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CA-3228580-C - LIPID COMPRISING DOCOSAPENTAENOIC ACID

CA3228580CCA 3228580 CCA3228580 CCA 3228580CCA-3228580-C

Abstract

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

Inventors

  • James Robertson Petrie
  • Surinder Pal Singh
  • Pushkar Shrestha
  • Jason Timothy McAllister
  • Robert Charles de Feyter
  • Malcolm David Devine

Assignees

  • GRAINS RESEARCH AND DEVELOPMENT CORPORATION
  • COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION
  • NUSEED NUTRITIONAL AUSTRALIA PTY LTD

Dates

Publication Date
20260505
Application Date
20150618
Priority Date
20141218

Claims (20)

  1. 92370127 148 CLAIMS: 1. Extracted Brassica napus seedoil, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, 6 fatty acids which comprise linoleic acid (LA) and -linolenic acid (GLA), 3 fatty acids which comprise -linolenic acid (ALA), docosapentaenoic acid (DPA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), wherein the level of palmitic 5 acid in the total fatty acid content of the extracted seedoil is between 2% and 16%, wherein the level of myristic acid (C14:0) in the total fatty acid content of the extracted seedoil, if present, is less than 1%, wherein at least 70% of the DPA esterified in the form of triacylglycerols (TAG) is in the sn-1 or sn-3 position of the TAG, and wherein ALA, SDA, ETA, EPA, DPA and eicosatrienoic acid (ETrA) are each present at a level in the total fatty acid content, each level being expressed as a percentage of the total fatty 10 acid content, whereby the sum of the percentages for DPA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA and ETrA, expressed as a percentage, is between 15.3% and 60.5%.
  2. 2. The seedoil of claim 1, wherein the sum of the percentages for DPA and DHA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA, DHA and ETrA, expressed as a percentage, is between 17% and 55%. 15
  3. 3. The seedoil of claim 1 or claim 2 which has one or more of the following features: i) the level of palmitic acid in the total fatty acid content of the extracted seedoil is between 2% and 15%, ii) the level of myristic acid in the total fatty acid content of the extracted seedoil is about 0.1%, iii) the level of oleic acid in the total fatty acid content of the extracted seedoil is between 1% and 20 30%, iv) the level of LA in the total fatty acid content of the extracted seedoil is between 4% and 35%, v) the level of ALA in the total fatty acid content of the extracted seedoil is between 4% and 40%, vi) the level of GLA in the total fatty acid content of the extracted seedoil is less than 4%, vii) the level of SDA in the total fatty acid content of the extracted seedoil is less than 8%, 25 viii) the level of ETA in the total fatty acid content of the extracted seedoil is less than 4%, ix) the level of ETrA in the total fatty acid content of the extracted seedoil is less than 4%, x) the level of total saturated fatty acids in the total fatty acid content of the extracted seedoil is between 4% and 25%, xi) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted seedoil 30 is between 4% and 40%, CA 3228580 92370127 149 xii) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted seedoil is between 20% and 75%, xiii) the level of total 6 fatty acids in the total fatty acid content of the extracted seedoil is between 20% and 35%, xiv) the level of new 6 fatty acids in the total fatty acid content of the extracted seedoil is less than 5 6%, xv) the level of total 3 fatty acids in the total fatty acid content of the extracted seedoil is between 36% and 65%, xvi) the level of new 3 fatty acids in the total fatty acid content of the extracted seedoil is between 21% and 45%, 10 xvii) the ratio of total 6 fatty acids: total 3 fatty acids in the fatty acid content of the extracted seedoil is between 0.1 and 1, xviii) the ratio of new 6 fatty acids: new 3 fatty acids in the fatty acid content of the extracted seedoil is between 0.02 and 0.1, xix) the total fatty acid in the extracted seedoil has less than 1.5% C20:1, 15 xx) the TAG content of the seedoil is at least 80%, xxi) the seedoil comprises diacylglycerol (DAG), which DAG comprises DPA, xxii) the extracted seedoil comprises less than 1% non-esterified fatty acids and/or phospholipid, and xxiii) at least 80% of the DPA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG. 20
  4. 4. The extracted seedoil according to any one of claims 1 to 3, wherein the level of GLA in the total fatty acid content of the extracted seedoil is less than 4%.
