifolius oil, Camelina sativa oil, Crambe abyssinica oil, Miscanthus x giganteus oil or Miscanthus sinensis oil. In a further embodiment, the oil is rapeseed oil, mustard oil (B. juncea), soybean oil (Glycine max), Camelina sativa oil, or Arabidopsis thaliana oil. In an alternative embodiment, the oil is a plant oil other than A. thaliana oil and / or other than C. sativa oil. In an embodiment, the plant oil is an oil other than G. max oil (soybean). In an embodiment, the oil was obtained from a plant grown under standard conditions, for example, as described in example 1, or from a plant grown in a field or greenhouse under standard conditions.

In a further aspect of the present invention, there is provided a method for producing an extracted plant lipid or microbial lipid, comprising the steps of:

i) obtaining a plant part, preferably a Brassica seed or a Camelina sativa seed, or microbial cells containing a lipid, the lipid comprising fatty acids in esterified form, and wherein the fatty acids include oleic acid, palmitic acid, ω6 fatty acids, which include linoleic acid (LA), ω3 fatty acids, which include α-linolenic acid (ALA) and docosapentaenoic acid (DPA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), moreover, that the level of DPC in the total fatty acid content of the lipid from the plant part or microbial cells is from about 7 to 35%, and ii) extraction of the lipid from the plant part or microbial cells,

- 8 037184 wherein the level of duodenum in the total fatty acid content of the extracted lipid is from about 7 to 35%. In an embodiment, the level of DPA in the total fatty acid content of the extracted lipid is from about 7 to 20%, or from 20.1 to 35%. In an embodiment, the WPC level is 7 to 20%, or 20.1 to 30%, preferably 20.1 to 35%, more preferably 30 to 35%. In an embodiment of the invention, the level of DPA in the total fatty acid content of the extracted lipid is 8 to 20% or 10 to 20%, preferably 11 to 20% or 12 to 20%.

In an embodiment of the above aspect of the invention, there is provided a method for producing an extracted plant lipid or microbial lipid, comprising the steps of:

i) obtaining a plant part, preferably Brassica seed, C. sativa seed or microbial cells containing a lipid, the lipid containing fatty acids in esterified form, and wherein the fatty acid composition of the lipid comprises oleic acid, palmitic acid, ω6 fatty acids, including linoleic acid (LA), ω3 fatty acids, including α-linolenic acid (ALA) and docosapentaenoic acid (DPA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), moreover that (i) the level of DPC in the total fatty acid content of the extracted lipid is from 7 to 30% or from 7 to 35%, preferably from 30 to 35%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is from 2 up to 16%, (iii) the level of myristic acid (C14: 0) in the total fatty acid content of the extracted lipid is less than 6%, preferably less than 1%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is 1 to 30%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is 4 to 35%, (vi) the level of α-linolenic acid (ALA) in the total content fatty acids of the extracted lipid is 4 to 40%, (vii) the level of eicosatrienoic acid (EtrA) in the total fatty acids of the extracted lipid is less than 4%, (viii) the total level of saturated fatty acids in the total fatty acids of the extracted lipid is between 4 to 25%, (ix) the ratio of the total ω6 fatty acids: the total ω3 fatty acids in the fatty acid content of the extracted lipid is between 0.05 and 1, (x) the triacylglyceride (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DPA esterified in the TAG form is at the sn-1 or sn-3 position of the TAG, and ii) lipid extraction from the plant part, the DPA level in the total content and the fatty acids of the extracted lipid is from about 7 to 30%, or from 7 to 35%, preferably from 30 to 35%. Preferably, at least 81 or at least 90% of the DPA esterified in the TAG form is at the sn-1 or sn-3 position of the TAG.

In another aspect of the present invention, there is provided a method for producing an extracted lipid, comprising the steps of:

i) obtaining lipid-containing cells, preferably a plant part containing cells or microbial cells, more preferably Brassica seed or C. sativa seed, wherein the lipid contains fatty acids in esterified form and wherein the fatty acids comprise docosapentaenoic acid (DPA), moreover, that at least 35% of the DPC esterified in the form of triacylglyceride (TAG) is esterified at the sn-2 position of the TAG, and ii) extraction of the lipid from the cells, with at least 35% of the DPC esterified in the form of triacylglyceride (TAG), in total the fatty acid content of the extracted lipid, esterified at the sn-2 position of the TAG. In an embodiment, the extracted lipid obtained by the method is further characterized by one or more or all of the following: (i) it contains fatty acids including oleic acid, palmitic acid, ω6 fatty acids including linoleic acid (LA), ω 3 fatty acids , including α-linolenic acid (ALA) and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), (ii) at least about 40, at least about 45, at least at least about 48%, from 35 to about 60%, or from 35 to about 50%, DPC esterified in the form of triacylglyceride (TAG) esterified at the sn-2 position of the TAG, and (iii) the level of DPC in the total fatty acid content of the extracted lipid is from about 1 to 35%, or from about 7 to 35%, or from about 20.1 to 35%. In embodiments of this aspect, the level of DPC in the total fatty acid content of the extracted lipid is about 7, about 8, about 9, about 10, about 12, about 15, about 18, about 20, about 22, about 24, about 26, about 28 about 30%, about 7 to about 28%, about 7 to about 25%, about 10 to 35%, about 10 to about 30%, about 10 to about 25%, about 10 to about 22 %, about 14 to 35%, about 16 to 35%, about 16 to about 30%, about 16 to about 25%, or about 16 to about 22%. In preferred embodiments, the extracted lipid is characterized by (i) and (ii), (i) and (iii) or (ii) and (iii), more preferably all of (i), (ii) and (iii). Preferably, the extracted lipid is additionally characterized by a palmitic acid level in the total fatty acid content of the extracted lipid which is from about 2 to 16%, the level of myristic acid (C14: 0) in the total fatty acid content of the extracted lipid, if present, is less than 1%.

In an embodiment of the above aspect of the invention, there is provided a method for producing an extracted lipid, comprising the steps of:

i) obtaining lipid-containing cells, preferably a plant part containing cells or microbial cells, more preferably Brassica seed or C. sativa seed, wherein the lipid contains fatty acids in esterified form, and wherein the fatty acids include docosapentaenoic acid (DPA) and additionally include oleic acid, palmitic acid, ω6 fatty acids including linoleic acid (LA), ω3 fatty acids including α-linolenic acid (ALA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), and eicosatetraenoic acid (ETA), wherein (i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (ii) the level of myristic acid (C14: 0) in the total fatty acid content of the extracted lipid is less than 1% , (iii) the level of oleic acid in the total fatty acid content of the extracted lipid is 1 to 30%, (iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is 4 to 35%, (v) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is 4 to 40%, (vi) the level of eicosatrienoic acid (EtrA) in the total content the fatty acid of the extracted lipid is less than 4%; (vii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is 4 to 25%, (viii) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is 0.05 to about 1% , (ix) the content of triacylglyceride (TAG) in the lipid is at least about 70%, and (x) at least 35% DPA esterified in the form of triacylglyceride (TAG) esterified at the sn-2 position of the TAG, and ii) extraction a lipid from a plant part, wherein at least 35% of the DPA esterified in the form of triacylglyceride (TAG), in the total fatty acid content of the extracted lipid, is esterified at the sn-2 position of the TAG.

The step of obtaining plant parts or microbial cells may include harvesting plant parts, preferably seed, from plants that form plant parts, isolating microbial cells from cultures of such cells, or obtaining plant parts or microbial cells by purchase from a manufacturer or supplier, or by import. The method may include the step of determining the fatty acid composition of a lipid in a sample of plant parts or microbial cells or an extracted lipid.

In a preferred embodiment, the extracted lipid obtained by the method of the invention exhibits, if appropriate, one or more of the features defined herein, eg, as defined above for the first two aspects.

Embodiments of the above aspects of the invention are described in more detail below. The skilled artisan will understand that none of the features described in the embodiments of the invention that are broader than the corresponding feature in the above aspect apply to this aspect.

In an embodiment, the plant portion is a seed, preferably an oilseed. Examples of such seeds include, but are not limited to, seed from Brassica spp., Gossypium hirsutum, Linum usitatissimum, Helianthus spp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium s. , Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa or Crambe abyssinica, preferably Brassica seed, C. sativa seed or G. max (soybean) seed, more preferably Brassica napus, B. juncea or C. sativa seed ... In an embodiment, the plant part is a seed, preferably an oilseed crop such as Brassica species such as Brassica napus or Brassica juncea, Gossypium hirsutum, Linum usitatissimum, Helianthus species, Carthamus tinctorius, Glycine max, Zea mays, Elaesis guineenis, Nicotiana bentham angustifolius, Camelina sativa or Crambe abyssinica, preferably with the seed of Brassica napus, B juncea or C. sativa. In an embodiment, the seed is rape seed, mustard seed, soybean seed, Camelina sativa seed, or Arabidopsis thaliana seed. In an alternative embodiment, the seed is a seed other than A. thaliana and / or C. sativa seed. In an embodiment, the seed is a seed other than a soybean seed. In an embodiment, the plant portion is a seed of the Brassica species. The plant part is preferably a seed of the Brassica species or a seed of Camelina sativa. In an embodiment, the seed was obtained from a plant grown under standard conditions, for example, as described in example 1, or from a plant grown in a field or in a greenhouse under standard conditions.

In another embodiment, the seed contains at least about 18, at least about 22, at least about 26 mg, about 18 to about 100 mg, about 22 to about 70 mg, about 80 mg,

10 037184 about 30 to about 80 mg, or about 24 to about 50 mg of WPC per gram of seed.

In a further embodiment, a plant part, such as a seed, contains exogenous polynucleotides encoding one of the following sets of enzymes:

i) ω3 desaturase, Δ6 desaturase, Δ5 desaturase, Δ6 elongase and Δ5 elongase, ii) Δ15 desaturase, Δ6 desaturase, Δ5 desaturase, Δ6 elongase and Δ5 elongase, iii) Δ12 desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, iv) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

v) ω3 desaturase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, vi) Δ15 desaturase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, vii) Δ12 desamurase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, viii) Δ12 desaturase, ω3 desaturase and / or Δ15 desaturase, Δ8 desaturase, Δ5 desamurase, Δ9 elongase and Δ5 elongase, each the polynucleotide is operably linked to one or more promoters capable of directing the expression of said polynucleotides in a cell of a plant part.

In a further embodiment, a plant part such as a seed or recombinant cells such as microbial cells comprises exogenous polynucleotides encoding one of the following sets of enzymes;

i) ω3-desaturase and / or Δ 15-desaturase, Δ6-desaturαza, Δ5-desaturαza, Δ6-elongαza and Δ5-elongase, ii) Δ12-desaturase, Δ6-desaturase, Δ5-desaturαza, Δ6-elongase and Δ5-elongase , iii) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongaza, iv) ω3-desaturase and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturαza, Δ9-elongase and Δ5-elongase,

v) Δ12-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongαse, vi) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturαse, Δ9-elongαse, and Δ5-elongαse, each polynucleotide being operably linked to one or more promoters capable of directing the expression of said polynucleotides in a plant part cell or cells.

In an embodiment, if a part of a plant or a cell contains a lipid that contains fatty acids in esterified form, the fatty acids containing docosapentaenoic acid (DPA) and / or docosahexaenoic acid (DHA), and at least 35% DPA and / or DHA (if it is present) esterified in the form of a triacylglyceride (TAG), and is charged at the sn2 position of the TAG, then a part of the plant, such as a seed, or recombinant cells, such as microbial cells, contains an exogenous polynucleotide encoding 1-acylglyceride-3-phosphate acyltransferase (LPAAT ), and, in this case, the polynucleotide is operably linked to one or more promoters that are capable of directing the expression of the polynucleotide in a cell of a plant part or cells. In a further embodiment, the cell contains exogenous polynucleotides encoding one of the following sets of enzymes:

i) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), ω3-desaturase, Δ6-desaturαse, Δ5-desaturase, Δ6-elongαse and Δ5-elongase, ii) 1-acylglyceride-3-phosphatacyltransferase (D15-desaturase) Δ6-desaturase, Δ5-desaturase, Δ6-elongαse and Δ5-elongase, iii) 1-acylglyceride-3-phosphatacyltransferase (LPAAT), Δ12-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongαse and Δ5-elongase, ) 1-acylglyceride-3-phosphatacyltransferase (LFAAT), Δ2-desaturαse, ω3-desaturαse and / or Δ15desaturase, Δ6-desaturαse, Δ5-desaturase, Δ6-elongαse and Δ5-elongαse,

v) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), ω3-desaturαse, Δ8-desaturαse, Δ5-desaturase, Δ9-elongαse and Δ5-elongase, vi) 1-acylglyceride-3-phosphatacyltransferase), Δ15 Δ8-desaturαse, Δ5-desaturase, Δ9-elongαse and Δ5-elongase, vii) 1-acylglyceride-3-phosphatacyltransferase (LPAAT), Δ12-desaturase, Δ8-desaturαse, Δ5-desaturase, Δ9-elongase and optional elongase, Δ5- Δ4-desaturase, viii) 1-acylglyceride-3-phosphatacyltransferase (LFAAT), Δ12-desaturαse, ω3-desaturαse and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturαse, Δ9-elongase and Δ5-elongase, each the polynucleotide is operably linked to one or more promoters capable of directing the expression of said polynucleotides in a cell. Preferably, the LFAAT can use a polyunsaturated C22 fatty acyl-CoA substrate such as DPA-CoA.

Preferably, a plant or part thereof, such as a seed, or a microbial cell does not contain a polynucleotide encoding a Δ4 desaturase, or does not contain a Δ4 desaturase polypeptide.

- 11 037184

In an embodiment, Δ12-desaturase additionally has ω3-desaturase and / or Δ15desaturase activity, i.e. activities are provided by a single polypeptide. Alternatively, Δ12 desaturase has no ω3 desaturase activity and no Δ15 desaturase activity, i. E. Δ12 desaturase is a separate polypeptide from a polypeptide having ω3 desaturase and / or Δ15 desaturase activity.

In yet another embodiment, a plant part, such as a seed, or recombinant cells, such as microbial cells, has one or more or all of the following traits:

i) Δ12 desaturase converts oleic acid to linoleic acid in one or more cells of a plant part or in recombinant cells with an efficiency of at least about 60, at least about 70, at least about 80%, about 60 to about 95% , from about 70 to about 90%, or from about 75 to about 85%, ii) ω3 desaturase converts ω6 fatty acids to ω3 fatty acids in one or more cells of a plant part or in recombinant cells with an efficiency of at least about 65, at least about 75, at least about 85%, about 65 to about 95%, about 75 to about 91%, or about 80 to about 91%, iii) Δ6 desaturase converts ALA to CDA in one or more cells parts of a plant or in recombinant cells with an efficiency of at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70%, from about 30 to about 70%, about 35 to about 60%, or about 50 to about 70%, iv) Δ6-dec amurase converts linoleic acid to γ-linolenic acid in one or more cells of a plant part or in recombinant cells with an efficiency of less than about 5, less than about 2.5, less than about 1%, about 0.1 to about 5%, about 0.5 to about 2.5%, or about 0.5 to about 1%,

v) Δ6-elongase converts SDC to ETC in one or more cells of a plant part or in recombinant cells with an efficiency of at least about 60, at least about 70, at least about 75%, from about 60 to about 95%, from about 70 to about 80%, or about 75 to about 80%, vi) Δ5-desamurase converts ETA to EPA in one or more cells of a plant part or in recombinant cells with an efficiency of at least about 60, at least about 70, at least about 75, at least about 80, at least about 90%, about 60 to about 95%, about 70 to about 95%, or about 75 to about 95%, vii) Δ5-elongase converts EPA to DPC in one or more cells of a part of a plant or in recombinant cells with an efficiency of at least about 80, at least about 85, at least about 90%, from about 50 to about 90%, or from about 85 to about 95%, ix ) efficiency of conversion of oleic acid to duodenum in one or more cells of a plant part or in recombinant cells is at least about 10, at least about 15, at least about 20, at least about 25%, about 20, about 25, about 30%, about 10 to about 50%, about 10 to about 30 %, about 10 to about 25%, or about 20 to about 30%,

x) the efficiency of converting LA to DPC in one or more cells of a plant part or in recombinant cells is at least about 15, at least about 20, at least about 22, at least about 25, at least about 30%, about 25, about 30, about 35%, about 15 to about 50%, about 20 to about 40%, or about 20 to about 30%, xi) the efficiency of converting ALA to DPC in one or more cells of a plant part or recombinant cells is at least about 17, at least about 22, at least about 24, at least about 30%, about 30, about 35, about 40%, from about 17 to about 55%, from about 22 to about 35%, or about 24 to about 35%, xi) one or more cells of the plant part or recombinant cells contain at least about 25, at least about 30%, about 25 to about 40%, or about 27 , 5 to about 37.5% more fatty acids ω3 than the corresponding cells without exogenous polynucleotides, xii) Δ6-desaturase is preferable significantly reduces the degree of saturation of α-linolenic acid (ALA) relative to linoleic acid (LA), xiii) Δ6-elongase additionally has a Δ9-elongase activity, xiv) Δ12-desaturase additionally has a Δ15-desaturase activity, xv) Δ6-desamurase additionally possesses ∆8-desamurase activity, xvi) ∆8-desamurase additionally has a ∆6-desamurase activity or does not have a ∆6-desamurase activity, xvii) ∆ 15-desaturase additionally has an ω3-desamurase activity against GLA, xvii i) ω3-desamurase additionally has a ∆15 activity -desaturase in relation to LA, xix) ω 3-desaturase decreases the saturation of LA and / or GLA, xx) ω3-desamurase preferably reduces the saturation of GLA relative to LA, xxi) one or more or all desaturase, preferably Δ6-desamurase and / or Δ5-desaturase, region

- 12 037184 give a higher activity on the acyl-CoA substrate than on the corresponding acylphosphatidylcholine (acyl-PC) substrate, xxii) Δ6-desaturase has a higher Δ6-desaturase activity against ALA than

LA as a fatty acid substrate, xxiii) Δ6-desaturase has a higher Δ6-desaturase activity against ALA-CoA as a fatty acid substrate than against ALA attached to the sn-2 position of PC as a fatty acid substrate, xxiv) Δ6-desaturase has a at least 2 times higher Δ6-desaturase activity, at least 3 times higher activity, at least 4 times higher activity, or at least 5 times higher activity against ALA as a substrate, compared to LA, xxv) Δ6-desaturase has a higher activity against ALA-CoA as a fatty acid substrate than against ALA attached to the sn-2 position of PC as a fatty acid substrate, xxvi) Δ6-desaturase has at least 5 times more high activity of Δ6-desaturase or at least 10 times higher activity against ALA-CoA as a fatty acid substrate than with respect to ALA attached to the sn-2 position of PC, to as a fatty acid substrate, xxvii) desaturase is a front-end desaturase, xxviii) Δ6-desaturase has no detectable Δ5-desaturase activity against ETA.

In yet another embodiment, a plant part, such as a seed, preferably a Brassica seed or a C. sativa seed, or a recombinant cell, such as a microbial cell, has one or more or all of the following traits:

i) Δ12 desaturase contains the amino acid sequence shown in SEQ ID NO: 4, a biologically active fragment thereof, or an amino acid sequence at least 50% identical to SEQ ID NO: 4, ii) ω3 desaturase contains the amino acid sequence shown in SEQ ID NO: 6, its biologically active fragment, or an amino acid sequence at least 50% identical to SEQ ID NO: 6, iii) Δ6-desaturase contains the amino acid sequence shown in SEQ ID NO: 9, its biologically active fragment, or the sequence amino acids at least 50% identical to SEQ ID NO: 9, iv) Δ6-elongase contains the amino acid sequence shown in SEQ ID NO: 16, a biologically active fragment thereof, such as SEQ ID NO: 17, or an amino acid sequence of at least 50% identical to SEQ ID NO: 16 and / or SEQ ID NO: 17,

v) Δ5-desaturase contains the amino acid sequence shown in SEQ ID NO: 20, a biologically active fragment or amino acid sequence at least 50% identical to SEQ ID NO: 20, and vi) Δ5-elongase contains the amino acid sequence shown in SEQ ID NO: 25, its biologically active fragment, or amino acid sequence at least 50% identical to SEQ ID NO: 25.

In an embodiment, a plant part, such as a seed, or recombinant cells, such as microbial cells, further comprises (at) an exogenous polynucleotide encoding diacylglyceride acyltransferase (DHAT), monoacylglyceride acyltransferase (MGAT), glyceride-3-phosphatacyltransferase Α-3-phosphate acyltransferase (LPKAT), preferably LPKAT, which can use a polyunsaturated fatty acyl-CoA substrate C22, such as DPA-CoA. acyl-CoA: lizofosfatidilholinatsiltransferazu (LFHAT), phospholipase A2 (WLL _2), phospholipase C (PLC), phospholipase D (PLD), PSD-choline diatsilglitseridholin phosphotransferase (HFT), phosphatidylcholine diacylglycerol acyltransferase (PDAT), phosphatidylcholine: diacylglycerol choline phosphotransferase ( PDCT), acyl-CoA synthetase (ACS), or a combination of two or more of these enzymes.

In another embodiment, the plant part, such as a seed, or recombinant cells, such as microbial cells, further comprises an introduced mutation or an exogenous polynucleotide that, in the cell of the plant part, negatively regulates the production and / or activity of an endogenous enzyme selected from FAE1, DHAT, MGAT, GPAT, LPKAT, LPKHAT, RLK ₂ , PLC, PLD, CPT, PDAT, thioesterase, for example FATB or Δ12 desaturase, or a combination of two or more of these enzymes.

In a further embodiment, at least one or preferably all of the promoters are seed specific promoters. In an embodiment, at least one or all of the promoters are derived from an oil biosynthesis or accumulation gene, such as a gene encoding oleosin, or from protein storage genes in a seed, such as a gene encoding conlinin.

- 13 037184

In another embodiment, the promoter (s) directing the expression of exogenous polynucleotides that encode the Δ5 elongase initiates the expression of the polynucleotides in the developing plant seed or recombinant cells, such as microbial cells, before or peaking before the promoter (s), directing (and) the expression of exogenous polynucleotides encoding Δ12-desaturase and ω3-desaturase.

In a further embodiment, the exogenous polynucleotides are covalently linked into a DNA molecule, preferably a T-DNA molecule integrated into the genome of cells of a plant part or recombinant cells, such as microbial cells, the number of such DNA molecules integrated into the genome of cells of a plant part or recombinant cells, preferably is no more than one, two or three, or is two or three.

In yet another embodiment, the plant part contains at least two different exogenous polynucleotides, each of which encodes a Δ6 desaturase with the same or a different amino acid sequence.

In a further embodiment, the total oil content of the plant part containing exogenous polynucleotides is at least about 40, or at least about 50, at least about 60, at least about 70%, about 50 to about 80%, or about 80 to about 100% of the total oil content in the corresponding part of the plant, which lacks exogenous polynucleotides. In a further embodiment, the weight of the seed containing exogenous polynucleotides is at least about 40, at least about 50, at least about 60, at least about 70%, about 50 to about 80%, or about 80 to 100% of the mass of the corresponding seed, which does not contain exogenous polynucleotides.

In another embodiment, the lipid is in the form of an oil, preferably an oilseed oil, with at least about 90, or at least about 95, at least about 98%, or from about 95 to about 98% by weight of the lipid triacylglycerides.

In a further embodiment, the method further comprises treating the lipid to increase the duodenum level as a percentage of the total fatty acid content. For example, treatment includes hydrolysis of esterified fatty acids to form free fatty acids or transesterification. For example, a lipid such as rapeseed oil can be treated to convert the fatty acids in the oil to alkyl esters, such as methyl or ethyl esters, which can then be fractionated to enrich the lipid or WPC oil. In embodiments, the fatty acid composition of the lipid after such treatment comprises at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90% DPC. In an embodiment of the invention, the level of DHA in the total fatty acid content of the lipid after treatment is less than 2.0% or less than 0.5%, preferably it is not detectable in the lipid.

In addition, there is provided a lipid or oil containing a lipid, such as free fatty acids or alkyl esters, obtained using the method of the invention.

In another aspect of the present invention, there is disclosed a method for preparing methyl or ethyl esters of polyunsaturated fatty acids, comprising reacting triacylglycerides with methanol or ethanol in an extracted plant lipid or during an extraction process, respectively, wherein the extracted plant lipid comprises esterified fatty acids in the form of TAG, and wherein the fatty acids include oleic acid, palmitic acid, ω6 fatty acids including linoleic acid (LA), ω3 fatty acids including α linolenic acid (ALA), and docosapentaenoic acid (DPA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), while the level of DPA in the total fatty acid content of the extracted lipid is from about 7 to 35%, preferably from 20.1 to 30% or from 20.1 to 35%, thus obtaining methyl or ethyl esters of poly unsaturated fatty acids.

In another aspect of the present invention, there is provided a method for preparing methyl or ethyl esters of docosapentaenoic acid (DPA), comprising reacting triacylglycerides (TAG) with methanol or ethanol in an extracted plant lipid or during an extraction process, respectively, wherein the extracted plant lipid contains fatty acids in esterified form, and the fatty acids include docosapentaenoic acid (DPA), with at least 35% of the DPA esterified in the TAG form being esterified at the sn-2 position of the TAG, thereby obtaining methyl or ethyl esters of polyunsaturated fatty acids.

In a preferred embodiment, the lipid used in the method according to the above two aspects has one or more of the characteristics defined herein in the context of the extracted lipid or oil of the invention.

In another aspect of the present invention, there is disclosed an oil plant or part thereof, such as a seed, preferably a Brassica plant or a C. sativa plant containing a lipid in a seed, or a microbial cell comprising:

a) a lipid containing fatty acids in esterified form and

- 14 037184

b) exogenous polynucleotides encoding one of the following sets of enzymes:

i) Δ12-desaturase, ω3-desaturase and / or λ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and

Δ5-desaturase or ii) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and

Δ5-elongase, iii) ω3-desaturase and / or Δ 15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, or iv) ω3-desaturase and / or Δ 15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, each polynucleotide is operably linked to one or more seed-specific promoters capable of directing the expression of said polynucleotides in the developing plant seed, or to one or more promoters capable of directing the expression of said polynucleotides in microbial cell, and, in this case, fatty acids include oleic acid, palmitic acid, ω6 fatty acids, including linoleic acid (LA) and optionally γ-linolenic acid (GLA), ω3 fatty acids, including α-linolenic acid (ALA), stearidonic acid (SDA), and docosapentaenoic acid (DPA) and optionally eicosapentaenoic acid (EPA) and / or eicosatetraenoic acid (ETA), while the level of DPA in the total fatty acids of the lipid of the semen or microbial cell with puts from 7 to 35%. In a preferred embodiment of this aspect, DHA is present at a level of less than 2 or less than 0.5% of the total fatty acid of the seed lipid and the extracted lipid, and more preferably is not determined in the total fatty acid of the lipid.

In another aspect, the present invention provides a cell, preferably a cell in or from a plant, such as an oilseed plant, or part thereof, such as a seed, or an oilseed plant or part thereof, preferably a Brassica plant or a C. sativa plant, or a microbial cell, containing:

a) fatty acids in esterified form, the fatty acids comprising docosapentaenoic acid (DPA) and wherein at least 35% of the DPA esterified as triacylglyceride (TAG) is esterified at the sn-2 position of the TAG, and

b) exogenous polynucleotides encoding one of the following sets of enzymes:

i) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), ω3-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, ii) 1-acylglyceride-3-phosphate acyltransferase (LFA-desaturase), Δ15 Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, iii) 1-acylglyceride-3-phosphatacyltransferase (LPAAT), Δ12-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, iv ) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ12-desaturase, ω3-desaturase and / or Δ15desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

v) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), ω3-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, vi) 1-acylglyceride-3-phosphatacyltransferase (LFA-desaturase), Δ 15 , Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, vii) 1-acylglyceride-3-phosphatacyltransferase (LPAAT), Δ12-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, viii) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, each polynucleotide being functionally linked to one or more promoters capable of directing the expression of said polynucleotides in a cell. Preferably, the LFAAT can use a C22 polyunsaturated fatty acyl-CoA substrate, such as DPA-CoA, and the level of DPA in the total fatty acid content of the extracted lipid is from about 1 to 35%, or from about 7 to 35%, or from about 20.1 to 35 %. In embodiments, at least about 40, at least about 45, at least about 48%, 35 to 60%, or 35 to 50% DPA and / or DHA esterified as triacylglyceride (TAG) esterified in position sn-2 TAG.

In preferred embodiments of each of the above two aspects of the invention, Δ15 desaturase is fungal Δ15 desaturase, and ω3 desaturase is fungal ω3 desaturase.

In a preferred embodiment, an oilseed plant, microbial cell or cell according to the invention possesses, as appropriate, one or more of the characteristics defined herein, for example, as defined above for an extracted plant lipid, an extracted microbial lipid, or a method for producing them.

Examples of oilseed plants include, but are not limited to, Brassica spp., Gossypium hirsutum, Linum usitatissimum, Helianthus spp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana,

- 15 037184

Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium species, Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa or Crambe abyssinica. In an embodiment, the oilseed plant is a Brassica plant, a C. sativa plant, or a G. max plant (soybean). In an embodiment, the oil plant is a rapeseed plant, B. juncea, Glycine max, Camelina sativa, or Arabidopsis thaliana. In an alternative embodiment, the oilseed plant is a plant other than A. thaliana and / or other than C. sativa. In an embodiment, the oilseed plant is a plant other than G. max (soybean). Preferably the plant is a Brassica or Camelina sativa plant. In an embodiment, the oilseed plant is in the field, or has been grown in the field, or has been grown in a greenhouse under standard conditions, for example, as described in Example 1.

In an embodiment, one or more desaturases used in the method of the invention or present in a cell or plant or part thereof according to the invention are capable of using an acyl-CoA substrate. In a preferred embodiment, one or more of Δ6-desaturase, Δ5-desamurase and Δ8-desaturase, if present, are capable of using an acyl-CoA substrate, preferably each of i) Δ6-desamurase and Δ5-desaturase, or ii) Δ5-desaturase, and Δ8-desaturase is able to utilize the acyl-CoA substrate. In an embodiment, Δ12-desaturase and / or ω3-desaturase are capable of using an acyl-CoA substrate. The acyl-CoA substrate is preferably ALK-CoA for Δ6 desaturase, ETC-CoA for Δ5 desaturase, ETRK-CoA for Δ8 desaturase, oleoyl-CoA for Δ8 desaturase, or one or more of LA-CoA, GLK-CoA and ARA -KoA for ω3-desaturase.

In an embodiment, the mature, harvested seed of the plant contains WPC in an amount of at least about 28 mg per gram of seed, preferably at least about 32 mg per gram of seed, at least about 36 mg per gram of seed, at least about 40 mg per gram of seed, more preferably at least about 44 mg per gram of seed, or at least about 48 mg per gram of seed, about 80 mg per gram of seed, or about 30 to about 80 mg for 1 g of seed.

In a further aspect of the present invention, there is disclosed a Brassica napus, Bjuncea or Camelina sativa plant that is capable of producing a WPC-containing seed, wherein the harvested mature seed of the plant contains WPC in an amount of at least about 28 mg per gram of seed, preferably at least about 32 mg per gram of seed, at least about 36 mg per gram of seed, at least about 40 mg per gram of seed, more preferably at least about 44 mg per gram of seed, or at least about 48 mg per gram of seed, about 80 mg per gram of seed, or about 30 to about 80 mg per gram of seed.

In another aspect of the present invention, a plant cell of the invention is disclosed comprising the exogenous polynucleotides disclosed herein.

Also disclosed is a plant part, preferably a seed, or recombinant cells such as microbial cells that exhibit one or more of the following traits:

i) obtained from a plant according to the invention, ii) contains a lipid disclosed herein, or iii) can be used in a method according to the invention.

In yet another aspect, the present invention discloses a mature, harvested seed of Brassica napus, B. juncea or Camelina sativa containing WPC having a moisture content of about 4 to about 15 wt%, preferably about 6 to about 8 wt. % or about 4 to about 8 wt.%, more preferably from about 4 to about 6 wt.%, and the content of WPC in the seed is at least about 28 mg per gram of seed, preferably at least about 32 mg per 1 g seed, at least about 36 mg per gram of seed, at least about 40 mg per gram of seed, more preferably at least about 44 mg per gram of seed, or at least about 48 mg per gram of seed, about 80 mg per gram of seed, or from about 30 to about 80 mg per gram of seed.

In an embodiment, a cell according to the invention, an oil plant according to the invention, a Brassica napus, B. juncea or Camelina sativa plant according to the invention, a part of a plant according to the invention, or a seed according to the invention can be used to produce an extracted lipid having one or more or all of the characteristics defined in this document.

In yet another aspect of the present invention, there is disclosed a method for producing a plant or cell according to the invention that can be used to obtain an extracted lipid according to the invention, the method comprising

a) analysis of the level of duodenum in a lipid produced by one or more parts of a plant, such as seeds, or recombinant cells, such as microbial cells, from several plants or recombinant cells, such as microbial cells, each plant or recombinant cell, such as microbial a cell contains one or more exogenous polynucleotides encoding one of the following sets of enzymes;

i) ω3 desaturase, Δ6 desaturase, Δ5 desaturase, Δ6 elongase and Δ5 elongase, ii) Δ15 desaturase, Δ6 desaturase, Δ6 desaturase, Δ6 elongase and Δ5 elongase, iii) Δ12 desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

- 16 037184 iv) Δ12-desaturase, ω3-desaturase or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and

Δ5-elongase,

v) ω3 desaturase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, vi) Δ15 desaturase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, vii) Δ12 desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, viii) Δ12-desaturase, ω3-desaturase or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase or ix) ω3- desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

x) Δ 15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, xi) Δ12-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, each polynucleotide being functionally linked with one or more promoters capable of directing the expression of said polynucleotides in a plant part cell or a recombinant cell, and

b) identifying from a plurality of plants or recombinant cells of a plant or recombinant cell that can be used to obtain the extracted plant lipid or cell lipid according to the invention in one or more parts thereof, and

c) optionally, obtaining progeny of plants or recombinant cells from the identified plant or recombinant cell, or their seed.

In an embodiment, the plant or recombinant cell further comprises an exogenous polynucleotide encoding LFAAT as defined herein.

Preferably, the offspring plant is at least a second or third generation relative to the identified plant, and is preferably homozygous for one or more polynucleotides. More preferably, one or more polynucleotides are present in the progeny plant at only one insertion locus. Thus, the invention provides a method that can be used as a screening method for identifying a plant or its seed from a plurality of transformed plants or candidate seeds, the identified plant or its progeny plant producing a lipid of the invention, preferably in a seed. Such a plant or progeny plant or its seed is selected if it produces a lipid according to the invention, in particular, with a specified level of duodenum, or is not selected if it does not produce a lipid according to the invention.

In an embodiment, exogenous polynucleotides that are present in a cell, such as a microbial cell, or a plant or part thereof, as defined herein, become stably integrated into the genome of a cell, plant, or plant part, such as a seed. Preferably, the exogenous polynucleotide (s) becomes stably integrated into the genome of a cell, plant, or plant part, such as a seed, at one locus in the genome, and is preferably homozygous for insertion. More preferably, the plant, plant part or seed is further characterized in that it contains no exogenous polynucleotides other than one or more T-DNA molecules.

Thus, no exogenous vector sequences are integrated into the genome, except for the T-DNA sequences.

In an embodiment, prior to step a), the method comprises introducing one or more exogenous polynucleotides into one or more plant cells.

Additionally, there is provided a plant obtained using the method according to the invention and the seeds of such a plant.

In an embodiment, the plant of the invention has both male and female fertility, preferably with both male and female fertility levels that are at least 70% relative to the corresponding wild-type plant, or are preferably the same. In an embodiment, pollen produced by a plant of the invention or a plant derived from a seed of the invention is 90-100% viable as determined by viability staining. For example, pollen viability can be assessed as described in Example 1.

In another aspect of the present invention, a method for producing a seed is disclosed, comprising:

a) growing a plant according to the invention or a plant that forms a part according to the invention, preferably in the field as part of a population of at least 1000 or 2000 or 3000 such plants or on an area of at least 1 or 2 or 3 hectares with a standard planting density , or, alternatively, in a greenhouse with standard conditions,

b) harvesting the seed from the plant or plants, and

c) optionally, extraction of the lipid from the seed, preferably to obtain an oil with a total WPC yield of at least 60, or 70, or 80 kg WPC per hectare.

In an embodiment, a plant, plant cell, plant part or seed, or a recombinant cell of the invention has one or more of the following characteristics:

i) the oil is as disclosed herein, or ii) a plant part or seed or recombinant cell can be used in the method of the invention

- 17 037184 niyu.

For example, the seed can be used to produce a plant according to the invention. The plant can be grown in the field or in a greenhouse under standard conditions, for example, as described in example 1.

A further aspect of the present invention discloses a lipid or oil produced or obtained using the method of the invention from a cell of the invention, an oil plant of the invention, a plant of the Brassica species, Brassica napus, B. juncea, G. max or Camelina sativa of the invention, a plant part of the invention, a seed of the invention or a plant, a plant cell, a plant part or a seed of the invention. Preferably, the lipid or oil is purified to remove contaminants such as nucleic acid (DNA and / or RNA), protein and / or carbohydrate, or pigments such as chlorophyll. In addition, the lipid or oil can be purified to increase the TAG content, for example, by removing free fatty acids (FFA) or phospholipid.

In an embodiment, the lipid or oil is obtained by extracting oil from an oilseed crop. Examples of oilseed oil include, but are not limited to, rapeseed oil (Brassica napus, subspecies Brassica rapa), mustard oil (Brassica juncea), another Brassica species oil, sunflower oil (Helianthus annus), linseed oil (Linum usitatissimum), soybean oil ( Glycine max), safflower oil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotiana tabacum), peanut oil (Arachis hypogaea), palm oil, cottonseed oil (Gossypium hirsutum), coconut oil (Cocos nucifera), oil avocado (Persea americana), olive oil (Olea europaea), cashew oil (Anacardium occidentale), macadamia oil (Macadamia intergrifolia), almond oil (Prunus amygdalus), or Arabidopsis seed oil (Arabidopsis thaliana).

In an embodiment, the cell (recombinant cell) according to the invention or used according to the invention is a microbial cell, such as a cell suitable for fermentation, preferably an oily microbial cell capable of accumulating triacylglycerides to a level of at least 25% by weight. The preferred fermentation processes are anaerobic fermentation processes, as are well known in the art. Suitable fermenting cells, usually microorganisms, are capable of fermenting, i. E. convert sugars such as glucose or maltose, directly or indirectly, into desired fatty acids. Examples of fermenting microorganisms include fungal organisms such as yeast. As used herein, yeast includes Saccharomyces spp., Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and preferably Yarrowia lipolytica.

In a further aspect of the present invention, there is disclosed a fatty acid produced or obtained using the method according to the invention from a cell according to the invention, an oil plant according to the invention, a plant of the Brassica species, Brassica napus, B. juncea, G. max or Camelina sativa according to the invention, a plant part according to the invention, a seed according to the invention, or a plant, plant cell, part of a plant or seed according to the invention. Preferably the fatty acid is DPA. The fatty acid can be present in a fatty acid mixture, the fatty acid composition of which is as described herein, or the mixture can be enriched such that the fatty acid, preferably DPA, constitutes at least 40% or at least 90% of the content of fatty acids in the mixture. In an embodiment, the fatty acid is unesterified. Alternatively, the fatty acid is esterified using, for example, methyl, ethyl, propyl or butyl groups.

Further disclosed is a cake obtained from a seed according to the invention or obtained from a plant according to the invention. Preferred cake includes, but is not limited to, cake from Brassica napus, B. juncea, Camelina sativa, or Glycine max. In an embodiment, the cake contains exogenous (s) polynucleotide (s) and / or genetic constructs as disclosed herein. In a preferred embodiment, the cake retains a portion of the lipid or oil produced in the seed from which the cake is derived, but at a low level (eg, less than 2 wt%) after most of the lipid or oil has been extracted. The cake can be used as animal feed or as an ingredient in food production.

