Synthesis of long-chain polyunsaturated fatty acids by recombinant cell
The present invention relates to methods of synthesizing long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, in recombinant cells such as yeast or plant cells. Also provided are recombinant cells or plants which produce long-chain polyunsaturated fatty acids. Furthermore, the present invention relates to a group of new enzymes which possess desatorase or elongase activity that can be used in methods of synthesizing long-chain polyunsaturated fatty acids.
1. A Brassica plant cell for producing docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the plant cell comprising
a polynucleotide which encodes a Δ4 desaturase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;
a polynucleotide which encodes a Δ5 desaturase which has the amino acid sequence set forth as SEQ ID NO:18, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;
a polynucleotide which encodes a Δ6 desaturase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and
a polynucleotide which encodes a Δ5/Δ6 bifunctional elongase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell.
2. The Brassica plant cell of claim 1 which is a Brassica napus plant cell.
3. A Brassica napus seed comprising the Brassica napus plant cell of claim 2 .
4. The Brassica plant cell of claim 1 , further comprising an exogenous desaturase which desaturates an acyl-CoA substrate.
5. The Brassica napus plant seed of claim 3 , further comprising an exogenous desaturase which desaturates an acyl-CoA substrate.
6. A Brassica plant cell comprising
a polynucleotide which encodes a Δ4 desaturase which has the amino acid sequence encoded by a Δ4 desaturase gene from Pavlova lutheri , operably linked to a promoter which directs expression of the polynucleotide in the plant cell;
a polynucleotide which encodes a Δ5 desaturase which has the amino acid sequence set forth as SEQ ID NO: 18, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;
a polynucleotide which encodes a Δ6 desaturase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and
a polynucleotide which encodes a Δ5/Δ6 bifunctional elongase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell.
7. The Brassica plant cell of claim 6 which is a Brassica napus plant cell.
8. A Brassica napus seed comprising the Brassica napus plant cell of claim 7 .
9. The Brassica napus seed of claim 8 comprising docasapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in seed.
10. The Brassica plant cell of claim 6 , further comprising an exogenous desaturase which desaturates an acyl-CoA substrate.
11. A Brassica seed comprising the Brassica plant cell of claim 10 .
12. The Brassica plant cell of claim 6 , wherein the Δ6 desaturase is a Δ5/Δ6 bifunctional desaturase.
13. The Brassica plant cell of claim 10 , wherein the Δ6 desaturase is a Δ5/Δ6 bifunctional desaturase.
14. A Brassica seed comprising the Brassica plant cell of claim 13 .
15. A Brassica plant cell comprising
a polynucleotide which encodes a Δ4 desaturase which has the amino acid sequence encoded by a Δ4 desaturase gene from Pavlova lutheri , operably linked to a promoter which directs expression of the polynucleotide in the plant cell;
a polynucleotide which encodes a Δ5 desaturase which has the amino acid sequence set forth as SEQ ID NO:18, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;
a polynucleotide which encodes a Δ6 desaturase operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and
a polynucleotide which encodes a Δ6 elongase which has the amino acid sequence set forth as SEQ ID NO:31, operably linked to a promoter which directs expression of the polynucleotide in the plant cell.
16. The Brassica plant cell of claim 15 which is a Brassica napus plant cell.
17. A Brassica napus seed comprising the Brassica napus plant cell of claim 16 .
18. The Brassica napus seed of claim 17 comprising docasapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the seed.
19. The Brassica plant cell of claim 15 , further comprising an exogenous desaturase which desaturates an acyl-CoA substrate.
20. A Brassica seed comprising the Brassica plant cell of claim 19 .
21. The Brassica plant cell of claim 15 , wherein the Δ6 desaturase is a Δ5/Δ6 bifunctional desaturase.
22. The Brassica plant cell of claim 19 , wherein the Δ6 desaturase is a Δ5/Δ6 bifunctional desaturase.
23. A Brassica seed comprising the Brassica plant cell of claim 22 .
24. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising
i) obtaining the Brassica napus seed of claim 3 , and
ii) extracting triacylglycerols from the seed.
25. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising
i) obtaining the Brassica napus seed of claim 9 , and
ii) extracting triacylglycerols from the seed.
26. The process of claim 25 which further comprises purifying the extracted triacylglycerols.
27. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising
i) obtaining the Brassica napus seed of claim 14 , and
ii) extracting triacylglycerols from the seed.
28. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising
i) obtaining the Brassica napus seed of claim 18 , and
ii) extracting triacylglycerols from the seed.
29. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising
i) obtaining the Brassica napus seed of claim 20 , and
ii) extracting triacylglycerols from the seed.
30. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising
i) obtaining the Brassica seed of claim 23 , and
ii) extracting triacylglycerols from the seed.
31. The Brassica plant cell of claim 1 , wherein at least one of the desaturases desaturates an acyl-CoA substrate.
32. The Brassica napus plant seed of claim 3 , wherein at least one of the desaturases desaturates an acyl-CoA substrate.
33. The Brassica plant cell of claim 6 , wherein the Δ6 desaturase desaturates an acyl-CoA substrate.
34. The Brassica plant cell of claim 15 , wherein the Δ6 desaturase desaturates an acyl-CoA substrate.