  5. 5. The extracted seedoil according to any one of claims 1 to 4, wherein the level of total saturated fatty acids in the total fatty acid content of the extracted seedoil is between 6% and 12%.
  6. 6. The extracted seedoil according to any one of claims 1 to 5, wherein the ratio of total ω6 fatty 25 acids: total ω3 fatty acids in the fatty acid content of the extracted seedoil is between 0.1 and 1.
  7. 7. The extracted seedoil according to any one of claims 1 to 6, wherein at least 80% of the DPA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG. CA 3228580 92370127 150
  8. 8. The extracted seedoil according to any one of claims 1 to 7, wherein the seedoil was obtained from Brassica napus seed harvested from a population of at least 1,000 Brassica napus plants grown in a field.
  9. 9. The extracted seedoil according to any one of claims 1 to 8, wherein docosahexaenoic acid (DHA) is either absent from the total fatty content or is present at less than 2% in the total fatty acid content of the 5 seedoil.
  10. 10. A composition comprising the seedoil according to any one of claims 1 to 9, wherein the seedoil has not been blended with another lipid, and wherein the composition comprises a suitable carrier.
  11. 11. A process for producing extracted Brasicca napus seedoil, comprising the steps of: i) obtaining transgenic Brassica napus seed comprising seedoil, comprising fatty acids in an 10 esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 polyunsaturated fatty acids which comprise linoleic acid (LA), ω3 polyunsaturated fatty acids which comprise α-linolenic acid (ALA), docosapentaenoic acid (DPA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), wherein the level of palmitic acid in the total fatty acid content of the seedoil is between 2% and 16%, wherein the level of myristic acid (C14:0) in the total fatty acid content of the seedoil, if present, 15 is less than 1%, wherein at least 70% of the DPA esterified in the form of triacylglycerols (TAG) in the seedoil is in the sn-1 or sn-3 position of the TAG, and wherein ALA, SDA, ETA, EPA, DPA and eicosatrienoic acid (ETrA) are each present at a level in the total fatty acid content, each level being expressed as a percentage of the total fatty acid content, whereby the sum of the percentages for DPA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA and ETrA, expressed as 20 a percentage, is between 15.3% and 60.5%, wherein the seed is transgenic for one or more exogenous polynucleotides encoding at least a Δ12-desaturase, an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase and a Δ5-elongase, and wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in a cell of the seed, and ii) extracting seedoil from the seed. 25
  12. 12. The process of claim 11, wherein the total fatty acid content in the seedoil has less than 1.5% C20:1.
  13. 13. The process of claim 11 or claim 12, wherein docosahexaenoic acid (DHA) is either absent or present at less than 2% in the total fatty acid content of the extracted seedoil.
  14. 14. The process according to any one of claims 11 to 13 which further comprises treating the seedoil 30 to increase the level of DPA as a percentage of the total fatty acid content, wherein the treatment comprises one or more of fractionation, distillation or transesterification. CA 3228580 92370127 151
  15. 15. The process of claim 14, wherein the transesterification comprises the production of methyl- or ethyl-esters of DPA.
  16. 16. A transgenic Brassica napus seed cell comprising seedoil as defined in any one of claims 1 to 9, wherein the cell is transgenic for one or more exogenous polynucleotides encoding a Δ12-desaturase, an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase and a Δ5-elongase, wherein each 5 polynucleotide is operably linked to one or more seed-specific promoters that are capable of directing expression of said polynucleotides in a cell of a developing seed of a B. napus plant.
  17. 17. The seed cell of claim 16, wherein the total fatty acid content in the seedoil has less than 1.5% C20:1.
  18. 18. Seedmeal obtained from seed comprising a cell according to claim 16 or claim 17. 10
  19. 19. A composition comprising the cell according to claim 16 or claim 17 and a suitable carrier.
  20. 20. A feedstuff comprising one or more of the seedoil according to any one of claims 1 to 9, the cell according to claim 16 or claim 17, the seedmeal of claim 18, or the composition of claim 10 or claim 19.