In another aspect of the present invention, a composition is disclosed comprising one or more of a lipid or oil of the invention, a fatty acid of the invention, a cell of the invention, an oil plant of the invention, a plant of the Brassica species, Brassica napus, B. juncea, Glycine max or Camelina sativa according to the invention, a part of a plant according to the invention, a seed according to the invention or a cake according to the invention. In embodiments, the composition comprises a carrier suitable for pharmaceutical, food or agricultural use, a seed treatment compound, fertilizer, other food or nutritional ingredient, or additional protein or vitamins.

In addition, food (feed), cosmetics or chemicals are disclosed containing one or more of a lipid or oil according to the invention, a fatty acid according to the invention, cells according to the invention, an oilseed plant according to the invention, plants of the species Brassica, Brassica napus, B. juncea, Glycine max or Camelina sativa according to the invention, a plant part according to the invention, a seed according to the invention, a cake according to the invention, or a composition according to the invention. The preferred food pi

- 18 037184 Tania is an infant formula containing a lipid or oil according to the invention.

In another aspect of the present invention, there is disclosed a method for preparing a food product, preferably an infant formula, comprising mixing one or more of a lipid or oil according to the invention, a fatty acid according to the invention, a cell according to the invention, an oil plant according to the invention, a plant of the Brassica species, Brassica napus, B juncea, Glycine max or Camelina sativa according to the invention, a plant part according to the invention, a seed according to the invention, a cake according to the invention, or a composition according to the invention with at least one other nutritive ingredient. The method may include the steps of mixing, heat treating, baking, extruding, emulsifying, or otherwise treating the food, or packaging the food or analyzing the lipid or oil content of the food.

In another aspect of the present invention, there is disclosed a method of treating or preventing a condition in which PUFA, preferably DPC, have a beneficial effect, said method comprising administering to a subject one or more of a lipid or oil of the invention, a fatty acid of the invention, a cell of the invention, an oil plant crops according to the invention, plants of the species Brassica, Brassica napus, B. juncea, Glycine max or Camelina sativa according to the invention, plant parts according to the invention, seed according to the invention, cake according to the invention, compositions according to the invention or feed according to the invention. In a preferred embodiment, the PUFA is administered in the form of a pharmaceutical composition containing an ethyl ester of the PUFA. The subject can be human or animal.

Examples of conditions in which PUFAs have beneficial effects include, but are not limited to, elevated serum triglyceride levels, elevated serum cholesterol levels, such as elevated LDL cholesterol levels, cardiac arrhythmias, angioplasty, inflammation, asthma, psoriasis, osteoporosis, kidney stones, AIDS, multiple sclerosis, rheumatoid arthritis, Crohn's disease, schizophrenia, cancer, fetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, adrenoleukodystrophy, coronary artery disease, heart disease, hypertension, obesity, , chronic obstructive pulmonary disease, ulcerative colitis, restenosis after angioplasty, eczema, hypertension, platelet aggregation, gastrointestinal bleeding, endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronic fatigue after viral infections, or eye disease.

Also disclosed is the use of one or more of a lipid or oil according to the invention, a fatty acid according to the invention, a cell according to the invention, an oil plant according to the invention, a plant of the species Brassica, Brassica napus, B. juncea, Glycine max or Camelina sativa according to the invention, a plant part according to the invention, the seed according to the invention, the oil cake according to the invention, the composition according to the invention or the feed according to the invention for the manufacture of a medicament for the treatment or prevention of a condition in which PUFA, preferably DPC, have a beneficial effect.

Formulation of a medicament may include mixing an oil of the invention with a pharmaceutically acceptable carrier to treat a condition disclosed herein. The method may include, firstly, refining and / or transesterification of the oil, and / or fractionating the oil in order to increase the WPC level. In a specific embodiment, the method comprises treating a lipid or oil, such as rapeseed oil, to convert the fatty acids in the oil to alkyl esters, such as methyl or ethyl esters. Further processing, such as fractionation or distillation, can be used to enrich the WPC lipid or oil. In a preferred embodiment, the medicament contains ethyl esters of duodenum. In an even more preferred embodiment, the level of ethyl esters of DPA in the drug is from 30% to 50%. The drug may additionally contain EPA ethyl esters, for example, from 30 to 50% or at least 90% of the total fatty acid content of the drug. Such drugs are suitable for administration to human or animal subjects in the treatment of medical conditions as disclosed herein.

In another aspect of the present invention, there is disclosed a method of trading seed comprising obtaining a seed according to the invention and trading the resulting seed for monetary gain.

In an embodiment, obtaining seed comprises cultivating the plants of the invention and / or harvesting seed from the plants.

In another embodiment, obtaining the seed further comprises placing the seed in a container and / or storing the seed.

In a further embodiment, obtaining the seed further comprises transporting the seed to another location.

In yet another embodiment, the method further comprises transporting the seed to another location after the seed is sold.

In a further embodiment, trading is carried out using electronic means, such as a computer.

In yet another aspect of the present invention, a method is disclosed for producing seed hoppers,

- 19 037184 including:

a) swathing, row composting and / or reaping of the ground parts of plants containing the seed according to the invention,

b) threshing and / or blowing of plant parts, separating the seed from the rest of the plant parts, and

c) sifting and / or sorting the seed separated in step b) and loading the sifted and / or sorted seed into hoppers, thus obtaining hoppers with seed.

In an embodiment, if appropriate, a lipid or oil, preferably seed oil, according to the invention or suitable according to the invention, contains fat levels as disclosed in the tables in the Examples section.

Any implementation in this document should be interpreted as applicable, mutatis mutandis, to any other implementation, unless specifically indicated otherwise.

The context of the present invention should not be limited to the specific options disclosed herein, which are provided for illustrative purposes only. Functionally equivalent products, compositions and methods are obviously within the context of the invention disclosed herein.

In this document, unless otherwise specifically indicated or the context clearly does not indicate otherwise, a reference to a single stage, composition of a substance, a group of stages or a group of compositions of a substance should be interpreted as including the singular and plural (i.e., one or more) such steps, compositions of matter, groups of steps, or groups of compositions of matter.

The invention is further disclosed by means of the following non-limiting examples with reference to the accompanying drawings.

Brief description of graphic materials

FIG. 1 - aerobic pathways of duodenal biosynthesis.

FIG. 2 is a map of the T-DNA insertion site from the left to right borders of pJP3416-GA7. PG indicates the right border; LH, left border; TEP, transcription / polyadenylation terminator region; PRO, promoter; coding regions are shown above the arrows, promoters and terminators below the arrows. Micpu-Δ6D, Micromonas pusilla Δ6 desaturase; Pyrco-Δ6E, Δ6-elongase Pyramimonas cordata; Pavsa-Δ5D, Pavlova salina Δ5-desaturase; Picpa-ω3D, ω3-desaturase of Pichia pastoris; P. salina Pavsa-Δ4D, Δ4 desaturase; Lack1-Δ12D, Lachancea kluyveri Δ12 desaturase; Pyrco-Δ5E, Δ5-elongase Pyramimonas cordata. HOC is the Agrobacterium tumefaciens nopaline synthetase transcription / polyadenylation terminator region; FP1, a truncated Brassica napus napin promoter; FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine max lectin transcription / polyadenylation terminator site; Cnl1 and Cnl2 denote the Linum usitatissimum conlinin 1 or conlinin 2 promoter or terminator. UPM denotes the site of attachment to the matrix of Rb7 from Nicotiana tabacum.

FIG. 3 (A) - basic structure of phytosterol with a ring and side chain numbering; (B) - chemical structures of some phytosterols.

FIG. 4 is a map of the T-DNA insertion site between the left and right borders of pJP3662. PG indicates the right border; LH, left border; TEP, transcription / polyadenylation terminator region, PRO, promoter; coding regions are indicated above the arrows, promoters and terminators below the arrows. Micpu-Δ6D, Micromonas pusilla Δ6-desaturase; Pyrco-Δ6E, Pyramimonas cordata Δ6-elongase; Pavsa-Δ5D, Pavlova salina Δ5-desaturase; Picpa-ω3D, Pichia pastoris ω3-desaturase; Lack1-Δ12D, Lachancea kluyveri Δ12-desaturase; Pyrco-Δ5E, Pyramimonas cordata Δ5-elongase. HOC is the Agrobacterium tumefaciens nopaline synthetase transcription / polyadenylation terminator region; FP1, a truncated napin promoter; Brassica napus FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine max lectin transcription / polyadenylation terminator site; Cnl1 refers to the Linum usitatissimum conlinin 1 promoter or terminator. UPM denotes the site of attachment to the Rb7 matrix of Nicotiana tabacum.

Key to sequence listing

SEQ ID NO: 1 is the nucleotide sequence of pJP3416-GA7. SEQ ID NO: 2 is the nucleotide sequence of pGA7-mod_B. SEQ ID NO: 3 — EPA codon optimized open reading frame for expression of Lachancea kluyveri Δ12 desaturase in plants. SEQ ID NO: 4-Lachancea kluyveri Δ12 desaturase;

SEQ ID NO: 5 — Codon-optimized open reading frame for expression of Pichia pastoris ω3 desaturase in plants;

SEQ ID NO: 6 Pichia pastoris ω3 desaturase;

SEQ ID NO: 7 — open reading frame encoding Micromonas pusilla Δ6 desaturase;

SEQ ID NO: 8 — Codon-optimized open reading frame for expression of Micromonas pusilla Δ6 desaturase in plants (version 2);

SEQ ID NO: 9 Micromonas pusilla Δ6 desaturase;

SEQ ID NO: 10 — open reading frame encoding Ostreococcus lucimarinus Δ6 desaturase;

SEQ ID NO: 11 — Codon-Optimized Open Reading Frame for Expression of Δ6-20 037184 Ostreococcus lucimarinus Desaturase in Plants;

SEQ ID NO: 12-Ostreococcus lucimarinus Δ6 desaturase;

SEQ ID NO: 13-Ostreococcus tauri Δ6 desaturase;

SEQ ID NO: 14 — open reading frame encoding Pyramimonas cordata Δ6 elongase;

SEQ ID NO: 15 — Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at the 3'-end and encoding functional elongase) (version 1);

SEQ ID NO: 16-Pyramimonas cordata Δ6 elongase;

SEQ ID NO: 17 — Truncated Pyramimonas cordata Δ6 elongase;

SEQ ID NO: 18 — open reading frame encoding Pavlova salina Δ5 desaturase;

SEQ ID NO: 19 — Codon-optimized open reading frame for expression of Pavlova salina Δ5 desaturase in plants;

SEQ ID NO: 20-Pavlova salina Δ5 desaturase;

SEQ ID NO: 21 — open reading frame encoding Pyramimonas cordata Δ5 desaturase;

SEQ ID NO: 22-Pyramimonas cordata Δ5 desaturase;

SEQ ID NO: 23 — open reading frame encoding Pyramimonas cordata Δ5 elongase;

SEQ ID NO: 24 — Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants;

SEQ ID NO: 25 - Pyramimonas cordata Δ5 elongase;

SEQ ID NO: 26 — open reading frame encoding Pavlova salina Δ4 desaturase;

SEQ ID NO: 27 — Codon-optimized open reading frame for expression of Pavlova salina Δ4 desaturase in plants;

SEQ ID NO: 28-Pavlova salina Δ4 desaturase;

SEQ ID NO: 29 — open reading frame encoding Isochrysis galbana Δ9 elongase;

SEQ ID NO: 30 — Codon Optimized Open Reading Frame for Expression of Emiliania huxleyi Δ9 Elongase in Plants;

SEQ ID NO: 32 — open reading frame encoding Pavlova pinguis Δ9 elongase;

SEQ ID NO: 33-Pavlova pinguis Δ9 elongase;

SEQ ID NO: 34 — open reading frame encoding Pavlova salina Δ9 elongase;

SEQ ID NO: 35-Pavlova salina Δ9 elongase;

SEQ ID NO: 36 — open reading frame encoding Pavlova salina Δ8 desaturase;

SEQ ID NO: 37-Pavlova salina Δ8 desaturase;

SEQ ID NO: 38 — V2 Viral Suppressor;

SEQ ID NO: 39 — open reading frame encoding viral suppressor V2;

SEQ ID NO: 40-LFKAT2 Arabidopsis thaliana;

SEQ ID NO: 41-LPKAT Limnanthes alba;

SEQ ID NO: 42-Saccharomyces cerevisiae LPKAT;

SEQ ID NO: 43-LFKAT Micromonas pusilla;

SEQ ID NO: 44-LPKAT Mortierella alpina;

SEQ ID NO: 45-LFKAT Braccisa napus;

SEQ ID NO: 46 — LFKAT Brassica napus;

SEQ ID NO: 47-ω3 desaturase of Phytophthora infestans;

SEQ ID NO: 48 - ω3 Thalassiosira pseudonana desaturase;

SEQ ID NO: 49-ω3 Pythium irregulare desaturase;

SEQ ID NO: 50-58 - Oligonucleotide primers / probes.

Detailed description of the essence of the invention

General methods and definitions.

Unless specifically defined otherwise, all technical and scientific terms used herein are to be understood as having the same meaning as usually given to them by those of ordinary skill in the art (e.g., in cell culture, molecular genetics, fatty acid synthesis, transgenic plants , recombinant cells, protein chemistry and biochemistry).

Unless otherwise indicated, the protein, cell culture and immunological methods used in the present invention are standard techniques well known to those skilled in the art. Such techniques are described and explained in literature, for example J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989 ), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all editions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual.

- 21 037184

Cold Spring Harbor Laboratory, (1988) and J.E. Coligan et al. (editors), Current Protocols in Immunology,

John Wiley & Sons (including all editions to date).

The term and / or, for example X and / or Y, is to be understood to mean either X and Y or X or Y, and is to be interpreted as explicitly supporting both or either of the meanings.

In this document, the term about, unless otherwise indicated, means +/- 10%, more preferably +/- 5%, more preferably +/- 1% of the stated value.

In this document, the word includes or variations, for example, includes or including, should be understood to mean the inclusion of the claimed element, integer or stage, or group of elements, integers or stages, but not exclusion of any other element, integer or stage, or group elements, integers or stages.

Some definitions.

As used herein, the terms extracted plant lipid and isolated plant lipid mean a lipid composition that is extracted, for example, by grinding, from a plant or part thereof, such as a seed. The extracted lipid can be a relatively crude composition obtained, for example, by grinding a plant seed, or a more purified composition, of which most, if not all of one or more or each of water, nucleic acids, proteins and carbohydrates derived from plant material removed. Examples of cleaning methods are disclosed below. In an embodiment, the extracted or isolated plant lipid comprises at least about 60, at least about 70, at least about 80, at least about 90, or at least about 95% (w / w) lipid in the composition. A lipid can be solid or liquid at room temperature, if it is a liquid, it is called oil. In an embodiment, the extracted lipid of the invention is immiscible with another lipid, eg, duodenum produced by another source (eg, duodenum from fish oil). In an embodiment, after extraction, one or more or all of the ratios of oleic acid: DPA, palmitic acid: DPA, linoleic acid: DPA, and total ω6 fatty acids: total ω3 fatty acids are substantially unchanged (for example, no more than 10 or 5% change), compared to the ratio in an intact seed or cell. In another embodiment, the extracted plant lipid is not processed by a procedure, such as hydrogenation or fractionation, that may alter one or more or all of the ratios of oleic acid: DPA, palmitic acid: DPA, linoleic acid: DPA, and total ω6 fatty acids: total ω3 fatty acids. acid, compared to the ratio in an intact seed or cell. If the extracted plant lipid of the invention is contained in an oil, the oil may additionally contain non-fatty acid molecules such as sterols.

As used herein, the terms extracted vegetable oil and isolated vegetable oil mean a substance or composition containing an extracted vegetable lipid or an isolated vegetable lipid that is liquid at room temperature. The oil is obtained from a plant or part of it, such as a seed. The extracted or isolated oil can be a relatively crude composition obtained, for example, by grinding a plant seed, or a more purified composition, of which most, if not all, of one or more or each of water, nucleic acids, proteins and carbohydrates originating from plant material are removed. The composition may contain other components, which may be lipid or non-lipid. In an embodiment, the oil composition comprises at least about 60, at least about 70, at least about 80%, at least about 90, or at least about 95% (w / w) of the extracted plant lipid. In an embodiment, the extracted oil of the invention is not miscible with another oil, such as WPC produced by another source (eg, WPC from fish oil). In an embodiment, after extraction, one or more or all of the ratios of oleic acid: DPA, palmitic acid: DPA, linoleic acid: DPA, and total ω6 fatty acids: total ω3 fatty acids do not change significantly (for example, no more than 10 or 5% change ), compared to the ratio in an intact seed or cell. In another embodiment, the extracted plant oil is not treated with a procedure, such as hydrogenation or fractionation, which may alter one or more or all of the ratios of oleic acid: DPA, palmitic acid: DPA, linoleic acid: DPA, and total ω6 fatty acids: total ω 3 fatty acids compared to the ratio in an intact seed or cell. The extracted vegetable oil of the invention may contain non-fatty acid molecules such as sterols.

As used herein, terms such as extracted microbial lipid or extracted microbial oil have the same meaning as the corresponding terms extracted vegetable lipid and extracted vegetable oil, respectively, with the main difference being the source of the lipid or oil.

In this document, an oil is a composition that contains mainly

- 22 037184 lipid and is liquid at room temperature. For example, the oil of the invention preferably contains at least 75%, at least 80%, at least 85%, or at least 90% by weight lipid. Typically, the refined oil contains at least 90% by weight triacylglycerides (TAG) based on the lipid content of the oil. Minor oil components such as diacylglycerides (DAGs), free fatty acids (FFAs), phospholipids, and sterols may be present as disclosed herein.

As used herein, the term fatty acid denotes a carboxylic acid (or organic acid), often with a long, aliphatic tail, saturated or unsaturated. Typically, fatty acids contain a chain of carbon-carbon bonds of at least 8 carbon atoms in length, more preferably at least 12 carbon atoms in length. Preferred fatty acids of the invention contain carbon chains of 18-22 carbon atoms long (C18, C20, C22 fatty acids), more preferably 20-22 carbon atoms (C20, C22) and most preferably 22 carbon atoms (C22). Most naturally occurring fatty acids contain an even number of carbon atoms, since they are biosynthesized with two-carbon acetate. Fatty acids can be in a free state (non-esterified) or in an esterified form, for example, as part of a triglyceride, diacylglyceride, monoacylglyceride, acyl-CoA (thioether) linkage, or other form of linkage. The fatty acid can be esterified as a phospholipid, for example, a form of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglyceride, phosphatidylinositol, or diphosphatidylglyceride. In an embodiment, the fatty acid is esterified with a methyl or ethyl group, such as methyl or ethyl ester of C20 or C22 PUFA. Preferred fatty acids are methyl or ethyl esters of EPA or DPA or EPA, DPA and DHA or EPA and DPA.

Saturated fatty acids do not contain double bonds or other functional groups along the chain. The term saturated means hydrogen, so that all carbon atoms (except for the carboxylic acid group [-COOH]) contain the maximum possible amount of hydrogen. In other words, the omega (ω) end contains 3 hydrogen atoms (CH3-), and each carbon atom within the chain contains 2 hydrogen atoms (-CH2-).

Unsaturated fatty acids are similar in form to saturated fatty acids, except that one or more alkene functional groups are present along the chain, with each alkene replacing a single -CH2-CH2- bond of the chain with a -CH = CH- double bond (i.e. . carbon is double bonded to another carbon atom). The next two carbon atoms in the chain, which are attached to each side of the double bond, can be present in the cis or trans configuration, preferably in the cis configuration. In an embodiment, the lipid or oil fatty acid composition of the invention comprises less than 1% fatty acids containing carbon double bonds in the trans configuration (trans fatty acids).

As used herein, the term monounsaturated fatty acid means a fatty acid that contains at least 12 carbon atoms in the carbon chain and only one alkene group (carbon-carbon double bond) in the chain. As used herein, the terms polyunsaturated fatty acid or PUFA mean a fatty acid that contains at least 12 carbon atoms in its carbon chain and at least two alkene groups (carbon double bonds).

As used herein, the terms long chain polyunsaturated fatty acid and LCPUFA denote a fatty acid that contains at least 20 carbon atoms in a carbon chain and at least two carbon-carbon double bonds, and thus includes ODCPUFA. As used herein, the terms very long chain polyunsaturated fatty acid and ODC-PUFA mean a fatty acid that contains at least 22 carbon atoms in the carbon chain and at least three carbon-carbon double bonds. Typically, the number of carbon atoms in a fatty acid carbon chain refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes the carbon atoms of the side groups. In one embodiment, the long chain polyunsaturated fatty acid is an ω3 fatty acid, i. E. contains desaturation (double carbon-carbon bond) at the position of the third carbon-carbon bond from the methyl end of the fatty acid. In another embodiment, the long chain polyunsaturated fatty acid is an ω6 fatty acid, i. E. contains desaturation (double carbon-carbon bond) at the position of the sixth carbon-carbon bond from the methyl end of the fatty acid. In another embodiment, the long chain polyunsaturated fatty acid is selected from the group consisting of arachidonic acid (ARA, 20: 4Δ5,8,11,14; ω6), eicosatetraenoic acid (ETA, 20: 4Δ8,11,14,17, ω3), eicosapentaenoic acid (EPA, 20: 5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA, 22: 5Δ7,10,13,16,19, ω3) or docosahexaenoic acid (DHA, 22: 6Δ4,7 , 10,13,16,19, ω3). In addition, DC-PUFA can be dihomo-γ-linoleic acid (DGLA) or eicosatrienoic acid (ETRA, 20: 3Δ11,14,17, ω3). It will be obvious that the DC-PUFA obtained in accordance with the invention can be a mixture of any or all of the

- 23 037184 of the above compounds, and may contain other DC-PUFA or derivatives of any of the listed DC-PUFA. In a preferred embodiment, the ω3 fatty acids are at least DPA or DPA and DHA, or EPA, DPA and DHA, or EPA and DPA. In an embodiment, DPA is present at a level of from about 7 to 30 or 35%, and DHA is present or absent, or, if present, is present at a level of less than 2.0%, preferably less than 1.0%, more preferably less than 0.5% of the total fatty acid composition in the composition, and most preferably is absent or undetectable. This can be due to the lack of Δ4-desaturase activity in the cell. In an embodiment, the DPA level exceeds EPA, more preferably exceeds the level of each of EPA and DHA, most preferably exceeds the total level of EPA and DHA. In this embodiment, DHA may be absent or, if present, is present at a level of less than 0.5% of the total fatty acid composition.

Additionally, as used herein, the terms long-chain polyunsaturated fatty acid (LTC-PUFA) and very long-chain polyunsaturated fatty acid (LTC-PUFA) denote a fatty acid that is free (non-esterified) or esterified, for example, as part of a triglyceride ( triglyceride), diacylglyceride, monoacylglyceride, linkage with acyl-CoA (thioether) or other form of linkage. In a triglyceride, DC-PUFA or ODC-PUFA, such as DPA, may be esterified at the sn-1/3 or sn-2 positions, or the triglyceride may contain two or three acyl groups selected from the acyl groups of DC-PUFA and ODCPUFA. For example, a triglyceride can contain DPA at both the sn-1 and sn-3 positions. The fatty acid can be esterified as a phospholipid, for example, a form of phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylglyceride, phosphatidylinositol, or diphosphatidylglyceride. Therefore, DC-PUFA can be present as a mixture of forms in cell lipid or purified oil or lipid extracted from cells, tissues or organisms. In preferred embodiments of the invention, an oil is disclosed containing at least 75% or at least 85% triacylglycerides, the remainder being other lipid forms, such as those mentioned above, in which at least said triacylglycerides containing DC-PUFA are present. Subsequently, the oil can be further refined or treated, for example by hydrolysis with a strong base to release free fatty acids, or transesterification, distillation, and the like.

As used herein, the expression total ω6 fatty acids or total ω6 fatty acids or the like denotes the sum of all ω6 fatty acids, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as defined by the context, expressed as a percentage of the total fatty acid content. Said ω6 fatty acids include (if present) LA, GLA, DGLA, ARA, EDA, and ω6-DPA, but exclude any ω 3 fatty acids and monounsaturated fatty acids. All ω6 fatty acids present in plants, seeds, lipids or oils according to the invention are included in the class of polyunsaturated fatty acids (PUFAs).

As used herein, the expression new ω6 fatty acids or new ω6 fatty acid content or the like denotes the sum of all ω6 fatty acids, excluding LA, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as defined by the context, expressed as a percentage of total fatty acids. Such new ω6 fatty acids are fatty acids produced in cells, plants, plant parts and seeds of the invention by expression of genetic constructs (exogenous polynucleotides) introduced into cells, and include (if present) GLA, DGLA, ARA, EDA and ω6-DPA, but exclude LA and any ω3 fatty acids and monounsaturated fatty acids. Representative total ω6 fatty acids and new ω6 fatty acids are determined from the conversion of fatty acids in the sample to FAME and the results of GC analysis as disclosed in Example 1.

As used herein, the expression total ω3 fatty acids or total ω3 fatty acids or the like denotes the sum of all ω3 fatty acids, esterified and non-esterified, in an extracted lipid, oil, recombinant cell, plant part or seed, as defined by the context, expressed as a percentage of the total fatty acid content. Such ω 3 fatty acids include (if present) ALA, SDA, ETRA, ETA, EPA, DPA, and DHA, and exclude any ω6 fatty acids and monounsaturated fatty acids. All ω 3 fatty acids present in plants, seeds, lipids or oils according to the invention belong to the class of polyunsaturated fatty acids (PUFAs).

As used herein, the expression new ω3 fatty acids or new ω3 fatty acid content or the like denotes the sum of all ω 3 fatty acids, excluding ALA, esterified and non-esterified, in an extracted lipid, oil, recombinant cell, plant part or seed, as defined by the context, expressed as a percentage of the total

- 24 037184 fatty acids. Such new ω3 fatty acids are ω3 fatty acids produced in cells, plants, plant parts and seeds of the invention through the expression of genetic constructs (exogenous polynucleotides) introduced into cells, and include (if present) SDA, ETRK, ETC, EPA , DPA and DHA, but exclude ALA and any ω6 fatty acids and monounsaturated fatty acids. Representative total ω3 fatty acids and new ω3 fatty acids are determined from the conversion of fatty acids in the sample to FAME and the results of GC analysis as described in Example 1.

As will be understood by the skilled artisan, the term obtaining a plant part as a step in a method of the invention may include obtaining one or more plant parts for use in the method. Receiving a plant part involves harvesting a plant part from a plant, for example, using a combine, or purchasing a plant part or obtaining a plant part from a supplier. In another example, obtaining a plant part may be purchasing a plant from someone else who harvested a plant part.

The proteins desaturase, elongase and acyltransferase and the genes encoding them that can be used in accordance with the invention are any of those known in the art, or their homologues or derivatives. Examples of such genes and the size of the encoded proteins are shown in table. 1. Desaturase enzymes, which have been shown to participate in the biosynthesis of DC-PUFA, all belong to the group of so-called front-end desaturases. Preferred proteins or combinations of proteins are those encoded by the genetic constructs provided herein as SEQ ID NOs: 1 and 2.

As used herein, the term frontend desaturase denotes a member of a class of enzymes that introduces a double bond between a carboxyl group and an already present unsaturated part of the acyl chain of lipids, and is structurally characterized by the presence of an N-terminal cytochrome b5 domain, together with a typical fatty acid desaturase domain, which contains three highly conserved histidine boxes (Napier et al., 1997).

The activity of any of the elongases or desaturases for use in the invention can be tested by expressing the gene encoding the enzyme in a cell, such as, for example, a plant cell, or preferably in somatic embryos or transgenic plants, and determining whether the cell has an increased ability to produce DC -PUFA versus a comparable cell, embryo or plant in which the enzyme is not expressed.

In one embodiment, one or more desaturases and / or elongases for use in the invention may be isolated from microalgae, i. E. their amino acid sequence is identical to a polypeptide that can be isolated from microalgae.

Although some enzymes are specifically described herein as bifunctional, the absence of such a term does not necessarily mean that a particular enzyme has no activity other than that specifically defined.

Table 1

Cloned genes involved in the biosynthesis of DC-PUFA

Enzyme Type of organism View Access numbers Protein size (ak) Links A4 desaturase The simplest Eugiena gracilis AY278558 541 Meyer et al., 2003 Seaweed Pavlova lutherii AY332747 445 Tonon et aL, 2003 Isochrysis galbana AAV33631 433 Pereira et al., 2004b Pavlova salina AAY15136 447 Zhou et at, 2007 Traustohitry diium Thraustochytrium aureum AAN75707 AAN75708 AAN75709 AAN75710 515 Absent Thraustochytrium species ATCC21685 AAM0968 8 519 Qiuet al., 2001 A5-desaturase Mammal not Homo sapiens AF199596 444 Cho etai; 1999b Leonard et al ,, 2000b Nematode Caenorhabditis elegans AFI 1440, NM_06935 0 447 Michaelson et al. 1998b; Watts and Browse, 1999b '

- 25 037184

Mushrooms Mortierella alpina AF067654 446 Michaelson et al., 1998a; Knutzon etal, 1998 Pythium irregplare AF419297 456 Hong et al., 2002a Dictyostelivm discoideum AB022O97 467 Saito et al, 2000 Saprolegnia diclina 470 WO02081668 Diatoms Phaeodactylum tricornutum AY082392 469 Domergue et al, 2002 Seaweed Thraustochytrium species AF489588 439 Qiu etaL, 2001 Thraustochytrium aureum 439 WO02081668 Isochrysis galbana 442 WO0208I668 Moss Marchanlia polymorpha AY583465 484 Kajikawa et al, 2004 Δ6-desaturase Mammals Homo sapiens NM01340 2 444 Cho etal, 1999a; Leonard et al, 2000 Mus musculus NM_01969 nine 444 Cho etal, 1999a Nematode Caenorhabditis elegans Z70271 443 Napier etal, 1998 Plants Borago officinales U79010 448 Sayanova et al., 1997 Echium AY055117 AY055118 Garcia-Maroto et al., 2002 Primula vialii AY234127 453 Sayanova et al, 2003 Anemone leveillei AF536525 446 Whitney et al., 2003 Mosses Ceratodon purpureas AJ250735 520 Sperling et al., 2000 Marchanlia polymorpha AY583463 481 Kajikawa et al., 2004 Physcomitrella patens CAA11033 525 Girkeetal, 1998 Mushrooms Mortierella alpina AF110510 AB020032 457 Huang et al. 1999; Sakuradani et al., 1999 Pythium irregulare AF419296 459 Hong et al., 2002a Mucor circinelloides AB052086 467 NCBi * Rhizopus species AY320288 458 Zhang et aL, 2004 Saprolegnia diclina 453 WO0208I668 Diatoms Phaedactylum tricornutum AY082393 477 Domergue a al, 2002 Bacteria Synechocysiis LI 1421 359 Reddy et al., 1993 Seaweed Thraustochytrium aureum 456 WO02081668 Bifunctional Δ5 / Δ6 desaturase Fish Danio rerio AF309556 444 Hastings et al., 2001 C20 Δ8 desaturase Seaweed Euglena gracilis AFI 39720 419 Wallis and Browse, 1999 Plants Borago officinales AAG43277 446 Sperling et al, 2001 A6-elongase Nematode Caenorhabdiiis elegans NM_06928 8 288 Beaudoin et al., 2000 Mosses Physcomitrella patens AF428243 290 Zank et al., 2002 Marchanlia polymorpha AY583464 290 Kajikawa et al., 2004

- 26 037184

Mushrooms Mortierella aipina AF206662 318 Parker-Barnes et al., 2000 Seaweed Pavlova lutheri ** 501 WO 03078639 Thraustochytrium AX951565 271 WO 03093482 view Thraustochylrium * * AX214454 271 WO 0159128 PNzhkelongaza Mammals Homo sapiens AF231981 299 Leonard et al., 2000b; Leonard et al., 2002 Rattus norvegicus AB071985 299 Inagaki et al., 2002 Rattus norvegicus ** AB071986 267 Inagaki et al., 2002 Mus musculus AFI70907 279 Tvrdik et al., 2000 Mus musculus AF17O908 292 Tvrdik et al., 2000 Fish Danio rerio AF532782 291 (282) Agaba et a |. _t 2004 Danio rerio ** NMJ9953 2 266 Lo et al., 2003 Worm Caenorhabditis elegans Z68749 309 Abbott et al., 1998 Beaudoin et al., 2000 Seaweed Thraustochytrium aureum ** AX464802 272 WO 0208401-A2 Pavlova lutheri ** 320 WO 03078639 D9-elongase Seaweed Isochrysis galbana AF390174 263 Qi et al., 2002 Euglena gracilis 258 WO 08/128241 D5-elongase Seaweed Ostreococcus tauri AAV67798 300 Meyer et al., 2004 Pyramimonas cordala 268 WO 2010/057246 view Pavlova CCMP459 AAV33630 277 Pereira et al., 2004b Pavlova salina AAY15135 302 Robert et al ,, 2009 Diatoms Thalassiosira pseudonana AAV67800 358 Meyer et al., 2004 Fish Oncorhynchus mykiss CAM55862 295 WO 06/008099 Moss Marchantia polymorpha BAE71129 348 Kajikawa et al., 2006

* http://o.ncbi.nlm.nih.gov/.

** Function not proven / demonstrated.

Desaturases.

As used herein, the term desaturase refers to an enzyme that is capable of introducing a carbon-carbon double bond into the acyl group of a fatty acid substrate, which is usually in esterified form, for example, acyl-CoA esters. The acyl group can be esterified to a phospholipid, for example phosphatidylcholine (PC), or to an acyl carrier protein (BHA), or, in a preferred embodiment, to CoA. Desaturases can be broadly classified into three groups, respectively. In one embodiment, the desaturase is a front-end desaturase.

As used herein, Δ4 desaturase refers to a protein that undergoes a desaturation reaction with the introduction of a carbon-carbon double bond at the 4th carbon-carbon bond position from the carboxyl terminus of a fatty acid substrate. Δ4-desaturase is at least capable of transforming duodenum to DHA. Preferably, the Δ4 desaturase is capable of converting DPA-CoA to DHA-CoA, i. E. it is acyl-CoA desaturase. In an embodiment, Δ4 desaturase is capable of converting duplex esterified at the sn-2 position of PC to DHA-PC. Preferably, Δ4 desaturase has a higher activity against DPA-CoA than against DPA-PC. The desaturation stage with DHA formation from the duodenum is catalyzed by Δ4-desaturase in non-mammalian organisms, and the gene encoding this enzyme has been isolated from the freshwater protozoan Euglena gracilis and the marine species Thraustochytrium (Qiu et al., 2001; Meyer et al., 2003). In one embodiment, the Δ4 desaturase comprises an amino acid sequence as set forth in SEQ ID NO: 28 or a Thraustochytrium Δ4 desaturase, a biologically active fragment thereof, or an amino acid sequence at least 80% identical to SEQ ID NO: 28. In an embodiment implementations of a plant, a part of a plant (such as a seed) or a cell according to the invention or used according to the invention, which produce high levels of DPA, for example, DPA is from 5 to 35% of the total content of extracted fatty acids, do not contain the gene encoding functional Δ4 desaturase.

As used herein, Δ5-desaturase refers to a protein that performs a desaturation reaction by introducing a carbon-carbon double bond at the 5th carbon-carbon bond position from the carboxyl terminus of the fatty acid substrate. In an embodiment, the fatty acid substrate is ETA and the enzyme produces EPA. Preferably, the Δ5 desaturase is capable of transforming ETA-CoA to EPA-CoA, i. E. is acyl-CoA desaturase. In an embodiment, Δ5-desaturase is capable of transforming ETC esterified at the sn-2 position of PC. Prefer

- 27 037184 Thus, Δ5-desaturase has a higher activity against ETC-CoA than against ETC-PC. Examples of Δ5-desaturases are given in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a) and also in tab. 1 of this document. In one embodiment, the amino acid sequence of the Δ5 desaturase is as set forth in SEQ ID NO: 20, a biologically active fragment thereof, or an amino acid sequence at least 80% identical to SEQ ID NO: 20. In another embodiment, the amino acid sequence of the Δ5 desaturase is as set forth in SEQ ID NO: 22, a biologically active fragment thereof, or an amino acid sequence that is at least 53% identical to SEQ ID NO: 22. In another embodiment, the Δ5 desaturase is derived from the species Thraustochytrium or Emiliania huxleyi.

As used herein, Δ6-desaturase refers to a protein that performs a desaturation reaction by introducing a carbon-carbon double bond at the 6th carbon-carbon bond position from the carboxyl terminus of the fatty acid substrate. In an embodiment, the fatty acid substrate is ALA and the enzyme produces SDA. Preferably, the Δ6 desaturase is capable of converting ALA-CoA to SDK-CoA, i. E. is acyl-CoA desaturase. In an embodiment, Δ6 desaturase is capable of converting ALA esterified at the sn-2 position of PC. Preferably, Δ6 desaturase has a higher activity against ALA-CoA than against ALCP. In addition, Δ6-desaturase can have a Δ5-desaturase activity, and at the same time be called a Δ5 / Δ6 bifunctional desaturase, provided that it has a higher Δ6 desaturase activity against ALA than the activity of Δ5-desamurase against ETA. Examples of Δ6 desaturases are given in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a), as well as in tab. 1 of this document. Preferred Δ6 desaturases are derived from Micromonas pusilla, Pythium irregulare or Ostreococcus taurii.

In an embodiment, Δ6-desaturase is further characterized by the presence of at least two, preferably all three, and preferably in a plant cell, of the following: i) a higher activity of Δ6-desaturase towards α-linolenic acid (ALA, 18: 3Δ9,12,15 , ω3) than in relation to linoleic acid (LC, 18: 2Δ9,12, ω6) as a fatty acid substrate; ii) a higher activity of Δ6-desaturase in relation to ALA-CoA as a fatty acid substrate than in relation to ALA attached at position sn-2 of PC, as a fatty acid substrate; and iii) the activity of Δ8-desaturase against ETRK. Examples of such Δ6-desaturases are given in table. 2.

In an embodiment, Δ6-desaturase has a higher activity against the ω3 substrate than against the corresponding ω6 substrate, and has activity against ALA to form octadecatetraenoic acid (stearidonic acid, SDA, 18: 4Δ6,9,12,15, ω3) , with an efficiency of at least 30%, more preferably at least 40%, or most preferably at least 50%, when expressed from an exogenous polynucleotide in a recombinant cell, such as a plant cell, or at least 35% when expressed in a yeast cell ... In one embodiment, Δ6-desaturase has a higher activity, for example, at least about 2 times the activity of Δ6-desaturase for ALA than for LA as a fatty acid substrate. In another embodiment, Δ6 desaturase has a higher activity, for example, at least about 5 times the activity of Δ6 desaturase, or at least 10 times higher activity against ALA-CoA as a fatty acid substrate than against ALA. attached at the sn-2 position of PC as a fatty acid substrate. In another embodiment, Δ6 desaturase is active against both ALA-CoA and ALA fatty acid substrates attached at the sn-2 position of PC.

table 2

Desaturases that have been shown to be active on the acyl-CoA substrate

Enzyme Type of organism View Access numbers Protein size (ak) Links Db desaturase Seaweed Mantoniella squamata CAQ30479 449 Hoffinann et al., 2008 Ostreococcus tauri AAW7O159 456 Doipergue et al., 2005 Micromonas pusilla EEH58637 Petrie et al., 2010a (SEQ ID

- 28 037184

NO: 13) A5-desaturase Seaweed Mantoniella squamata CAQ30478 482 Hoffmann et al., 2008 Plant Anemone leveillei Is absent Sayanova et al., 2007 ω3-desaturase Mushrooms Pythium aphanidermatum FW362186.1 359 Xue et al., 2012; WO2008 / 054565 Mushrooms (oomycete) Phytophthora sojae FW362214.1 363 Xue et al., 2012; WO2008 / 054565 Mushrooms (oomycete) Phytophthora rumorum FW362213.1 361 Xue et a!., 2012; WO2008 / 054565

In one embodiment, Δ6-desaturase has no detectable Δ5-desaturase activity against ETA. In another embodiment, the amino acid sequence of the Δ6 desaturase is represented by SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 13, a biologically active fragment thereof, or an amino acid sequence at least 77% identical to SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 13. In another embodiment, the amino acid sequence of the Δ6-desaturase is SEQ ID NO: 12 or SEQ ID NO: 13, a biologically active fragment or amino acid sequence thereof that is at least 67% identical to one or both of SEQ ID NO: 12 or SEQ ID NO: 13. In addition, Δ6 desaturase may have Δ8 desaturase activity.

