Synthesis of long-chain polyunsaturated fatty acids by recombinant cell

• US 9,970,033 B2
• Filed: 07/07/2017
• Issued: 05/15/2018
• Est. Priority Date: 04/22/2004
• Status: Active Grant

• Current Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Original Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Inventors: Singh, Surinder Pal, Robert, Stanley Suresh, Nichols, Peter David, Blackburn, Susan Irene Ellis, Zhou, Xue-Rong, Petrie, James Robertson, Green, Allan Graham
• Primary Examiner(s): O HARA, EILEEN B
• Application Number:
  US15/644,656
• Publication Number:
  US 20170321234A1
• Time in Patent Office: 312 Days
• Field of Search: None
• CPC Class Codes: A01H 5/10 Seeds
• CPC Class Codes: A23K 10/12 by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• CPC Class Codes: A23K 10/30 from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C11C 3/12 by hydrogenation
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 114/19004 DELTA8-fatty-acid desaturase (1.14.19.4)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

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.

Claims

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 Δ

4 desaturase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;

a polynucleotide which encodes a Δ

5 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 Δ

6 desaturase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and

a polynucleotide which encodes a Δ

5/Δ

6 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.

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.

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.

6. A Brassica plant cell comprising

a polynucleotide which encodes a Δ

4 desaturase which has the amino acid sequence encoded by a Δ

4 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 Δ

5 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 Δ

6 desaturase, operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and

a polynucleotide which encodes a Δ

5/Δ

6 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 Δ

6 desaturase is a Δ

5/Δ

6 bifunctional desaturase.

13. The Brassica plant cell of claim 10, wherein the Δ

6 desaturase is a Δ

5/Δ

6 bifunctional desaturase.

14. A Brassica seed comprising the Brassica plant cell of claim 13.

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.

33. The Brassica plant cell of claim 6, wherein the Δ

6 desaturase desaturates an acyl-CoA substrate.

15. A Brassica plant cell comprising

a polynucleotide which encodes a Δ

4 desaturase which has the amino acid sequence encoded by a Δ

4 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 Δ

5 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 Δ

6 desaturase operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and

a polynucleotide which encodes a Δ

6 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 Δ

6 desaturase is a Δ

5/Δ

6 bifunctional desaturase.

22. The Brassica plant cell of claim 19, wherein the Δ

6 desaturase is a Δ

5/Δ

6 bifunctional desaturase.

23. A Brassica seed comprising the Brassica plant cell of claim 22.

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.

34. The Brassica plant cell of claim 15, wherein the Δ

6 desaturase desaturates an acyl-CoA substrate.