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1. WO2011008803 - NOVEL Δ9-ELONGASE FOR PRODUCTION OF POLYUNSATURATED FATTY ACID-ENRICHED OILS

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[ EN ]

WHAT IS CLAIMED IS:

1. An isolated nucleic acid or fragment thereof comprising or complementary to an isolated nucleotide sequence encoding a polypeptide having elongase activity, wherein the amino acid sequence of the polypeptide has at least 68% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 20.

2. An isolated nucleotide sequence or fragment thereof comprising or complementary to at least 68% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19.

3. The isolated nucleotide sequence of claim 1 or 2 wherein the isolated nucleotide sequence encodes a functionally active elongase which utilizes a polyunsaturated fatty acid as a substrate.

4. The isolated nucleotide sequence of claim 1 or 2 wherein the isolated nucleotide sequence is from a Euglenoid sp.

5. The isolated nucleotide sequence of claim 4 wherein the isolated nucleotide sequence is from Euglena deses Ehr. CCMP 2916.

6. A purified polypeptide encoded by the isolated nucleotide sequence of claim 1 or 2.

7. A purified polypeptide which elongates polyunsaturated fatty acids containing unsaturation at the carbon 9 position and has at least 68% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 20.

8. An expression vector comprising a nucleotide sequence operably linked to a regulatory sequence, wherein the nucleotide sequence comprises or is complementary to at least 68% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19.

9. A host cell comprising the expression vector of claim 8.

10. The host cell of claim 9 wherein the host cell is selected from the group consisting of a eukaryotic cell and a prokaryotic cell.

11. The host cell of claim 10 wherein the cukaryotic cell is selected from the group consisting of: a mammalian cell, an insect cell, a plant cell, and a fungal cell.

12. The host cell of claim 11 wherein the plant cell is from an oilseed plant selected from the group consisting of: soybean, Brassica species, safflower, sunflower, maize, cotton, and flax.

13. A plant cell, plant seed, plant, or plant tissue comprising the expression vector of claim 8, wherein expression of the nucleotide sequence of the expression vector results in production of at least one polyunsaturated fatty acid by the plant cell, plant seed, plant, or plant tissue.

14. The plant cell, plant seed, plant or plant tissue of claim 13 wherein the polyunsaturated fatty acid is selected from the group consisting of ω6-eicosadienoic acid (ω6-EDA), ω3-eicosatrienoic acid (ω3-ETrA), and combinations thereof.

15. A method of producing a Δ9-elongase, the method comprising the steps of:

a) isolating a nucleotide sequence comprising or complementary to at least 68% of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 19;

b) constructing an expression vector comprising i) the isolated nucleotide sequence operably linked to ii) a regulatory sequence; and

c) introducing the expression vector into a host cell for a time and under conditions sufficient for production of the Δ9-elongase.

16. The method of claim 15 wherein the host cell is selected from the group consisting of a eukaryotic cell and a prokaryotic cell.

17. The method of claim 16 wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell, and a fungal cell.

18. The method of claim 17 wherein the plant cell is from an oilseed plant selected from the group consisting of soybean. Brassica species, safflower, sunflower, maize, cotton, and flax.

19. A method for producing a polyunsaturated fatty acid comprising the steps of:

a) isolating a nucleotide sequence comprising or complementary to at least 68% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19;

b) constructing an expression vector comprising i) the isolated nucleotide sequence operably linked to ii) a regulatory sequence;

c) introducing the expression vector into a host cell for a time and under conditions sufficient for expression of a Δ9-elongase; and

d) exposing the expressed Δ9-elongase to a substrate polyunsaturated fatty acid in order to convert the substrate polyunsaturated fatty acid to a first product polyunsaturated fatty acid.

20. The method of claim 19 wherein the substrate polyunsaturated fatty acid is linoleic acid (LA) and the first product polyunsaturated fatty acid is ω6-eicosadienoic acid (ω6-EDA).

21. The method of claim 19 wherein the substrate polyunsaturated fatty acid is α-linolenic acid (ALA) and the first product polyunsaturated fatty acid is ω3-eicosatrienoic acid (ω3-ETrA).

22. The method of claim 19 further comprising the step of exposing the first product polyunsaturated fatty acid to at (east one desaturase. at least one additional elongase. or combinations thereof, in order to convert the first product polyunsaturated fatty acid to a second or subsequent product polyunsaturated fatty acid.

23. The method of claim 22 wherein the second or subsequent product polyunsaturated fatty acid is selected from the group consisting of dihomo-γ-linoienic acid (DGLA). ω3-eicosatetraenoic acid (ω3-ETA). arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA). docosahexaenoic acid (DHA). and combinations thereof.

24. A method for producing a polyunsaturated fatty acid in a host cell comprising the steps of:

a) isolating a nucleotide sequence comprising or complementary to at least 68% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19;

b) constructing an expression vector comprising i) the isolated nucleotide sequence operably linked to ii) a regulatory sequence:

c) introducing i) the expression vector and ii) at least one additional recombinant DNA construct comprising an isolated nucleotide sequence encoding a Δ8-desaturase and operably linked to at least one regulatory sequence, into a host cell for a time and under conditions sufficient for expression of a Δ9-elongase and the Δ8-desaturase; and

d) exposing the expressed Δ9-elongase and the Δ8-desaturase to a substrate polyunsaturated fatty acid selected from the group consisting of linoleic acid (LA). α-linolenic acid (ALA), and combinations thereof, in order to convert the substrate polyunsaturated fatty acid to a first product polyunsaturated fatty acid.

25. The method of claim 24 wherein the first product polyunsaturated fatty acid is selected from the group consisting of dihomo-γ-linolensc acid (DGLA), ω3-eicosatetraenoic acid (ω3-ETA). and combinations thereof.

26. The method of claim 24 further comprising the step of exposing the first product polyunsaturated fatty acid to at least one additional desaturase or to at least one additional elongase in order to convert the first product polyunsaturated fatty acid to a second or subsequent polyunsaturated fatly acid.

27. The method of claim 26 wherein the second or subsequent

polyunsaturated fatty acid is selected from the group consisting of arachidonic acid (ARA). eicosapentaenoic acid (EPA). docosapentaenoic acid (DPA). docosahexaenoic acid (DHA). and combinations thereof.

28. The method of claim 24 wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

29. The method of claim 28 wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell, and a fungal cell.

30. The method of claim 29 wherein the plant cell is from an oilseed plant selected from the group consisting of soybean, Brassica species, safflower. sunflower, maize, cotton, and flax.

31. The method of claim 24 further comprising introducing into the host cell a recombinant DNA construct comprising i) an isolated nucleotide sequence encoding a Δ5-desaturase operably linked to ii) a regulatory sequence.

32. A method for producing a transgenic plant comprising transforming a plant cell with at least one isolated nucleotide sequence or fragment thereof of claim 2 and regenerating a transgenic plant from the transformed plant cell.

33. The method of claim 32 wherein the plant cell is from an oilseed pianl selected from the group consisting of soybean. Brassica species, safflower, sunflower, maize, cotton, and flax.

34. A transgenic seed obtained from the transgenic plant made by the method of claim 32.

35. A transgenic seed comprising the expression vector of claim 8.