As used herein, Δ8 desaturase refers to a protein that performs a desaturation reaction by introducing a carbon-carbon double bond at the 8th carbon-carbon bond position from the carboxyl terminus of the fatty acid substrate. Δ8-Desaturase is at least capable of converting ETRC to ETA. Preferably, the Δ8 desaturase is capable of converting ETRK-CoA to ETC-CoA, i. E. is acyl-CoA desaturase. In an embodiment, Δ8 desaturase is capable of converting ETRK esterified at the sn-2 position of PC. Preferably, Δ8 desaturase has a higher activity against ETRK-CoA than against ETPK-PC. In addition, Δ8 desaturase may have a Δ8 desaturase activity, and be called a Δ6 / Δ8 bifunctional desaturase, provided that it has a higher Δ8 desaturase activity for ETRK than the activity of Δ6 desaturase for ALA. Examples of Δ8-desaturases are given in table. 1. In one embodiment, the amino acid sequence of the Δ8 desaturase is as shown in SEQ ID NO: 37, a biologically active fragment thereof, or an amino acid sequence at least 80% identical to SEQ ID NO: 37.

As used herein, ω3 desaturase refers to a protein that undergoes a desaturation reaction by introducing a carbon-carbon double bond at the position of the 3rd carbon-carbon bond from the methyl end of the fatty acid substrate. Thus, ω3 desaturase can convert LA to ALA and GLA to SDA (all C18 fatty acids), or DGLA to ETA and / or ARA to EPA (C20 fatty acids). Some ω3 desaturases (group I) are active only on C18 substrates, such as plant and cyanobacterial ω3 desaturases. Such ω3 desaturases are also Δ15 desaturases. Other ω 3 desaturases are active on C20 substrates without activity (group II) or with some activity (group III) on C18 substrates. Such ω3 desaturases are also Δ 17 desaturases. Preferred ω3 desaturases are type III group which converts LA to ALA, GLA to SDA, DGLA to ETA and ARA to EPA, for example Pichia pastoris ω3 desaturase (SEQ ID NO: 6). Examples of ω3 desaturases include those described by Pereira et al. (2004a) (Saprolegnia diclina ω3-desaturase, group II), Horiguchi et al. (1998) Berberich et al. (1998) and Spychalla et al. (1997) (C. elegans ω3-desaturase, group III). In a preferred embodiment, the ω3 desaturase is fungal ω3 desaturase. In this document, fungal ω3 desaturase means ω3 desaturase, which is obtained from a fungal source, including an oomycete source, or a variant thereof with an amino acid sequence at least 95% identical to it. Genes encoding numerous ω3 desaturases have been isolated from fungal sources such as, for example, Phytophthora infestans (accession number CAJ30870, WO2005083053; SEQ ID NO: 47), Saprolegnia diclina (accession number AAR20444, Pereira et al., 2004a and US 7211656), Pythium irregulare (WO2008022963, group II; SEQ ID NO: 49), Mortierella alpina (Sakuradani et al., 2005; accession number BAD91495; WO2006019192), Thalassiosira pseudonana (Armbrust et al., 2004; accession number XP_0022912316; WO20050 NO: 48), Lachancea kluyveri (also known as Saccharomyces kluyveri; Oura et al., 2004; accession number AB118663). Xue et al. (2012) describes ω3 desaturases from oomycetes Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum, capable of efficiently converting fatty acid substrates ω6 into the corresponding ω3 fatty acids, with preference for C20 substrates, i.e. having a more pronounced activity of Δ 17-desaturase than the activity of Δ 15-desaturase. These enzymes do not possess the activity of Δ12 desaturase, but are capable of using fatty acids in the acyl-CoA and phospholipid fraction as substrates.

In a more preferred embodiment, the fungal ω3 desaturase is Pichia pastoris ω3-29 037184 desaturase / Δ15 desaturase (also known as Komagataella pastoris) (Zhang et al., 2008;

access number EF116884; SEQ ID NO: 6) or a polypeptide that is at least 95% identical thereto.

In an embodiment, ω3 desaturase is capable of at least one of the following transformations: ARA to EPA, DGLA to ETC, GLA to SDA, ARA to EPA and DGLA to ETC, ARA to EPA and GLA to SDK, or all three of them.

In one embodiment, ω3 desaturase has Δ 17 desaturase activity on a C20 fatty acid that contains at least three carbon-carbon double bonds, preferably ARA. In another embodiment, ω3 desaturase has Δ 15 desaturase activity on a C18 fatty acid that contains three carbon-carbon double bonds, preferably GLA. Preferably both activities are present.

As used herein, Δ12-desaturase refers to a protein that performs a desaturation reaction with the introduction of a carbon-carbon double bond at the 12th carbon-carbon bond position from the carboxyl terminus of the fatty acid substrate. Δ12-Desaturases usually convert oleoylphosphatidylcholine or oleoyl-CoA to linoleoylphosphatidylcholine (18: 1-PC) or linoleoyl-CoA (18: 1KoA), respectively. The subclass using a substrate associated with PC is called phospholipid-dependent Δ12 desaturases, the second subclass is called acyl-CoA-dependent Δ12 desaturases. Plant and fungal Δ12 desaturases generally belong to the first subclass, while animal Δ12 desaturases belong to the second subclass, for example, Δ12 desaturases, encoded by genes cloned from insects by Zhou et al. (2008). Many other Δ12 desaturase sequences can be easily identified by searching the sequence databases.

As used herein, Δ15 desaturase refers to a protein that performs a desaturation reaction by introducing a carbon-carbon double bond at the 15th carbon-carbon bond position from the carboxyl terminus of the fatty acid substrate. Numerous genes encoding Δ15 desaturases have been cloned from plant and fungal species. For example, US5952544 discloses nucleic acids encoding plant Δ15 desaturases (FAD3). These enzymes contain amino acid motifs that were characteristic of plant Δ15-desaturases. WO200114538 discloses a gene encoding soybean FAD3. Many other Δ 15-desaturase sequences can be easily identified by searching the sequence databases.

As used herein, Δ17 desaturase refers to a protein that performs a desaturation reaction by introducing a carbon-carbon double bond at the 17th carbon-carbon bond from the carboxyl terminus of the fatty acid substrate. Δ17-Desaturase is also regarded as ω3 desaturase if it acts on the C20 substrate, with the introduction of desaturation at the position of the ω3 bond.

In a preferred embodiment, Δ12 desaturase and / or λ15 desaturase is fungal Δ12 desaturase or fungal λ15 desaturase. In this document, fungal Δ12 desaturase or fungal Δ15 desaturase means D12 desaturase or Δ15 desaturase obtained from a fungal source, including an oomycete source, or a variant thereof, the amino acid sequence of which is at least 95% identical thereto. Genes encoding numerous desaturases have been isolated from fungal sources. US 7211656 discloses a Δ12 desaturase from Saprolegnia diclina. In WO2009016202 discloses fungal desaturases from Helobdella robusta, Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosiga brevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola, Naegleria gruben, Nectria haematococca, Nematostella vectensis, Phycomyces blakesleeanus, Trichoderma resii, Physcomitrella patens, Postia placenta, Selaginella moellendorffii and Microdochium nivale. WO2005 / 012316 discloses a Δ12 desaturase from Thalassiosira pseudonana and other fungi. WO2003 / 099216 discloses genes encoding fungal Δ12 desaturase and Δ15 desaturase isolated from Neurospora crassa, Aspergillus nidulans, Botrytis cinerea and Mortierella alpina. WO2007133425 discloses fungal Δ15 desaturases isolated from: Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans, Neurospora crassa, Fusarium graminearum, Fusarium moniliforme and Magnaporthe grisea. The preferred Δ12 desaturase has been isolated from Phytophthora sojae (Ruiz-Lopez et al., 2012).

An excellent subclass of fungal Δ12 desaturases and fungal Δ15 desaturases are bifunctional fungal Δ12 / Δ15 desaturases. The genes encoding them were cloned from Fusarium monoliforme (accession number DQ272516, Damude et al., 2006), Acanthamoeba castellanii (accession number EF017656, Sayanova et al., 2006), Perkinsus marinus (WO2007042510), Claviceps purpurea (accession number EF5368 Meesapyodsuk et al., 2007) and Coprinus cinereus (accession number AF269266, Zhang et al., 2007).

In another embodiment, ω3 desaturase has at least some activity, preferably greater activity, on the acyl-CoA substrate than on the corresponding acyl-PC substrate. As used herein, the corresponding acyl-PC substrate means phosphatidylcholine (PC) esterified with a fatty acid at the sn-2 position, in which the fatty acid is the same fatty acid as in the acyl-CoA substrate. For example, the acyl-CoA substrate may be ARA-CoA, and the corresponding acyl-PC substrate is sn-2 ARK-30 037184

FH. In an embodiment, the activity is at least twice as high. Preferably, ω3 desaturase has at least some activity on the acyl-CoA substrate and the corresponding acyl-PC substrate, and has activity on the C18 and C20 substrates. Examples of such ω3 desaturases are known among the cloned fungal desaturases listed above.

In another embodiment, ω3 desaturase comprises an amino acid sequence represented by SEQ ID NO: 6, a biologically active fragment thereof, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 12, preferably at least 90% or at least 95% identical to SEQ ID NO: 6.

In yet another embodiment, the desaturase for use in the present invention has a higher activity on the acyl-CoA substrate than the corresponding acyl PC substrate. In another embodiment, the desaturase for use in the present invention has a higher activity on the acyl-PC substrate than the corresponding acylCoA substrate, but has some activity on both substrates. As outlined above, the corresponding substrate acyl-PC is a fatty acid-charged at position sn-2 phosphatidylcholine (PC), in which the fatty acid is the same as the fatty acid in the acylCoA substrate. In an embodiment, higher activity means at least twice the activity. In an embodiment, the desaturase is Δ5 or Δ6 desaturase, or ω3 desaturase, examples of which are disclosed, without limitation, in table. 2. To test which substrate the desaturase acts on, namely acyl-CoA or acyl-PC, yeast cell assays can be performed as described in Domergue et al. (2003 and 2005). In addition, the ability of desaturase to act on the acyl-CoA substrate can be expected if the elongase, which is expressed with desaturase, exhibits an enzymatic conversion efficiency in plant cells of at least about 90% if the elongase catalyzes the elongation of the desaturase product. On this basis, Δ5-desaturase and λ4-desaturase expressed from the GA7 construct (see example 2, FIG. 2 and SEQ ID NO: 1) and their variants (example 3) are capable of desaturation of the corresponding acyl-CoA substrates, ETA- CoA and DPK-CoA.

Elongazy.

Biochemical evidence suggests that fatty acid elongation has 4 stages: condensation, reduction, dehydration, and second reduction. In the context of the present invention, elongase means a polypeptide that catalyzes the condensation step in the presence of other members of the elongation complex under suitable physiological conditions. It has been shown that heterologous or homologous expression in the cell of only the condensing component (elongase) of the protein elongation complex is required for elongation of the corresponding acyl chain. Therefore, the administered elongase can successfully recruit reducing and dehydrating activity from the transgenic host in order to effect successful acyl elongation. It is believed that the condensation component is responsible for the specificity of the elongation reaction with respect to the chain length and the degree of desaturation of fatty acid substrates. In addition, this component is believed to be the rate limiting component in the elongation reaction.

As used herein, the Δ5-elongase is at least capable of converting EPA to DPC. Examples of Δ5 elongases include those disclosed in WO2005 / 103253. In one embodiment, the Δ5-elongase has EPA activity to form DPC with an efficiency of at least 60%, more preferably at least 65%, more preferably at least 70%, or most preferably at least 80 or 90%. In a further embodiment, the amino acid sequence of the Δ5-elongase is represented by SEQ ID NO: 25, a biologically active fragment thereof, or an amino acid sequence at least 47% identical to SEQ ID NO: 25. In a further embodiment, the Δ6-elongase is derived from Ostreococcus taurii, or Ostreococcus lucimarinus (US2010 / 088776).

As used herein, the Δ6-elongase is at least capable of converting SDA to ETC. Examples of Δ6-elongases include those listed in table. 1. In one embodiment, elongase comprises an amino acid sequence as set forth in SEQ ID NO: 16, a biologically active fragment thereof (eg, a fragment disclosed as SEQ ID NO: 17), or an amino acid sequence at least 55% identical to one or both from SEQ ID NO: 16 or SEQ ID NO: 17. In an embodiment, the Δ6 elongase is obtained from Physcomitrella patens (Zank et al., 2002; accession number AF428243) or Thalassiosira pseudonana (Ruiz-Lopez et al., 2012).

As used herein, the Δ9-elongase is at least capable of converting ALA to ETrK. Examples of Δ9-elongases include those given in table. 1. In one embodiment, the amino acid sequence of the Δ9-elongase is represented by SEQ ID NO: 43, a biologically active fragment thereof, or an amino acid sequence at least 80% identical to SEQ ID NO: 43. In another embodiment, the Δ9-elongase comprises the amino acid sequence represented in SEQ ID NO: 31, a biologically active fragment thereof or an amino acid sequence that is at least

- 31 037184 81% identical to SEQ ID NO: 31. In another embodiment, the Δ9 elongase comprises the amino acid sequence shown in SEQ ID NO: 33, a biologically active fragment thereof, or an amino acid sequence at least 50% identical to SEQ ID NO : 33. In another embodiment, the amino acid sequence of the Δ9-elongase is represented by SEQ ID NO: 35, a biologically active fragment thereof, or an amino acid sequence at least 50% identical to SEQ ID NO: 35. In a further embodiment, the Δ9-elongase has more high activity in relation to the substrate ω6 than the corresponding substrate ω3, or vice versa.

As used herein, the term has a higher activity on substrate ω6 than the corresponding substrate ω3 denotes a relative enzyme activity on substrates that differs in ω3 desaturase activity. Preferably, the ω6 substrate is LA and the ω3 substrate is ALA.

Elongase with Δ6-elongase and Δ9-elongase activity is at least capable of (i) converting SDC to ETC and (ii) converting ALA to ETRK, and also has a higher Δ6-elongase activity than Δ9-elongase activity. In one embodiment, the elongase has an efficiency for converting SDA to ETC that is at least 50%, more preferably at least 60%, and / or an efficiency for converting ALA to form ETRC that is at least 6% or more preferably at least 9%. In another embodiment, the elongase has a Δ6 elongase activity at least about 6.5 times the λ9 elongase activity. In a further embodiment, elongase has no detectable Δ5-elongase activity.

Other enzymes.

Transgenes introduced into a recombinant cell, such as a microbial cell, or a transgenic plant or part thereof, may additionally encode LFAAT. As used herein, the term 1acylglyceride-3-phosphate acyltransferase (LPAT), also called lysophosphatidic acid acyltransferase or acyl-CoA-lysophosphatidate acyltransferase, denotes a protein that sn-acylates sn-1-acylglyceride-1-3-phosphatidate-P position sn-2, with the formation of phosphatidic acid (PA). Thus, the term 1-acylglyceride-3-phosphate acyltransferase activity means the acylation of (sn-1 G-3-P) at the sn-2 position to form PK (EC 2.3.1.51). Preferred PPATs are those that can use the polyunsaturated C22 acyl-CoA as a substrate to transfer the polyunsaturated C22 acyl group to the sn-2 position of the PPA to form FA. In an embodiment, the polyunsaturated C22 acyl-CoA is DPA-CoA. Examples of such LFKAT are shown in Example 6 and can be tested as disclosed herein. In an embodiment, the amino acid sequence of LPKAT useful in accordance with the invention is represented by any of SEQ ID NOs: 40-46, a biologically active fragment thereof, or an amino acid sequence at least 40% identical to any of SEQ ID NOs: 40-46. In another embodiment, the LPAAT does not contain the amino acid sequence depicted in any of SEQ ID NO: 44. In a preferred embodiment, the LPAAT useful according to the invention, which can use a polyunsaturated fatty acyl-CoA substrate C22, preferably DPA-CoA, contains the sequence amino acids shown in any of SEQ ID NOs: 41, 42 and 44, a biologically active fragment or amino acid sequence thereof, at least 40% identical to any one or more of SEQ ID NOs: 41, 42 and 44. In a preferred embodiment An LPAAT suitable according to the invention, which can use a polyunsaturated fatty acyl-CoA substrate C22, preferably DPA-CoA, contains an amino acid sequence represented by any one of SEQ ID NO: 41 or 42, a biologically active fragment thereof or an amino acid sequence of at least 40% identical to any one or more of SEQ ID NOs: 41 and 42. In an embodiment of the invention tenia LFAAT is preferably the LFAAT Mortierella alpina, the amino acid sequence of which is given as SEQ ID NO: 44, or another LFAAT that is capable of using DPA-CoA as a substrate for transferring DPA into LPA, with the formation of a PK containing DPA at position sn-2 ...

Transgenes introduced into a recombinant cell, transgenic plant, or part of it can additionally encode DHAT. As used herein, the term diacylglyceride acyltransferase (EC 2.3.1.20; DHAT) means a protein that transfers a fatty acyl group from acyl-CoA to a diacylglyceride substrate to form triacylglyceride. Therefore, the term diacylglyceride acyltransferase activity refers to the transfer of acyl-CoA to diacylglyceride to form triacylglyceride. There are three known types of DGAT, designated DGAT1, DGAT2 and DGAT3, respectively. DHAT1 polypeptides usually contain 10 transmembrane domains, DHAT2 usually contain 2 transmembrane domains, while DHAT3 is usually soluble. Examples of DHAT1 polypeptides include those encoded by the DHAT1 genes from Aspergillus fumigatus (accession number XP_755172), Arabidopsis thaliana (CAB44774), Ricinus communis (AAR11479), Vernicia fordii (ABC94472), Vernonia galonym2. Caenorhabditis elegans (AAF82410), Rattus norvegicus (NP_445889), Homo sapiens (NP_036211), as well as their variants and / or mu

- 32 037184 tants. Examples of DG AT2 polypeptides include those encoded by DG AT2 genes from Arabidopsis thaliana (accession number NP_566952), Ricinus communis (AAY16324), Vernicia fordii (ABC94474), Mortierella ramanniana (AAK84179), Homo sapiens, Q96Turus taurus ), Mus musculus (AAK84175), Micromonas CCMP1545, as well as their variants and / or mutants. Examples of DGAT3 polypeptides include those encoded by the DGAT3 genes from peanuts (Arachis hypogaea, Saha, et al., 2006), as well as variants and / or mutants thereof.

Polypeptides / peptides.

The terms polypeptide and protein are generally used interchangeably.

A polypeptide or class of polypeptides can be determined by the degree of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by a greater% identity of one reference amino acid sequence than another. The% polypeptide identity to the reference amino acid sequence is usually determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with gap opening penalty = 5 and gap extension penalty = 0.3. The query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a stretch of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length and the GAP analysis aligns the two sequences over a stretch of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a stretch of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a stretch of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their full length. A polypeptide or class of polypeptides may have the same enzymatic activity or a different activity, or may not have the activity of the reference polypeptide. Preferably, the polypeptide has an enzymatic activity of at least 10, at least 50, at least 75, or at least 90% of the activity of the reference polypeptide.

A biologically active fragment is a portion of a polypeptide as defined herein that retains the defined activity of a full-length reference polypeptide, for example, having desaturase and / or elongase activity or other enzymatic activity. Biologically active fragments herein exclude a full-length polypeptide. Biologically active fragments can be part of any size as long as they retain a certain activity. Preferably, the biologically active fragment retains at least 10, at least 50, at least 75, or at least 90% of the activity of the full-length protein.

With respect to a particular polypeptide or enzyme, it should be understood that% identity greater than those disclosed herein includes preferred variants. Thus, if appropriate, in light of the minimum% identity, it is preferred that the polypeptide / enzyme contains an amino acid sequence that is at least 60, more preferably at least 65, more preferably at least 70, more preferably at least 75, more preferably at least 76, more preferably at least 80, more preferably at least 85, more preferably at least 90, more preferably at least 91, more preferably at least 92 , more preferably at least 93, more preferably at least 94, more preferably at least 95, more preferably at least 96, more preferably at least 97, more preferably at least 98, more preferably at least 99, more preferably at least 99.1, more preferably at least 99.2, more n preferably at least 99.3, more preferably at least 99.4, more preferably at least 99.5, more preferably at least 99.6, more preferably at least 99.7, more preferably at least 99.8% and even more preferably at least 99.9% identical to SEQ ID NO.

Amino acid sequence variants / mutants of the polypeptides disclosed herein can be prepared by introducing suitable nucleotide modifications into the nucleic acid disclosed herein or by in vitro synthesis of the target polypeptide. Such variants / mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. The combination of deletion, insertion and substitution can be carried out in such a way as to obtain the final construct, provided that the final peptide product has the desired enzymatic activity.

Mutant (modified) peptides can be obtained using any technique known in the art. For example, a polynucleotide disclosed herein can be processed by in vitro mutagenesis or DNA shuffling techniques, as detailed by Harayama (1998).

- 33 037184

Screening of products derived from altered / modified DNA can be easily performed using the methods disclosed herein to determine whether they have, for example, desaturase or elongase activity.

When constructing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the characteristic (IC) that is being modified. The sites for mutation can be modified individually or serially, for example, by: (1) replacing initially selected conservative amino acids, and then more radical substitutions, depending on the results achieved, (2) removing the target residue, or (3) inserting other residues, adjacent to the hosted site.

Deletions in the amino acid sequence generally range from about 1 to 15 residues, more preferably from about 1 to 10 residues, and usually from about 1 to 5 contiguous residues.

In substitution mutants, at least one amino acid residue in the polypeptide molecule has been removed and another residue inserted in its place. The most interesting positions for mutagenesis by substitution include sites that are not conserved in natural desaturases or elongases. These sites are preferably replaced in a relatively conservative manner in order to maintain enzyme activity. Such conservative substitutions are illustrated in table. 3 under the heading examples of substitutions.

In a preferred embodiment, the mutant / variant polypeptide contains only or no more than one or two or three or four conservative amino acid modifications as compared to the naturally occurring polypeptide. Details of conservative amino acid modifications are given in table. 3. As will be understood by the skilled artisan, such minor modifications can reasonably be predicted as not affecting the activity of the polypeptide when expressed in a recombinant cell.

Table 3

Examples of amino acid substitutions

Original residue Examples of substitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gin; his Asp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pto, ala His (H) asn; gin Pe (I) leu; val; ala Leu (L) ile; val; met; ala; phe Lys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S) thr Thr (T) ser Tip (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe; ala

Polynucleotides.

In addition, the invention provides the use of polynucleotides, which can be, for example, a gene, an isolated polynucleotide, a chimeric genetic construct such as a T-DNA molecule or a chimeric DNA. It can be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, as well as combined with carbohydrate, lipids, protein or other materials to carry out the specific activity disclosed herein. The term polynucleotide is used herein interchangeably with the term nucleic acid molecule.

In an embodiment, the polynucleotide is unnatural. Examples of unnatural polynucleotides include, but are not limited to, mutated (e.g., using the methods described herein) and polynucleotides in which an open reading frame encoding a protein

- 34 037184 is operably linked to a promoter to which it is not naturally associated (such as in the constructs described herein).

In this document, the term gene is used in the broadest context and includes deoxyribonucleotide sequences containing a transcribed region and, in the case of translation, a protein coding region of a structural gene, including sequences located adjacent to the coding region at the 5 'and 3' ends at a distance of at least about 2000 base pairs at either end that are involved in gene expression. In this regard, a gene contains control signals, for example promoters, enhancers, termination and / or polyadenylation signals, which are naturally associated with a given gene, or heterologous control signals, in which case the gene is called a chimeric gene. Sequences that are located at the 5 'end of the protein coding region and that are present on the mRNA are referred to as 5' untranslated sequences. Sequences that are located 3 'or downstream of the protein coding region and that are present on the mRNA are referred to as 3' untranslated sequences. The term gene includes cDNA and genomic forms of a gene. The genomic form or clone of a gene contains a coding region that can be interrupted by non-coding sequences called introns or intermediate regions or intermediate sequences. Introns are segments of a gene that are transcribed into nuclear RNA (heterogeneous nuclear RNA, hnRNA). Introns can contain regulatory elements such as enhancers. Introns are removed or spliced into a nuclear or primary transcript; thus, introns are absent in the messenger RNA (mRNA) transcript. The function of mRNA during translation is to specify the sequence or order of amino acids in the nascent polypeptide. The term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins disclosed herein and a complementary nucleotide sequence to any of the above.

As used herein, chimeric DNA or chimeric genetic construct or similar expression means any DNA molecule that is not a naturally occurring DNA molecule in its natural location, also referred to herein as a DNA construct. Typically, a chimeric DNA or chimeric gene contains regulatory and transcribed or protein-coding sequences that are not found to be operably linked to each other in nature, i. E. which are heterologous in relation to each other. Accordingly, a chimeric DNA or chimeric gene may contain regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from a single source, but organized differently from those found in nature.

Endogenous gene refers to a naturally occurring gene in its natural position in the genome of an organism. As used herein, a recombinant nucleic acid molecule, a recombinant polynucleotide or variations thereof means a nucleic acid molecule that is constructed or modified using recombinant DNA technology, The terms foreign polynucleotide or exogenous polynucleotide or heterologous polynucleotide and the like mean any nucleic acid introduced into a cell using experimental manipulations. Foreign or exogenous genes can be genes inserted into a non-natural organism, natural genes that are introduced at a new location within a natural host, or chimeric genes. A transgene is a gene that has been introduced into the genome by a transformation procedure. The terms genetically modified, transgenic and their variations include the introduction of genes into cells by transformation or transduction, mutation of genes in cells and modification or modulation of gene regulation in the cell or organisms with which these actions were performed, or their progeny. A genomic region herein refers to a position within a genome at which a transgene or group of transgenes (also referred to herein as a cluster) is inserted into a cell or its ancestor. Such regions include only nucleotides that are introduced by human intervention, for example, by the methods disclosed herein.

The term exogenous in the context of a polynucleotide means a polynucleotide present in a cell in an altered amount from its natural state. In one embodiment, the cell is a cell that does not naturally contain a polynucleotide. However, the cell can be a cell that contains a non-endogenous polynucleotide, which results in a change in the level of production of the encoded polypeptide. An exogenous polynucleotide includes polynucleotides that are not separated from other components of a transgenic (recombinant) cell or noncellular expression system in which it is present, and polynucleotides produced in such cells or noncellular systems that have subsequently been purified from at least some other components ... An exogenous polynucleotide (nucleic acid) can be a contiguous chain of nucleotides found in nature, or contain two or more contiguous nucleotide regions from different sources (natural and / or synthetic), connected in such a way as to form a single polynucleotide. Typically, such chimeric polynucleotides contain at least an open reading frame encoding a polypeptide operably linked to a promoter suitable for directing transcription of the open reading frame in a target cell.

With respect to the disclosed polynucleotides, it should be understood that higher% identity than those disclosed above will include preferred variants. Thus, if appropriate, in light of the minimum% identity, the polynucleotide preferably contains a polynucleotide sequence that is at least 60, more preferably at least 65, more preferably at least 70, more preferably at least 75, more preferably at least 80, more preferably at least 85, more preferably at least 90, more preferably at least 91, more preferably at least 92, more preferably at least 93, more preferably at at least 94, more preferably at least 95, more preferably at least 96, more preferably at least 97, more preferably at least 98, more preferably at least 99, more preferably at least by 99.1, more preferably at least 99.2, more preferably at least 99.3, more preferably at least 99.4, more preferably at least 99.5, more preferably at least 99.6, more preferably at least 99.7, more preferably at least 99.8 and even more preferably at least 99.9% identical to the corresponding SEQ ID NO.

Polynucleotides may contain, as compared to naturally occurring molecules, one or more mutations, which are deletions, insertions or substitutions of nucleotide residues. Polynucleotides that contain mutations relative to the reference sequence can be natural (i.e., isolated from a natural source) or synthetic (e.g., obtained by site-directed mutagenesis or DNA shuffling from a nucleic acid, as disclosed above). Thus, it is obvious that the polynucleotides can be obtained from a natural source or can be recombinant. Preferred polynucleotides are those containing coding regions that are codon-optimized for translation in plant cells, as is known in the art.

Recombinant vectors.

Recombinant expression can be used to produce recombinant cells or plants or plant parts of the invention. Recombinant vectors contain heterologous polynucleotide sequences, i.e. polynucleotide sequences that are not naturally found contiguous to the polynucleotide molecules disclosed herein, which are preferably derived from species other than those from which the polynucleotide molecule (s) are derived. The vector can be RNA or DNA, and is usually a plasmid. Plasmid vectors usually contain additional nucleic acid sequences that allow for ease of selection, amplification and transformation of an expression cassette in prokaryotic cells, for example, pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, or preferably binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include sources of replication to allow autonomous replication of the vector, breeding marker genes preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing multiple insertion sites for nucleic acid sequences or genes encoded in a nucleic acid construct, and sequences that increase the activity of transformation of prokaryotic and eukaryotic (especially plant) cells. A recombinant vector may contain more than one polynucleotide disclosed herein, such as three, four, five, or six polynucleotides disclosed herein, in combination, preferably a chimeric genetic construct described herein, wherein each polynucleotide is operably linked to control sequences expressions that are active in the target cell. Preferably, the expression control of the sequences includes or is all of the heterologous promoters, i. E. heterologous with respect to the coding regions that they control. More than one polynucleotide disclosed herein, for example 3, 4, 5 or 6 polynucleotides, preferably 7 or 8 polynucleotides, each of which encodes a different polypeptide, preferably covalently linked in a single recombinant vector, preferably within a single T-DNA molecule, which can then be introduced as a single molecule into the cell to obtain a recombinant cell according to the invention, and is preferably integrated into the genome of the recombinant cell, for example, in a transgenic plant. Integration into the genome can be carried out in the nuclear genome or in the plastid genome of a transgenic plant. Thus, polynucleotides linked in this way will be inherited together as a single genetic locus in the offspring of the recombinant cell or plant. A recombinant vector or plant may contain two or more such recombinant vectors, each of which contains several polynucleotides, for example, each recombinant vector contains 3, 4, 5, or 6 polynucleotides.

Functionally linked in this document denotes a functional relationship

- 36 037184 between two or more segments of nucleic acid (eg DNA). This usually means a functional relationship between a transcriptional regulatory element (promoter) and a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide disclosed herein, if it stimulates or modulates the transcription of the coding sequence in a suitable cell. In general, transcriptional promoter regulatory elements that are operably linked to the transcribed sequence are physically adjacent to the transcribed sequence, i. E. they are cis-operable. However, some transcriptional regulatory elements, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequences for which they increase transcription.

If multiple promoters are present, each promoter can independently be the same or different. Preferably, at least 3 and up to a maximum of 6 different promoter sequences are used in the recombinant vector to direct the expression of exogenous polynucleotides.

In addition, recombinant molecules, such as chimeric DNA or genetic constructs, may contain: (a) one or more secretory signals encoding signal peptide sequences to allow an expressed polypeptide disclosed herein to be secreted from a cell that produces the polypeptide, or that provides localization of the expressed polypeptide, for example, for retention of the polypeptide in the endoplasmic reticulum (ER) of the cell or transfer to the plastid, and / or (b) fusion sequences that result in the expression of nucleic acid molecules as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion or localization of a polypeptide disclosed herein. Additionally, recombinant molecules can contain intermediate and / or untranslated sequences around and / or within the nucleic acid sequences of the nucleic acid molecules disclosed herein.

To facilitate the identification of transformants, it is desirable that the nucleic acid construct contains a selection or screening marker gene as, or in addition to, a foreign or exogenous polynucleotide. Marker gene refers to a gene that confers a distinct phenotype on cells expressing the marker gene, such that the transformed cells are different from cells lacking the marker. The breeding marker gene provides a trait that can be selected based on resistance to the breeding agent (eg, herbicide, antibiotic, radiation, heat, or other treatment that damages untransformed cells). A screenable marker gene (or reporter gene) provides a trait that can be identified by observation or testing, for example, by screening (for example, β-glucuronidase, luciferase, green fluorescent protein (GFP), or other enzyme activity not present in untransformed cells ). The marker gene and target nucleotide sequence must not be linked. In fact, the choice of marker is not critical as long as it is functional (ie, selective) in combination with cells of choice, such as a plant cell.

Examples of breeding markers are markers that confer antibiotic resistance, for example ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Examples of breeding markers for breeding transformant plants include, but are not limited to, the hyg gene, which encodes hygromycin B resistance; gene neomycin phosphotransferase (nptII), providing resistance to kanamycin, paromomycin, G418; a rat liver glutathione S-transferase gene conferring resistance to glutathione-derived herbicides such as those disclosed in EP 256223; a glutamine synthetase gene, when overexpressed, confers resistance to glutamine synthetase inhibitors such as phosphinothricin, for example, disclosed in WO 87/05327, an acetyltransferase gene from Streptomyces viridochromogenes, conferring resistance to the selection agent phosphinothricin, for example, cod. enolshikimate-3-phosphate synthase (ESPS), which mediates the tolerability of N-phosphonomethylglycine, for example, disclosed by Hinchee et al. (1988), or preferably a bar gene conferring bialaphos resistance, for example disclosed in WO91 / 02071.

Preferably, the nucleic acid construct is stably incorporated into the genome of a cell, for example a plant cell. Accordingly, the nucleic acid may contain suitable elements that allow the molecule to be incorporated into the genome, preferably the sequence of the right and left boundaries of the T-DNA molecule, or the construct is placed in a suitable vector that can be incorporated into the chromosome of the cell.

Expression.

As used herein, an expression vector is a DNA vector that is capable of transforming a host cell and expressing one or more of said polynucleotide molecules. The expression vectors of the present invention can direct the expression of a gene in plant cells or in recombinant cells such as microbial cells. Expression vectors,

- 37 037184 suitable in accordance with the invention, contain regulatory sequences, for example sequences for control of transcription, sequences for translation control, sources of replication and other regulatory sequences, which are compatible with the recombinant cell and which direct the expression of polynucleotide molecules of the present invention. In particular, polynucleotides or vectors suitable in accordance with the present invention contain sequences for controlling transcription. Transcriptional control sequences are sequences that direct the initiation, elongation, and termination of transcription. Particularly important sequences for transcriptional control are those that direct the initiation of transcription, for example, promoter and enhancer sequences. Suitable transcriptional control sequences include any transcriptional control sequence that can function in at least one of the recombinant cells of the present invention. The choice of regulatory sequences used depends on the target organism, such as a plant, and / or the target organ or target tissue. Such regulatory sequences can be obtained from any eukaryotic organism, such as plants or plant viruses, or can be chemically synthesized. Many such transcriptional control sequences are known to those skilled in the art. Particularly preferred sequences for controlling transcription are promoters that actively direct transcription in plants, in a constitutive or stage and / or tissue specific manner, depending on the use of the plant or its parts.

A variety of vectors suitable for the stable transfection of plant cells or the production of transgenic plants are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors contain, for example, one or more cloned plant genes under transcriptional control of 5 'and 3' regulatory sequences and a dominant selection marker. Additionally, such expression vectors in plants may contain a promoter regulatory region (e.g., a regulatory region that drives an inducible or constitutive, environmental or developmental stage or cell or tissue specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

A number of constitutive promoters have been described that are active in plant cells. Suitable promoters for constitutive expression in plants include, but are not limited to, the 35S Cauliflower Mosaic Virus (CaMV) promoter, the 35S Pinus Mosaic Virus (FMV), and the light inducible promoter from the small subunit of ribulose 1,5-bis-phosphate carboxylase.

For the purpose of expression in the original tissues of the plant, such as leaf, seed, root or stem, the promoters used in accordance with the present invention preferably exhibit relatively high expression in these specific tissues. Many examples are well known in the art. In addition, various plant gene promoters that are regulated in response to environmental, hormonal, chemical and / or developmental signals may be used to express genes in plant cells, or organ-specific promoters may be preferred.

As used herein, the term seed-specific promoter or variations thereof means a promoter that preferentially directs gene transcription in the developing seed of a plant, preferably a Brassica, Camelina sativa or G. max plant, over other plant tissues. In an embodiment, the seed-specific promoter is expressed at least 5-fold more actively in the developing seed of the plant relative to the leaves and / or stems of the plant, and is preferably expressed more actively in the embryo of the developing seed than in other plant tissues. Preferably, the promoter only directs the expression of the target gene in the developing seed, and / or expression of the target gene in other parts of the plant, such as leaves, cannot be detected by Northern blotting and / or RT-PCR. Typically, the promoter controls gene expression during seed growth and development, in particular during the synthesis and accumulation phase of storage substances in the seed. Such promoters can direct the expression of a gene in the entire storage organ of a plant, or only in a part thereof, for example the seed coat or cotyledon (s), preferably in embryos, in dicotyledonous plant seeds, or in the endosperm or aleurone layer of monocotyledonous plant seeds.

Preferred seed-specific expression promoters include: i) promoters from genes encoding enzymes involved in fatty acid biosynthesis and accumulation in seeds, for example fatty acid desaturase and elongase, ii) promoters from genes encoding storage proteins for storage in the seed, and iii) promoters from genes encoding enzymes involved in the biosynthesis of carbohydrates and their accumulation in seeds. Suitable seed-specific promoters are the oilseed napin gene promoter (US 5608152), the Vicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO98 / 45461), the Phaseolus vulgaris phaseolia promoter (US All Brass 5504,200), the (WO91 / 13980) or the LeB4 legumin promoter from Vicia faba (Baum

- 38 037184 lein et al., 1992), and promoters that provide seed-specific expression in monocots such as maize, barley, wheat, rye, rice, and the like. As suitable, the following promoters should be mentioned: the promoter of the Ipt2 or Ipt1 gene of barley (WO95 / 15389 and WO95 / 23230) or the promoters disclosed in WO99 / 16890 (promoters from the gene of barley hordein, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, maize zein gene, oat glutelin gene, sorghum casirin gene, rye sekalin gene). Other promoters include those disclosed by Broun et al. (1998) Potenza et al. (2004), US20070192902 and US20030159173. In an embodiment, the seed-specific promoter is preferably expressed in certain portions of the seed, such as the embryo, cotyledon (s), or endosperm. Examples of such specific promoters include, but are not limited to, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000), the bean phytohemagglutinin promoter (Perrin et al., 2000), the conlinin 1 and conlinin 2 promoters. for genes encoding flax 2S storage proteins (Cheng et al., 2010), the FAE1 gene promoter from Arabidopsis thaliana, the BnGLP promoter of the Brassica napus globulin-like protein gene, and the LPXR promoter of the peroxiredoxin gene from Linum usitatissimum.