Description

LIPID COMPRISING DOCOSAPENTAENOIC ACID This is a divisional application of Canadian Patent Application Serial No. 2,953,008, filed on June 18, 2015. FIELD OF THE INVENTION The present invention relates to lipid compnsmg docosapentaenoic acid, 5 obtained from plant cells or microbial cells, and processes for producing and using the lipid. BACKGROUND OF THE INVENTION Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) are now widely 10 recognized as important compounds for human and animal health. These fatty acids may be obtained from dietary sources or by conversion of linoleic (LA, 18 :2ro6) or alinolenic (ALA, 18:3ro3) fatty acids, both of which are regarded as essential fatty acids in the human diet. While humans and many other vertebrate animals are able to convert LA or ALA, obtained from plant sources to C22 they carry out this conversion 15 at a very low rate. Moreover, most modem societies have imbalanced diets in which at least 90% of polyunsaturated fatty acids (PUF A) are of the ro6 fatty acids, instead of the 4:1 ratio or less for ro6:co3 fatty acids that is regarded as ideal (Trautwein, 2001). The immediate dietary source of LC-PUFAs such as eicosapentaenoic acid (EPA, 20:5ro3), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA, 22:6co3) for 20 humans is mostly from fish or fish oil. Health professionals have therefore recommended the regular inclusion of fish containing significant levels of LC-PUFA into the human diet. Increasingly, fish-derived LC-PUFA oils are being incorporated into food products and in infant formula, for example. However, due to a decline in global and national fisheries, alternative sources of these beneficial health-enhancing 25 oils are needed. Flowering plants, in contrast to animals, lack the capacity to synthesise polyunsaturated fatty acids with chain lengths longer than 18 carbons. In particular, crop and horticultural plants along with other angiosperms do not have the enzymes needed to synthesize the longer chain co3 fatty acids such as EPA, docosapentaenoic 30 acid (DPA, 22:5co3) and DHA that are derived from ALA. An important goal in plant biotechnology is therefore the engineering of crop plants which produce substantial quantities ofLC-PUFA, thus providing an alternative source of these compounds. LC-PUF A Biosynthesis Pathways 35 Biosynthesis of LC-PUFAs in organisms such as microalgae, mosses and fungi usually occurs as a series of oxygen-dependent desaturation and elongation reactions Date Rec;ue/Date Received 2024-02-08 CA 02953008 2016-12-20 2 (Figure 1 ). The most common pathway that produces EPA in these organisms includes a Ll6-desaturation, .tl6-elongation and Ll5-desaturation (termed the .tl6-desaturation pathway) whilst a less common pathway uses a .tl9-elongation, Ll8-desaturation and .tl5- desaturation (termed the .tl9-desaturation pathway). These consecutive desaturation 5 and elongation reactions can begin with either the ro6 fatty acid substrate LA, shown schematically as the upper left part of Figure 1 (ro6) or the ro3 substrate ALA through to EPA, shown as the lower right part of Figure 1 (ro3). If the initial Ll6-desaturation is performed on the ro6 substrate LA, the LC-PDF A product of the series of three enzymes will be the ro6 fatty acid ARA. LC-PDFA synthesising organisms may 10 convert ro6 fatty acids to ro3 fatty acids using an ro3-desaturase, shown as the Ll 17- desaturase step in Figure 1 for conversion of arachidonic acid (ARA, 20:4ro6) to EPA. Some members of the ro3-desaturase family can act on a variety of substrates ranging from LA to ARA. Plant ro3-desaturases often specifically catalyse the MSdesaturation of LA to ALA, while fungal and yeast ro3-desaturases may be specific for 15 the Ll17-desaturation of ARA to EPA (Pereira et al., 2004a; Zank et al., 2005). Some reports suggest that non-specific ro3-desaturases may exist which can convert a wide variety of ro6 substrates to their corresponding ro3 products (Zhang et al., 2008). The conversion of EPA to DHA in these organisms occurs by a Ll5-elongation of EPA to produce DPA, followed by a Ll4-desaturation to produce DHA (Figure 1). In 20 contrast, mammals use the so-called "Sprecher" pathway which converts DP A to DHA by three separate reactions that are independent of a Ll4-desaturase (Sprecher et al., 1995). The front-end desaturases generally found in plants, mosses, microalgae, and lower animals such as Caenorhabditis elegans predominantly accept fatty acid 25 substrates esterified to the sn-2 position of a phosphatidylcholine (PC) substrate. These desaturases are therefore known as acyl-PC, lipid-linked, front-end desaturases (Domergue et al., 2003). In contrast, higher animal front-end desaturases generally accept acyl-CoA substrates where the fatty acid substrate is linked to CoA rather than PC (Domergue et al., 2005). Some microalgal desaturases and one plant desaturase are 30 known to use fatty acid substrates esterified to CoA (Table 2). Each PDF A el