Production of omega 3 long chain polyunsaturated fatty acids in oilseed crops by a thraustochytrid PUFA synthase
This disclosure concerns recombinant host organisms genetically modified with a polyunsaturated fatty acid (PUFA) synthase system and one or more accessory proteins that allow for and/or improve the production of PUFAs in the host organism. The disclosure also concerns methods of making and using such organisms as well as products obtained from such organisms.
1. A genetically modified Brassica plant that produces oil comprising eicosapentaenoic acid (C20:5, n-3) (EPA) in amount between 0.01% and 10% by weight of total fatty acids, the plant comprising:
a first polynucleotide encoding a first polypeptide of a polyunsaturated fatty acid (PUFA) synthase system comprising SEQ ID NO:1;
a second polynucleotide encoding a second polypeptide of the PUFA system comprising SEQ ID NO:4;
a third polynucleotide encoding a third polypeptide of the PUFA system consisting of SEQ ID NO:14; and
a fourth polynucleotide encoding a phosphopantetheinyl transferase (PPTase) from a Nostoc sp.,
wherein each of the polynucleotides is operably linked to a promoter functional in the plant.
2. A cell, tissue, or part of the genetically modified plant of claim 1 .
3. The genetically modified plant of claim 1 , wherein the promoter is a seed-specific promoter or a leaf-specific promoter.
4. The genetically modified plant of claim 1 , wherein the promoter is a seed-specific promoter, and wherein the polynucleotides are operably linked to a transcription termination sequence.
5. The genetically modified plant of claim 1 , wherein the promoter is selected from the group consisting of PvDlec2; LfKCS3; FAE1; BoACP; BnaNapinC; ubiquitin; CsVMV; SSPRO2745.1; and SSPRO2743.1 promoters.
6. The genetically modified plant of claim 1 , wherein the oil produced by the plant further comprises DHA (docosahexaenoic acid (C22:6, n-3)) in an amount between 0.01% and 15% by weight of total fatty acids.
7. The genetically modified plant of claim 6 , wherein the oil produced by the plant comprises between 0.05% and 10% DHA by weight of total fatty acids.
8. The genetically modified plant of claim 7 , wherein the oil produced by the plant comprises between 0.05% and 5% DHA by weight of total fatty acids.
9. The genetically modified plant of claim 1 , wherein the oil produced by the plant comprises an amount of EPA between 0.05% and 5% by weight of total fatty acids.
10. The genetically modified plant of claim 9 , wherein the oil produced by the plant comprises an amount of EPA between 0.05% and 1% by weight of total fatty acids.
11. The genetically modified plant of claim 1 , wherein the plant or cell, tissue, seed, or part thereof comprises a ratio of EPA:DHA (docosahexaenoic acid (C22:6, n-3)) of between 1:1 and 1:30 by weight of total fatty acids.
12. The genetically modified plant of claim 11 , wherein the oil produced by the plant comprises a ratio of EPA:DHA of between 1:1 and 1:3 by weight of total fatty acids.
13. A seed obtained from the genetically modified plant of claim 1 , the seed comprising the first, second, third, and fourth polynucleotides.
14. A commodity product obtained from the genetically modified plant of claim 1 , wherein the commodity product is selected from the group consisting of a refined oil, an unrefined oil, a raw oil, a feed or meal composition, and a functional food product, the commodity product comprising the first, second, third, and fourth polynucleotides.
15. A method for obtaining oil comprising at least one PUFA, the method comprising recovering oil from the genetically modified plant of claim 1 .
16. The method according to claim 15 , wherein the oil is recovered from a cell, tissue, seed, or part of the genetically modified plant.
17. The genetically modified plant of claim 1 , wherein the first polynucleotide is at least 70% identical to at least one of SEQ ID NO:2 and SEQ ID NO:3, wherein the second polynucleotide is at least 70% identical to at least one of SEQ ID NO:5 and SEQ ID NO:6, and wherein the third polynucleotide is at least 70% identical to SEQ ID NO:13.
18. The genetically modified plant of claim 1 , wherein the first polynucleotide is SEQ ID NO:3, wherein the second polynucleotide is SEQ ID NO:6, and wherein the third polynucleotide is SEQ ID NO:13.
19. The genetically modified plant of claim 1 , wherein the polynucleotides are arranged in a head-to-tail configuration in the same nucleic acid molecule.