The 5 'untranslated leader sequence can be derived from a promoter selected to express a heterologous gene sequence of a polynucleotide of the present invention, or is preferably heterologous with respect to the region encoding the enzyme that is produced and can be specifically modified, if desired, in such a way as to increase translation mRNA. For a review of the optimization of transgenic expression see Koziel et al. (1996). Additionally, 5 'untranslated regions can be obtained from RNA of plant viruses (among others, tobacco mosaic virus, tobacco engraving virus, maize mosaic virus, alfalfa mosaic virus), from suitable eukaryotic genes, plant genes (the leader of the binding protein a / b chlorophyll of wheat and maize), or from a synthetic gene sequence. The present invention is not limited to constructs in which the untranslated region is derived from the 5 'untranslated sequence accompanying the promoter sequence. Additionally, the leader sequence can be derived from an unrelated promoter or coding sequence. Leader sequences useful in the context of the present invention include the maize Hsp70 leader (US 5362865 and US 5859347) and the tobacco mosaic virus (TMV) omega element.

Termination of transcription is provided by a 3 'untranslated DNA sequence operably linked in the chimeric vector to the target polynucleotide. The 3 'untranslated region of the recombinant DNA molecule contains a polyadenylation signal that functions in plants to allow the addition of adenylate nucleotides to the 3' end of RNA. 3 'Untranslated regions can be obtained from various genes that are expressed in plant cells. A 3 'untranslated region of nopaline synthetase, 3' an untranslated region from a small subunit of the Rubisco gene of pea, 3 'an untranslated region from a soybean 7S storage protein gene or a flax conlinin gene are commonly used in this case.

3 'Transcribed, untranslated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Recombinant DNA technologies can be used to enhance expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of a polynucleotide molecule within a host cell, the efficiency with which such polynucleotide molecules are transcribed, the efficiency with which the resulting copies are translated, and the efficiency of post-translational modifications. Recombinant methods suitable for increasing the expression of polynucleotide molecules disclosed herein include, without limitation, integrating a polynucleotide molecule into one or more chromosomes of a host cell, introducing stabilizing sequences into mRNA, replacing or modifying transcription control signals (e.g., promoters, operators , enhancers), substitutions or modifications of translation control signals (e.g., ribosome binding sites, Schein-Dalgarno sequences), modification of polynucleotide molecules to match codon usage in the host cell, and deletion of sequences that destabilize transcripts.

Transgenic plants.

The term plant as a noun herein denotes whole plants, but when used adjectively (vegetable) denotes any substance in which it is present, derived from, derived from, or associated with a plant, for example plant organs (such as leaves, stems, roots , flowers), single cells (such as pollen), seeds, plant cells, etc. The term plant part refers to all parts of a plant that contain plant DNA, including vegetative structures such as leaves or stems, roots, flower organs or structures, pollen, seed, parts of a seed such as an embryo, endosperm, germ plate or seed coat, plant tissue such as vascular tissue, cells and their progeny, as long as the plant part synthesizes the lipid of the invention.

A transgenic plant, a genetically modified plant or a variation thereof means a plant that contains a genetic construct (transgene) not found in a wild-type plant of the same species, variety or variety. Transgenic Plants in the Context of the Present Invention

39 037184 include plants and their progeny that have been genetically modified using recombinant techniques to produce a lipid or at least one polypeptide disclosed herein in a target plant or plant organ. Transgenic plant cells and transgenic plant parts have a corresponding meaning. A transgene as used herein has its ordinary meaning in the art of biotechnology and contains a genetic sequence that has been obtained or modified by DNA or RNA recombination technology and has been introduced into a plant cell. The transgene may contain genetic sequences derived from a plant cell, which may be of the same species, variety or variety as the plant cell into which the transgene is introduced, or a different species, variety or variety, or from a non-plant cell. Typically, the transgene is introduced into a cell, for example a plant, by human manipulation, such as transformation, and any method at the discretion of a person skilled in the art can be used.

The terms seed and grain are used interchangeably in this document. A grain denotes a mature seed, such as a seed harvested during harvest, or a seed that is still on the plant but ready to be harvested, and may also denote a seed after it has swollen or germinated according to the context. A mature seed or seed usually contains less than about 18-20% moisture, preferably less than 10% moisture. The moisture content of a Brassica seed, such as a mature rapeseed, is typically about 4-8% or 6-8%, preferably about 4 to about 6%. A developing seed herein means a seed before maturity, usually found in the reproductive structures of a plant after fertilization or flowering, but may also mean such pre-maturity seeds that are isolated from the plant.

As used herein, the term obtaining a plant part or obtaining a seed means any means for obtaining a plant part or seed, respectively, including harvesting plant parts or seeds from plants in a field or enclosed space such as a greenhouse or growth chamber, or purchasing or obtaining plant parts or seeds from the supplier. Typical growing conditions in a greenhouse include daytime temperatures of 22-24 ° C and night temperatures of 16-18 ° C with natural sunlight. The seed may be suitable for planting, i.e. capable of germinating and forming a progeny plant, or, alternatively, treated in such a way that it can no longer germinate, for example, chopped, ground or ground seed, which is suitable for use in food preparation or nutrition, or for lipid extraction according to the invention.

As used herein, the term plant storage organ means a part of a plant specialized in storing energy in the form of, for example, proteins, carbohydrates, fatty acids and / or oils. Examples of plant storage organs are seed, fruit, tuberous roots and tubers. The preferred storage organ of the plant is the seed.

Plants or plant parts according to the invention or used according to the invention are preferably phenotypically normal. As used herein, the term phenotypically normal means a genetically modified plant or plant organ, especially a storage organ such as a seed, tuber or fruit, which does not have a substantially reduced ability to grow and reproduce as compared to an unmodified plant or plant organ. In an embodiment, a genetically modified plant or plant organ that is phenotypically normal has the ability to grow or reproduce substantially the same as an isogenic plant or organ that does not contain exogenous polynucleotide (s). Preferably, the biomass, growth rate, germination rate, storage organ size, pollen viability, fertility of male and female plants, seed size and / or the number of viable seeds formed is at least 90% of that of a plant that does not contain the specified exogenous polynucleotide, when grown in identical conditions. Preferably, the viability of the pollen of a plant of the invention or of plants obtained from a seed of the invention is about 100% relative to that of the corresponding wild-type plant. This term does not cover traits of a plant that may differ from a wild-type plant, but which do not adversely affect the usefulness of the plant from a commercial point of view, for example, the phenotype of the sapling leaf ballerina.

Plants disclosed or included for use in the practice of the present invention include monocotyledonous plants and dicotyledonous plants. In preferred embodiments, the plants of the present invention are crops (eg, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or peas) or other legumes. Plants can be grown to produce edible roots, tubers, leaves, stems, flowers, or fruit. Plants can be vegetable or ornamental plants. Plants according to the invention or suitable according to the invention may be corn (Zea mays), rapeseed (Brassica napus, subspecies Brassica rapa), mustard (Brassica juncea), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa ), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybeans (Glycine max), tobacco (Nicotiana tabacum), potatoes (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),

- 40 037184 sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea species), coconut (Cocos nucifera), pineapple (Anana comosus), citrus tree (Citrus species), cocoa (Theobroma cacao), tea ( Camellia senensis), banana (Musa species), avocado (Persea americana), fig tree (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occ ), macadamia (Macadamia intergrifolia), almonds (Prunus amygdalus), sugar beets (Beta vulgaris), oats or barley.

In a preferred embodiment, the plant is an angiosperm plant.

In an embodiment, the plant is an oilseed plant, preferably an oilseed crop. As used herein, an oilseed plant is a plant species used for the commercial production of oils from plant seeds. The oilseed plant can be oilseed rape (eg rapeseed), maize, sunflower, soy, sorghum, flax (flaxseed), or sugar beet. In addition, the oilseed plant may be other Brassica, cotton, peanuts, poppy seeds, mustard, castor bean, sesame, sunflower, safflower, Camelina, Crambe, or a nut-producing plant. The plant can produce high levels of oil in the fruit, such as olive, oil palm, or coconut. Garden plants to which the present invention can be applied are lettuce, endive or cruciferous vegetables, including cabbage, broccoli or cauliflower. The present invention can be applied to tobacco, pumpkin, carrot, strawberry, tomato or pepper.

In yet another preferred embodiment, the non-transgenic plant used to produce the transgenic plant of the invention produces an oil, especially in the seed, that contains: i) less than 20, less than 10, or less than 5% 18: 2 fatty acids and / or ii) less than 10 or less than 5% fatty acids 18: 3.

In a preferred embodiment, the transgenic plant, or part of it, is homozygous for all and every gene (exogenous polynucleotide) that has been introduced (transgene), such that its offspring are not segregated for the desired phenotype. In addition, the transgenic plant may be heterozygous for the introduced transgene (s), preferably uniformly heterozygous for the transgene, for example in the F1 progeny that is grown from hybrid seed. Such plants can provide benefits such as hybrid vigor, well known in the art, or can be used in breeding or backcrossing plants.

If appropriate, the transgenic plant or part of it may contain additional transgenes encoding enzymes that are involved in the production of DC-FFA, such as, without limitation, Δ6-desaturase, Δ9-elongase, Δ8-desaturase, Δ6-elongase, Δ5- desaturase, ω3-desaturase, Δ5-elongase, diacylglyceride acyltransferase, LPKAT, Δ 17-desaturase, Δ15-desaturase and / or Δ12-desaturase. Examples of such enzymes with one or more of these activities are known in the art and include those disclosed herein. In specific examples, the transgenic plant at least contains a set of exogenous polynucleotides encoding:

a) Δ5-desaturase, Δ6-desaturase, Δ5-elongase and Δ6-elongase,

b) Δ5-desaturase, Δ8-desaturase, Δ5-elongase and Δ9-elongase,

c) Δ5-desaturase, Δ6-desaturase, Δ5-elongase, Δ6-elongase and Δ15-desaturase,

d) Δ5-desaturase, Δ8-desaturase, Δ5-elongase, Δ9-elongase and Δ15-desaturase,

e) Δ5-desaturase, Δ6-desaturase, Δ5-elongase, Δ6-elongase and Δ17-desaturase,

f) Δ5-desaturase, Δ8-desaturase, Δ5-elongase, Δ9-elongase and Δ17-desaturase,

g) ω3-desaturase or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

h) ω3-desaturase or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase,

i) Δ12-desaturase, ω3-desaturase or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

j) Δ12-desaturase, ω3-desaturase or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase,

j) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), ω3-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

k) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

l) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ12-desaturase, Δ6-desaturase, Δ5desaturase, Δ6-elongase and Δ5-elongase,

m) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ12-desaturase, ω3-desaturase and / or Δ15desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase,

o) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), ω3-desaturase, Δ8-desaturase, Δ5desaturase, Δ9-elongase and Δ5-elongase,

n) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ15-desaturase, Δ8-desaturase, Δ5- 41 037184 desaturase, Δ9-elongase and Δ5-elongase,

p) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ12-desaturase, Δ8-desaturase, Δ5desaturase, Δ9-elongase and Δ5-elongase, or

c) 1-acylglyceride-3-phosphate acyltransferase (LFAAT), Δ12-desaturase, ω3-desaturase and / or Δ15desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase.

In an embodiment, the exogenous polynucleotides encode a set of polypeptides that are Pythium irregulare Δ6 desaturase, Thraustochytrid Δ5 desaturase or Emiliana huxleyi Δ5 desaturase, Physcomitrella patens Δ6 elongase, Phraustochytrella patens Δ5 elongase, Thraustochytrid Δ5 elongase, or Phythostra O5 elongase, or O5 elongase. infestans or ω3-desaturase Pythium irregulare.

In an embodiment, the plants according to the invention or used according to the invention are grown in the field, preferably as a population of at least 1,000, 1,000,000 or 2,000,000 plants that are substantially the same, or on an area of at least 1 or 2 hectares. Planting density varies according to plant species, plant variety, climate, soil conditions, fertilization rates and other factors, as is known in the art. For example, rapeseed is usually grown at a planting density of 1.2-1.5 million plants per hectare. Plants are harvested as is known in the art, which may involve swathing, row composting and / or harvesting the fruit of the plants, followed by threshing and / or blowing the plant material to separate the seed from the rest of the plant, often in the form of chaff. Alternatively, the seed crop can be harvested from the plants in the field in a single process, namely combining.

Plant transformation.

Transgenic plants can be obtained using methods known in the art, such as those generally described in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

In this document, the terms stably transforming, stably transformed and their variations mean the integration of exogenous nucleic acid molecules into the cell genome in such a way that they are passed on to progeny cells during cell division, without the need for positive selection for their presence. Stable transformants or their progeny can be selected by any means known in the art, such as Southern blots on chromosomal DNA or in situ hybridization of genomic DNA. Preferably, the transformation of plants is carried out as described in the examples in this description.

Agrobacterium mediated transfer is a widely used system for introducing genes into plant cells because DNA can be introduced into cells in whole plant tissues or plant organs or explants in tissue culture for the purpose of transient expression or stable integration of DNA into the plant cell genome. The use of Agrobacterium mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, US 5177010, US 5104310, US 5004863 or US 5159135), including flower immersion techniques using Agrobacterium or other bacteria that can transfer DNA into plant cells. The region of DNA to be transferred is determined by the sequences of the boundary, with an intermediate DNA (T-DNA) usually inserted into the plant genome. Further, the integration of T-DNA is a relatively precise process, leading to several reorganizations. In those plant varieties in which Agrobacterium-mediated transformation is effective, it is the mode of choice due to the easy and definite nature of gene transfer. Preferred Agrobacterium transformation vectors are capable of replication in E. coli, as well as Agrobacterium, allowing for the necessary manipulations as described (Klee et al., In Plant DNA Infectious Agents, Hohn and Schell, eds., Springer-Verlag, New York, p. 179-203 (1985)).

Acceleration methods that can be used include, for example, ballistic transfection and the like. One example of a method for delivering transforming nucleic acid molecules into plant cells is ballistic transfection. This method is reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microparticles) in it can be coated with nucleic acids and delivered to cells by a driving force. Examples of particles include tungsten, gold, platinum, and the like. A particular advantage of ballistic transfection, in addition to being an effective means of reproducible transformation of monocots, is that neither protoplast isolation nor susceptibility to Agrobacterium infection is required.

In another alternative embodiment, the plastids can be stably transformed. Methods disclosed for transforming plastids in higher plants include delivering with a gene gun a DNA particle containing a selection marker and targeting the DNA to the plastid genome by homologous recombination (US 5451513, US 5545818, US 5877402, US 5932479 and WO 99/05265) ...

- 42 037184

Other methods of converting cells can also be used and include, without limitation, the introduction of DNA into plants by direct transfer of DNA into pollen, direct injection of DNA into the reproductive organs of the plant, or direct injection of DNA into cells of immature embryos, followed by rehydration of the dried embryos.

Regeneration, development and cultivation of plants from single transformants of the plant protoplast or from various transformed explants are well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)). Such a regeneration and growth process usually includes the stages of selection of transformed cells, culturing such individual cells through the usual stages of embryonic development, including the stage of rooting a seedling. Transgenic embryos and seeds are regenerated in a similar way. The resulting transgenic rooted shoots are subsequently planted in a suitable medium for plant growth, such as soil.

The development or regeneration of plants containing a foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinating to provide homozygous transgenic plants. Alternatively, pollen obtained from regenerated plants is crossed with seed-grown plants of agronomically important lines. On the other hand, pollen from such important lines is used to pollinate regenerated plants. The transgenic plant of the present invention containing the desired exogenous nucleic acid is cultured using methods well known to one of ordinary skill in the art.

To confirm the presence of transgenes in transgenic cells and plants, amplification by polymerase chain reaction (PCR) or Southern blot analysis can be performed using methods known to those of skill in the art. Transgenic expression products can be detected in any of a variety of ways, depending on the nature of the product, and include Western blotting and enzymatic analysis. Once the transgenic plants are obtained, they can be grown to obtain plant tissues or parts containing the desired phenotype. Plant tissue or plant parts can be harvested and / or seed harvested. The seed can serve as a source of growing additional plants with tissues or parts that have the desired characteristics.

A transgenic plant obtained using Agrobacterium or other transformation methods usually contains a single genetic locus on a single chromosome. Such transgenic plants may be called hemizygous for the introduced gene (s). More preferred is a transgenic plant homozygous for the introduced gene (s); those. a transgenic plant that contains two additional genes, one gene at the same locus on each chromosome of a pair of chromosomes. A homozygous transgenic plant can be obtained by self-fertilizing a hemizygous transgenic plant, germinating a portion of the resulting seeds, and analyzing the resulting plants for the presence of the target gene.

In addition, it should be understood that two different transgenic plants that contain two independently segregating exogenous genes or loci can be crossed (mated) to produce offspring that contains both sets of genes or loci. Self-fertilization of a suitable F1 offspring can produce plants homozygous for both exogenous genes or loci. Backcrossing with the mother plant and killing the non-transgenic plant is also considered vegetative breeding. Descriptions of other breeding methods that are commonly used for different traits and yields can be found in Fehr, Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Increased levels of exogenous RNA and stabilized expression.

Silence suppressors.

In an embodiment, the plant cell, plant, or plant part comprises an exogenous polynucleotide encoding a silencing suppressor protein.

Post-transcriptional gene silencing (PTSG) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs for degradation. PTSH occurs in plants or fungi that are stably or temporarily transformed by foreign (heterologous) or endogenous DNA, and leads to a decrease in the accumulation of RNA molecules, the sequence of which is similar to the introduced nucleic acid.

It has been widely studied that co-expression of the silencing suppressor with the target transgene will increase the levels of RNA present in the cell, which is transcribed from the transgene. Although this is a proven fact for cells in vitro, significant side effects have been observed in many co-expression studies in whole plants. More specifically, as described in Mallory et al. (2002), Chapman et al. (2004), Chen et al. (2004), Dunoyer et al. (2004), Zhang et al. (2006), Lewsey et al. (2007) and Meng et al. (2008) plants expressing silencing suppressors, generally under the control of constitutive promoters, are often phenotypically abnormal to the point of being unsuitable for commercial production.

- 43 037184

It has recently been found that the levels of RNA molecules can be increased and / or the levels of RNA molecules can be stabilized over multiple generations by limiting the expression of the silencing suppressor by the plant seed or part of it (WO2010 / 057246). As used herein, a silencing suppressor protein or BSS is any polypeptide that can be expressed in a plant cell that increases the level of the expression product of another transgene in the plant cell, especially in subsequent generations from the initially transformed plant. In an embodiment, BCC is a viral silencing suppressor or a mutant thereof. A large number of viral silencing suppressors are known in the art and include, but are not limited to, P19, V2, P38, Pe-Po, and RPV-P0. In an embodiment, the viral silencing suppressor comprises an amino acid sequence represented by SEQ ID NO: 38, a biologically active fragment thereof, or an amino acid sequence at least 50% identical to SEQ ID NO: 38, which further has a silencing suppressor activity.

As used herein, the terms expression stabilizing, stably expressed, stabilized expression and their variations mean the level of the RNA molecule substantially the same or higher than the level in the offspring plants over the next generations, for example at least 3, at least 5 or at least 10 generations, compared to isogenic plants that do not contain an exogenous polynucleotide encoding a silencing suppressor. However, this term (s) does not exclude the possibility of some decrease in the levels of the RNA molecule in the next generations, compared with the previous generation, for example, a decrease of at least 10% per generation.

The suppressor can be selected from any source, for example, a plant, virus, mammal, and the like; see WO2010 / 057246 for a list of viruses from which a suppressor can be derived and designating a protein (eg, B2, P14, etc.) or coding region for a suppressor from each particular virus. Multiple copies of the suppressor can be used. Various suppressors can be used together (eg in tandem).

RNA molecules.

Essentially any RNA molecule that is desired to be expressed in a plant seed can be co-expressed with a silencing suppressor. The encoded polypeptides can be involved in the metabolism of oil, starch, carbohydrates, nutrients, and the like. or may be responsible for the synthesis of proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugar, carbohydrates, flavors, aromas, toxins, carotenoids, hormones, polymers, flavonoids, stored proteins, phenolic acids, alkaloids, lignin , tannins, cellulose, glycoproteins, glycolipids, etc., preferably TAG biosynthesis or assembly.

In a specific example, plants produce increased levels of enzymes to produce oil in plants, for example Brassica species such as rapeseed, or sunflower, safflower, flax, cotton, soybean, Camelina, or maize.

Production levels of DC-PUFA.

The levels of DC-PUFA or a combination of DC-PUFA produced in a recombinant cell or plant part, such as a seed, are of great importance. Levels can be expressed as a composition (percentage) of total fatty acids, which are specific DC-PUFAs or a group of related DC-PUFAs, for example ω3 DC-PUFA or ω6 DC-PUFA, or ODC-PUFA, or others, which may be determined by methods known in the art. In addition, the level can be expressed as the content of DC-PUFA, for example, as the percentage of DC-PUFA in the dry weight of the material containing recombinant cells, for example, the percentage of the weight of the seed, which is DC-PUFA. It should be understood that the level of DC-PUFA produced in an oilseed crop can be significantly higher in terms of DC-PUFA content than in a vegetable or seed that was not grown for oil production, although both may have a similar composition of DC-PUFA and both can be used as sources of DC-PUFA for human or animal consumption.

DC-PUFA levels can be determined by any of the methods known in the art. In a preferred method, the total lipid is recovered from cells, tissues or organisms and the fatty acid is converted to methyl esters prior to analysis by gas chromatography (GC). Such methods are described in Example 1. The position of the peak on the chromatogram can be used to identify each specific fatty acid, and the area of each peak is integrated to determine the amount. In this document, unless otherwise indicated, the percentage of a particular fatty acid in a sample is determined by expressing the peak area of the indicated fatty acid as a percentage of the total fatty acid peak area in the chromatogram. Essentially, this corresponds to the weight percentage (w / w). The identity of the fatty acids can be confirmed by GC-MS. Total lipids can be isolated by methods known in the art to purify fractions, eg, TAG fractions. For example, thin layer chromatography (TLC) can be performed on an analytical scale to separate TAG from other lipid fractions such as DAG, acyl-CoA or phospholipid, in order to determine the fatty acid composition specifically for TAG.

In one embodiment, the total sum of ARA, EPA, DNA and DHA in the fatty acids of the extracted lipid is about 21 to about 40% of the total fatty acids in the cell. In a further embodiment, the total fatty acids in the cell contain less than 1% C20: 1. In preferred embodiments, the extractable TAG in the cell contains fatty acids in the amounts specified herein. Each of the possible combinations of lipid defining traits disclosed herein is also included.

Additionally, the level of DC-PUFA production in a recombinant cell, plant, or plant part, such as a seed, can be expressed as the percent conversion of a particular fatty acid substrate to one or more fatty acid products, which is further referred to herein as conversion efficiency or enzyme efficiency. This parameter is based on the fatty acid composition of a lipid extracted from a cell, plant, plant part or seed, i.e. the amount of DC-PUFA formed (including other DC-PUFA derived from it), expressed as a percentage of one or more fatty acid substrates (including all other fatty acids derived from it). The general formula for the percent conversion: 100 x (the sum of the percentages of the DC-PUFA product and all products derived from it) / (the sum of the percent of the fatty acid substrate and all products derived from it). With respect to DPC, for example, this can be expressed as the ratio of the level of DPC (as a percentage of the total fatty acids in the lipid) to the level of the fatty acid substrate (e.g. TC, LA, ALA, SDA, ETA, or EPA) and all products including DPC. obtained from the substrate. The percent conversion or conversion efficiency can be expressed for a single enzymatic step in the pathway, as well as for part or all of the pathway.

Specific conversion efficiencies are calculated herein using the following formulas.

1. OK in WPC = 100 x (% DHA +% WPC) / (sum% for OK, LC, GLA, DGLK, ARK, EDK, ALA, SDK, ETRK, ETC, EPA, DPK and DHA).

2. LA in WPC = 100 x (% DHA + WPC) / (sum% for LA, GLA, DGLA, ARA, EDC, ALA, SDK, ETRK, ETK, EPA, DPA and DHA).

3. ALA in WPC = 100 x (% DHA +% WPC) / (sum% for ALA, SDK, ETRK, ETC, EPA, WPC and DHA).

4. EPA in DPA = 100 x (% DHA + DPA) / (sum% for EPA, DPA and DHA).

5. DPA in DHA (efficiency of Δ4-desaturase) = 100 x (% DHA) / (sum% for DPA and DHA).

6. Efficiency of Δ12-desaturase = 100 x (sum% for LA, GLA, DGLA, ARA, EDK, ALA, SDK, ETRK, ETC, EPA, DPK and DHA) / (sum% for OK, LC, GLA, DGLK, ARK, EDK, ALK, SDK, ETRK, ETC, EPK, DPK and DHK).

7. Efficiency of ω3-desaturase = 100 x (sum% for ALA, SDK, ETRK, ETK, EPA, DPA and DHA) / (sum% LA, GLA, DGLK, ARA, EDK, ALK, SDK, ETRK, ETC, EPA , DPK and DHA).

8. OK in ALK = 100 x (sum% for ALK, SDK, ETRK, ETK, EPK, DPK and DHK) / (sum% for OK, LC, GLK, DGLK, ARK, EDK, ALK, SDK, ETRK, ETC , EPA, DPA and DHA).

9. Efficiency of Δ6-desaturase (on ω3 substrate ALA) = 100 x (sum% for SDA, ETC, EPA, DPA and DHA) / (% ALA, SDC, ETRC, ETC, EPA, DPA and DHA).

10. Efficiency of Δ6-elongase (on ω3 substrate SDK) = 100 x (sum% for ETC, EPA, DPA and DHA) / (sum% for SDC, ETC, EPA, DPA and DHA).

11. The efficiency of Δ5-desaturase (on ω3 substrate ETA) = 100 x (sum% for EPA, DPA and DHA) / (sum% for ETA, EPA, DPA and DHA).

12. Efficiency of Δ5-elongase (on ω3 EPA substrate) = 100 x (sum% for DPA and DHA) / (sum% for EPA, DPA and DHA).

The fatty acid composition of the lipid, preferably seed oil, according to the invention is also characterized by the ratio ω6 fatty acids: ω3 fatty acids in total fatty acids, for any total ω6 fatty acids: total ω3 fatty acids or for new ω6 fatty acids: ω3 new fatty acids. The terms total ω6 fatty acids, total ω3 fatty acids, new ω6 fatty acids and new ω 3 fatty acids have the meanings defined herein. The ratios are calculated based on the fatty acid composition of a lipid extracted from a cell, plant, plant part or seed lipid, in a manner exemplified herein. It is desirable for the lipid to have a higher level of ω3 fatty acids than ω6 fatty acids, and thus an ω6: ω3 ratio of less than 1.0 is preferred. A ratio of 0.0 indicates the complete absence of certain ω6 fatty acids; a ratio of 0.03 has been achieved. Such low ratios can be achieved by the combined use of Δ6 desaturase with preference for substrate ω3, together with ω3 desaturase, especially fungal ω3 desaturase such as Pichia pastoris ω3 desaturase exemplified herein.

Additionally, the yield of DC-PUFA per seed weight can be calculated based on the total oil content of the seed and the% WPC in oil. For example, if the oil content of rapeseed is about 40% (w / w) and about 12% of the total fatty acids in the oil is WPC, then the WPC content of the seed is about 4.8%, or about 48 mg per 1 g seed. With the content of the WPC

- 45 037184 about 21% rapeseed or Camelina sativa seed has a DPK level of about 84 mg per gram of seed. The present invention thus discloses the plants of Brassica napus, B. juncea and Camelina sativa, and the seeds obtained therefrom, which contain at least about 80 mg or at least about 84 mg of WPC per gram of seed. The moisture content in the seed is standard for the mature seed harvested during the harvest after drying (4-15% moisture). The invention also discloses a method for producing an oil comprising obtaining a seed and extracting oil from a seed, using the oil, and methods for producing a seed comprising harvesting seeds from the plants of the invention.

In addition, the amount of WPC produced per hectare can be calculated if the seed yield per hectare is known or can be estimated. For example, rapeseed in Australia usually yields about 2.5 tonnes of seed per hectare, which with an oil content of 40% yields about 1000 kg of oil. With a WPC content of 20.1% in the final oil, this provides about 200 kg WPC per 1 ha. If the oil content is reduced by 50%, this still provides about 100 kg of WPC per hectare.

Evidence to date suggests that some desaturases expressed heterologously in yeast or plants have relatively low activity in combination with some elongases. This situation can be improved by giving desaturase the ability to use the acyl-CoA form of the fatty acid as a substrate in the synthesis of DC-PUFA, and this is considered preferable in recombinant cells, especially in plant cells. A particularly preferred combination for efficient DPC synthesis is fungal ω3 desaturase, such as, for example, Pichia pastoris ω3 desaturase (SEQ ID NO: 6), which is a Δ6 desaturase with preference for ω3 acyl substrates, such as Micromonas pusilla Δ6 desaturase (SEQ ID NO: 9), or variants thereof with at least 95% amino acid sequence identity.

As used herein, the term substantially free means that the composition (e.g., lipid or oil) contains a small amount (e.g., less than about 0.5%, less than about 0.25%, less than about 0.1% or less than about 0.01%) or does not contain a specific component. In an embodiment, substantially free means that the component cannot be detected using a routine assay method, for example, a particular fatty acid (e.g., ω6 doocosapentaenoic acid) cannot be detected using gas chromatography, as outlined in Example 1.

In an embodiment of the invention, the extracted lipid, extracted oil, plant or part thereof, such as a seed (according to the invention or used in the process / method according to the invention), food or composition according to the invention does not contain all-cis-6,9,12,15 , 18geneicosapentaenoic acid (n-3 HPA).

Obtaining oils.

Methods that are routinely practiced in the art can be used to isolate, process, and analyze oils produced by cells, plants, seeds, and the like, according to the present invention. Typically, plant seeds are thermally treated, pressed and extracted to produce a crude oil that is further degummed, refined, bleached and deodorized. In general, seed grinding techniques are known in the art. For example, oilseeds can be softened by spraying them with water to increase the moisture content, for example to 8.5%, and rolled with a smooth roller with 0.23-0.27 mm gaps. Depending on the type of seed, you may not need to add water before grinding. Applying heat inactivates enzymes, facilitates further cell rupture, pooling of oil droplets, and agglomeration of protein particles, all of which simplify the extraction process.

In an embodiment, most of the seed oil is released when passed through a screw press. The cake leaving the screw press is further extracted, for example, with hexane, using a column with an accompanying heat control. Alternatively, the crude oil obtained from the pressing operation can be passed through a slotted wire-top sedimentation tank to remove solids that enter the oil during the pressing operation. The clarified oil can be passed through a frame and filter press to remove any remaining fine solids. If desired, the oil after the extraction process can be combined with the clarified oil to form a blended crude oil.

Once the solvent has been stripped from the crude oil, the compressed and extracted portions are combined and processed with conventional oil processing procedures. As used herein, the term refined as used in connection with a lipid or oil of the invention generally means that the extracted lipid or oil has undergone one or more processing steps that increase the purity of the lipid / oil component. For example, the purification step can include one or more or all of the group consisting of degumming, deodorizing, bleaching, drying, and / or fractionating the extracted oil. However, as used herein, the term purified does not include a transesterification process or other process that modifies the fatty acid composition of a lipid or oil of the invention to increase WPC content as a percentage of total content.

- 46 037184 fatty acids. In other words, the fatty acid composition of the purified lipid or oil is substantially the same as the composition of the crude lipid or oil.

Degumming.

Degumming is an early stage in the refining of oils, and its main purpose is to remove most phospholipids from the oil, which may be present as about 1-2% of the total extracted lipid. The addition of ~ 2% of water, usually containing phosphoric acid, to the crude oil at 70-80 ° C leads to the separation of most phospholipids, accompanied by trace metals and pigments. The insoluble material that is removed is mainly a mixture of phospholipids and triacylglycerides, and is also known as lecithin. Degumming can be performed by adding concentrated phosphoric acid to the crude seed oil to convert the incapable of hydration phosphatides into a hydratable form and form metal chelates that are present in minor amounts. The resin is separated from the seed oil by centrifugation.

Alkaline refining.

Alkaline refining is one of the refining methods for processing crude oil, sometimes also referred to as neutralization. It usually follows degumming and precedes bleaching. After degumming, the seed oil can be processed by adding enough alkali solution to titrate all fatty acids and phosphoric acids, and then removing the resulting soap. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. This process is usually carried out at room temperature to remove the free fatty acid fraction. The soap is removed by centrifugation or extraction in a soap solvent and the neutralized oil is washed with water. If necessary, excess alkali in the oil can be neutralized with a suitable acid such as hydrochloric acid or sulfuric acid.

Whitening.

Bleaching is a cleaning method in which oils are heated to 90-120 ° C, holding at this temperature for 10-30 minutes in the presence of bleaching clay (0.2-2.0%) and in the absence of oxygen, in a nitrogen atmosphere or steam or in a vacuum. This stage of oil treatment is designed to remove unwanted pigments (carotenoids, chlorophyll, gossypol, etc.), and the process further removes oxidation products, trace metals, sulfur compounds and traces of soap.

Deodorization.

Deodorization is the treatment of oils and fats at high temperatures (200-260 ° C) and low pressure (0.1-1 mm Hg). This is usually achieved by injecting steam into the seed oil at a rate of about 0.1 ml / min / 100 ml of seed oil. After about 30 minutes of irrigation, the seed oil is allowed to cool under vacuum. The seed oil is usually transferred to a glass container and sparged with argon and then stored at low temperatures. This treatment improves the color of the seed oil and removes most volatile substances or odors, including residual free fatty acids, monoacylglycerides and oxidation products.

Winterization.

Winterization is a process sometimes used in the commercial production of oils for the purpose of separating oils and fats into solid (stearin) and liquid (olein) fractions by crystallization at temperatures below ambient temperature. It was originally applied to cottonseed oil to produce a solid-free product. Typically, this method is used to reduce the saturated fatty acid content of the oil.

Transesterification.

In this document, transesterification means a method of exchanging fatty acids within and between TAGs or transferring fatty acids to another alcohol to form an ester. This may include the primary release of fatty acids from the TAG in the form of free fatty acids or the direct production of fatty acid esters, preferably methyl esters or fatty acid ethyl esters. During the transesterification reaction of the TAG with an alcohol such as methanol or ethanol, the alkyl group of the alcohol forms an ester bond with the acyl groups (including DPA) of the TAG. When combined with a fractionation method, transesterification can be used to modify the fatty acid composition of lipids (Marangoni et al., 1995). For transesterification, chemical (for example, catalyzed by a strong acid or base) or enzymatic agents can be used, the latter including lipases, which can be specific for the position (sn-1/3 or sn-2 specific) of the fatty acid in the TAG, or with preference for some fatty acids versus others (Speranza et al., 2012). Fractionation of fatty acids in order to increase the concentration of DC-PUFA in oil can be carried out by any of the methods known in the art, such as, for example, freeze crystallization, complexation using urea, molecular distillation, supercritical fluid extraction, countercurrent chromatography, and

- 47 037184 the formation of complexes with the silver ion. Complexation with urea is the preferred method due to its simplicity and effectiveness in reducing the level of saturated and monounsaturated fatty acids in oil (Gamez et al., 2003). First, TAGs in oil are cleaved into their constituent fatty acids, often in the form of fatty acid esters, by hydrolysis under catalysis conditions with an acid or base, whereby 1 mol of TAG reacts with at least 3 mol of alcohol (for example, ethanol for ethyl esters or methanol for methyl esters), and using an excess of alcohol to allow the separation of the formed alkyl esters and glycerol, which is also formed, or with the help of lipases. Such free fatty acids or fatty acid esters, which usually remain unchanged in the fatty acid composition after treatment, can then be mixed with an ethanolic urea solution to form complexes. Saturated and monounsaturated fatty acids readily complex with urea, crystallize upon cooling, and can subsequently be removed by filtration. The fraction that has not formed complexes with urea is thus enriched with DC-PUFA.

Food (feed).

The present invention includes compositions that can be used as food (feed). For the purposes of the present invention, food (feed) includes any food or preparation for human or animal consumption that, when ingested: (a) serves the purpose of nourishing or tissue formation or energy supply; and / or (b) maintains, restores, or promotes proper nutritional status or metabolic function. Food products (feed) according to the invention include nutritional compositions for infants and / or young children, such as, for example, infant formula and cake according to the invention.

Food (feed) according to the invention includes, for example, a cell according to the invention, a plant according to the invention, a part of a plant according to the invention, a seed according to the invention, an extract according to the invention, a product of a method according to the invention, a product of a fermentation process according to the invention, or a composition together with a suitable (and ) by the bearer (s). The term carrier is used in its broadest sense and includes any component that may or may not be nutritive. As will be understood by the skilled artisan, the carrier must be suitable for use (or used in a sufficiently low concentration) in the food (feed) such that it does not adversely affect the organism consuming the food (feed).

The food product (feed) of the present invention includes an oil, fatty acid ester or fatty acid obtained directly or indirectly using the methods, cells or plants disclosed herein.

Additionally, the composition can be in solid or liquid form. In addition, the composition may contain edible micronutrients, protein, carbohydrates, vitamins and / or minerals in amounts desired for a particular application. The amounts of these ingredients will vary depending on whether the composition is intended for use in healthy individuals or for use in individuals with special needs, such as individuals suffering from metabolic disorders and the like.

Examples of suitable carriers of nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates, and proteins. Examples of such edible fats include, but are not limited to, coconut oil, borage oil, mushroom oil, black currant oil, soybean oil, and mono- and diglycerides. Examples of such carbohydrates include, but are not limited to, glucose, edible lactose, and hydrolyzed starch. Additionally, examples of proteins that can be used in the nutritional composition of the invention include, but are not limited to, soy proteins, electrodialysis treated whey, electrodialysis treated skim milk, milk whey, or hydrolysates of these proteins.

Regarding vitamins and minerals, the following can be added to the food (feed) compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, vitamins A, E, D , C and B vitamins. In addition, other such vitamins and minerals can be added.

The components used in the food (feed) compositions of the present invention can be semi-refined or refined. By semi-purified or purified is meant material that is obtained by purification of natural material or de novo synthesis.

Additionally, the food (feed) composition of the present invention can be added to food even if no dietary supplements are needed. For example, the composition can be added to any type of food, including but not limited to margarine, modified butter, cheeses, milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meat, fish and drinks.

Additionally, fatty acids produced in accordance with the present invention or host cells transformed to contain and express target genes can be used as food additives for animals to modify the composition of fatty acids in tissue, egg or milk. animal to more desirable for human consumption by humans or animals. Examples of such animals include sheep, cattle, horses, poultry such as chickens, and the like.

In addition, the food (feed) according to the invention can be used in aquaculture to increase the levels of fatty acids in the body of fish or crustaceans, such as, for example, shrimp, for human or animal consumption. Preferably the fish is salmon.

Preferred food (feed) according to the invention are plants, seeds and other plant parts, for example leaves and stems, which can be directly used as food or feed for humans or animals. For example, animals can graze directly on such plants grown in the field, or they can be fed more precise amounts through controlled feeding. The invention includes the use of such plants and plant parts as food for increasing the levels of DC-PUFA in humans and animals.

In an embodiment of the invention, the food product is an infant formula containing a lipid or oil according to the invention. As used herein, infant formula refers to a non-naturally occurring composition that at least partially satisfies an infant's nutritional needs. Infant refers to a human subject ranging in age from birth to no more than one year of age, and includes age-adjusted infants from 0 to 12 months. Adjusted age refers to the chronological age of the infant minus the amount of time the infant was born prematurely. Thus, the adjusted age represents the age of the infant as if it were born full-term. In this document, non-naturally occurring means that the product is not found in nature, but is created by human intervention. In this document, the infant formula of the invention excludes pure human breast milk (Koletzko et al., 1988) and pure milk produced by animals, although the infant formula of the invention may contain components derived from milk such as milk protein or carbohydrates such as whey protein or lactose. The infant formula of the invention excludes naturally occurring meats such as veal, fur seal meat, whale meat, or fish, although the infant formula of the invention may contain components from these sources such as proteins. The infant formula of the invention always contains the lipid containing the WPC of the invention, preferably at a level of 0.05 to about 0.5% by weight, based on the total fatty acid content. DPA can be present as a TAG, as a phospholipid, or as a non-esterified fatty acid, or a mixture thereof. The lipid or oil of the invention can be incorporated into infant formula using methods known in the art. For example, a skilled artisan can readily prepare an infant formula of the invention, generally using the methods described in WO 2008/027991, US20150157048, US2015094382 and US20150148316, in which WPC is added in addition to or instead of one or more polyunsaturated fatty acids described in specified documents.

