US 10,648,046 B2Granted

Enzymes and methods for producing omega-3 fatty acids

AssigneesCommonwealth Scientific and Industrial Research Organisation, Grains Research and Development Corporation

InventorsJames Robertson Petrie, Anne Maree Mackenzie, Qing Liu, Pushkar Shrestha, Peter David Nichols, Susan Irene Ellis Blackburn, Maged Peter Mansour, Stanley Suresh Robert, Dion Matthew Frederick Frampton, Xue-Rong Zhou, Surinder Pal Singh, Craig Christopher Wood

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Abstract

The present invention relates generally to the field of recombinant fatty acid synthesis, particularly in transgenic plants. The application describes genes involved in fatty acid synthesis and provides methods and vectors for the manipulation of fatty acid composition of plant oils. In particular, the invention provides constructs for achieving the integration of multiple heterologous genes involved in fatty acid synthesis into the plant genome, such that the resulting plants produce altered levels of polyunsaturated fatty acids. Also described are methods for enhancing the expression of fatty acid biosynthesis enzymes by co-expressing a silencing suppressor within the plant storage organ.

Claims24

Dependent claims

A process for producing seed comprising eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), the process comprising the steps of

(i) growing a Brassica napes plant comprising exogenous polynucleotides encoding a microalgal Δ6-desaturase which has greater Δ6-desaturase activity on α-linolenic acid (ALA) joined to CoA (ALA-CoA) as fatty acid substrate than on ALA joined to the sn-2 position of phosphatidylcholine (PC) as the fatty acid substrate, a Δ6-elongase, a microalgal Δ5-desaturase, a microalgal Δ5-elongase, and a Pavlova spp. Δ4-desaturase, wherein each polynucleotide is operably linked to one or more promoters that direct expression of said polynucleotides during the development of the seed, to produce seed that comprises a total fatty acid content comprising ALA, stearidonic acid (SDA), eicosatetraenoic acid (ETA), EPA, DPA and DHA, wherein the ALA, SDA, ETA, EPA, DPA and DHA are each present at a level in the total fatty acid content of the seed, each level being expressed as a percentage of the total fatty acid content, such that the sum of the percentages for DPA and DHA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA and DHA, expressed as a percentage, is at least 15.4%, and
(ii) harvesting seed from the plant, thereby producing the seed comprising EPA, DPA and DHA.

A process for producing seed comprising eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), the process comprising the steps of

(i) growing a Brassica napes plant comprising exogenous polynucleotides encoding a microalgal Δ6-desaturase which has greater Δ6-desaturase activity on α-linolenic acid (ALA) joined to CoA (ALA-CoA) as fatty acid substrate than on ALA joined to the sn-2 position of phosphatidylcholine (PC) as the fatty acid substrate, a Δ6-elongase, a microalgal Δ5-desaturase, a microalgal Δ5-elongase, and a Pavlova spp. Δ4-desaturase, wherein each polynucleotide is operably linked to one or more promoters that direct expression of said polynucleotides during the development of the seed, to produce seed that comprises a total fatty acid content comprising ALA, stearidonic acid (SDA), eicosatetraenoic acid (ETA), EPA, DPA and DHA, wherein the ALA, SDA, ETA, EPA, DPA and DHA are each present at a level in the total fatty acid content of the seed, each level being expressed as a percentage of the total fatty acid content, such that the sum of the percentages for EPA, DPA and DHA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA and DHA, expressed as a percentage, is at least 17.3%, and
(ii) harvesting seed from the plant, thereby producing the seed comprising EPA, DPA and DHA.

A process for producing oil comprising eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), the process comprising the steps of

(i) obtaining Brassica napus seed comprising a total fatty acid content and exogenous polynucleotides encoding a microalgal Δ6-desaturase which has greater Δ6-desaturase activity on α-linolenic acid (ALA) joined to CoA (ALA-CoA) as fatty acid substrate than on ALA joined to the sn-2 position of phosphatidylcholine (PC) as the fatty acid substrate, a Δ6-elongase, a microalgal Δ5-desaturase, a microalgal Δ5-elongase, and a Pavlova spp. Δ4-desaturase, wherein each polynucleotide is operably linked to one or more promoters that direct expression of said polynucleotides during the development of the seed, wherein the total fatty acid content comprises ALA, stearidonic acid (SDA), eicosatetraenoic acid (ETA), EPA, DPA and DHA, wherein the ALA, SDA, ETA, EPA, DPA and DHA are each present at a level in the total fatty acid content of the seed, each level being expressed as a percentage of the total fatty acid content, such that the sum of the percentages for DPA and DHA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA and DHA, expressed as a percentage, is at least 15.4%, and
(ii) extracting oil from the seed, thereby producing the oil comprising EPA, DPA and DHA.

A process for producing oil comprising eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), the process comprising the steps of

(i) obtaining Brassica napus seed comprising a total fatty acid content and exogenous polynucleotides encoding a microalgal Δ6-desaturase which has greater Δ6-desaturase activity on α-linolenic acid (ALA) joined to CoA (ALA-CoA) as fatty acid substrate than on ALA joined to the sn-2 position of phosphatidylcholine (PC) as the fatty acid substrate, a Δ6-elongase, a microalgal Δ5-desaturase, a microalgal Δ5-elongase, and a Pavlova spp. Δ4-desaturase, wherein each polynucleotide is operably linked to one or more promoters that direct expression of said polynucleotides during the development of the seed, wherein the total fatty acid content comprises ALA, stearidonic acid (SDA), eicosatetraenoic acid (ETA), EPA, DPA and DHA, wherein ALA, SDA, ETA, EPA, DPA and DHA are each present at a level in the total fatty acid content of the seed, each level being expressed as a percentage of the total fatty acid content, such that the sum of the percentages for EPA, DPA and DHA divided by the sum of the percentages for ALA, SDA, ETA, EPA, DPA and DHA, expressed as a percentage, is at least 17.3%, and
(ii) extracting oil from the seed, thereby producing the oil comprising EPA, DPA and DHA.

Dependencies are inferred from the claim text.

Facts

Application: 15/642,223Filed 5 Jul 2017 · continuation
Priority: 18 Nov 2008
Published: US 2018/0087004 A129 Mar 2018
Granted: 12 May 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification56 codes

C12N 9/0083
Acting on paired donors with incorporation of molecular oxygen: miscellaneous
C12N 15/8247
Involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine: involving modified lipid metabolism, e.g. seed…
A61P 1/04
Drugs for disorders of the alimentary tract or the digestive system: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of…
A61P 3/02
Drugs for disorders of the metabolism: nutrients, e.g. vitamins, minerals

Family

Application61/199,669Us provisional application · filed 18 Nov 2008
Application61/270,710Us provisional application · filed 9 Jul 2009
PCT/AU2009/001488Parent of this continuation · filed 17 Nov 2009
Application14/332,202Parent of this continuation · filed 15 Jul 2014
US 8,809,559 B2Parent of this continuation · filed 19 Aug 2014
US 2018/0087004 A1Pre-grant publication · published 29 Mar 2018
US 9,938,486 B2Parent of this continuation
Application13/129,940Parent of this continuation
US 2012/0016144 A1Published application
US 2015/0018571 A1Published application
US 10,648,046 B2This patent

From the US filing history. Foreign family members aren’t listed.