20. The genetically modified plant of claim 1 , wherein the first polynucleotide and the fourth polynucleotide are arranged in a tail-to-tail configuration, and wherein the second polynucleotide and the third polynucleotide are arranged in a tail-to-tail configuration in the same nucleic acid molecule.
21. The genetically modified plant of claim 1 , wherein the first polynucleotide and the fourth polynucleotide are arranged in a head-to-head configuration, and wherein the second polynucleotide and the third polynucleotide are arranged in a head-to-head configuration in the same nucleic acid molecule.
22. The genetically modified plant of claim 1 ,
wherein the first polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:2 or SEQ ID NO:3,
wherein the second polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:5 or SEQ ID NO:6,
wherein the third polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:13, or
wherein the fourth polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:10.
23. An isolated nucleic acid molecule comprising:
a first polynucleotide encoding a first polypeptide of a polyunsaturated fatty acid (PUFA) synthase system comprising SEQ ID NO:1;
a second polynucleotide encoding a second polypeptide of the PUFA system comprising SEQ ID NO:4;
a third polynucleotide encoding a third polypeptide of the PUFA system consisting of SEQ ID NO:14; and
a fourth polynucleotide encoding the phosphopantetheinyl transferase (PPTase) from a Nostoc sp.,
wherein each of the polynucleotides is operably linked to a promoter functional in the plant.
24. The isolated nucleic acid molecule of claim 23 , wherein the first polynucleotide is at least 70% identical to at least one of SEQ ID NO:2 and SEQ ID NO:3, wherein the second polynucleotide is at least 70% identical to at least one of SEQ ID NO:5 and SEQ ID NO:6, wherein the third polynucleotide is at least 70% identical to SEQ ID NO:13, or wherein the fourth polynucleotide is at least 70% identical to SEQ ID NO:10.
25. The isolated nucleic acid molecule of claim 23 , wherein the first polynucleotide is SEQ ID NO:3, wherein the second polynucleotide is SEQ ID NO:6, wherein the third polynucleotide is SEQ ID NO:13, and wherein the fourth polynucleotide is SEQ ID NO:10.
26. The isolated nucleic acid molecule of claim 23 , wherein the polynucleotides are all arranged in a head-to-tail configuration.
27. The isolated nucleic acid molecule of claim 23 , wherein the first and fourth polynucleotides are arranged in a tail-to-tail configuration, and wherein the second and third polynucleotides are arranged in a tail-to-tail configuration.
28. The isolated nucleic acid molecule of claim 23 , wherein the first and fourth polynucleotides are arranged in a head-to-head configuration, and wherein the second and third polynucleotides are arranged in a head-to-head configuration.
29. A system for producing a genetically modified plant, the system comprising at least one isolated nucleic acid molecules, the system comprising:
a first polynucleotide encoding a first polypeptide of a polyunsaturated fatty acid (PUFA) synthase system comprising SEQ ID NO:1;
A second polynucleotide encoding a second polypeptide of the PUFA system comprising SEQ ID NO:4;
A third polynucleotide encoding a third polypeptide of the PUFA system consisting of SEQ ID NO:14; and
a fourth polynucleotide encoding a phosphopantetheinyl transferase (PPTase) from a Nostoc sp.
30. The system of claim 29 , wherein the fourth polynucleotide encodes the PPTase encoded by SEQ ID NO:10.
31. The system of claim 30 ,
wherein the first polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:2 or SEQ ID NO:3,
wherein the second polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:5 or SEQ ID NO:6,
wherein the third polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:13, and
wherein the fourth polynucleotide hybridizes under stringent conditions to the complement of SEQ ID NO:10.
32. The nucleic acid molecule of claim 23 , wherein the nucleic acid is a recombinant expression vector.
33. The nucleic acid molecule of claim 32 , wherein the nucleic acid is selected from the group consisting of SEQ ID NOs:15-38.
34. The system of claim 29 , wherein the polynucleotides are comprised in one or more recombinant expression vector(s).
35. The genetically modified plant of claim 1 , wherein the polynucleotide are operably linked to at least one regulatory element selected from the group consisting of a 5′ UTR, a 3′ UTR, and a transcription termination sequence.
36. The genetically modified plant of claim 1 , wherein the PPTase is the PPTase encoded by SEQ ID NO:10.
37. The genetically modified plant of claim 36 , wherein polynucleotide encoding the PPTase is SEQ ID NO:10.
38. The system of claim 29 , wherein each of the polynucleotides is operably linked to a promoter functional in a plant cell.