In one example, the infant formula contains DPA (i.e. omega-3 DPA as described herein), optionally with probiotics, especially polydextrose (PDT) and galactooligosaccharides (GOS), lactoferrin from a non-human source, and other long-chain polyunsaturated fatty acids (DC-PUFA). In some embodiments, the nutritional composition further comprises SDA and / or gamma-linolenic acid (GLA). In some embodiments, the infant formula contains up to 7 g / 100 kCal of fat or lipid source, more preferably from about 3 g / 100 kCal to about 7 g / 100 kCal of fat or lipid source, wherein the fat or lipid source comprises at least about 0.5 g / 100 kcal, and more preferably from about 1.5 g / 100 kcal to about 7 g / 100 kcal protein source or protein equivalent, more preferably from about 1 g / 100 kcal to about 7 g / 100 kcal source protein or source of protein equivalent; and at least about 5 g / 100 kcal carbohydrate, more preferably about 5 g to about 25 g / 100 kcal carbohydrate. Additionally, infant formula may contain one or more or all of 1) at least about 10 mg / 100 kcal lactoferrin, more preferably from about 10 mg / 100 kcal to about 200 mg / 100 kcal lactoferrin; 2) from about 0.1 g / 100 kcal to about 1 g / 100 kcal of a probiotic composition containing PDT and GOS; and 3) at least about 5 mg / 100 kcal of additional DC-PUFA (i.e., non-DPA DC-PUFA), including DHA, more preferably from about 5 mg / 100 kcal to about 75 mg / 100 kcal additional DC-PUFA, including DHA.

In an embodiment of the invention, the ratio of DPA: DHA in the total fatty acid content of the infant formula is from 1: 3 to 2: 1. In addition, EPA may be present, but is preferably absent. If present, the ratio of EPA: DPA in the total fatty acid content is preferably less than 1: 2, more preferably less than 1: 5. Additionally, ARA may be absent, but is preferably present, and preferably the ratio of ARA: DPA in the total fatty acid content is from 1: 3 to 2: 1. More preferably, the levels of each DC-PUFA in infant formula are approximately the same as found in any human breast milk, which naturally exhibits variations depending on maternal age, genetic factors, dietary intake and nutritional status; for example see Koletzko et al. (1988). In a preferred embodiment, the infant formula does not contain detectable levels of heneicosapentaenoic acid (HPA, 21: 5ω3).

- 49 037184

Infant formula can refer to, for example, liquids, powders, gels, pastes, solids, concentrates, suspensions or ready-to-use forms of enteral mixtures, oral mixtures, infant formula.

Probiotics useful in accordance herewith may include polydextrose, polydextrose powder, lactulose, lactosucrose, raffinose, glucooligosaccharides, inulin, fructooligosaccharide, isomaltooligosaccharides, oligosaccharides of soy, lactosucrose, xylooligosaccharide, chitooligosaccharides, mannooligosaccharide, aribinooligosaharid, sialliloligosaharid, fucooligosaccharides, galactooligosaccharide and gentsiooligosaharidy ...

In addition, lactoferrin can be incorporated into a nutritional formulation according to this document. Lactoferrins are approximately 80 kDa single chain polypeptides containing 1-4 glycans, depending on the species. The 3-D structure of lactoferrin in different species is highly similar, but not identical. Each lactoferrin contains two homologous parts, called the N- and C-lobes, which means the N-terminal and C-terminal part of the molecule, respectively.

The protein source or protein equivalent can be any source as used in the art, for example, skim milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and the like. Cow's milk protein sources useful herein include, but are not limited to, milk protein powder, milk protein concentrates, milk protein isolates, skim milk solids, skim milk, skimmed milk powder, whey protein, whey protein isolates, concentrates whey protein, sweet whey, sour whey, casein, sour casein, caseinate (eg sodium caseinate, sodium calcium caseinate, calcium caseinate), and any combination thereof.

Suitable carbohydrate sources can be any used in the art, for example lactose, glucose, fructose, corn syrup solids, maltodextrins, sucrose, starch, rice syrup solids, and the like. The amount of carbohydrate component in the nutritional formulation is at least about 5 g / 100 kcal and can typically range from about 5 g to about 25 g / 100 kcal. In some embodiments, the amount of carbohydrate is from about 6 g to about 22 g / 100 kcal. In other embodiments, the amount of carbohydrate is from about 12 g to about 14 g / 100 kcal. In some embodiments, corn syrup solids are preferred. In addition, it may be desirable to add hydrolyzed, partially hydrolyzed and / or highly hydrolyzed carbohydrates to the nutritional composition due to their easy digestibility. Specifically, hydrolyzed carbohydrates are less likely to contain allergenic epitopes. Non-limiting examples of carbohydrate materials suitable for use herein include hydrolyzed or intact, naturally occurring or chemically modified corn, tapioca, rice or potato starch, in waxy or non-waxy forms. Non-limiting examples of suitable carbohydrates include various types of hydrolyzed starch, namely hydrolyzed corn starch, maltodextrin, maltose, corn syrup, glucose, corn syrup solids, glucose, and various other glucose polymers and combinations thereof. Non-limiting examples of other suitable carbohydrates include those often referred to as sucrose, lactose, fructose, high fructose corn syrup, indigestible oligosaccharides such as fructooligosaccharides, and combinations thereof.

In addition, one or more vitamins and / or minerals may preferably be added to the infant formula in amounts sufficient to meet the daily nutritional needs of the subject. One of ordinary skill in the art will understand that the need for vitamins and minerals will vary, for example, according to the age of the child. The nutritional composition may optionally contain, but is not limited to, one or more of the following vitamins or their derivatives: vitamin B1 (thiamine, thiamine pyrophosphate, TPP, thiamine triphosphate, TTF, thiamine hydrochloride, thiamine mononitrate), vitamin B2 (riboflavin, flavin mononucleotide , FMN, adenine flavine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic acid mononucleotide, NikMN, pyridinic acid), precursor 3-precursor tryptophan, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate, panthenol), folate (folic acid, folacin, pteroglutamic acid), vitamin B12 (cobalamin, methylcobalaminosylamine, desocobalaminosylamino) biotins, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esters and other long-chain fatty acids, retinal, retinoic acid, retinol esters), vitamin D (calciferol, cholecalciferol, vitamin 3, 1.25, -dihydroxyvitamin D), vitamin E (α-tocopherol, α-tocopherol acetate, α-tocopherol succinate, α-tocopherol nicotinate, α-tocopherol), vitamin K (vitamin K1, phylloquinone, naphthoquinone, vitamin K2, menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8, menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone -10, menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol, β-carotene and any combinations thereof. In addition, the nutritional composition optionally may contain, but is not limited to, one or more of the following minerals or their derivatives: boron, calcium, calcium acetate, calcium gluconate, calcium chloride, calcium lactate, calcium phosphate, calcium sulfate, chloride, chromium, chromium chloride, chromium picolinate, copper, copper sulfate, copper gluconate, copper sulfate, fluoride, iron, iron carbonyl, iron (P), iron (III), iron (III) orthophosphate, trituration of iron, iron polysaccharide, iodide, iodine, magnesium, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese, molybdenum, phosphorus, potassium, potassium phosphate, potassium iodide, potassium chloride, potassium acetate, selenium, sulfur, sodium, sodium docusate, sodium chloride, sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate and mixtures thereof. Non-limiting examples of derivatives of mineral compounds include salts, alkali salts, esters, and chelates of any mineral compound. Minerals can be added to nutritional formulations in the form of salts such as calcium phosphate, calcium glyceryl phosphate, sodium citrate, potassium chloride, potassium phosphate, magnesium phosphate, iron (II) sulfate, magnesium sulfate, copper sulfate, manganese sulfate, and sodium selenate. Additional vitamins and minerals can be added as is known in the art.

In an embodiment of the invention, the infant formula according to the invention or obtained in accordance with the invention does not contain human or animal breast milk or an extract from it containing WPC.

In another embodiment, the level of omega-6 DPA in the total fatty acid content of the infant formula is less than 2, preferably less than 1%, or from 0.1 to 2%, more preferably absent.

Compositions.

The present invention further includes compositions, especially pharmaceutical compositions, containing one or more fatty acids and / or oils obtained using the methods of the invention, preferably in the form of ethyl esters of fatty acids.

The pharmaceutical composition may contain one or more fatty acids and / or oils, in combination with a standard, known, non-toxic pharmaceutically acceptable carrier, adjuvant or solvent such as phosphate buffered saline, water, ethanol, polyols, vegetable oils, wetting agent or emulsion for example a water / oil emulsion. The composition can be in liquid or solid form. For example, the composition can be in the form of a tablet, capsule, swallowable liquid or powder, injectable form, or in the form of a topical ointment or cream. The required fluidity can be maintained, for example, by maintaining the required particle size in the case of dispersions and the use of surfactants. In addition, it may be desirable to administer isotonic agents such as sugar, sodium chloride, and the like. In addition to such inert diluents, the composition may contain adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavors and aromas.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, esters of sorbitol and polyoxyethylene sorbitan, microcrystalline cellulose, aluminum meta-hydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared using methods well known in the art. For example, the fatty acids obtained in accordance with the present invention can be tableted with conventional tablet bases such as lactose, sucrose and corn starch, in combination with binders such as acacia, corn starch or gelatin, disintegrants such as potato starch or alginic acid, and lubricants such as stearic acid or magnesium stearate. Capsules can be prepared by combining these excipients in a gelatin capsule with antioxidants and suitable fatty acid (s).

For intravenous administration, fatty acids obtained in accordance with the present invention, or derivatives thereof, can be incorporated into commercial preparations.

Typical dosages of a particular fatty acid are from 0.1 mg to 20 g, when administered 1 to 5 times a day (up to 100 g daily), and preferably ranges from about 10 mg to about 1, 2, 5 or 10 g daily (in one or more doses). As is known in the art, it is desirable to administer at least about 300 mg / day of a fatty acid, especially DC-PUFA. However, it should be understood that any amount of fatty acid will be beneficial to the subject.

Possible routes of administration for the pharmaceutical compositions of the present invention include, for example, enteral (eg, oral and rectal) and parenteral. For example, a liquid preparation can be administered orally or rectally. Additionally, the homogeneous mixture can be completely dispersed in water, premixed under sterile conditions with physiologically acceptable diluents, preservatives, buffers or propellants to form a spray or inhaler.

The dosage of the composition for administration to a patient can be determined by one of ordinary skill in the art and will depend on various factors such as the patient's body weight, the patient's age, the patient's general health, the patient's history, the patient's immune status, and the like.

- 51 037184

Additionally, the compositions of the present invention can be used for cosmetic purposes. They can be added to existing cosmetic compositions in such a way that a mixture is formed, or the fatty acid obtained in accordance with the present invention can be used as the only active ingredient in the cosmetic composition.

Examples of

Example 1. Materials and methods.

Expression of genes in plant cells in a transient expression system.

Exogenous genetic constructs are expressed in plant cells in a transient expression system as essentially described by Voinnet et al. (2003) and Wood et al. (2009).

Analysis of Fatty Acids by Gas Chromatography (GC) FAMEs are analyzed by gas chromatography using an Agilent Technologies 7890A GC gas chromatograph (Palo Alto, CA, USA) equipped with an SGE-BPX70 column (70% cyanopropyl polysylphenylin-siloxane, inner diameter 0.25 mm, film thickness 0.25 mm) 30 m long, FID (Plasma Ionization Detector), split / splitless injector, as well as a serial Agilent Technologies 7693 autosampler and injector. Helium is used as a carrier gas. Samples are injected in a split method (50: 1 ratio) at an oven temperature of 150 ° C. After injection, the furnace temperature is maintained at 150 ° C for 1 min, then increased to 210 ° C at a rate of 3 ° C / min, again increased to 240 ° C at a rate of 50 ° C / min, and finally maintained for 1.4 min at 240 ° C. Peak areas are determined using Agilent Technologies ChemStation software (Rev B.04.03 (16), Palo Alto, CA, USA) based on the response of a known amount of external standard GLC-411 (Nucheck) and internal standard C17: 0-ME ...

Lipid analysis by liquid chromatography-mass spectrometry (LC-MS).

Total lipids are extracted from lyophilized developing seeds 12 days post-flowering (DPC) and mature seeds after adding a known amount of tri-C17: 0-TAG as an internal standard for quantification. The extracted lipids are dissolved in 1 ml of 10 mM butylated hydroxytoluene in butanol / methanol (1: 1, v / v) per 5 mg dry material and analyzed using an Agilent 1200 series liquid chromatograph with LC and electrospray ionization by LC-MS 6410b with three quadrupole lenses. Lipids were chromatographically separated using an Ascentis Express RP-Amide column (50 x 2.1 mm, 2.7 μm, Supelco) in a binary gradient mode with a flow rate of 0.2 ml / min. Mobile phases: A) 10 mM ammonium formate in a mixture of H ₂ O: methanol: tetrahydrofuran (50:20:30 v / v / v); C) 10 mM ammonium formate in a mixture of H ₂ O / methanol / tetrahydrofuran (5:20:75, v / v / v). Multiple Reaction Monitoring Lists (MMPs) are based on the following essential fatty acids: 16: 0, 18: 0, 18: 1, 18: 2, 18: 3, 18: 4, 20: 1, 20: 2, 20: 3 , 20: 4, 20: 5, 22: 4, 22: 5, 22: 6, using a collision energy of 30 V and a fragmenter of 60 V. neutral particles 22: 6. The amount of TAG is determined using a 10 μM external tristearin standard.

Determination of lipid profile by LC-MS.

The extracted total lipids were analyzed using an Agilent 1200 Series LC coupled to an electrospray ionization device (Agilent, Palo Alto, CA, USA). An injection of 5 μL of each total lipid extract was separated by chromatography using an Ascentis Express RP-Amide 50 χ 2.1 mm, 2.7 μm HPLC column (Sigma-Aldrich, Castle Hill, Australia) using a binary gradient at a flow rate of 0.2 ml / min. Mobile phases: A) 10 mM ammonium formate in H ₂ O: methanol: tetrahydrofuran (50:20:30, v / v / v); C) 10 mM ammonium formate in H ₂ O: methanol: tetrahydrofuran (5:20:75, v / v / v). Selected neutral lipids (TAG and DAG) and phospholipids (PL, including PC, FE, PI, PS, PA, FG) were analyzed by monitoring multiple reactions (MMP) using collision energy 30 V and fragmentation energy 60 V. Neutral lipids were tested for the following essential fatty acids: 16: 0 (palmitic acid), 18: 0 (stearic acid), 18: 1ω9 (oleic acid, OC), 18: 2ω6 (linoleic acid, LA), 18: 3ω3 ( α-linolenic acid, ALA), 18: 4ω3 (stearidonic acid, SDA), 20: 1, 20: 2, 20: 3, 20: 4ω3, 20: 5ω3, 22: 4ω3, 22: 5ω3, 22: 6ω3, while the phospholipids were scanned for C16, C ₁₈ , C ₂₀ and C ₂₂ molecules with 0-3, 0-4, 0-5, 4-6 double bonds, respectively.

The individual MMDs of TAGs were identified based on the ammoniated precursor ion and the product ion formed as a result of a 20: 1 neutron loss, SDC, EPA, and DHA. TAG and DAG were quantified using 50 μM tristearin and distearin as external standards. PL was quantitatively determined using 10 μM external standards di-18: 0-PC, di-17: 0-FA, di-17: 0-PE, 17: 0-17: 1-FG, di-18: 1-PI and di-17: 0-FS (Avanti Polar Lipids, Alabaster, Alabama, USA). For the selected TAG, DAG, and PL species, further confirmation was performed using MS / MS 6520 Q-TOF.

Determination of the fatty acid profile and oil content of the seed.

If it is necessary to determine the oil content of the seed, the seeds are dried in a desiccator for 24

- 52,037184 hours, and about 4 mg of semen is transferred into a 2 ml glass vial with a Teflon-coated screw cap. 0.05 mg of triheptadecanoin dissolved in 0.1 ml of toluene is added to the vial as an internal standard.

FAME of seed is obtained by adding 0.7 ml of 1N. methanol HCl (Supelco) in a vial containing semen material, vortexed briefly and incubated at 80 ° C for 2 hours.After cooling to room temperature, 0.3 ml of 0.9% (w / v) NaCl solution and 0.1 ml of hexane is added to the vial and mixed thoroughly for 10 minutes in a Heidolph Vibramax 110. The FAMEs are collected in a 0.3 ml glass insert and analyzed by plasma ionization detector (FID) GC as previously mentioned.

The peak area of an individual FAME is first corrected based on the peak area response of a known amount of the same FAME present in the commercial standard GLC-411 (NUCHEK PREP, INC., USA). GLC-411 contains equal amounts of 31 fatty acids (wt%) ranging from C8: 0 to C22: 6. In the case of fatty acids that are absent in the standard, the inventors used the peak area responses of the most similar FAME. For example, a 16: 1d9 FAME peak area response was used for 16: 1d7 and a C22: 6 FAME response was used for C22: 5.

The corrected peak area values are used to calculate the mass of each FAME in the sample versus the mass of the internal standard. The oil is stored primarily in the form of TAG and its mass is calculated based on the mass of the FAME. The total moles of glycerol are determined by calculating the moles of each FAME and dividing the total moles of FAME by three. The TAG content is calculated as the sum of glycerol and fatty acyl fragments using the ratio: wt% oil = 100 x ((41 x total moles FAME / 3) + (total grams of FAME - (15 x total moles FAME))) / gram of seed, where 41 and 15 are the molecular weights of the glyceride residue and methyl group, respectively.

Analysis of sterol content in oil samples.

Samples of about 10 mg of oil, together with an added aliquot of C24: 0 monol as an internal standard, are saponified with 4 ml of 5% KOH solution in 80% MeOH and heating to 80 ° C, incubating for 2 h at this temperature, in Teflon coated glass test tube with screw cap. After the reaction mixture was cooled, 2 ml of Milli-Q water was added and the sterols were extracted with 2 ml of hexane: dichloromethane (4: 1, v / v) with shaking and vortexing. The mixture is centrifuged, the sterol extract is recovered and washed with 2 ml of Milli-Q water. Then the sterol extract is separated after shaking and centrifugation. The extract is evaporated in a stream of nitrogen gas, and the sterols are silylated using 200 ml of N, O-bis- (trimethylsilyl) trifluoroacetamide (BSTFA) while heating to 80 ° C, keeping for 2 h at this temperature.

For sterol GC / GC-MS analysis, sterol-O-trimethylsilyl derivatives (sterol-OTMSi) are dried in a nitrogen gas stream on a thermoblock at 40 ° C and then redissolved in chloroform or hexane just prior to GC / GC-MS analysis. ... Sterol-OTMS derivatives were analyzed by gas chromatography (GC) using an Agilent Technologies 6890A GC gas chromatograph (Palo Alto, CA, USA) equipped with a Supelco Equity ™ -1 quartz glass capillary column (15 mx 0.1 mm ID, 0.1 μm film thickness), FID and split / non-split injector, and Agilent Technologies 7683B serial autosampler and injector. Helium is used as a carrier gas. Samples are injected in a non-splitting mode at an oven temperature of 120 ° C. After injection, the furnace temperature is increased to 270 ° C at a rate of 10 ° C / min, and finally to 300 ° C at a rate of 5 ° C / min. Peak areas are determined using Agilent Technologies ChemStation software (Palo Alto, CA, USA). GC results contain an error of ± 5% of the peak area values of the individual components.

GC-MS (GC-MS) analyzes are performed using Finnigan Thermoquest GCQ and Finnigan Thermo Electron Corporation GC / MS instruments, both equipped with a direct on-column sample injector, and Thermoquest Xcalibur software (Austin , Texas, USA). Each GC instrument is equipped with a capillary column with a polarity similar to that described above. Individual components are identified using mass spectrometry data and comparing retention time data with those obtained for authentic and laboratory standards. The complete blank assay procedure is performed in parallel with a batch of samples.

RT-PCR conditions.

Reverse transcriptase PCR (RT-PCR) amplification is typically performed using a Superscript III One-Step RT-PCR system (Invitrogen) in a volume of 25 μl, using 10 pmol forward primer and 30 pmol reverse primer, MgSO ₄ to a final concentration of 2 , 5 mM, 400 ng total RNA with buffer and nucleotide components according to manufacturer's instructions. Typical temperature conditions were: 1 cycle at 45 ° C for 30 min for reverse transcription to occur; then 1 cycle at 94 ° C for 2 min, and then 40 cycles with 94 ° C for 30 s, 52 ° C for 30 s, 70 ° C for 1 min; then 1 cycle

- 53 037184 at a temperature of 72 ° C for 2 min before cooling the reaction mixtures to 5 ° C.

Determination of the number of copies of transgenes using digital PCR.

To determine the copy number of the transgenes in the transgenic plant, the PCR method was used as described below. In addition, this method can be applied to determine if a plant is transgenic for the genetic constructs described herein. About a square centimeter of leaf tissue is obtained from each individual plant and placed in a collection microtube (Qiagen). Samples are then freeze dried for 24-48 hours. To disrupt the samples for DNA extraction, stainless steel beads are added to each dried sample and the tubes are shaken on a Qiagen tissue lysator. 375 μl of extraction buffer (0.1 M Tris-HCl, pH 8, 0.05 M EDTA, pH 8, and 1.25% VAT) is added to each tube, the mixtures are incubated at 65 ° C for 1 hour and then chill before adding 187 μl of 6 M ammonium acetate to each tube (4 ° C.) with vigorous stirring. Then the samples are centrifuged for 30 minutes at 3000 rpm. The supernatant from each tube is transferred to new microtubes, each containing 220 μl of isopropanol, to precipitate the DNA at room temperature for 5 minutes. DNA is harvested by centrifugation of tubes at 3000 rpm for 30 min, the DNA pellets are washed with 320 μl of 70% ethanol and dried before resuspending the DNA in 225 μl of water. The undissolved material is granulated by centrifugation at 3000 rpm for 20 minutes, and 150 μl of each supernatant is transferred to 96-well plates for long-term storage.

For efficient and quantitative digital PCR (ddPCR), DNA is digested with restriction enzymes prior to amplification reactions to ensure that multiple copies of transgenes or multiple insertions are physically separated. Thus, aliquots of the DNA preparations were digested with EcoRI and BamHI combined in a volume of 20 μl using 10x EcoRI buffer, 5 μl DNA and about 4 units of each enzyme per sample, incubated overnight at 37 ° C.

The primers used in these PCR reactions were designed using the Primer3 software to confirm that no interaction between the primers for the reference and target genes is predicted, or such interaction does not pose a problem under the conditions used. The reference gene in the analysis was the rapeseed gene Hmg (high mobility group), which is present in one copy in the rapeseed genome (Weng et al., 2004). Since rape is an allotetraploid, 4 copies of the Hmg gene were assumed, i.e. 2 alleles of each of the two genes, in Brassica napus. In the reactions of the reference gene, a pair of primers and a double-labeled probe were used as follows: sense primer, GCGAAGCACATCGAGTCA (SEQ ID NO: 50); antisense primer, Can12 GGTTGAGGTGGTAGCTGAGG (SEQ ID NO: 51); Hmg-P3 5'Hex / TCTCTAC / zen / CCGTCTCACATGACGC / 3IABkFQ / -3 '(SEQ ID NO: 52). The size of the amplification product was 73 bp.

In one amplification reaction of the target gene, in which the PPT selection marker gene was found for screening all transgenic plants, the sense primer was Can17, ATACAAGCACGGTGGATGG (SEQ ID NO: 53); antisense primer, Can18 TGGTCTAACAGGTCTAGGAGGA (SEQ ID NO: 54); probe, PPT-P3 5 '/ FAM / TGGCAAAGA / zen / GATTTCGAGCTTCCTGC / 3IABkFQ / -3' (SEQ ID NO: 55). The size of this target gene amplification product was 82 bp. In some cases, a second assay of the target gene was performed in parallel to detect partial T-DNA insertions. In this second assay, a region of the Δ6 desaturase gene was detected using a sense primer, Can23 CAAGCACCGTAGTAAGAGAGCA (SEQ ID NO: 56); an antisense primer, Can24 CAGACAGCCTGAGGTTAGCA (SEQ ID NO: 57); probe, D6des-P3 5 '/ FAM / TCCCCACTT / zen / CTTAGCGAAAGGAACGA / 3IABkFQ / -3' (SEQ ID NO: 58). The size of this target gene amplification product was 89 bp. In the reactions, 2 μL of digested DNA preparations were used as a template. The composition of the reaction mixture per sample: reference sense primer (10 pM), 1 μl; reference antisense primer (10 pM), 1 μl; reference gene probe (10 pM), 0.5 μl; sense primer of the target gene (10 pM), 1 μl; antisense primer of the target gene (10 pM), 1 μl; target gene probe (10 pM), 0.5 μl; ddPCR reagent mix, 12.5 μl; water 5.5 μl, in a total volume of 25 μl.

The mixtures were then placed in a QX100 droplet generator, which divided each sample into 20,000 droplets in the nL range. This was done in 8-well cartridges until all samples were processed and transferred to a 96-well PCR plate. The plate was then sealed with heat shrink pierce foil using a plate sealer. After that, the samples were treated with the following reaction conditions: 95 ° C, 10 min, rise at a rate of 2.5 ° C / s; then 39 cycles at 94 ° C, 30 s rise at a rate of 2.5 ° C / s; 61 ° C, 1 min, rise at a rate of 2.5 ° C / s; 98 ° С, 10 min, followed by cooling to 12 ° С. After amplification reactions of DNA in droplets, the plates were placed on a QX100 droplet reader, with which each droplet was analyzed individually using a two-color detection system (detection kit

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FAM or Hex). The digital PCR data of the droplets was viewed as a 1-D plot in which each sample droplet was plotted on a fluorescence intensity plot, or a 2-D plot on which fluorescence (FAM) versus fluorescence (Hex) was plotted for each droplet. The software measured the number of positive and negative droplets for each fluorophore (FAM or Hex) in each sample. Then, using the software, the fraction of positive droplets was approximated to the Poisson algorithm to determine the concentration of the target DNA molecule in copy units / μl of input. The copy number variation was calculated using the formula: CNV = (A / B) · Nb, where concentration A = concentration of the target gene, B = concentration of the reference gene and Nb = 4, the number of copies of the reference gene in the genome.

Pollen viability assessment.

Fluorescein diacetate (PDA) was dissolved in acetone at a concentration of 2 mg / ml to obtain a stock solution. PDA dilutions were prepared immediately prior to use by adding a PDA stock solution dropwise to 2 ml sucrose solution (0.5 M) until saturation indicated by the appearance of persistent turbidity.

Propidium iodide (PI) was dissolved in sterile distilled water at a concentration of 1 mg / ml to obtain a stock solution. Immediately before use, 100 μl of the stock solution was added to 10 ml of sterile distilled water to obtain a working solution. To check the ratio of viable and nonviable pollen, stock solutions of PY and PDA were mixed in a ratio of 2: 3.

Rape and mustard plants, transgenic and wild type, were grown under standard conditions in a greenhouse at 22 ± 2 ° C with a photoperiod of 16 h per day. Mature buds that were ready to open the next day were marked and harvested the next morning at 9-10 am. Pollen from the opened flowers were stained with PDA / PY and visualized with a Leica MZFLIII fluorescence microscope. Used an emission filter with a transmission in the long wavelength region of the spectrum GFP-2 510 nm (transmission of red and green light) with an excitation filter 480/40 nm to detect viable and non-viable pollen. The non-viable pollen that captured the PY dye was red under a fluorescence microscope, while the viable pollen when stained with PY and PDA had a bright green color.

Example 2. Stable expression of the transgenic DHA pathway in the seeds of Camelina sativa.

Binary vector pJP3416-GA7 (see FIG. 2 and SEQ ID NO: 1) is introduced into A. tumefaciens strain AGL1, and cells from a culture of transformed Agrobacterium are used to treat a flowering C. sativa plant using a submerged transformation method ( Lu and Kang, 2008). After the plants have grown and matured, the T1 seed is harvested from the treated plants, sown in the ground, and the resulting plants are sprayed with BASTA herbicide to select transgenic plants that express the selection marker gene bar on T-DNA pJP3416-GA7. Surviving herbicide-tolerant T1 plants are grown to maturity after being allowed to self-fertilize and the resulting T ₂ seed is harvested. Five transgenic plants were obtained, only three of them contained full T-DNA.

Lipid is extracted from a pool of about 20 seeds from each of the three plants, which contained total T-DNA. Two of the pooled samples contained very low, barely detectable levels of DHA, but the third pool of samples contained about 4.7% DHA. Thus, lipid was recovered from 10 individual T2 seeds of a given plant, and the fatty acid composition was analyzed by GC. Data on the composition of fatty acids of individual seeds for this transformed line are also shown in table. 4. Compiled data from profiles (Table 4) of the total lipid of seeds are shown in Table. five.

DHA was present in six out of 10 individual seeds. Four other seeds did not contain DHA and were adopted as null segregants without T-DNA based on the hemizygosity of the T-DNA insertion in the mother plant. The extracted lipid from the single seed with the highest DHA level contained 9.0% DHA, while the combined percentage for EPA, DPC and DHA was 11.4%.

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Table 4

Fatty acid composition of the total lipid of seeds from transgenic T2 Camelina sativa seeds transformed with T-DNA from pJP3416-GA7. Fatty acid compositions are given for a pooled batch (FD5.46) seed and for 10 individual seeds ranked (left to right) from highest to lowest ________________________ DHA ________________________

Fatty acid Pool FD5.46 # 2 #four #8 # 7 #nine #one # 3 #five # 6 #ten 14: 0 0 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 16: 0 11.6 12.1 12.3 12.1 13.2 123 12.8 11.9 11.4 11.5 11.7 16: 1 0.2 0.0 0.1 0.1 0.0 0.2 0.0 0.2 0.2 0.2 0.2 16: 3 0.3 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18: 0 3.7 3.3 3.2 3.2 3.0 3.1 3.2 3.3 3.1 3.2 3.2 18: 1 10.8 8.0 8.0 8.6 8.5 9.4 11.0 10.2 8.3 9.4 8.6 18: 1Δ11 1.7 1,3 1.4 1.4 1.7 1.4 1.5 1,3 1,3 1,3 1,3 18: 2 24.7 18.2 19.5 19.2 18.5 20.1 23.8 32.2 30.3 29.8 31.6 18: 3ω3 27.4 26.7 26.6 27.3 28.9 28.2 27.4 28.3 29.2 29.5 28.2 18: 3ω6 0.2 1.4 0.3 0.3 0,4 0.2 0.5 0.0 0.5 0,4 0.6 20: 0 1.6 1.4 1,3 1.4 1,2 1.4 1.4 1.8 2.1 1.9 2.0 18: 4ω3 2.2 6.8 6.4 5.7 7.2 5.7 4.1 0.0 0.0 0.0 0.0 20: 1Δ11 5.3 4.4 4.6 4.8 3.3 4.1 3.5 4.4 6.1 5.8 5.5 20: 1 iso 0,4 0.3 0.3 0.3 0.3 0.3 ο, ο 0.5 0.6 0.5 0.5 20: 2ω6 0.8 0.8 0.9 0.8 0.6 0.8 0.7 1,3 1.5 1.4 1.4 20: 3ω3 0.6 0.8 0.8 0.8 0.7 0.8 0.7 0.6 0.7 0.7 0.6 22: 0 0,4 0.5 0.5 0.5 0,4 0.5 0.5 0.6 0.6 0.6 0.6 20: 4ω3 0.2 0.3 0.3 0.3 0,4 0,4 0.5 0.0 0.0 0.0 0.0 22: 1 1.1 1.1 1,2 1.1 0.5 0.9 0.8 1.6 2.2 1.9 2.0 20: 5ω3 0.7 1,3 1.6 1.5 1.6 1.1 1.7 0.0 0.0 0.0 0.1 22: 2ω6 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.3 0.2 0.2 22: 4ω6 + 22: 3ω3 0.3 0.2 0.3 0.3 0.0 0.3 0.0 0,4 0.6 0.5 0.5 24: 0 0.3 0.3 0.3 0.3 0.0 0.3 0.0 0,4 0,4 0,4 0,4 24: 1 0.3 0,4 0,4 0.3 0.0 0.3 0.0 0.5 0.6 θ, 5 0.5 22: 5ω3 0.3 1.1 1,2 1.1 1.1 0.9 0.8 0.0 0.0 0.0 0.0 22: 6ω3 4.7 9.0 8.5 S3 83 7.1 4.9 0.0 0.0 0.0 0.0

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Table 5

Compiled data from the total lipid profiles of the seeds for the transgenic seed shown in table. 4. Calculations do not include minor fatty acids included in table. four

Parameter Pool FD5.46 # 2 #four #8 # 7 #nine #one # 3 #five # 6 #ten General SHZ (% of total LCD) 36.1 46 45.4 45 48.2 44.2 40.1 28.9 29.9 30.2 28.9 Common sb (% of total zhu 25.8 20.4 20,7 20.3 19.5 21.1 25 33,7 32.6 31.8 33.8 Ratio ®3 / ω6 1.40 2.25 2.19 2.22 2.47 2.09 1.60 0.86 0.92 0.95 0.86 The ratio ω6 / ω3 0.71 0.44 0.46 0.45 0.40 0.48 0.62 1.17 1.09 1.05 1.17 General new m3 (% of total LCD) 8.1 18.5 18 16.9 18.6 15.2 12 0 0 0 OD General new ®6 (% of total LCD} 1.1 2.2 1,2 1.1 one one 1,2 1.5 2,3 2 2.2 Ratio of new SOPs 7.36 8.41 15.00 15.36 18.60 15.20 10.00 0.05 The ratio of new ωό / ω3 0.14 0.12 0.07 0.07 0.05 0.07 0.10 22.00 Efficiency of converting OK to EPA 8.2% 15.6% 15.5% 15.1% 15.1% 12.8% 10.5% 0.0% 0.0% 0.0% 0.1% Efficiency of conversion of OA to DHA 6.7% 12.3% 11.6% 11.5% 11.4% 10.0% 7.0% 0.0% 0.0% 0.0% 0.0% The efficiency of the conversion of LA into EPA 9.2% 17.2% 17.1% 16.7% 16.2% 13.9% 11.4% 0.0% 0.0% 0.0% 0.2% Efficiency of conversion of LA to DHA 7.6% 13.6% 12.9% 12.7% 12.3% 10.9% 7.5% 0.0% 0.0% 0.0% 0.0% Efficiency of conversion of ALA to EPA 15.8% 24.8% 24.9% 24.2% 22.8% 20.6% 18.5% 0.0% 0.0% 0.0% 0.3% Efficiency of conversion of ALA to DHA 13.0% 19.6% 18.7% 18.4% 17.2% 16.1% 12.2% 0.0% 0.0% 0.0% 0.0% Total Saturated Fatty Acids 17.6 17.8 17.8 17.6 18 17.8 18.1 18.2 17.7 17.8 18.1 Total Monounsaturated Fatty Acids 19.8 15.5 16 16.6 14.3 16.6 16.8 18.7 19.3 19.6 18.6 Total polyunsaturated fatty acids 62.5 66.6 66.4 65.6 67.7 65.6 65.1 63 63.1 62.5 63.2 General C20 9.6 9.3 9.8 9.9 8.1 8.9 8.5 8.6 eleven 10.3 10.1 General C22 5.4 10.3 ten 9,7 9.4 8.3 5.7 0.6 0.9 0.7 0.7 Ratio General C20 / C22 1.78 0.90 0.98 1.02 0.86 1.07 14.9 14.33 12.22 14.71 14.43

The homozygous seed of this line is obtained in the T4 generation. Up to 10.3% DHA is produced in the FD5-46-18-110 event with an average of 7.3% DHA observed for the entire T4 generation. The next generation (T5) was obtained for the purpose of additional testing of the stability of PUFA production over several generations, especially DHA. It was found that the observed maximum DHA levels were stable in the fifth generation, even though the DHA content in the seed pool did not stabilize until the T4 generation due to the presence of numerous transgenic loci. Additionally, batches of T5 seed were germinated on MC in vitro media together with maternal C. sativa seed with no apparent difference in efficacy or germination rate. Further generations of the transgenic line (generations T6, T7, etc.) did not show a decrease in the level of DHA in the seed. The transgenic plants had full male and female fertility, and the pollen showed about 100% viability relative to wild-type plants. Analysis of oil content in seeds containing different levels of DHA did not reveal a correlation between DHA level and oil content, in contrast to the correlation observed for Arabidopsis thaliana.

In several further transgenic lines, the DHA content in individual seeds from independent events exceeded 12%. The transgenic: nil ratio for these lines was about 3: 1 to 15: 1. Analysis of characteristic profiles of fatty acids for samples with the highest DHA content from each construct revealed only 1.2-1.4% GLA in the absence of other new ω6 RNA. Conversely, it was found that new ω3 PUFA (SDA) ω3 DC-PUFA (ETC, EPA, DPK,

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DHA) accumulate up to 18.5% at a DNA level of 9.6% in the total fatty acid content. The desaturation rate Δ6 was 32%, and the EPA content was 0.8% of the total fatty acid content.

The Δ5 elongation efficiency was 93% and the Δ6 elongation efficiency was 60%. DHA was found in the polar lipid fraction of the GA7 seed lines.

It was noted that the observed segregation ratios (~ 3: 1 to ~ 15: 1) indicate that one or a maximum of two transgenic loci were required for DHA production in C. saliva at levels similar to those in fish oil. This is important for the ease of breeding the transgenic trait, as well as for the stability of the transgene.

Homozygous seed is planted in several greenhouses in order to obtain a total of over 600 individual plants. The oil was extracted from the seed using a variety of methods, including Soxhlet, acetone and hexane extraction.

^{A 13} C NMR analysis of regiospecificity for the seed oil of transgenic C. saliva was carried out in order to determine the positional distribution of ω3 DC-PUFA in TAG. An event with about equal EPA and DHA was chosen to maximize the response to these fatty acids, with a sn-1.3 to sn-2 ratio found at 0.75: 0.25 for EPA and 0.86: 0.14 for DHA, while the objective distribution should have been 0.66: 0.33. Those. 75% of EPA and 86% of DHA were located at the sn-1,3 position of TAG. This indicates that both fatty acids were preferentially localized to the TAG positions of C. saliva, although the preference for EPA was less pronounced than for DHA. The fact that DHA was preferentially found in sn-1,3 was similar to the results previously reported for A. thaliana seed (Petrie el al., 2012).

Since the number of independent transgenic lines obtained in the above-disclosed transformation experiments was low, further transformations of C. saliva were carried out using the GA7-modB motif construct (Example 3). A greater number of transformants were obtained, and homozygous lines producing DHA in an amount of more than 20.1% were identified.

Example 3. Modifications of T-DNA encoding DHA pathways in plant seeds.

To improve the level of DHA production in B. napus in comparison with the levels described in WO2013 / 185184, the binary vectors pJP3416-GA7-modA, pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7- modE and pJP3416-GA7-modF were constructed as described in WO2013 / 185184 and tested in transgenic plants. These binary vectors are constructs of pJP3416-GA7 and were designed to further enhance DHA synthesis in plant seeds, especially by improving the functions of Δ6 desaturase and Δ6 elongase. The accumulation of SDC was observed in some seeds transformed with the GA7 construct, due to the relatively low efficiency of Δ6 elongation compared to Δ5-elongase, therefore, among other modifications, the position of two elongase genes was changed in T-DNA.

The two sequences encoding the elongases in pJP3416-GA7 were swapped with TDNA, resulting in pJP3416-GA7-modA by first cloning a new P. cordala Δ6 elongase cassette between the SbfI sites of pJP3416-GA7 to replace the P. cordala Δ5 elongase cassette. This construct was further modified by exchanging the FP1 promoter driving Δ6-desaturase M, pusilla, with the conlinin Cnl2 promoter (pLuCnl2) to obtain pJP3416-GA7-modB. This modification was carried out in order to increase the expression of Δ6-desaturase, and thus the efficiency of the enzyme. It is believed that the Cn12 promoter may result in higher transgene expression in B. napus than the truncated napin promoter.

Eight transgenic events pJP3416-GA7-modB A. lhaliana and 15 transgenic events pJP3416-GA7-modG A. lhaliana were obtained. There is 3.4 to 7.2% DHA in the pJP3416-GA7-modB seed pool and 0.6 to 4.1% DHA in the T2 seed pool of pJP3416-GA7-modG. Some of the pJP3416-GA7-modB events with the highest level are sown on breeding media and surviving seedlings are selected for the next generation. The seed is analyzed for DHA content. Since the pooled T1 seeds represent transgenic segregated populations and include any null segregants, DHA levels in homozygous seed of offspring plants are expected to be higher, up to 30% of the total fatty acids in the seed oil. Other modified constructs were used to transform A. lhaliana. Although only a small number of transformed lines were obtained, none of them produced higher levels of DHA than the modB construct.

The pJP3416-GA7-modB construct was additionally used to generate transformed B. napus plants cv. Oscar and a series of varietal lines designated NX002, NX003, NX005, NX050, NX052 and NX054. A total of 1558 transformed plants were obtained, including 77 independent transformed plants (T0) for Oscar transformation, and 1480 independent plants for varietal lines, including 189 for NX005, which is a line with a high content of oleic acid in seed oil due to mutations in the FAD2 genes. Other cultivar lines contained higher levels of LA and ALA. Transgenic plants that showed more than 4 copies of T-DNA by digital PCR (Example 1) were discarded; about 25% of T0 plants were discarded according to this criterion. About 53% of transgenic T0 plants contained 1 or 2 copies of T-DNA, according to

- 58 037184 digital PCR method, 12% contained about 3 copies, and 24% contained 4 or more copies. The seed (T1 seed) was harvested from about 450 transgenic lines after self-pollination, achieved by isolating the plants with pouches during flowering to avoid crossbreeding. T1 seeds are harvested from the rest of the transgenic plants after maturation. About 1-2% of the plant lines were sterile in terms of male or female fertility and did not produce viable seeds; such T0 plants were discarded.

Seed pools (20 T1 seeds in each pool) were tested for DHA levels in the seed oil pool, and lines that showed the highest levels were selected. In particular, lines were selected with a DHA content of at least 2% of the total fatty acid content in the T1 seed pool. About 15% of transgenic lines were selected in this way; the remaining 85% were discarded. Some of them were designated as CT132-5 (Oscar variety), CT133.15-15, -24, -63, -77, -103, -129 and -130 (in NX005). Selected lines in NX050 included CT136-4, -8, -12, -17, -19, -25, -27, -49, and -51. Twenty seeds from the selected lines were allowed to swell, including CT132.5 and 11 CT133.15 seeds, and two days later, oil was extracted from half of the cotyledon of each of the individual seeds. The other half of the cotyledon with embryonic axes is kept and cultured on media to preserve specific lineages of offspring. The composition of fatty acids in the oil is determined; data for ST132.5 are given in table. 6. The level of DHA in 10 of the analyzed 20 seeds, according to GC analysis, is in the range of 7-20% of the total fatty acid content. Other seeds contained less than 7% DHA and may contain a partial (incomplete) copy of the T-DNA from pJP3416-GA7-modB. Apparently, the transgenic line contains multiple transgene inserts that have been genetically separated. Seeds of the transgenic line CT133.15 showed DHA levels in the range of 0-5%. Seeds without DHA were likely zero segregants. These data confirm that the modB construct functions properly to generate DHA in rapeseed.

Twenty or 40 individual seeds (T2 seeds) obtained from each of a plurality of T1 plants after self-pollination, from selected transformed lines, were individually tested for fatty acid composition. Seeds have been identified containing DHA levels above 20% (Table 7). Two representative samples, CT136-27-18-2 and CT136-27-18-19, contained 21.2 to 22.7% DHA, respectively. The total ω3 fatty acid content of these seeds was about 60% as a percentage of the total fatty acid content, and the ω6 content was less than 10%. Other sets of 20 or 40 T2 seeds from each of the T1 plants were tested for fatty acid composition. Seeds containing up to 34.3% DHA were identified, for example, among the CT136-27-47-25 seed (Table 9). The composition of fatty acids for seed oil obtained from CT136-27-47-25 is shown in table. 9. The fatty acid composition was 34.3% DHA, along with about 1.5% DPA, 0.6% EPA and 0.5% ETA. SDC was about 7.5%, ALA was about 21.9%, and LA was about 6.9%. New ω6 PUFAs demonstrated 1.1% GLA, in the absence of detectable ω6-C20 or -C22 DC-PUFAs. Total saturated fatty acids: 9.6%; monounsaturated fatty acids, 12.5%; total PUFA, 75.2%; total ω6 PUFA (including LA), 7.2%; total ω3 PUFA, 66.9%; the ratio of the total content of ω6: ω3 fatty acids, 9.3: 1; new ω6: new ω3 fatty acids, 37: 1. The efficiency of each of the oleic acid to DHA fermentation steps was as follows: Δ12 desaturase, 90%; Δ15 / ω3 desaturase 89%; Δ6-desaturase, 67%; Δ6 elongase, 83%; Δ5-desaturase, 99%; Δ5-elongase, 98%; Δ4 desaturase, 96%. The overall conversion efficiency of oleic acid to DHA was about 50%. Thus, it was evident that seeds producing DHA in the range of 20.1-35% of the total fatty acids in seed oil could be identified and selected, including seeds containing from 20.1 to 30% DHA or from 30 to 35% DHA in total fatty acids.

Oil content in some seeds was reduced from about 44% in wild-type seeds to about 31-39% in some of the DHA-producing seeds, but was similar to wild-type levels in other DHA-producing seeds.

Various transformed plant lines that produced DHA at levels of at least 10% in T2 seed were crossed and self-pollinated F1 offspring to produce F2 offspring homozygous for multiple T-DNA insertions. The seed oil from the homozygous seed was analyzed and found that DHA accounts for 30 or 35% of the total fatty acids in the seed oil.

TAGs in oil obtained from CT136-27-18-2 and CT136-27-18-19 were analyzed by the regiospecific ¹³ C NMR method for the positional distribution of DHA in the glycerol base of TAG molecules. DHA was predominantly attached at the sn-1,3 position. More than 70%, in fact more than 90% of the DHA was in the sn-1,3 position.

In several further transgenic lines, the DHA content in individual seeds from independent events exceeded 12%. The ratio of transgenic: null for these lines was about 3: 1, which corresponded to one transgenic locus, or 15: 1, which corresponded to two transgenic loci. Analysis of characteristic fatty acid profiles for samples from each construct with the highest DHA levels revealed only 1.2-1.4% GLA in the absence of other detectable

- 59 037184 new ω6 PUFA. In contrast, the new ω3 PUFA (SDA) and ω3 DC-PUFA (ETC, EPA, DPA, DHA) accumulated to a total level of 25.8% for the modF construct and 21.9% for the modG construct, compared with 18.5 % for GA7-transformed seed. DHA levels in oil from these seeds were 9.6%, 12.4%, and 11.5%, respectively. The degree of Δ6-desaturation was lower in GA7-transformed seeds than in modF- and modG-transformed seeds (32% versus 47% and 43%), which led to a decrease in ALA levels in modF and modG seeds, compared to GA7. Another significant difference was the accumulation of EPA in the modF seed (3.3% versus 0.8% in the other two transgenic seeds), and this was reflected in the decrease in the degree of Δ5-elongation observed in the modF seed (80%) compared to GA7 seeds. and modG (93 and 94%). There was a slight increase in the degree of Δ6-elongation in these seeds (66% versus 60 and 61%), although the amount of SDC actually increased due to slightly more active Δ6-desaturation. DHA was found in the polar lipid fraction of the GA7 seed lines.

The composition of fatty acids in the T1 seed lipid was analyzed for 70 independent transgenic plants of B. napus cultivar line NX54 transformed with the modB T-DNA construct. One of these transgenic plants was found to produce a seed containing DPA, but no DHA was present in the seed oil. In the T1 seed of this line (ST-137-2), about 4% of the duodenum was produced, in the absence of detectable DHA in the T1 seed pool. The inventors have investigated the question of whether this is caused by inactivation of the Δ4desaturase gene in a particular insertional T-DNA as a result of spontaneous mutation. Analysis by PCR and DNA sequencing showed the presence of a deletion, which was identified as deleted nucleotides 12988-15317 T-DNA motif GA7-modB (SEQ ID NO: 2). The deleted nucleotides correspond to a portion of the Linus Cn12 promoter directing the expression of the Δ4 desaturase coding region, as well as the Δ4 desaturase coding region itself, explaining why seeds transformed with T-DNA and containing the deletion did not produce DHA.

About 50 T1 seeds from this transgenic line germinated, and one emerging cotyledon from each was analyzed for the composition of fatty acids in the residual oil. Selected seedlings showing more than 5% WPC were subsequently grown to maturity and the T2 seed was harvested. The composition of fatty acids in the seed pool is illustrated in table. 8, with more than 7% of the duodenum being observed in these lines. The T4 seed was obtained from the CT-137-2 DPK line of B. napus and analyzed for the fatty acid profile. Up to 13% DPC was observed in the pool of mature seed samples.

The seed oil, which contained about 10% WPC, was treated with a mild alkali to hydrolyze the fatty acids.

Another transgenic line, designated B0003-514, showed about 10-16% duodenum in T2 seed. Seed was selected containing 15.8% DPA, 0.2-0.9% DHA and 0.1-2.5% EPA. The T2 seed population showed a segregation ratio of 1: 2: 1 for high content: low content: no duodenum, indicating the presence of a single genetic locus for the production of DPA in the indicated transgenic line.

The oil was extracted using a screw press from seed samples producing LCPUFA, thus obtaining a cake.

Construction design.

Although the focus of this experiment was on demonstrating DHA and WPC production in oilseed species, the results noted above were also interesting from a structural design perspective. First, switching the localization of the region encoding the Δ6 and Δ5 elongase in the modF design leads to the intended profile modifications when more EPA accumulates due to the lower degree of Δ5 elongation.

A concomitant increase in the degree of Δ6 elongation was observed, but did not lead to lower SDR levels. This was the result of an increase in the degree of Δ6 desaturation in the transformed modF seed caused by the addition of an additional M. pusilla Δ6 desaturase expression cassette, as well as the replacement of the truncated napin promoter (FP1) with the more active flax conlinin 2 promoter. The slightly less pronounced increase in the degree of Δ6-desaturation observed in the case of the modG construct was caused by capitalization on the cassette of Δ5-elongase with high expression in GA7. Switching the positions of the regions encoding Δ6-desaturase and Δ5-elongase resulted in a higher degree of Δ6-desaturation. The Δ5-elongase activity did not decrease in this case due to the replacement of the FP1 promoter with the Cn12 promoter.

These data confirm that the modB, modF and modG constructs are efficient in terms of DHA production in Camelina seed, relative to Arabidopsis and rapeseed.

The inventors believed that, in general, the efficiency of rate-limiting enzyme activity in the biochemical pathway of DHA could be higher in multicopy T-DNA transformants compared to single-copy T-DNA transformants, or could be increased by insertion of several genes into T-DNA. encoding an enzyme that can be restrictive in the biochemical pathway. Evidence of the possible importance of multicopy transformants was observed in Arabidopsis seeds transformed with construct GA7, in which the event with the highest DHA yield contained three T-DNA insertions in the host genome. Several genes may be identical, or preferably are different variants that encode the same polypeptide or are under the control of different promoters with overlapping expression patterns. For example, increased expression can be achieved by expressing several regions encoding Δ6 desaturase, even if the same protein is produced. For example, in pJP3416-GA7-modF and pJP3416-GA7-modC, two versions of M. pusilla Δ6 desaturase were present and expressed by different promoters. In the coding sequences, codons were used in different ways and thus their nucleotide sequences were different in order to reduce the potential for silencing or the effects of co-suppression, but resulted in the production of the same protein.

Table 6

Lipid fatty acid composition during germination of B. napus T1 transgenic seeds containing T-DNA from the GA7-modB construct. Lipids additionally contained 0.1-0.3% of each of C16: 1, C16: 3, C24: 0 and

C24: 1 and did not contain C20: 1Δ11

Seed about fe chg h, oe ϋ 0U “Ί 0.1 4.21.8

0.1 4.74.0

0.1 3.71.8

0.1 4.62.9

0.1 4.01.7

0.1 3.5 1.6

0.1 6.01.7

0.1 4.92.7

0.1 3.92.4

0.1 3.72.1

0.1 5.73.8

0.1 4.62.4

0.1 4.34.2

0.1 6.34.0

0.1 5.13.3

0.1 4.44.0

0.2 7.24.9

0.1 4.02.3

oo yu az oo z ϋ z s

29.9 2.5 9.9 0.1 23.0 2.3 7.4 0.3 55.1 1.9 4.7 0.2 22.1 1.8 6.6 0.4 27, 4 2.1 8.1 0.3 59.8 2.0 4.3 0.1 16.6 2.6 23.9 1.0 12.9 1.4 11.7 0.3 41.6 1 , 7 21.5 0.0 30.9 1.7 19.2 0.4 41.2 2.4 26.7 2.1 25.5 1.7 16.1 0.3 19.4 1.6 9.2 0.1 10.5 2.3 8.4 0.3 16.8 2.4 11.2 0.3 16.2 1.5 11.6 0.2 15.0 2.1 8, 9 0.3 64.8 1.2 7.2 0.1

38.4 0.5 0.8 1.0 29.3 1.0 4.3 1.1 15.2 0.8 1.8 1.4 26.5 1.0 7.2 1.0 26, 4 0.6 2.8 1.0 18.5 0.6 0.5 1.3 23.2 0.6 5.4 0.8 34.3 0.9 5.0 0.9 23.4 0 , 7 0.0 1.2

23.6 0.7 2.1 1.1 7.2 1.3 0.3 1.2 28.9 0.8 3.9 1.1

45.5 1.0 0.2 1.1 31.1 1.3 3.9 0.8 28.8 1.0 4.5 0.9 33.5 0.9 2.8 1.1

25.9 1.4 5.1 0.9 12.5 1.0 3.5 1.5

0.1 2.1 0.32.8

0.1 1.9 0.46.9

0.1 0.3 0.5 11.3

0.1 0.8 0.5 11.2

0.1 1.5 0.37.6

0.0 0.7 0.36.0

0.2 0.6 0.4 2.6

0.2 2.4 0.5 4.1

0.1 2.2 0.40.0

0.1 1.5 0.4 3.6

0.2 0.3 0.84.8

0.1 1.9 0.43.9

0.1 5.2 0.4 2.6

0.1 2.3 0.64.6

0.1 2.1 0.63.2

0.2 3.7 0.44.6

0.0 1.6 0.84.9

0.1 0.0 0.70.0

0.3 0.1 0.5 3.9 1.0 0.0 1.7 9.5 0.0 0.0 0.0 0.0 1.9 0.0 1.7 8.7

1.5 0.0 1.8 12.2 0.0 0.0 0.0 0.0

1.1 0.0 1.7 9.9

1.3 0.0 1.8 13.8 0.0 0.1 0.0 0.0 0.6 0.0 0.7 6.9 0.0 0.0 0.0 0.0 0, 6 0.0 1.1 6.2 0.3 0.2 0.4 3.4

1.8 0.1 2.5 18.1 1.5 0.1 1.8 15.1 0.7 0.1 1.3 12.1 2.1 0.0 2.2 15.0 0, 0 0.0 0.0 0.0

Table 7

Composition of lipid fatty acids in T2 transgenic seeds of B. napus containing T-DNA from the GA7-modB construct

Sample (seed T2) ST136-27-18-1 ST136-27-18-2 ST136-27-18-3 ST136-27-18-4 ST136-27-18-5 ST136-27-18-6 ST136-27 -18-7 ST136-27-18-8 ST136-27-18-9 ST136-27-18-10 ST136-27-18-11 ST136-27-18-12 ST136-27-18-13 ST136-27- 18-14 ST136-27-18-15 ST136-27-18-18 ST136-27-18-19 ST136-27-18-20

5.0 2.6 25.4 3.6 6,7 0.2 37.5 1.4 1.0 0.1 2.1 0.8 0,4 0.9 10.2 53.4 7.1 0.13 60.5 7.1 2.8 16.9 4.3 5.5 0,4 29.1 5.4 0.8 0.1 1,2 0.5 0.5 1.9 21.2 59.8 6.1 0.10 66.0 5.4 2.5 26.5 3.8 6.4 0,4 26.4 4.7 1.0 0.1 0.7 1.1 0.6 1,2 17.3 52.0 6.9 0.13 58.9 5.3 2.4 34,7 4.0 5.9 0.3 30.3 1,3 1.1 0.1 1.1 1.5 0.3 0,4 9.3 44.4 6.3 0.14 50.7 4.8 2.7 34.5 • 3.8 5.6 0.3 23.5 3.9 1,2 0.1 0.7 1.1 0.5 1.1 14.2 45.1 6.0 0.13 51.1 5.0 2.1 54.3 3.8 5.7 0.2 18.2 0.6 1.5 0.1 1.1 0.7 0.1 0.2 4.4 25.5 6.1 0.24 31.5 5.3 2.1 43.8 4.2 5.6 0,4 18.3 2.2 1,3 0.2 0.6 1.5 0,4 0.5 11.6 35.2 6.2 0.18 41.4 5.4 2.7 25.8 4.1 6,7 0,4 26.6 5.7 1.0 0.1 0.6 1,3 0.6 1.2 15.8 51.9 7.1 0.14 59.0 4.6 1.6 53.8 3.7 17.5 0.5 9.2 0.5 1.6 0.3 0.6 0,4 0.1 oh, 1 3.7 14.5 18.3 1.26 32.8 4.8 2.4 44.1 3.7 5.4 0,4 19.1 2,3 1.1 0.1 0.6 1.5 0.5 0.8 11.4 36.1 5.9 0.16 42.0 5.1 2.2 48.3 4.1 10.9 0.7 12.5 1,2 1,3 0.2 0.5 1.5 0.3 0.3 9.1 25.3 11.8 0.47 37.1 5.3 2.7 23.3 3.7 6.0 0,4 27.9 4.9 0.9 0.1 0.7 1,3 0.8 1.5 18.5 55,7 6.6 0.12 62.2 5.5 3.4 30.7 5.6 5.1 0,4 23.1 3.5 1.1 0.1 1,2 1.1 0.6 1,2 14.9 45.8 5.5 0.12 51.3 5.4 2,3 23.9 3.5 6.0 0,4 30.1 3.7 1.0 0.1 1.0 0.7 0.6 U 18.2 55.5 6.6 0.12 62.1 5.0 2,3 45.4 4.0 5.3 0,4 16.2 2,3 1,2 0.1 0.5 1.9 0.6 0.7 12.3 34.4 5.8 0.17 40.3 5.1 2,3 29.0 3.6 5.7 0,4 26.5 3.8 1.1 0.2 0.8 0.8 0.6 1.0 17.4 50.8 6.3 0.12 57.1 5.8 2,3 19.7 4.2 6,7 0.7 23,7 7,7 0.9 0.1 0,4 0.7 0.6 1.7 22.7 57.6 7.5 0.13 65.1 5.7 2.9 23.2 4.0 5.6 0.3 35.8 2.4 1.0 oh, 1 1.3 1.1 0.5 1.0 13.0 55.1 6.1 0.11 61.2

ARA (C20: 4ω6) and DPKω6 were not found in any of the samples. In addition, the samples contained 0.1% C14: 0, about 0.2% or 0.3% C16: 1, about 0.1-0.3% C16: 3, from about 0.7% to 1.0 % C20: 0, about

0.3% C22: 0, with some samples containing trace amounts (<0.1%) C20: 1Δ13, C22: 3ω3, C24: 0 and C24: 1

- 61 037184

Table 8

Lipid fatty acid composition in transgenic T2 seeds of B. napus transformed with T-DNA from the GA7-modB construct, with a mutation in the Δ4-desaturase gene. Lipids additionally contained about 0.1% 14: 0, 0.2% 16: 3, 0.2-0.4% GLA, 0.1% 20: 1Δ13, 0.3-0.4% 22: 0, and ARC, DPAω6 (22: 5ω6), 16: 2 and 22: 1 were not detected

© 0 £ C "five 0 05 C “Y and and GP 1 g and S 9 <h * and Ό e * and Joint venture nine S and one and SP £ and 9 rj ri and SP 9 ri η and e and count V SP i ri η and Joint venture and 3 and © © mid and ST-137-2-34 5.3 0.2 3.7 26.8 3.1 12.4 29.1 0.8 2.5 0.8 oh, 1 0.0 1.1 1.7 0.8 0.0 0.1 0.1 0.1 10.0 0.0 ST-137-2-38 5.3 0.2 4.2 24.4 3.0 12.6 29.4 0.9 2.5 0.8 0.1 0.0 1,3 2.2 0.9 0.0 0.1 0.2 0.1 10.8 0.0 ST-137-2-48 5.0 0.2 4.2 24.1 3.1 11.9 31.0 0.9 2.4 0.9 0.1 0.0 1.5 2.0 1.0 0.0 0.1 0.1 0.1 10.5 0.0 ST-137-2-51 5.7 0.2 4.6 22.3 3.4 12.3 34.5 1.0 2.0 0.8 0.1 0.0 1.9 1,2 0.5 0.0 0.1 0.2 0.2 7,7 0.0 ST-137-2-59 5.4 0.2 3.9 25.7 3.4 12.9 27.8 0.9 2.6 0.8 0.1 0.0 1.0 1.9 0.9 0.0 0.1 0.2 0.1 11.0 0.0

Table 9

Fatty acid composition of oil from T2 seed of B. napus transformed with T-DNA from GA7-modB motif

The seed oil samples additionally contained 0.1% C14: 0; 0.2% C16: 1; 0.1% C20: 3ω6; did not contain C22: 1 and C22: 2ω6; contained 0.1% C24: 0 and 0.2% C24: 1, 2.6% other fatty acids.

Example 4. Analysis of TAG from transgenic seed of A. thaliana, producing DHA.

The positional distribution of DHA in TAG from transformed A. thaliana seed was determined by NMR. The total lipid is extracted from about 200 mg of semen, first by crushing it under a layer of hexane, followed by transferring the crushed semen into a glass tube containing 10 ml of hexane. The tube is heated to about 55 ° C in a water bath, then vortexed and centrifuged. The hexane solution is removed and the procedure is repeated with additional portions of 4 x 10 ml. The extracts are combined, evaporated using a rotary evaporator, and the TAG in the extracted lipid is purified from polar lipids by passing through a short silica column using 20 ml of 7% diethyl ether in hexane. The positional distribution of the acyl group on the purified TAG is quantified as previously described (Petrie et al., 2010a and b).

The analysis demonstrated that most of the DHA in the total seed oil is located at the sn-1/3 positions of the TAG, and a small amount is found at the sn-2 position. In contrast, TAGs from ARA-producing seeds demonstrated that 50% of ARA (20: 4Δ ^5,8,11,4 ) are located at the sn-2 position of transgenic rapeseed oil, while the expected value for a random distribution is only 33% (Petrie et al., 2012).

Additionally, the total lipid from the transgenic seed of A. thaliana was analyzed by LC-MS with a triple quadrupole lens to determine the main DHA-containing species of triacylglyceride (TAG). It was found that the TAG varieties with the highest DHA content are DHA-18: 3-18: 3 (TAG 58:12; the nomenclature does not describe positional distribution), and in second place is DHA-18: 3-18: 2 (TAG 58:11). Tri-DHA TAG (TAG 66:18) is observed in total seed oil, albeit with low detectable levels. Other major DHA-containing TAG varieties included DHA-34: 3 (TAG 56: 9), DHA-36: 3 (TAG 58: 9), DHA-36: 4 (TAG 58:10), DHA-36: 7 ( TAG 58:13) and DHK-38: 4 (TAG 60:10). The identity of the two main DHA-containing TAGs was further confirmed by quadrupole time-of-flight (Q-TOF) MS / MS.

Example 5. Analysis of the content and composition of sterols in oils.

Phytosterols from 12 vegetable oil samples purchased from commercial sources in Australia were characterized by GC and GC-MS methods as derivatives of O-trimethylsilyl ether (OTMSi-ether), as disclosed in example 1. Sterols were identified by retention data, interpretation of mass spectra and comparison with literature data and data of mass spectroscopy of laboratory standards. The amount of sterols is determined using an internal standard 5b (H) cholan-24-ol. The basic structure of phytosterol and the chemical structure of some of the identified sterols are illustrated in FIG. 3 and in table. ten.

Analyzed vegetable oils of sesame (Sesamum indicum), olives (Olea europaea), sunflower (Helianthus annus), castor oil plant (Ricinus communis), rapeseed (Brassica napus), safflower (Carthamus tinctorius), peanuts (Arachisoga hypimea), flax and soy (Glycine max). In order of decreasing relative content, in all oil samples, the main phytosterols were β-sitosterol (range 28-55% of total sterols), Δ5-avenasterol (isofucosterol) (3-24%), campeste

- 62 037184 roll (2-33%), Δ5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%) and Δ7-avenasterol (0.1-5%). Several other minor sterols have been identified: cholesterol, brassicasterol, chalinasterol, campestanol, and eburicol. Additionally, four C29: 2 and two C30: 2 sterols have been identified, but further research is needed to definitively identify these minor components. In addition, several other unidentified sterols were present in some of the oils, but due to their very low content, the mass spectra were not intense enough to allow identification of their structure.

Table 10

IUPAC names / systematic names of identified sterols

Sterol No. Common name (s) IUPAC / systematic name one cholesterol cholest-5-en-C β-ol 2 brassicasterol 24-megylcholecta-5,22E-diene-3β-ol 3 chalinasterol / 24-methylenecholesterol 24-methylcholesta-5.24 (28) E-diene 3β-οπ four campesterol / 24-methylcholesterol 24-methylcholest-5-en-3 β-ol five campestanol / 24-methylcholestanol 24-methylcholestan-3 β-ol 7 Δ5 -stigmasterol 24-ethylcholesta-5,22E-diene ^ -ol nine ergost-7-en-Z β-ol 24-methylcholecτ-7-en-Zβ-ol eleven eburicol 4,4,14-trimethylergosta-8.24 (28) - ΛΗβΗ-3β-οπ 12 β-sitosterol / 24-ethylcholesterol 24-ethylcholest-5-en-3 β-ol thirteen O5-avenasterol / isofucosterol 24-ethylcholecta-5 _J 24 (28) Z-diene-Zβol 19 P7-stigmasterol / stigmast-7-en-Zb-ol 24-ethylcholest-7-en-3 β-ol twenty O7-avenasterol 24-ethylcholesta-7.24 (28) -diene-3 βol

The sterol content, expressed as mg / g oil, in decreasing order of quantity, was as follows: rapeseed oil (6.8 mg / g), sesame oil (5.8 mg / g), linseed oil (4.8-5.2 mg / g), sunflower oil (3.7-4.1 mg / g), peanut oil (3.2 mg / g), safflower oil (3.0 mg / g), soybean oil (3.0 mg / g ), olive oil (2.4 mg / g), castor oil (1.9 mg / g). The composition of sterols in% and the total content of sterols are shown in table. eleven.

In general, among all seed oil samples, β-sitosterol was the major phytosterol (range 30-57% of total sterols). For oils, a wide range of the proportion of other major sterols was observed: campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%), Δ7 stigmasterol (1-18%). The different types of oils contained a different sterol profile, with some profiles being quite different. In the case of rapeseed oil, it contained the highest proportion of campesterol (33.6%), while samples of other species generally contained lower levels, for example up to 17% in peanut butter.

Safflower oil contained a relatively high proportion of Δ7-stigmasterol (18%), while the content of this sterol was usually low in other oils, up to 9% in sunflower oil. Since they are specific to each species, the sterol profiles can thus be used as an aid to identify specific vegetable or vegetable oils and to check whether they are genuine or counterfeit with other oils.

Compared two samples from sunflower and safflower, in each case one of them was obtained by cold pressing of seeds and was unrefined, while the other was not obtained by cold pressing and was refined. Although some differences were observed, the two sources of oils had similar sterol composition and total sterol content, indicating that processing and refining had little effect on these two parameters. The sterol content varied threefold between samples and ranged from 1.9 to 6.8 mg / g. Rapeseed oil had the highest and castor oil had the lowest sterol content.

Example 6. Increased accumulation of DHA and DPA in the sn-2 position of TAG.

The authors of the present invention believed that the accumulation of DHA and / or DPA at the sn-2 position in TAG could be increased by co-expression of 1-acylglyceride-3-phosphate acyltransferase (LPAAT) with the biosynthetic pathway of DHA or DPA, for example, as provided by the GA7 construct or its options. Preferred LPAATs are those that can act on the polyunsaturated C22

- 63 037184 fatty acyl-CoA as a substrate, preferably DHA-CoA and / or DPA-CoA, especially those that can use both DHA-CoA and DPA-CoA as substrates, leading to an increase in the insertion of the C22 polyunsaturated chain at position sn-2 LPH with the formation of PC, relative to endogenous LPAAT. Cytoplasmic LPAAT enzymes often exhibit a variety of substrate preferences, especially if species synthesize and accumulate unusual fatty acids in TAG. It was shown that LFAAT2 from Limnanthes douglasii uses erucoyl-CoA (C22: 1-CoA) as a substrate for PC synthesis, in contrast to LFAAT 1 of the same species, which could not utilize substrate C22 (Brown et al., 2002).

Table 11

The content and composition of sterols in the analyzed vegetable oils

Common name for sterol Sesame Olive Sunflower Castor oil plant Rape Safflower Arachne Flax (flax seed) Soy cholesterol 0.2 0.8 0.2 0.1 0.3 0.2 0.2 0,4 0.2 brassicasterol 0.1 0.0 0.0 oh, 3 0.1 0.0 0.0 0.2 0.0 chalinasterol / 24-methylenecholesterol 1.5 0.1 0.3 1.1 2.4 0.2 0.9 1.5 0.8 campesterol / 24-methylcholesterol 16.2 2.4 7.4 8.4 33.6 12.1 17.4 15.7 16.9 campestanol / 24-methylcholestanol 0.7 0.3 0.3 0.9 0.2 0.8 0.3 0.2 0.7 C29: 2 * 0.0 0.0 0.1 0.0 0.1 0.5 0.0 U od D5-stigmasterol 6.4 1,2 7.4 18.6 0.7 7.0 6.9 5.1 17.6 unknown 0.5 1,3 0.7 0.8 0.7 0.7 0,4 0.7 1,3 ergost-7-en-Zr-ol 0.1 0.1 1.9 0.2 0,4 2.7 1.4 1.4 1.0 unknown 0.0 1,3 0.9 1,2 0.9 1.8 1,2 0.7 0.7 eburicol 1.6 1.8 4.1 1.5 1.0 1.9 1,2 3.5 0.9 β-sitosterol / 24-ethylcholesterol 55.3 45.6 43.9 37.7 50.8 40.2 57.2 29.9 40.2 D5-avenasterol / isofucosterol 8.6 16.9 7.2 19.3 4.4 7.3 5.3 23.0 3.3 triterpene alcohol 0.0 2.4 0.9 0.0 0.0 1.6 0.0 0.0 0.9 triterpene alcohol 0.0 0.0 0.7 0.0 0.0 2.8 0.0 0.0 0.0 D7-stigmasterol / stigmast-7-en-Zr-ol 2.2 7.1 9.3 2,3 0.9 10.5 1.1 7.9 5.6 D7-avenasterol 1,3 0.1 4.0 0.6- 0.2 2.0 0.7 0,4 0.6 Total sterols (mg / g oil) 5.8 2.4 4.1 1.9 6.8 3.2 3.2 4.8 3.0

C29: 2 * refers to a C29 sterol with two double bonds.

Known LPKATs were considered and many were selected for testing, including those that were not expected to increase the incorporation of DHA at the sn-2 position, as controls. Known LPKATs included: LPKAT2: Arabidopsis thaliana (SEQ ID NO: 40, accession number ABG48392, Kim et al., 2005), LPKAT Limnanthes alba (SEQ ID NO: 41, accession number AAC49185, Lassner et al., 1995), Slc1p Saccharomyces cerevisiae (SEQ ID NO: 42, accession number NP_010231, Zou et al., 1997), LPKAT 1 Mortierella alpina (SEQ ID NO: 44, accession number AED33305; US 7879591) and LPKAT Brassica napus (SEQ ID NO: 45 and SEQ ID NO: 46, accession numbers ADC97479 and ADC97478, respectively).

Arabidopsis LPKAT2 (also designated LPAT2) is an endoplasmic reticulum localized enzyme that has been shown to be active on substrates C16 and C18, but activity on substrates C20 or C22 has not been tested (Kim et al., 2005). LPKAT2 Limnanthes alba demonstrated the insertion of the C22: 1 acyl chain at the sn-2 position of PK, although the ability to use DHA or DPC as a substrate has not been tested (Lassner et al., 1995). The selected LPKAT S. cerevisiae Slc1p showed activity in the form of the use of 22: 1-CoA in addition to 18: 1-CoA as substrates, indicating broad substrate specificity based on chain length (Zou et al., 1997). Again, DHA-CoA, DPA-CoA, and other DC-PUFAs have not been tested as substrates. It was previously demonstrated that Mortierella LPKAT has activity against the fatty acid substrates EPA and DHA in the transgenic Yarrowia lipolytica (US 7879591), but their activity in plant cells is unknown.

Additional LFKAT have been identified by the inventors. Micromonas pusilla is a microalga that produces and accumulates DHA in oil, although the positional distribution of DHA to TAG has not been confirmed in this species. Micromonas pusilla LPKAT (SEQ ID NO: 43, accession number XP002501997) was identified by searching the genomic sequences of Micromonas pusilla using Arabidopsis LPKAT2 as the FLK-ST query sequence. Several candidate sequences emerged and the sequence XP002501997 was synthesized for testing against C22 DC-PUFA. LPKAT Ricinus communis was annotated as a putative LPKAT in the castor bean genome sequence (Chan et al., 2010). Four candidates for LPKAT from the castor bean genome were synthesized and tested on crude leaf lysates of infiltrated N. benthamiana leaf tissue. The candidate sequence disclosed herein demonstrated APCAT activity.

A number of LPKAT candidates have been aligned with the well-known LPKAT on a phylogenetic tree. It should be noted that the putative LFKAT Micromonas is not grouped with the putative C22 LFKAT of Micromonas, but has a diverging sequence.

As a primary analysis of various LPATs for their ability to use DHCKoA and / or DPK-CoA as a substrate, chimeric genetic constructs were obtained for constitutive expression of exogenous LPATs in N. benthamiana leaves, each under the control of the 35S promoter, as disclosed below: 35S: Arath -LFAT2 (Arabidopsis ER LFKAT); 35S :: Limal-LFKAT (LFKAT Limnanthes

- 64 037184 alba); 35S: Sacce-Slc1p (LPKAT S, cerevisiae); 35S: Micpu-LFKAT (LFKAT Micromonas pusilla); 35S: Moral-LFKAT1 (LFKAT Mortierella alpina); 35S: Brana-LPAAT1.13 (Brassica napus LPAAT1.13); 35S: Brana-LPAAT1.5 (Brassica napus LPAAT1.5). The 35S: p19 constructs, lacking exogenous LPKAT, were used in the experiment as a control: it was included in each N. benthamiana inoculation. Each of these constructs was introduced with Agrobacterium into the leaves of N. benthamiana as disclosed in Example 1, and 5 days after infiltration, the treated leaf zones were excised and pulverized to obtain a leaf lysate. Each lysate contains exogenous LPKAT, as well as endogenous enzymes for the synthesis of LPA. Reaction mixtures were prepared in vitro, separately adding ¹⁴ C-labeled OK and DHA to lysates. The reaction mixtures were incubated at 25 ° C, and the level of incorporation of ¹⁴ C-labeled fatty acids into FA was determined by TLC. The ability of each LPKAT to use DHA relative to ARA and C18 fatty acids was evaluated. It was found that LFKAT of meadow foam (Limnanthes alba), Mortierella, and Saccharomyces have activity against the substrate DHA, and radiolabeled PK occurs in the case of these, but not other LFKATs. The activity of all LPKAT was confirmed by control consumption of oleic acid.

To test the activity of LPKAT in seeds, a number of protein-coding sequences or LPKAT were inserted into a binary vector under the control of the conlinin promoter (pLuCn12). The resulting genetic constructs containing the chimeric genes Cn12Arath-LPKAT (negative control), Cn12: Limal-LPKAT, Cn2: Sacce-Slc1p and Cn12Moral LPKAT, respectively, were then used to transform A. thaliana plants producing DHA in seed to obtain stable transformants expressing LPKAT and the transgenic DNA pathway in a seed-specific form to test whether the incorporation of DHA at the sn-2 position of TAG will increase. In addition, the constructs are used to transform B. napus and C. sativa plants, which already contain the GA7 construct and its variants (examples 2 and 3), in order to obtain offspring carrying maternal and LFKAT genetic constructs. An increase in the incorporation of DHA and / or DPA at the sn-2 position of TAG relative to incorporation in plants without LPKAT-encoding transgenes was tested. The oil content is also increased in the seeds, especially in the case of seeds producing higher levels of DHA, neutralizing the tendency observed in Arabidopsis seed, as disclosed in example 2.

The seed-specific pCn12: Moral-LPAAT1 construct was used to transform an already transgenic Arabidopsis thaliana line that was homozygous for T-DNA from construct GA7 and whose seed contained about 15% DHA in seed lipids (Petrie et al., 2012). For this, the gene for the selection marker kanamycin was used in the pCn12: Moral-LPAAT1 construct, which was different from the gene for the selection marker bar, which was already present in the transgenic line. Transgenic seedlings resistant to kanamycin were selected and grown to maturity in a greenhouse. The T2 seed was harvested and the composition of fatty acids in the total lipids of the seeds was analyzed by GC (Table 12). Three phenotypes were observed among 33 independently transformed lines. In the first group (6/33 lines), the content of duodenum was significantly increased to a significantly higher level than the level of DHA, up to about 10.6% in the total lipids of seeds. This was due to DHA, the content of which was significantly reduced in this group of lines. In two lines from this first group, the total content of DPA + DHA was reduced, but not in the other 4 lines. In the second group (5/33), the levels of DPA and DHA were equal, with about the same total DPA + DHA content as in the seed of the mother plant. In the third group, the levels of DPA and DHA were similar to those in the seeds of the mother plants. One of the possible explanations for the increased level of duodenum in the first and second groups is that LFAAT displaces Δ4-desaturase on the substrate duplex-CoA and mainly incorporates duplex in PC and further in TAG, relative to Δ4-desaturation. A second possible explanation is that Δ4 desaturation is partially inhibited.

Seeds of Arabidopsis plants transformed with T-DNA construct GA7 were harvested, further transformed with the vector pCn12 :: Moral-LPAAT, and oil was extracted from the seeds. Then, the TAG fraction was isolated from the extracted oil by TLC methods and isolated from a TLC plate. The resulting TAG samples and seed oil samples prior to fractionation were analyzed by digestion with Rhizopus lipase to determine the positional distribution of DHA. The lipase is specific for acyl groups esterified at the sn-1 or sn-3 position of the TAG. This was done by emulsifying each lipid sample in 5% gum arabic using a sonicator and adding a solution of Rhizopus lipase in 0.1 M Tris-HCl, pH 7.7, containing 5 mM CaCl ₂ , incubating the mixtures at 30 ° C and continuous shaking. The reaction in each reaction mixture was stopped by adding chloroform: methanol (2/1, v / v) and one volume of 0.1 M KCl to each mixture. The lipid was extracted into the chloroform fraction, and the relative amounts of sn-2 MAG, sn-1/3 PPC, DAG and TAG components of the obtained lipid were determined by separation on a TLC plate impregnated with 2.3% boric acid using hexane / diethyl ether / acetic acid (50 / 50/1, v / v). Lipid bands were visualized by spraying with 0.01% primulin in acetone / water (80/20, v / v) on a TLC plate with visualization under UV light. Separate bands of lipid ID 65 037184 were identified based on the spots of lipid standards applied to the same TLC plate. TLC lipid bands were collected in glass vials, and methyl esters of fatty acids were obtained from them using 1N. methanol HCl (Supelco), incubated at 80 ° C for 2 h. The fatty acid composition of individual lipids was analyzed by GC.

This analysis demonstrated that DHA in the seeds of GA7-transformed mother plants (lines 22-2-1-1 and 22-2-38-7) was predominantly esterified at the sn-1 or sn-3 position of TAG. Conversely, lipids in NY11 and NY15 seed transformed with both GA7 construct and the transgene encoding LPAAT were enriched with DHA in sn-2, with 35% DHA in one lineage and 48% DHA in another line esterified at position sn -2 TAG, i.e. after lipase digestion, DHA was present as sn-2-MAT (Table 13). Similar results were obtained for the seed of B. napus and B. juncea transformed with both T-DNA from the GA7-modB construct and the gene encoding LPAAT and producing DHA, as well as for the seed of B. napus and B. juncea, which produces duodenum.

In order to determine whether there is a preference for Mortierella LPAAT or another LPAAT for DPA-CoA or DHA-CoA, in vitro reaction mixtures were prepared by adding separately ¹⁴ Labeled-DPA-CoA or -DHA-CoA to the leaf lysates of N. benthamiana, temporarily expressing a candidate LPAAT under the control of a constitutive promoter, as described above. The reaction mixtures were incubated at 25 ° C, and the levels of incorporation of ¹⁴ C-labeled fatty acids in PC were determined by TLC lipid analysis. The ability of each LPAAT to use DHA-CoA relative to DPA-CoA was assessed. The genes encoding LFAAT with a proven high DHA-incorporating activity of LFAAT were used to obtain transformed DHA-producing rape and seed plants.

Genes encoding LFAAT with high activity of using DPA-CoA were used to transform DPA-producing plants and seeds in order to increase the amount of DPA esterified at the sn-2 position of TAG.

Table 12

Fatty acid composition (% of total fatty acids) of transgenic A. thaliana seeds transformed with LFAAT1 construct, as well as T-DNA from GA7 construct for DHA production. C20: 4ω6 was not detected in seeds. In addition, the seeds contained 0.3-0.9% C22: 0 and 0.4-1.5% C22: 1

about and o “5 0 90 V 45 ech 90 and 1 90 V 1 "in υ o o hh V one 9S , -4 S th one MS © d) Y ·*: OU 1 about MS ID © WITH 3 eh hh 1 ri r "and NY-1 9.3 3.2 9.1 6.8 9.4 0.5 23.8 1.6 4D 7.9 5.1 0.6 0.9 0.6 1,2 7.9 4.5 NY-2 10.7 3.3 6.5 4.4 7.6 0.3 28.1 1.9 4.3 8.5 3.7 0.7 1.1 1.1 1.4 1.1 11.6 NY-3 9.3 2.8 6.3 3.4 10.3 0.2 32.8 2.2 2.7 6.2 3.6 1.1 1.9 1.4 0.7 1.0 10.7 NY-4 11.4 3.5 4.5 3.1 7.0 0.3 32.5 2.1 4.7 5.5 2,3 1.0 1.9 0.8 1.1 0.9 143 NY-5 14.6 4.5 7.0 7,7 6,7 0.3 20,7 2.2 5.7 5.4 4.8 0,4 0.9 0.8 1,2 1.0 11.7 NY-6 7.8 2.7 12.5 2.2 18.0 od 24.9 1.8 0.7 15.5 3.1 1.4 1,2 0.5 0.3 3.0 0.8 NY-7 9.3 2.9 6,7 3.8 9.2 0.2 31.5 2.1 3.2 7.5 3.7 0.9 1.6 1,3 0.8 1.1 10.9 NY-8 8.8 3.2 8.2 5.5 11.0 0.3 25.3 1.9 3.0 8.3 5.4 1.0 1,2 0.8 0.8 6.1 6.0 NY-9 12.3 3.7 5.0 4.6 7.1 0.2 28.3 2,3 4.2 5.6 3.8 0.8 1.6 0.7 1.1 1,2 13.8 NY-10 8.6 3.2 8.5 3.1 9,7 0.3 31.5 1.6 3.4 8.7 2.8 1.0 1,3 0.9 1.1 10.6 1.0 NY-11 11.5 3.2 4.5 2.5 7.1 0.3 33.3 2.1 3.9 5.7 1.9 0.9 2.0 0.7 0.8 1.0 15.6 NY-12 8.7 3.2 7.5 5.1 8.5 0.2 26.8 2.0 3.7 8.7 5.1 0.9 1,2 1.1 1,2 10.0 2.6 NY-13 11.5 3.4 5.2 3.4 8.3 0.3 30.0 2.2 5.0 6.2 3.2 0.9 1.7 1.5 1.1 1.0 11.6 NY-14 9.2 2.9 6.6 2.0 10.3 0.2 34,7 1.9 3.3 7,7 1.6 1,2 1.8 1,2 0.9 0.8 11.1 NY-15 10.9 3.3 4.6 2.7 7.0 0.3 34.1 1.9 5.1 5.5 2.0 0.9 1.8 0.8 1.0 1.0 14.7 NY-16 10.5 3.4 6.0 4.6 7.8 0.3 30.3 1.8 4.4 5.4 2.9 0.7 1.5 0.9 1.1 thirteen 14.2 NY-17 9.1 2.4 5.9 2.5 10.4 0.2 35.4 1.6 3.6 6.4 2.1 1.1 1.9 1,2 1.0 0.9 11.7 NY-18 9,7 3.6 8.8 6.2 12.1 0.3 21.0 1.9 4.0 8.3 5.9 0.8 0.9 0.6 1.0 5.7 5.1 NY-19 8.4 3.1 12.0 3.1 14.6 0.2 28.8 1.7 1.6 11.3 3.2 1.0 1.4 0.6 0.6 3.9 1,2 NY-20 10.1 3.2 5.4 3.3 8.9 0.3 32.8 2.1 4D 5.5 2.8 1.0 1.9 1.1 0.9 M 12.1 NY-21 10.5 3.6 5.6 3.8 8.2 0.3 31.9 2.0 4.6 5.9 2.8 0.9 1.7 0.8 1, o 0.9 12.5 NY-22 8.4 3.3 7.4 2,3 9.4 0.2 33.5 1.8 3.4 8.8 2.2 1,2 1.7 1,3 1.0 5.5 6.1

Table 13

The presence of DHA at the sn-2 position of TAG or in the total oil of transgenic A. thaliana seeds transformed with the Cn12 :: Moral-LFAAT gene, as well as T-DNA from the GA7 construct, demonstrating the positional distribution of DHA in TAG. In addition, the fatty acids TAG and sn-2 MAG contained 00.4% of each of 14: 0, 16: 1ω13t, 16: 2, 16: 3, 22: 0, and 24: 0. C20: 3ω6, C20: 4ω6 were not found in seeds

Sample © £ 0 § 45 and © five and & and gh f and 'g a and 3 and © © ech and 05 and < © rh and < © rh and rh © rh and 3 **> © gh υ one © rh and ri hh and No. 3 "l © rh and 8 "Λ hh hh and one hh hh and 3 rh g "and 3 h " hh hh and 22-2-1 * 1 TAG 12.2 0.4 4.4 6.4 3.9 7.2 0.8 28.8 1.6 4.3 9.7 2.3 0.7 1.3 1.0 0.6 2, 1 0.0 0.7 10.1 0.6 0.1 0.3 8.3 2.5 10.1 0.7 53.9 0.2 6.5 0.3 0.1 0.1 0.3 0.2 0.0 3, 8 0.0 2.3 9.1 2-MAG DHA in sn-2 = 30% 22-2-38-7 oil 10.0 0.2 3.7 6.0 2.7 6.4 0.4 33.8 1.6 3.7 11.3 1.8 0.8 1.3 0.9 0.6 1, 2 0.7 11.6 0.5 0.1 0.3 9.7 2.4 11.1 0.6 60.0 0.1 3.6 0.3 0.1 0.1 0.4 0.2 0.0 2, 1 0.1 1.3 6.7 2-MAG DHA in sn-2 = 19% Additionally, η disbanded by the gene encoding the LPAAT Mortierella alpina. ΝΥ11-ΤΑΓ 11.0 0.2 3.4 6.0 2.8 9.2 0.3 34.8 1.6 3.6 6.3 1.8 1.0 1.8 0.7 0.6 0, 9 0.0 0.1 0.6 12.2 0.7 0.1 0.2 6.7 1.1 11.8 0.3 49.8 0.2 3.7 0.5 1.5 0.3 1.6 0.6 0.1 0, 8 0.1 0.2 1.6 17.8 2-MAG DHA in sn-2 = 48% NY-15-oil 11.0 0.0 3.3 4.6 2.8 6.9 0.3 33.6 2.0 5.1 5.5 2.1 0.9 1.9 0.7 0.6 0, 9 0.4 0.9 14.9 0.8 0.1 0.3 6.4 1.3 11.4 0.3 50.2 0.2 4.9 0.4 1.4 0.2 1.5 0.6 0.1 0, 9 0.0 0.2 1.6 16.7 2-MAG DHA in sn-2 = 37%

Example 7. Further analysis of transgenic seeds of Camelina sativa.

- 66 037184

Total lipid content.

Seed of C. sativa, homozygous for T-DNA from construct GA7 and containing DHA in total fatty acids, was analyzed for total lipid composition and as indicated below. The seeds were subjected to two successive solvent extraction steps, first with hexane and then with chloroform / methanol. No antioxidants were added during extraction and analysis. The Soxhlet extraction method, which is commonly used to extract lipids from semen by prolonged heating and refluxing a lipid / solvent mixture, was not used in this case due to the potential degradation or oxidation of ω3 PUFAs such as DHA.

Hexane was used as a solvent for the first extraction because it is the industry standard for oilseeds. In addition, it preferentially extracts TAG-containing oil due to its solvating properties and relatively weak solubilization of polar lipids, especially at room temperature. Transformed and control Camelina seeds (130 and 30 g, respectively) were moistened with hexane and ground using an electric agate mortar and pestle (Retsch Muhle, Germany). The mixtures were transferred to separatory funnels and extracted four times using a total volume of 800 ml of hexane, including static extraction overnight for the third extraction. For each extraction, the extracts were filtered through a GFC glass filter under vacuum to remove fines and then evaporated on a rotary evaporator at 40 ° C. under vacuum. The extracts were pooled to give TAG-rich hexane extracts.

After extraction with hexane, the remaining seed cake was further extracted using chloroform-methanol (XM, 1: 1 v / v) in the same manner as the extraction with hexane. Then the cake was removed by filtration, and the combined extracts were evaporated on a rotary evaporator. The pooled crude CM of total lipid was then dissolved using a single phase methanol-chloroform-water mixture (2: 1: 0.8, v / v / v). Fractions were separated by addition of chloroform-water (final solvent ratio, 1: 1: 0.9 v / v / v methanol / chloroform / water). The purified lipid in each extract, dispersed in the lower chloroform phase, was concentrated using rotary evaporation to obtain XM extracts rich in polar lipids. The lipid content of each of these extracts was determined gravimetrically.

To analyze the composition of fatty acids, aliquots of hexane and XM extracts were trans-methylated according to the method of Christie et al. (1982) to obtain methyl esters of fatty acids (FAME) using methanol-chloroform-conc. hydrochloric acid (3 ml, 10: 1: 1, 80 ° C, 2 h). FAME was extracted with hexane-chloroform (4: 1, 3 x 1.8 ml). In addition, samples of seed cake (1-2 g) remaining after extraction with hexane and XM were trans-methylated for gravimetric measurement of any residual lipid in the form of FAME. The total lipid content in the seed was calculated by summing the lipid content in the hexane and HM extracts and the FAME content in the transmethylated oil cake after solvent extraction.

The transgenic seeds contained slightly lower total lipids, at 36.2% of the seed weight, compared to wild-type Camelina sativa seeds, 40.9% of the seed weight. For seeds, including oilseeds, total lipids were defined as the sum of solvent-extracted lipid obtained by sequential extraction with hexane and then chloroform-methanol and residual lipid released by transmethylation of the extracted oil cake after solvent extraction, as described by way of example herein. Such total lipids consisted mostly of fatty acid lipids such as triacylglycerides and polar lipids, and small amounts of fatty acid free lipids such as phytosterols and fatty alcohols, which may be present in free non-esterified form or may be esterified with fatty acids. acids. In addition, any sterol esters or wax esters and hydrocarbons such as carotenoids, eg β-carotene, were also included in the solvent-extractable lipid, if present. They were included in the general gravimetric determination and were shown in the TLC-FID analysis (Table 14).

Of the total lipids, 31-38% of the lipids by weight of the seed was extracted with hexane for transgenic and control seeds, respectively, which corresponds to 86 and 92% of the total lipids in the seeds. With the help of XM extraction, an additional 4.8 and 2.4% (based on the weight of the seed) were recovered, mainly the extract rich in polar lipids from the transgenic and control seeds, respectively. Residual lipid released by transmethylation of oilseed cake remaining after extraction with solvents was 0.3 and 0.4% of the seed weight, respectively. Those. the first and second solvent extractions together extract 99% of the total lipid content in the seed (i.e. 36.2 or 40.9% of the seed mass, which is mainly a lipid containing fatty acids, for example triglycerides and polar lipids consisting of from glyco- and phospholipids (see the next section - analysis of lipid classes)).

Analysis of lipid classes.

- 67 037184

The lipid classes in the hexane and XM extracts were analyzed by TLC with detection using a plasma ionization detector (TLC-FID; Iatroscan Mark V, Iatron Laboratories, Tokyo, Japan) using hexane / diethyl ether / glacial acetic acid (70:10 : 0.1, v / v / v) as a solvent system for developing, in combination with Chromarod S-III silicon dioxide on quartz sticks. Fitting calibration curves were obtained using representative standards obtained from Nu-Chek Prep, Inc. (Elysian, Minnesota, USA). The data were processed using the SIC-480II software (SISC version: 7.0-E). Different types of phospholipids were separated using a purified phospholipid fraction obtained after column chromatography on silica, with development of rods in chloroform / methanol / glacial acetic acid / water (85: 17: 5: 2, v / v / v) before detection by PID.

To separate TAG, glycolipid and phospholipid fractions from XM extracts, silica gel 60 (100-200 mesh) (0.3-1 g) was used in a short glass column or a Pasteur pipette filled with glass wool to purify 10 mg of purified XM lipid extract. The residual TAG fraction in the XM extract was eluted using 20 ml of 10% diethyl ether in hexane, glycolipids were eluted with 20 ml acetone, and phospholipids were eluted in two stages, first with 10 ml of methanol and then with 10 ml of methanol-chloroform-water (5: 3: 2). This second elution increases the yield of phospholipids. The yield of each fraction was determined gravimetrically and the purity was checked by TLC-FID. All extractions and fractions were stored in dichloromethane at -20 ° C until further analysis by GC and GC-MS.

The TAG-rich hexane extracts from each of the transgenic and control seeds contained about 96% TAG. XM extracts contained residual amounts of TAG 44 and 13 wt% XM extracts for transgenic and wild-type seeds, respectively. In contrast to the hexane extracts, the XM extracts were rich in polar lipids, namely, phospholipids and glycolipids, the content of which corresponded to 50 and 76 wt% of the XM extracts for transgenic and control seeds, respectively (Table 14). The main phospholipid was phosphatidylcholine (PC), which accounted for 70-79% of total phospholipids, followed by phosphatidylethanolamine (PE, 7-13%), with relatively low levels of phosphatidinic acid (PC, 2-5%) and phosphatidylserine (PS, <2 %). There are procedural advantages in filing a response to the final expert opinion before 7 July 2015.

Fatty acid composition.

In general, for seeds producing DHA and / or DPA, the inventors observed a fatty acid composition of total lipids in the seed, as measured by direct transmethylation of total lipids in the seed, similar to the lipid composition of the TAG fraction. This was a result of the fact that over 90% of the total lipids present in the seed were in the TAG form.

The composition of fatty acids of various classes of lipids in hexane and XM extracts was determined by gas chromatography (GC) and GC-MS using an Agilent Technologies 6890A GC instrument (Palo Alto, California, USA) equipped with a Supelco Equity ™ fused silica capillary column - 1 (15 m χ 0.1 mm id, 0.1 μm film thickness, Bellefont, PA, USA), PID, split / no split injector, Agilent Technologies 7683B series autosampler and injector. The carrier gas was helium. The samples were injected in a non-splitting mode at an oven temperature of 120 ° C. After injection, the furnace temperature was increased to 270 ° C at a rate of 10 ° C / min, and finally to 300 ° C at a rate of 5 ° C / min. Eluted compounds were quantified using Agilent Technologies ChemStation software (Palo Alto, California, USA). GC results contained an error of no more than ± 5% of the peak area of the individual compounds.

Table 14

Composition of the lipid class (% of total lipids obtained for each stage of extraction) in hexane and XM extracts of transgenic and control Camelina sativa seed. ES, EV and HC did not separate

Lipid class Transgenic seeds Control Seeds Hexane hmm Hexane XM ES / EV / HC * 1.0 1.4 1.0 1.4 TAG 95.6 44.2 96.0 13.1 SZhK 0.9 1,3 0.8 1.4 But** 0.9 1.1 0.8 1,2 ST 0.5 0.7 0,4 0,4 MAG 0.7 1.1 0.8 6.2 FL 0.3 50.3 0.3 76.3 Total 100.0 100.0 100.0 100.0

Abbreviations: sterol esters (ES), wax esters (EV), hydrocarbons (HC), triacylglycerides (TAG), free fatty acids (FFA), not defined (n / a), sterols (ST), monoacylglycerides (MAG), polar lipids (PL), consisting of glycolipids and phospholipids; ES, EV and UV from this system

- 68,037184 threads eluted together; may contain fatty alcohols and diacylglycerides (DAG).

GC-MS analyzes were performed on a Finnigan Trace ultra quadrupole GC-MS instrument (model: ThermoQuest Trace DSQ, Thermo Electron Corporation). The data were processed using ThermoQuest Xcalibur software (Austin, Texas, USA). The GC instrument was equipped with an on-column injector and an HP-5 Ultra Agilient J&W capillary column (50 m χ 0.32 mm id, 0.17 μm film thickness, Agilent Technologies, Santa Clara, CA, USA) with polarity similar to that described above. Individual components were identified using mass spectrum data and comparing retention times with those obtained for authentic and laboratory standards. A blank analysis according to the complete method was carried out in parallel with a series of samples.

The data on the composition of fatty acids in different classes of lipids in the extracts are given in table. 15. In the DHA-producing seed of Camelina, DHA is distributed into the main lipid fractions (TAGs, phospholipids and glycolipids) in a proportion ranging from 1.6 to 6.8%, with an inverse correlation between the proportions of DHA and ALA. The TAG-rich hexane extract from the transgenic seed contained 6.8% DHA and 41% ALA (Table 15). The CM extract, rich in polar lipids, contained 4.2% DHA and 50% ALA, i.e. relatively less DHA and more ALA. Residual TAGs from the polar lipid-rich CM extract contained 6% DHA and 40% ALA. The glycolipid fraction isolated from the HM extract contained 3% DHA and 39% ALA, and the phospholipid fraction contained the lowest DHA level (1.6%) and the highest ALA levels (54%). Transgenic Camelina seed contained higher levels of ALA and lower levels of LA (linoleic acid, 18: 2ω6) compared to control seeds in the main lipid classes (TAGs, glycolipids, and phospholipids). The shares of ALA and LA were: ALA 39-54% and LA 4-9% for transgenic seeds and ALA 12-32% and LA 20-29% for control seeds. The relative level of erucic acid (22: 1ω9) was lower in all fractions from transgenic seeds than in control seeds, for example, 1.3% versus 2.7% in hexane extracts (Table 15).

The composition of the sterols in the seeds.

To determine the content and composition of sterols in the extracted lipids, samples containing about 10 mg of total lipids from the TAG-rich hexane extract and the polar lipid-rich XM extract were saponified using 4 ml of 5% KOH in 80% MeOH and heated for 2 h at 80 ° C. C in a Teflon-lined glass vial with a screw cap. After cooling the reaction mixtures, 2 ml of Milli-Q water was added, sterols and alcohols were extracted three times with 2 ml of hexane: dichloromethane (4: 1, v / v), shaking and stirring by vortex stirring. The mixtures were centrifuged and each organic phase extract was washed with 2 ml of Milli-Q water, shaking and centrifugation. After the selection of the upper sterol-containing organic layer, the solvent was evaporated using a stream of nitrogen gas, and sterols and alcohols were silylated using 200 μL of bis (trimethylsilyl) trifluoroacetamide (BSTFA, Sigma-Aldrich), heating for 2 h at 80 ° С in a sealed GC vial. In this way, free hydroxyl groups were converted into their trimethylsilyl ethers. The sterol- and alcohol-OTMSi derivatives were dried under a stream of nitrogen gas on a heating block (40 ° C) and redissolved in dichloromethane (DCM) immediately prior to GC / GC-MS analysis as described above.

Table 15

Fatty acid composition (% total fatty acids) of lipid extracts and fractions from transgenic and control seeds of C. sativa

Transgenic seeds Control Seeds Fatty acid Hexane XM Cake Hexane XM Cake TAG Are common TAG GL FL Residual TAG Are common TAG GL FL Residual 16: 1ω7 0.1 0.2 0.1 0.2 0.1 0.2 oh, 1 0.2 0.2 - - 0.3 16: 0 6.2 12.8 6.8 21.3 19.4 10.4 6,7 12.8 7.8 29.6 13.7 10.3 18: 4ω3 3.7 h, h 3.4 2.1 2.9 3.6 - - - 18: 2ω6 7.1 3.9 8.8 7.2 3.7 8.8 22.2 28.4 29.4 20.8 29.3 27.9 18: 3ω3 41.9 50.3 39.9 38.6 54.1 38.9 32.0 20.6 19.7 13.0 12.3 20.0 18: 1ω9 11.1 4.7 9.6 7.2 2.8 8.1 14.0 25.4 13.3 14.7 35,7 14.3 18: 1 (07 1.4 2,3 2.1 3.7 3.4 2.8 1.0 1.5 2.2 4.0 2.8 2.2 18: 0 3.2 4.0 3.0 4.5 5.7 3.1 3.0 2.7 2.9 5.7 3.6 2.7 20: 5 (03 0,4 0.2 0.3 - 0.3 - - - 20: 4 (03 0,4 0,4 0,4 0.2 0.3 - - - - 20: 2 (06 0.7 0.7 0.8 0.6 0,4 0.7 1.8 0.8 2.1 1,2 - 1.8 20: 3 (03 0.8 1,2 0.9 0.6 1,3 0.5 0.9 0.3 - - 0,4

20: 1ω9 / 11 11.6 6.1 10.9 5.1 1,3 8.4 12.5 3.0 11.1 4.2 1.7 9.4 20: 1ω7 0.6 0.8 1.4 0.6 0.2 1.1 0.6 0.6 2.0 1,3 - 1.8

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20: 0 1,3 0.8 1.4 0.6 0.1 1.4 1.5 0.7 2.0 1.4 - 1.8 22: 6ω3 6.8 4.2 6.1 3.0 1.6 5.4 - - - - - 22: 5ω3 oh h 1.1 0,4 0.6 1.4 0.3 - - - - - - 22: 1ω9 1,3 1.0 1.8 0.6 0.1 1.5 2.7 0.7 3.6 0.9 - 2.9 22: 0 oh, 3 0.2 oh h 0.6 ο, ι 0.7 0.3 0.2 0.7 0.8 0.8 24: 1ω9 0.3 0,4 0,4 0.6 0.3 0.6 0.3 0.6 0.7 0.9 0.5 ι, ο 24: 0 0.1 0,4 0.2 0.9 0,4 1.1 Ο, ι 0,4 0.5 1.4 0,4 1,3 others * 0,4 ι, ο ι, ο 1.4 0.5 1.8 0.3 1.1 0.9 0.1 1.1 Total 100 100 100 100 100 100 100 100 100 100 100 100

Abbreviations: triacylglycerides (TAG), glycolipids (GL), phospholipids (PL);

general: an extract rich in polar lipids containing GL and PL from the HM extract;

TAG, GL, and PL were separated by chromatography on a silica column from XM extracts;

* total content of minor fatty acids.

The main sterols in both transgenic and control seeds were 24-ethylcholesterol (sitosterol, 43-54% of total sterols), 24-methylcholesterol (campesterol, 20-26%) with lower cholesterol levels (5-8%), brassicasterol (2-7%), isofucosterol (Δ5-avenasterol, 4-6%), stigmasterol (0.5% -3%), cholest-7-en-3v-ol, (0.2-0.5% ), 24-methylcholestanol (campestanol, 0.4-1%) and 24dehydrocholesterol (0.5-2%) (Table 16). These nine sterols are responsible for 86-95% of total sterols, while the rest of the sterol components are identified only partially by the number of carbon atoms and double bonds. The overall sterol profiles were similar for transgenic and control seeds for both hexane and XM extracts.

Analysis of fatty alcohols.

Fatty alcohols from seeds were derived and analyzed for sterols. Fatty alcohol series from C16-C22, with concomitant iso-branched fatty alcohols, have been identified in both hexane and XM extracts. Similar profiles were observed for transgenic and control seeds, with some variation in the proportion of observed individual components. Phytol, formed from chlorophyll, was the main aliphatic alcohol and was responsible for 47 and 37% of the total fatty alcohols in the hexane fractions from transgenic and control seeds, respectively. Odd chain alcohols were present at higher levels in the XM extract (37-38% of the total fatty alcohols) than in the hexane extract (16-23%). Iso-17: 0 (16-38%) prevailed over 17: 0 (0.3-5.7%). The content of other alcohols with an odd chain length was 19: 0 (4.5-6.5%). Other alcohols found included iso-16: 0, 16: 0, iso-18: 0, 18: 1, 18: 0, with minor levels of iso20: 0, 20: 1, 20: 0, and iso-22 were also present: 0, 22: 1 and 22: 0.

Discussion.

The results show that milling with a mechanized mortar and pestle with repeated extraction with hexane at room temperature was effective in recovering most of the TAG-containing oil from transgenic seeds. In addition to transgenic seed oil containing moderate levels of DHA, transgenic seeds also contained significantly higher ALA levels in the major lipid classes (triacylglycerides, glycolipids, and phospholipids) compared to control seeds. This showed that the activity of Δ15-desaturase was significantly increased in transgenic seeds during seed development. Interestingly, there were some slight differences in the composition of fatty acids and the proportions of DHA in various extracts and fractions, with DHA levels being higher in the TAG-rich hexane extract and TAG from the HM extract (6-6.8%) and lower in the polar lipid fractions ( 3% in glycolipids and 1.6% in phospholipids). The 16: 0 level was higher in the polar lipid, glycolipid, and phospholipid fractions in the XM extracts (19-21%) compared to the TAG-rich hexane extract and the TAG from the XM extract (6-7%).

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Table 16

Sterol composition (% of total sterols) in transgenic and control Camelina seeds

Transgenic seeds Control Seeds Sterols Hexane XM Hexane hmm 24-dehydrocholesterol 0.8 1.8 0.5 1.4 Cholesterol 5.7 7.6 4.7 7.2 Brassicasterol 4.4 6.5 1.9 4.2 Cholest-7-en-Z β-ol 0.2 0.5 0.3 0,4 Campesterol 24.5 20.8 25.7 21,7 Campestanol 0,4 1.1 0,4 0.9 Sigmasterol 1.0 2.6 0.5 1.6 Sitosterol 54.3 43,7 53.8 42.9 D5-avenasterol 4.2 5.2 4.7 5.5 Amount 95.5 89.6 92.6 85.9 Others UNI С28 Idb 0.6 1,2 0.7 1.2, UN2 C29 Idb 1,2 2.0 1,2 2.4 UN3 C29 2db 0.9 1.8 1,3 2.4 UN4 C28 Idb 0.3 0.9 0.6 1.1 UN5C302db 1,2 1.8 1.4 1.8 UN6C29 ldb + C30 2db 0.3 2.7 2.2 5.2 The sum of others 4.5 10.4 7.4 14.1 Total 100 100 100 100

Abbreviations: N / A means unknown sterol;

the number after C indicates the number of carbon atoms; db stands for the number of double bonds.

The sterol composition in transgenic seeds and control seeds was similar to that found in refined Camelina oil (Shukla et al., 2002), the same basic sterols were present, indicating that the added genes did not affect sterol synthesis in seeds. The cholesterol level in Camelina oil was higher than that found in most vegetable oils. Included was brassicasterol, which is a characteristic sterol found in the Brassicaceae family, which includes Camelina sativa.

Example 8. Production of DC-PUFA in seeds of Brassica juncea.

Transgenic Brassica juncea plants were generated using the GA7-modB construct (Example 3) to generate DHA as described below. Seeds of B. juncea, cultivar sensitive to long daylight hours, were sterilized using chlorine gas as described by Kereszt et al. (2007). Sterilized seeds were germinated MS media with 1/2 activity (Murashige and Skoog, 1962) solidified with 0.8% agar, adjusted to pH 5.8, and grown at 24 ° C and the fluorescent light (50 MKE / m ² s ) with a photoperiod of 16/8 h (light / dark) for 6-7 days. Cotyledon stalks with a stem of 2-4 mm were isolated from these seedlings under aseptic conditions and used as explants. Agrobacterium tumefaciens strain AGL1 was transformed with the binary construct GA7. The Agrobacterium culture was seeded and treated for infection as described by Belide et al. (2013). For all transformations, about 50 freshly isolated cotyledon petioles were infected with 10 ml of A. tumefaciens culture for 6 min. Infected petioles were blotted with sterile filter paper to remove excess A. tumefaciens and transferred to coculture media (MS containing 1.5 mg / L BA, 0.01 mg / L NCC, and 100 μM acetosyringone, also containing L-cysteine (50 mg / L), ascorbic acid (15 mg / L) and MES (250 mg / L)). All plates were sealed with microporous tape and incubated in the dark at 24 ° C for 48 hours of co-culture. Then the explants were transferred to a pre-breeding medium (MS agar containing 1.5 mg / L BA, 0.01 mg / L NCC, 3 mg / L AgNO ₃ , 250 mg / L cefotaxime, and 50 mg / L tymentin) and cultured for 4-5 days at 24 ° C with a photoperiod of 16/8 h, after which the explants were transferred to the selection medium (MS agar containing 1.5 mg / L BA, 0.01 mg / L NCC, 3 mg / L AgNO ₃ , 250 mg / L cefotaxime, 50 mg / L tymentin and 5 mg / L PPT) and cultured for 4 weeks at 24 ° C with a photoperiod of 16/8 h. Explants with green callus were transferred to a medium for shoot regeneration (MC agar containing 2.0 mg / l BA, 3 mg / l AgNO ₃ , 250 mg / l cefotaxime, 50 mg / l thimene-71 037184 tin and 5 mg / l PPT) and cultured for another 2 weeks. Small buds of regenerating shoots were transferred to MS medium without hormones (MS agar containing 3 mg / L AgNO ₃ , 250 mg / L cefotaxime, 50 mg / L tymentin, and 5 mg / L PPT) and cultured for another 2-3 weeks.

Potentially transgenic shoots at least 1.5 cm in size were isolated and transferred to rooting medium (MS agar containing 0.5 mg / L NCC, 3 mg / L AgNO ₃ , 250 mg / L cefotaxime and 50 mg / L tymentin) and cultured for 2-3 weeks. Transgenic shoots with a large number of roots, confirmed by PCR, were transferred to soil in a greenhouse and grown with a photoperiod of 16/8 h (light / dark) at 22 ° C. Three confirmed transgenic plants were obtained. The transformed plants were grown in a greenhouse, allowed to self-pollinate, and harvested T1 seeds. The lipid fatty acid composition was analyzed in the T1 seed pools from each of the transformed T0 plants, and the presence of 2.8% DPA and 7.2% DHA was found in one line, designated JT1-4, while the other line, designated JT1-6, showed 2.6% WPC.

Seed oil from individual T1 seeds was analyzed for fatty acid composition; some of the data are given in table. 17. Several T1 seeds produced DHA at levels ranging from 10% to about 21% of total fatty acids, including JT1-4-A-13, JT1-4-A-5, and JT1-4-B-13. Surprisingly and unexpectedly, it was found that some of the T1 seeds contained DPA at levels from 10% to about 18% of the total fatty acids, with no detectable DHA (<0.1%). The inventors concluded that the Δ4 desaturase gene in the T-DNA inserted in these plants was inactivated by a spontaneous mutation similar to that described in Example 2. T1 seeds were germinated, and one emerging cotyledone from each was analyzed for the composition of fatty acids in the remaining oil. The rest of each seedling was stored and grown to maturity to produce a T2 seed.

Transgenic plants that were homozygous for single T-DNA insertions were identified and selected. Plants from one selected line, designated JT1-4-17, contained a single T-DNA insertion and produced DHA and only low levels of DPC, while plants from a second selected line, designated JT1-4-34, also contained a single T-DNA insertion. but produced DPA without producing DHA. The authors of the invention concluded that the original transformant contained two separate T-DNAs, one of which provided the production of DHA, and the other provided the production of DPA without DHA. B. juncea plants producing DHA in the seed were crossed with plants producing DPC in the seed. The F1 offspring included plants that were heterozygous for both T-DNA insertions. In the seeds of these offspring plants, the production of about 20% DHA and about 6% DPA was observed, with a total DHA + DPA content of 26%. F1 plants self-pollinated, and offspring homozygous for both T-DNA insertions were expected to produce up to 35% DHA and duodenum.

About 18% of the duodenum was observed in the lipid of the T3 progeny seed pool, designated JT1-4-34-11. Likewise, about 17.5% DHA was observed in the lipid of the seed pool of the T3 progeny JT1-4-17-20. The fatty acid composition of the JT1-4 T1 seed pool, T1 single seed, T2 seed pool, T2 single seed and T3 seed pool, T3 single seed is shown in Table 1. 18-21. JT1-4 T3 segregants JT-1-4-34-11 contained 18% DPA in the T3 seed pool, and a single seed from that particular segregante contained about 26% DPA, in each case as a percentage of total fatty acids.

The following parameters were calculated for seed oil containing 17.9% DNA: total saturated fatty acids, 6.8%, total monounsaturated fatty acids, 36.7%; total polyunsaturated fatty acids, 56.6%, total ω6 fatty acids, 7.1%; new ω6 fatty acids, 0.4% of which was GLA; total ω3 fatty acids, 46.5%; new ω3 fatty acids, 24.0%; the ratio of total ω6: total ω3 fatty acids, 6.5; the ratio of new ω6: new ω3 fatty acids, 60; efficiency of conversion of oleic acid to LA under the action of Δ12-desaturase, 61%; efficiency of conversion of ALA to SDA under the action of Δ6-desaturase, 51%; the efficiency of converting SDA into ETC acid under the action of Δ6-elongase, 90%; the efficiency of conversion of ETC to EPA under the action of Δ5-desaturase, 87%; the efficiency of conversion of EPA to DPC under the action of Δ5-elongase, 98%.

To obtain more transgenic B. juncea plants with the modB construct, the transformation was repeated five times, and 16 putative transgenic shoots / seedlings were regenerated. T1 semen analysis was performed to determine the content of DPA and DHA.

In order to obtain another seed containing DPC and not containing DHA, the genetic construct, which was a variant of the ModB construct without the Δ4 desaturase gene, was obtained as follows. Two DNA fragments were synthesized, an EPA-DPK 1 fragment and an EPKDRK 2 fragment (Geneart, Germany) with suitable restriction sites. An intermediate cloning vector, pJP3660, was obtained by cloning the AatII-MluI fragment of the EPA-DPK 1 fragment into the AscIAatII sites of the vector designated 11ABHZHC_GA7-frag_d6D_pMS, which vector was previously used in the construction of GA7-modB containing the Δ6 desaturase cassette. Next, pJP3661 was obtained by cloning the PmeI-PspOMI pJP3660 fragment into the PmeI-PspOML modB sites. Thereafter, the DPK vector, pJP3662 (Fig. 4), was collected by cloning the BsWI-PspOMI fragment of the EPA-DPK 2 fragment into the sites

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BsiWI-PspOMI pJP3661. This vector contained genes for fatty acid biosynthesis, encoding enzymes that convert oleic acid to DPAω3 and the corresponding ω6 fatty acid. The resulting construct was used to transform B. juncea and B. napus. The seed of the offspring was obtained, containing up to 35% of the WPC in the total content of fatty acids in the lipid of the seed.

When analyzed by NMR of the oil extracted from the seeds of a plant producing DHA, at least 95% of the observed DHA was present at the sn-1,3 position of the TAG molecules.

Table 17

Composition of fatty acids in seed oil from B. juncea T1 seeds transformed with T-DNA from GA7

Seed T1 No. f and § f and £ and her and <"Uh do and f 8 do and 3 and < © сч 40 S hh f and t 8 φ η and 3 f and and w> f GH and 3 l and 3 f and that and f gh and JT1-4-A-1 5.0 0.2 2.7 23.5 3.4 17.0 0.7 24.8 0.7 2.0 1.1 0.2 0.8 4.0 0.6 2.4 9.9 JT1-4-A-2 4.3 0.3 2.6 37.2 3.2 11.0 0.3 22.1 0.7 0.9 1,3 0.2 1.4 3.2 0.3 9.4 0.0 JT1-4-A-3 5.6 0.3 2.7 20.8 3.7 16.0 0.6 24.4 0.7 2.0 0.9 0.2 1.1 4.5 0.7 3.1 11.4 JT1-4-A-4 4.6 0,4 2.8 36.2 3.4 10.6 0.3 24.5 0.8 9.9 1.7 0.2 0.3 0.5 0.0 2.5 0.0 JT1-4-A-5 5.0 0.2 3.2 20.3 3.6 13.7 0.7 25.9 0.7 2.0 0.9 0.2 1,3 4.4 1.5 1.6 13.5 JT1-4-A-6 4.8 0,4 3.4 37.9 3.7 7.4 0,4 19.9 0.9 1.4 1.4 0.1 0.8 1.9 0,4 13.9 0.0 JT1-4-A-7 5.6 0.3 3.0 26.2 4.0 8.9 0.3 26.6 0.6 1.8 1.0 0.1 1.8 3.7 1,3 2.2 n, s JT1-4-A-8 4.8 0,4 2.9 40.3 3.4 7.8 0.3 22.2 0.8 1.4 1,3 0.1 0.8 2.4 0,4 9.6 0.0 JT1-4-A-9 7.1 0.3 3.6 17.7 4.3 17.9 0.7 23.1 1.0 2.1 0.8 0.2 1.5 3.6 0.8 2.0 11.9 JT1-4-A-10 5.1 0.2 4.2 22.3 3.4 19.5 0.7 21,7 0.8 1.5 0.9 0.2 1.7 7.8 0.9 1.0 6.5 JT1-4-A-11 5.0 0.5 2.8 37.6 4.0 7.1 0,4 19.2 0.7 1.9 1.4 0.2 0.5 1.6 0.3 15.5 0.0 JT1-4-A-12 5.2 0.3 3.0 28.2 4.0 9.2 0.3 27.4 0.6 1.9 0.9 0.1 1.5 3.2 1.1 1.8 10.2 JT1-4-A-13 5.4 0.2 3.0 16.7 4.1 9.9 0.6 29.9 0.7 2.2 1.0 0.2 1.7 2.0 1.1 2.0 17.9 JT1-4-A-14 5.1 0,4 3.1 30.0 4.0 11.5 0.3 27,7 0.7 2.2 1.0 0.1 0.6 2.4 0.8 1,3 7.8 JT1-4-A-15 5.1 0,4 2.5 34.2 3.6 6.9 0.6 20.4 0.7 1.6 1.1 0.2 0.6 4.7 0.9 15.2 0.0 JT1 ^ -B-1 5.5 0.2 2.7 18.9 4.0 17.6 0.8 24.1 0.8 2.2 1.0 0.2 1,2 4.6 0.9 2.2 11.5 JT1-4-B-2 5.5 0.2 2.7 20.2 4.0 14.3 0.5 25.5 0.7 1.7 0.9 0.2 1.6 8.7 1,3 2.2 8.5 JT1-4-B-3 5.3 0.3 3.6 34.1 3.5 35.0 0.6 9.3 0.8 0.2 1.4 0,4 0.6 0.9 0.1 0.3 2.1 JT1-4-B-4 5.3 0.3 3.1 25.2 3.6 17.0 0.7 24.1 0.7 1.9 1.0 0.2 0.8 4.3 0.5 2,3 7.8 JTI-4-B-5 5.5 0.5 2.2 30.1 4.6 10.2 0.5 21,7 0.6 1.4 1.1 0.2 0.9 2.4 0.5 16.1 0.0 JT1-4-B-8 6.2 0.5 1.9 33.1 4.0 30.0 0.5 12.7 0.6 0.3 1,3 0,4 1.4 0.9 0.1 4.4 0.0 JT1-4-B-13 5.6 0.3 2.8 20.9 3.9 11.9 0,4 27.0 0.7 2.0 1.0 0.2 1.7 2,3 0.7 4.1 13.5

The seed oil samples additionally contained 0.1% C14: 0; 0.1-0.2% C16: 3; 0.0-0.1% of each of C20: 1Δ13, C20: 3ω6 and C20: 4ω6; 0.3-0.4% C22: 0; did not contain C22: 1 and C22: 2ω6; contained 0.2% C24: 0 and 0.2-0.4% C24: 1.

Table 18

Lipid fatty acid composition from T1 seeds (pool) B. juncea transformed with T-DNA from GA7-modB. Lipids additionally contained about 0.1% of each of 14: 0, 16: 3, 20: 1d13 and 16: 2, and 22: 1 was not detected

Seed

JT1-2

JT1-3

JT1-4

JT1-5

JT1-6 © - ®b!

f f di eesho

G U U 2 Q

U

4.2 0.3 2.5 42.4 3.2 27.7

4.5 0.3 2.7 44.6 3.126.8

5.1 0.3 3.2 26.8 3.5 17.4

4.7 0.4 2.4 41.6 3.4 28.4

4.8 0.4 2.3 37.3 3.3 30.2

0.1 16.4 0.6 0.0 1.20.1

0.1 14.8 0.7 0.0 1.20.1

0.5 22.8 0.7 2.5 1.10.2

0.1 15.8 0.7 0.0 1.20.1

0.4 13.2 0.7 0.2 1.40.3

0.0 0.0 0.0 0.3 0.00.0

0.0 0.0 0.0 0.3 0.00.0

0.0 0.0 1.2 0.3 2.90.7

0.0 0.0 0.0 0.3 0.00.0

0.0 0.0 0.7 0.3 0.60.1? about -? ?

M «L V} F ri ri count uchri hh hh O and ghsch i and ai

0.0 0.0 0.2 0.4 0.00.0

0.0 0.0 0.2 0.4 0.00.0

0.0 0.1 0.2 0.3 2.87.2

0.0 0.0 0.2 0.4 0.00.0

0.0 0.3 0.2 0.5 2.60.0

Table 19

Composition of fatty acids in T1 seed oil (single) B. juncea transformed with T-DNA from GA7-modB

about $ f MS "H 3 3 f but < f 8 3 3 I 3 3 f that OO < f < F Seed T1 No. and chi and and rh and about and and and and JT1-4-A-1 5.0 0.2 2.7 23.5 3.4 17.0 0.7 24.8 0.7 2.0 1.1 0.2 0.8 4.0 0.6 2.4 9.9 JT1-4-A-2 4.3 0.3 2.6 37.2 3.2 11.0 0.3 22.1 0.7 0.9 1,3 0.2 1.4 3.2 oh h 9.4 0.0 JT1-4-A-3 5.6 0.3 2.7 20.8 3.7 16.0 0.6 24.4 0.7 2.0 0.9 0.2 1.1 4.5 0.7 3.1 11.4 JT1-4-A-4 4.6 0,4 2.8 36.2 3.4 10.6 0.3 24.5 0.8 9.9 1.7 0.2 0.3 0.5 0.0 2.5 0.0 JT1-4-A-5 5.0 0.2 3.2 20.3 3.6 13.7 0.7 25.9 0.7 2.0 0.9 0.2 1,3 4.4 1.5 1.6 13.5 JT1-4-A-6 4.8 0,4 3.4 37.9 3.7 7.4 0,4 19.9 0.9 1.4 1.4 0.1 0.8 1.9 0,4 13.9 0.0 JT1-4-A-7 5.6 oh h 3.0 26.2 4.0 8.9 0.3 26.6 0.6 1.8 1.0 0.1 1.8 3.7 1,3 2.2 11.3 JT1-4-A-8 4.8 0,4 2.9 40.3 3.4 7.8 0.3 22.2 0.8 1.4 1,3 0.1 0.8 2.4 0,4 9.6 0.0

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JT1-4-A-9 7.1 0.3 3.6 17.7 4.3 17.9 0.7 23.1 1.0 2.1 0.8 0.2 1.5 3.6 0.8 2.0 11.9 JT1-4-A-10 5.1 0.2 4.2 22.3 3.4 19.5 0.7 21,7 0.8 1.5 0.9 0.2 1.7 7.8 0.9 1.0 6.5 JT1-4-A-11 5.0 0.5 2.8 37.6 4.0 7.1 0,4 19.2 0.7 1.9 1.4 0.2 0.5 1.6 0.3 15.5 0.0 JT1-4-A-12 5.2 0.3 3.0 28.2 4.0 9.2 0.3 27.4 0.6 1.9 0.9 0.1 1.5 3.2 1.1 1.8 10.2 JT1-4-A-13 5.4 0.2 3.0 16.7 4.1 9.9 0.6 29.9 0.7 2.2 1.0 0.2 1.7 2.0 1.1 2.0 17.9 JTM-A-14 5.1 0,4 3.1 30.0 4.0 11.5 0.3 27,7 0.7 2.2 1.0 0.1 0.6 2.4 0.8 1,3 7.8 JT1-4-A-15 5.1 0,4 2.5 34.2 3.6 6.9 0.6 20.4 0.7 1.6 1.1 0.2 0.6 4.7 0.9 15.2 0.0 JT1-4-B-1 5.5 0.2 2.7 18.9 4.0 17.6 0.8 24.1 0.8 2.2 1.0 0.2 1,2 4.6 0.9 2.2 11.5 JT1-4-B-2 5.5 0.2 2.7 20.2 4.0 14.3 0.5 25.5 0.7 1.7 0.9 0.2 1.6 8.7 1,3 2.2 8.5 JT1-4-B-3 5.3 oh, 3 3.6 34.1 3.5 35.0 0.6 9.3 0.8 0.2 1.4 0,4 0.6 0.9 0.1 0.3 2.1 JT1-4-B-4 5.3 0.3 3.1 25.2 3.6 17.0 0.7 24.1 0.7 1.9 1.0 0.2 0.8 4.3 0.5 2,3 7.8 JT1-4-B-5 5.5 0.5 2.2 30.1 4.6 10.2 0.5 21,7 0.6 1.4 1.1 0.2 0.9 2.4 0.5 16.1 0.0 JT1-4-B-6 5.6 0.3 2.5 19.5 3.8 15.2 0.5 27,7 0.6 2.1 0.9 0.2 1.1 3.7 0.6 3.3 11.1 JT1-4-B-7 5.9 0.5 2.0 29.9 4.0 11.2 0.3 26.2 0.6 11.5 1.4 0.2 0.3 0,4 0.0 4.1 0.1 JT1-4-B-8 6.2 0.5 1.9 33.1 4.0 30.0 0.5 12.7 0.6 oh h 1,3 0,4 1.4 0.9 0.1 4.4 0.0 JT1-4-B-9 4.9 0.2 3.4 24.6 3.0 18.5 0.3 26.2 0.8 1,3 1.1 0.2 2.0 5.5 0.6 0.8 5.2 JTM-B-10 5.2 0.3 2.7 19.0 4.0 12.0 0.6 30.5 0.7 1.6 1.0 0.2 1.7 4.9 1.1 3.0 10.2 JT1-4-B-11 4.8 0.2 3.0 23,7 3.1 18.1 0.6 23.5 0.7 1.6 1,2 0.2 1.5 4.5 0.8 1.6 9.6 JT1-4-B-12 5.0 0.2 2.6 19.6 3.4 12.5 0.6 26.9 0.8 3.1 1.1 0.2 0.9 5.6 0.9 3.5 11.7 GG1 ^ -V-13 5.6 0.3 2.8 20.9 3.9 11.9 0,4 27.0 0.7 2.0 1.0 0.2 1.7 2,3 0.7 4.1 13.5 JT1-4-B-14 5.1 0.3 3.1 25.5 3.3 16.7 0.7 23.9 0.8 1.8 1,2 0.2 0.9 2.6 0,4 2.9 9.2 JT1-4-B-15 5.6 0.3 2.7 19.5 4.1 14.0 0.8 24.6 0.7 2.7 0.9 0.2 0.7 9.4 1,3 2.5 8.5

The seed oil samples additionally contained 0.1% C14: 0; 0.1-0.2% C16: 3; 0.0-0.1% each of C20: 1Δ13, C20: 3ω6 and C20: 4ω6; 0.3-0.4% C22: 0; did not contain C22: 1 and C22: 2ω6; contained 0.2% C24: 0 and 0.2-0.4% C24: 1.

Table 20

Fatty acid composition of oil from single seeds of T2 Bjuncea transformed with T-DNA from GA7 modB. Lipids additionally contained 0.1-0.2% C16: 1Δ9, C16: 3 and C20: 2ω6, 0.5-0.6% C20: 0, did not contain C20: 3ω6, C20: 4ω6 and C22: 2ω6

Seed No.

4.4 1.7 36.3 2.9 5.6 1.9 39.1 3.1

5.5 1.8 42.3 3.2 5.6 1.5 36.8 3.7 4.6 1.7 36.3 2.7 4.9 1.8 38.3 3.1 4, 7 1.7 36.2 3.0 4.8 2.2 41.0 3.0 5.8 1.7 36.6 3.7 4.8 2.1 47.1 2.9 5.1 1 , 7 37.4 3.3 4.7 1.8 37.3 2.7 4.9 2.0 37.9 3.0 4.7 1.6 35.7 3.2 4.7 1.8 37.6 3.4 5.3 1.6 35.3 3.5

4.9 1.7 39.4 3.3 5.0 1.8 38.5 3.1 5.1 1.8 39.5 2.9 4.8 1.8 38.2 3.2 5, 0 2.0 39.7 2.9 4.7 1.6 36.0 3.3

6.2 2.1 32.0 4.4

3

7: 7

00 "OS and and C and

8.3 0.5 22.0 1.4 8.4 0.4 18.9 1.2

9.9 0.3 24.0 5.9 9.4 0.3 19.6 0.6 7.2 0.3 22.6 1.0 7.4 0.3 20.2 0.8 8, 2 0.4 20.9 0.7 9.8 0.2 27.0 4.2 9.1 0.3 21.3 0.9

7.4 0.2 23.9 4.8 7.7 0.3 20.7 0.9

7.9 0.4 20.6 1.1 7.1 0.4 20.1 1.1

6.9 0.3 22.4 0.7 7.8 0.3 23.7 0.6

8.1 0.5 21.1 0.8 7.7 0.3 21.1 0.7

7.8 0.4 20.5 0.8 9.0 0.2 22.2 0.6

7.8 0.4 21.1 0.7 7.9 0.4 20.2 0.7

8.3 0.3 23.7 0.6

7.2 0.6 19.4 1.2

1.2 0.4 0.3 4.2

1.3 0.5 0.3 2.5

1.5 0.2 0.4 0.5

1.4 1.4 0.3 1.9

1.5 0.7 0.3 2.1

1.3 0.8 0.3 2.7

1.3 0.9 0.3 2.9

1.8 0.3 0.3 0.5

1.4 0.8 0.3 1.5

1.7 0.2 0.3 0.5

1.4 0.8 0.3 2.5

1.3 0.5 0.3 4.3

1.3 0.6 0.3 4.1

1.4 1.3 0.3 3.0

1.5 1.2 0.2 1.7

1.2 0.7 0.3 3.1

1.4 0.8 0.3 2.0

1.3 0.8 0.2 2.3

1.5 1.0 0.3 1.7

1.4 0.7 0.3 2.1

1.3 0.7 0.3 2.3

1.5 1.2 0.3 1.7

1.2 0.6 0.4 2.2

0.6 0.1 0.1 0.0 0.4 0.1 0.2 0.0 0.0 0.0 0.2 0.0 0.3 0.2 0.2 0.0 0, 3 0.1 0.2 0.0 0.5 0.2 0.2 0.0 0.5 0.2 0.2 0.0 0.0 0.1 0.2 0.0 0.3 0 , 1 0.2 0.0 0.0 0.0 0.2 0.0 0.4 0.1 0.2 0.0 0.6 0.1 0.1 0.0 0.5 0.1 0.1 0.0 0.5 0.2 0.1 0.0 0.3 0.2 0.1 0.0 0.5 0.2 0.1 0.0 0.3 0.2 0, 1 0.0 0.3 0.2 0.1 0.0 0.2 0.1 0.2 0.0 0.4 0.2 0.1 0.0 0.3 0.2 0.1 0 , 0 0.3 0.2 0.1 0.0 0.5 0.3 0.2 0.0

1.8 0.3 12.1 0.0 1.5 0.4 12.6 0.0 0.4 0.4 1.5 0.0 1.6 0.4 13.1 0.0 2, 2 0.3 14.4 0.0 1.7 0.3 13.7 0.0 2.0 0.3 14.2 0.0 0.7 0.3 2.2 0.0 1.2 0 , 4 12.7 0.0 0.5 0.3 1.5 0.0 1.6 0.4 13.6 0.0 2.2 0.3 12.3 0.0 2.1 0.3 12.6 0.0 1.9 0.3 14.0 0.0 1.8 0.3 11.4 0.0 1.9 0.3 13.9 0.0 1.7 0.3 12, 3 0.0 2.0 0.3 13.1 0.0 1.6 0.3 10.2 0.0 1.7 0.3 13.3 0.0 1.9 0.3 12.2 0 , 0 1.8 0.3 12.7 0.0 1.6 0.4 17.6 0.0

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Table 21

Fatty acid composition of oil from single seeds of T3 B. juncea transformed with T-DNA from GA7 modB. The seeds additionally contained 0.1-0.2% each of C16: 3, C20: 1Δ13, C20: 2ω6. I have not found a wife C20: 3ω6, C20: 4ω6, C22: 2ω6, C22: 5ω6 and C22: 6ω3 ιλ

ΙΛ.

Y__Y_Y__Y__Y_Y__Y__Y__Y__Y__Y__Y__Y_Y__Y__Y__Y_Y__Y.

one 4.8 0,4 2.8 38.4 3.7 5.7 0,4 18.0 0.7 1, o 1.5 1.1 oh, 3 1.4 0,4 0.3 1.4 0.5 2 4.3 0,4 3.0 43.3 3.6 5.2 0.2 18.5 0.7 0.8 1.7 1.4 0.3 1,2 0.3 0.2 1,2 0.3 12.4 3 4.6 0,4 2.8 33.1 4.1 5.1 0,4 18.5 0.7 1,2 1.4 1.1 0.3 1.6 0.5 0.3 1.4 0,4 20.8 four 4.5 0,4 2.9 39.5 3.3 6.3 0,4 18.5 0.8 1,2 1.5 1.0 0.3 1.7 0.3 0.2 1.8 0.3 14.2 five 4.9 0.5 2.8 32.2 3.9 4.7 0.3 20,7 0.8 1,2 1.4 2.0 0.3 1.4 0.5 0.3 1,2 0,4 19.4 6 4.3 0.3 3.0 38.1 3.2 5.8 03 19.4 0.7 1.1 1.5 1,2 0.3 1.5 0,4 0.2 1,3 0,4 14?) 7 5.4 0.5 3.2 29.3 4.0 4.6 0,4 18.6 0.9 1.7 1,3 1,2 0,4 1.6 0.7 0.3 1.4 0.5 22.9 8 5.2 0.5 3.7 34.5 4.1 4.5 0.3 17.2 1.0 1.4 1.4 1.5 0,4 1.4 0.6 0.3 1,2 0.5 19.4 nine 5.3 0.5 3.4 33.4 3.7 4.6 0.3 17.6 0.9 1.7 1,2 1.1 0,4 1.5 0.6 0.2 1,2 0.5 20,7 ten 4.6 0,4 3.0 39.5 3.5 5.1 0.3 17.8 0.8 0.8 1.6 1.4 0,4 1,3 0,4 0.3 1,3 0,4 eleven 4.3 0,4 3.1 41,7 3.5 5.6 0.2 19.0 0.7 0.9 1.6 1,3 0.3 1.4 0.3 0.2 1.5 0.3 12.7 12 4.8 0.5 2.8 33.8 4.0 5.3 0,4 18.2 0.7 1.4 1,3 1,2 0.3. 1.6 0.6 0.3 1,3 0,4 20.1 thirteen 4.4 0,4 3.5 40.3 3.5 5.2 0.2 19.1 0.7 1.0 1.5 1.6 0.3 1.4 0,4 0.2 1.4 0.3 13.8 14 4.8 0,4 3.2 36.1 3.7 5.9 0.3 19.9 0.7 1.4 1,3 1.1 0.3 1.9 0.5 0.2 1.7 0.3 15.4 1S 4.0 0.3 2.8 37.2 3.2 4.9 0.3 19.6 0.8 0.9 1.6 1.5 0,4 1,3 0.5 0.3 1.1 0,4 17.9 one" 4.5 0,4 3.8 36.7 3.2 4.5 0.2 19.0 0.9 1.1 1.4 1.8 0,4 1,2 0.5 0.2 1.0 0.5 17.8 17 5.2 0,4 2.8 27.8 3.7 5.3 0.5 18.3 0.8 1.7 1,3 1.0 0,4 1.9 0.7 0.3 1.7 0.5 24.7 18 5.4 0.6 2.8 31.7 4.1 4.6 0.3 18.5 0.8 1,3 1,3 1.4 0,4 1.4 0.6 0.2 1,3 0,4 21.8 19 6.4 0.6 2.7 30.3 3.5 4.1 0,4 16.1 0.8 2.1 1.1 0.9 0,4 1.4 0.7 0.2 1.1 0.5 25.8 twenty 4.3 0.3 3.2 39.2 3.3 5.7 0.2 20.1 0.7 0.9 1.6 1.7 0.3 1,3 0.3 0.2 1,3 0.3 14.1

Example 9. Further analysis of transformed plants and field trials.

Hybridization analysis by Southern blotting was performed for selected T2 plants of B. napus transformed with T-DNA from the GA7-modB construct. DNA extracted from plant tissue samples was digested with several restriction enzymes for hybridization analysis by Southern blotting. A radioactive probe corresponding to a portion of the T-DNA was hybridized to the spots, which were washed under stringent conditions, and the spots were contacted with the film to detect hybridization bands. Some of the samples showed single hybridization bands for each of the mixtures after restriction enzyme digestion, which corresponded to single insertions of T-DNA in plants, while others showed two bands, and the following again showed several T-DNA bands, which corresponded to 4-6 insertions. The number of hybridization bands observed in Southern blot analysis correlated well with the copy number of T-DNA in transgenic plants as determined by digital PCR, up to a copy number of about 3 or 4. With a copy number greater than about 5, digital PCR was less reliable. ...

Some of the selected lines were used as pollen donors when crossed with series of about 30 different varieties of B. napus with different background genotypes. Further backcrossing was performed to determine if multiple T-DNA insertions were genetically linked or not, and to allow segregation of genetically unrelated transgenic loci. Thus, lines were selected containing single transgenic loci.

One-primer PCR reactions were performed in transgenic lines using primers adjacent to the left and right boundaries of the T-DNA, and any lines showing the presence of reverse T-DNA repeats were discarded.

A number of transgenic lines showed delayed flowering, while others showed reduced seed production and thus reduced seed yield per plant after greenhouse cultivation, consistent with reduced male or female fertility. Investigated the morphology of the flower in these plants and observed that in some cases the opening of the seed pods and the release of pollen from the anthers was delayed in such a way that the stigmas of the pistil lengthened until the discs opened and the pollen was released, thus moving the anthers away from the stigmas. Full fertility could be restored by artificial pollination. In addition, the viability of pollen was determined at the moment of opening the seed pods and the release of pollen using staining dyes to determine the viability of PDA and PI (example 1), and it was shown that it was reduced in some lines, while in most transgenic lines the viability of pollen was about 100% of the viability in wild-type control plants. As a further analysis to identify the possible cause of the decrease in seed yield from some plants, the content and composition of fatty acids in flower buds, including the anthers and stigmas / pistils of some T3 and T4 plants, were analyzed. DHA was not detected in the extracted lipids, suggesting that genes in the genetic construct were not expressed in flower buds during plant development, and excluding this as a cause of reduced seed yield.

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Oil content was measured by NMR, and the level of DHA in total fatty acids was determined for T2 seeds. Transgenic lines containing less than 6% DHA were discarded. The number of T-DNA copies in leaf samples of plants of generations T1, T2 and T3 was determined by digital PCR (example 1).

Selected lots of T3 and T4 seeds were sown in the field at two sites in Victoria, Australia, each in rows 10 m long with a seeding density of 10 seeds / m. Selected lots of seeds included a line derived from B003-5-14 that showed DHA levels in the seed pool of about 8-11% and DHA levels for individual T2 seeds up to about 19%, with a T-DNA 3 copy number in the T0 plant. In addition, the selected seed lots included lines derived from B0050-27, which showed DHA levels in T2 seeds in excess of 20% and a T-DNA copy number of 1 or 2 in T2 plants. Seeds sown in the field germinated and sprouted at the same rate as wild-type seeds. Plants grown from most but not all seeded seed lots were phenotypically normal, for example, showed morphology, growth rate, plant height, male and female fertility, pollen viability (100%), seed formation, size and morphology of pods that were essentially the same as wild-type control plants grown under the same conditions. The seed yield per plant was similar to that of wild-type control plants grown under the same conditions. Other seed samples were sown in large areas for mass production of the selected transgenic lines. The total DHA content of the harvested seed was at least 30 mg / g seed.

Those skilled in the art will understand that numerous variations and / or modifications of the invention may be made as disclosed in specific embodiments without departing from the spirit or context of the invention in its broadest sense. The options presented are therefore to be considered in all respects as illustrative and not restrictive.

Any discussion of documents, acts, materials, devices, articles, and the like that is included in this document is intended solely for the purpose of providing context for the present invention. It should not be assumed that any or all of these issues form part of the prior art or constitute public knowledge in the field to which the present invention pertains, as it existed prior to the priority date of each of the claims of this application.

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Claims (28)

1. Extracted plant lipid containing fatty acids in esterified form, the fatty acids include oleic acid, palmitic acid, ω6 fatty acids including linoleic acid (LA), ω3 fatty acids including α-linoleic acid (ALA), docosapentaenoic acid ( DPA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), and the level of DPA in the total fatty acid content of the extracted lipid ranges from 7 to 35%. 2. Lipid according to claim 1, characterized in that it is characterized by at least one of the following features: i) the level of palmitic acid in the total fatty acid of the extracted lipid is about 2 to 15% or about 3 to about 10%, ii) the level of myristic acid (C14: 0) in the total fatty acid of the extracted lipid is about 0.1 %, iii) the level of oleic acid in the total fatty acid content of the extracted lipid is from about 1 to about 30%, from about 3 to about 30%, from about 6 to about 30%, from 1 to about 20%, from about 30 to about 60%, about 45 to about 60%, about 30%, or about 15 to about 30%, iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is about 4 to about 35%, about 4 to about 20%, about 4 to 17%, or about 5 to about 10%, v) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is from about 4 to about 40%, from about 7 to about 40%, from about 10 to about 35%, from about 20 to about 35%, about 4 to 16%, or about 2 to 16%, vi) the level of γ-linolenic acid (GLA) in the total fatty acids of the extracted lipid is less than 4, less than about 3, less than about 2, less than about 1, less than about 0.5%, 0.05 to 7%, 0.05 to 4%, 0.05 to about 3%, or 0.05 to about 2%, vii) stearidonic acid (SDA) level in the total fatty acid content of the extracted lipid is less than about 10, less than about 8, less than about 7, less than about 6, less 80 037184 than about 4, less than about 3%, from about 0.05 to about 7%, from about 0.05 to about 6%, from about 0.05 to about 4%, from about 0.05 to about 3%, about 0.05 to about 10%, or 0.05 to about 2%, viii) the level of eicosatetraenoic acid (ETA) in the total fatty acids of the extracted lipid is less than about 6, less than about 5, less than about 4, less than about 1, less than about 0.5%, from about 0.05 to about 6%, from about 0.05 to about 5%, from about 0.05 to about 4%, from about 0, 05 to about 3% or from 0.05 to about 2%, ix) the level of eicosatrienoic acid (ETRA) in the total fatty acids of the extracted lipid is less than 4%, less than about 2, less than about 1%, from 0, 05 to 4%, 0.05 to 3%, or 0.05 to about 2%, or 0.05 to about 1%, x) The level of eicosapentaenoic acid (EPA) in the total fatty acids of the extracted lipid is 4 to 15%, less than 4%, less than about 3, less than about 2%, from 0.05 to 10%, from 0.05 up to 5%, from 0.05 to about 3%, or from 0.05 to about 2%, xi) the level of docosahexaenoic acid (DHA) in the total fatty acid content of the extracted lipid is less than 2% or from 0.05 to about 2 %, xii) the lipid contains less than 0.1% ω6-docosapentaenoic acid (22: 5 d ^{4 7 10 13 16} ) in the total fatty acids, xiii) the level of total saturated fatty acids in the total fatty acids of the extracted lipid is from about 4 to about 25%, from about 4 to about 20%, from about 6 to about 20%, from about 6 to about 12%, xiv) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 4 to about 40%, about 4 to about 35%, about 8 to about 25%, 8 to about 22%, about 15 to about 40%, or about 15 days about 35%, xv) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is from about 20 to about 75%, from 30 to 75%, from about 50 to about 75%, about 60, about 65, about 70 , about 75% or about 60 to about 75%, xvi) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is from about 35 to about 50%, from about 20 to about 35%, from about 6 to 20% , less than 20, less than about 16, less than about 10%, from about 1 to about 16%, from about 2 to about 10%, or from about 4 to about 10%, xvii) the level of total ω6 fatty acids in the total content fatty acids of the extracted lipid is less than about 10, less than about 8, less than about 6%, less than 4%, about 1 to about 20%, about 1 to about 10%, 0.5 to about 8% or from 0.5 to 4%, while the indicated ω6 fatty acids are the sum of all ω6 fatty acids, except LA, in the extracted lipid, expressed as a percentage of the total sodium retention of fatty acids in the extracted lipid, xviii) the level of total ω 3 fatty acids in the total fatty acid content of the extracted lipid is from 36 to about 65%, from 36 to about 70%, from 40 to about 60%, from about 30 to about 60 %, about 35 to about 60%, 40 to about 65%, about 30 to about 65%, 35 to about 65%, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or about 70%, xix) the level of total ω 3 fatty acids in the total fatty acid content of the extracted lipid is from 21 to about 45%, from 21 to about 35%, from about 23 to about 35%, from about 25 to about 35%, about 27 to about 35%, about 23, about 25, about 27, about 30, about 35, about 40, or about 45%, wherein said ω3 fatty acids are the sum of all ω3 fatty acids other than ALA, in the extracted lipid, expressed as a percentage of the total fatty acids in the extracted lipid, xx) the ratio of total ω6 fatty acids to total ω3 fatty acids in the extract the lipid is from about 1.0 to about 3.0, from about 0.1 to about 1, from about 0.1 to about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1, about 0.10 to about 0.4, or about 0.2, xxi) ratio of total ω6 fatty acids (xvii) to total ω3 fatty acids (xix) in the extracted lipid is from about 1.0 to about 3.0, from about 0.02 to about 0.1, from about 0.1 to about 1, from about 0.1 to about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.02, about 0.05, about 0.1, about 0.2, or about 1.0, xxii) the total fatty acids in the extracted lipid contain less than 1.5% C20: 1, less than 1% C20: 1, or about 1% C20 : 1, xxiii) the content of triacylglyceride (TAG) in the extracted lipid is at least about 70, at least about 80, at least about 90, at least 95% (w / w), from about 70 d about 99% (w / w) or about 90 to about 99% (w / w), xxiv) lipid contains diacylglyceride (DAG), and DAG contains DPA, xxv) at least 70% esterified DHA and / or DPC in the TAG is in position sn-1 or sn-3 TAG and xxvi) the level of DPC in the total fatty acid content of the extracted lipid is about - 81 037184 lo 7, about 8, about 9, about 10, about 12, about 15, about 18, about 20, about 22, about 24, about 26, about 28, about 31, from about 7 to about 31%, about 7 to about 28%, about 10 to 35%, about 10 to about 30%, about 10 to about 25%, about 10 to about 22%, about 14 to 35%, about 16 to 35%, about 16 to about 30%, about 16 to about 25%, or about 16 to about 22%. 3. The lipid of claim 2, wherein the DHA level is less than 0.5% of the total fatty acid content of the extracted lipid. 4. Lipid according to any one of claims 1 to 3, characterized in that it is a component of an oil obtained from an oilseed crop. 5. Lipid according to claim 4, characterized in that the oil is obtained from plants of the genus Brassica or plants of the genus Camelina, in particular C. sativa. 6. A method of obtaining an extracted plant lipid according to any one of claims 1 to 5, comprising the steps of: I) obtaining a plant part containing a lipid comprising a fatty acid profile according to any one of claims 1 to 5, wherein the plant part contains exogenous polynucleotides encoding one of the following sets of enzymes: a) ω3-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, b) Δ 15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, c) Δ12-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, d) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, e) ω3-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, f) Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, g) Δ12-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, h) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, i) ω3-desaturase or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, or j) ω3-desaturase or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5- elongase, each polynucleotide is operably linked to one or more promoters capable of directing the expression of said polynucleotides in a cell of a plant part, and Ii) extraction of a lipid from a plant part, wherein the level of DPA in the total fatty acid content of the extracted lipid is from 7 to 35%, wherein one or more cells of the plant part contain at least about 25% more ω3 fatty acids than the corresponding cells without exogenous polynucleotides. 7. A method according to claim 6, wherein the plant portion is a seed. 8. A method according to claim 7, wherein the seed is the seed of Brassica napus, B. juncea or C. sativa. 9. A method according to any one of claims 6 to 8, characterized in that the plant part contains exogenous polynucleotides encoding Δ12 desaturase, ω3 desaturase and / or Δ15 desaturase, Δ6 desaturase, Δ5 desaturase, Δ6 elongase and Δ5- elongase. 10. A method according to any one of claims 6 or 9, characterized in that said enzymes have the following activities: i) Δ6-desaturase preferably desaturates α-linolenic acid (ALA) relative to linoleic acid (LA), ii) Δ6-elongase also has Δ9-elongase activity, iii) Δ12-desaturase also has Δ15-desaturase activity, iv) Δ6- desaturase also has Δ8-desaturase activity, v) Δ8-desaturase also has a Δ6-desaturase activity, vi) Δ 15-desaturase also has an ω3-desaturase activity against GLA, vii) ω3-desaturase also has a Δ15-desaturase activity against LA, viii) ω3-desaturase performs desaturation of LA and / or GLA, ix) ω3-desaturase performs desaturation of GLA relative to LA, x) one or more or all desaturase has a higher activity towards acylKoA than towards acyl-PC (acyl-phosphatidylcholine), xi) Δ6-desaturase has a higher activity of Δ6-desaturase towards ALA than towards LA , xii) Δ6-desaturase has a higher activity against ALA-CoA than against ALA attached to the sn-2 position of PC, xiii) Δ6-desaturase has at least 2 times higher Δ6-desaturase activity, at least 3 times higher activity, at least 4 times higher activity, or at least 5 times higher activity against ALA than against LA, - 82 037184 xiv) Δ6-desaturase has a higher activity against ALA-CoA than against ALA attached to the sn-2 position of PC, xv) Δ6-desaturase has at least 5-fold or 10-fold higher Δ6-desaturase activity against ALA-CoA than against ALA attached to the sn-2 position of PC, xvi ) desaturase is a front-end desaturase; and xvii) Δ6-desaturase has no detectable Δ5-desaturase activity against ETA. 11. The method according to any one of claims 6-10, characterized in that the exogenous polynucleotides are covalently linked into a T-DNA molecule, which is integrated into the genome of the cells of the plant part, and the number of such T-DNA molecules integrated into the genome of the cells of the plant part is one, two or three. 12. The method according to any one of claims 6-11, characterized in that it further comprises treating the extracted lipid to increase the level of DPA as a percentage of the total fatty acid content, and the treatment includes fractionation, distillation or transesterification, in particular, to obtain methyl or ethyl esters WPC or a combination of these methods. 13. Oilseed plant containing a lipid according to any one of claims 1 to 5, in a seed, or part thereof, comprising exogenous polynucleotides encoding one of the following sets of enzymes: i) Δ12-desaturase, ω3-desaturαse and / or Δ15-desaturαza, Δ6-desaturase, Δ5-desaturαza, Δ6-elongαse and Δ5-elongαse, ii) Δ12-desaturase, ω3-desaturαse and / or Δ15-desaturαse, Δ8- desaturase, Δ5-desaturαse, Δ9-elongase and Δ5-elongase, iii) ω3-desaturase and / or Δ15-desaturase, Δ6-desaturase, Δ5-desaturαse, Δ6-elongase and Δ5-elongase or iv) ω3-desaturase and / or Δ15 desaturase, Δ8 desaturase, Δ5 desaturαse, Δ9 elongase and Δ5 elongase, each polynucleotide being operably linked to one or more seed specific promoters capable of directing the expression of said polynucleotides in a developing plant seed. 14. The plant or part thereof according to claim 13, comprising exogenous polynucleotides encoding Δ12 desaturase, ω3 desaturase and / or Δ15 desaturase, Δ6 desaturase, Δ5 desaturase, Δ6 elongase and Δ5elongase. 15. A plant according to claim 13 or 14, which is Brassica napus, B. juncea or Camelina sativa, wherein the mature seed of said plant contains WPC in an amount of at least about 28 mg per gram of seed. 16. A plant cell according to any one of claims 13-15, containing exogenous polynucleotides encoding one of the following sets of enzymes: i) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ6-desaturase, Δ5-desaturαse, Δ6-elongαse and Δ5-elongαse, ii) Δ12-desaturase, ω3-desaturαse and / or Δ15-desaturase, Δ8 -desaturationαza, Δ5-desaturαza, Δ9-elongase and Δ5-elongase, iii) ω3-desaturase and / or Δ15-desaturase, Δ6-desaturase, Δ5-desaturαza, Δ6-elongase and Δ5-elongase, or iv) ω3-desaturase, and / or Δ 15-desaturase, Δ8-desaturase, Δ5-desaturαse, Δ9-elongase and Δ5-elongase, each polynucleotide being operably linked to one or more seed-specific promoters capable of directing the expression of said polynucleotides in a developing plant seed. 17. A plant seed which is obtained from a plant according to any one of claims 13 or 15 and contains a lipid according to any one of claims 1 to 5. 18. The seed of claim 17, which is a mature, harvested seed of Brassica napus, B. juncea, or Camelina sativa having a moisture content of about 4 to about 15% by weight, the WPC content of said seed being at least about 28 mg. for 1 g of seeds. 19. A method for producing an extracted plant lipid according to any one of claims 1 to 5, comprising the following steps: i) obtaining a seed according to claim 17 or 18; and ii) extracting the lipid from the seed. 20. A method for producing a plant containing a lipid according to any one of claims 1-5, comprising: a) quantifying the level of duodenum in a lipid that is formed in one or more parts of a plurality of plants, each plant containing at least one exogenous polynucleotide encoding one of the following sets of enzymes; i) ω3-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, ii) Δ 15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase, iii) Δ12-desaturase , Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongαza, iv) Δ12-desaturαza, ω3-desaturase and / or Δ 15-desaturase, Δ6-desaturase, Δ5-desaturαza, Δ6-elongαse - 83 037184 and Δ5-elongase, v) ω3 desaturase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, vi) Δ 15 desaturase, Δ8 desaturase, Δ5 desaturase, Δ9 elongase and Δ5 elongase, vii) Δ12 desaturase , Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, viii) Δ12-desaturase, ω3-desaturase and / or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5-elongase, ix ) ω3-desaturase or Δ15-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase and Δ5-elongase; or x) ω3-desaturase or Δ15-desaturase, Δ8-desaturase, Δ5-desaturase, Δ9-elongase and Δ5- elongase, each polynucleotide is operably linked to one or more promoters capable of directing the expression of said polynucleotides in a cell of a plant part, and b) identifying a plant among a plurality of plants, one or more parts of which contain a lipid according to any one of claims 1 to 5, and c) obtaining offspring from an identified plant or its seeds. 21. A method of obtaining seeds, including: a) growing in the field one or more plants according to any one of claims 13-15, which form seeds according to claim 17 or 18, and b) harvesting the seeds of these plants. 22. A method for producing a lipid according to any one of claims 1-5, comprising obtaining seeds by a method according to claim 21 and extracting a lipid from said seeds, which has a total WPC yield of at least 60 kg WPC per 1 ha. 23. Oil cake obtained from seeds according to claim 17 or 18, or from seeds obtained from a plant according to any one of claims 13-15. 24. A composition comprising a lipid according to any one of claims 1 to 5 in combination with a standard, known, non-toxic food or agricultural acceptable carrier, adjuvant or solvent such as phosphate buffered saline, water, ethanol, polyols, vegetable oils, a moisturizing agent or emulsion such as a water / oil emulsion, but also with an isotonic agent, sugar, sodium chloride, sweeteners, flavors and aromas, vitamins, fatty acids, proteins or vitamins, other food or feed ingredients, wherein said composition is in a form selected from tablets, capsules, swallowable liquid or powder, injectable form, or in the form of a topical ointment or cream for use in the food industry or agriculture. 25. Feed containing a lipid according to any one of claims 1 to 5, a plant or part of a plant according to any one of claims. 13-15, plant cells according to claim 16, seeds according to claim 17 or 18, cake according to claim 23, or a combination thereof. 26. A method of producing food, comprising mixing a lipid according to any one of claims 1-5, a plant or plant part according to any one of claims 13-15, plant cells of claim 16, seeds according to claim 17 or 18, or a cake according to claim 23 with at least one other feed ingredient. 27. A method of producing a food product, comprising mixing a lipid according to any one of claims 1-5, a plant or a part of a plant according to any one of claims 13-15, plant cells according to claim 16, seeds according to claim 17 or 18, or a cake according to claim .23 with at least one other food ingredient. 28. Use of a lipid according to any one of claims 1 to 5 for the preparation of a medicament for the treatment or prevention of a condition in which PUFAs have a beneficial effect.