Synthesis of long-chain polyunsaturated fatty acids by recombinant cell

• US 8,853,432 B2
• Filed: 06/10/2013
• Issued: 10/07/2014
• 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): Carr, Deborah D
• Application Number:
  US13/913,999
• Publication Number:
  US 20130296589A1
• Time in Patent Office: 484 Days
• Field of Search: 554/9
• US Class Current: 554/9
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/04 for urolithiasis
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07C 51/42 Separation; Purification; Stabilisation; Use of additives
• CPC Class Codes: C11B 1/025 : by enzymes or microorganisms, living or dead
• CPC Class Codes: C11B 1/06 : by pressing
• CPC Class Codes: C11B 3/006 : by extraction
• CPC Class Codes: C12N 15/8247 : involving modified lipid metabolism, e.g. seed oil composition
• 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 7/6427 : 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: 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 desaturase or elongase activity that can be used in methods of synthesizing long-chain polyunsaturated fatty acids.

Claims

1. A process for producing oil containing eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, comprising the steps of (i) obtaining a transgenic seed of an oilseed plant which comprises eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid in an esterified form as part of triglycerides, wherein the total fatty acid of the transgenic seed comprises at least 2.5% ω

3 C20 fatty acids (w/w) and wherein the docosapentaenoic acid is present at a level based on a conversion ratio of eicosapentaenoic acid to docosapentaenoic acid of at least 5% (w/w), (ii) extracting oil from the transgenic seed, and (iii) purifying the extracted oil or treating the extracted oil by hydrolysis, fractionation or distillation.

2. The process of claim 1, wherein the step of extracting the oil comprises crushing the seed.

3. The process of claim 1, wherein the total fatty acid of the seed comprises at least 9% C20 fatty acids (w/w).

4. The process of claim 1, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

5. The process of claim 1, wherein the total fatty acid of the seed comprises at least 2.1% eicosapentaenoic acid and less than 0.1% eicosatrienoic acid (w/w).

6. The process of claim 1, wherein at least 25% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

7. The process of claim 1, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

8. The process of claim 1, wherein the extracted oil comprises at least 50% triacylglycerols.

9. The process of claim 1, wherein the total fatty acid of the seed comprises at least 7.9% C20 fatty acids (w/w).

10. The process of claim 9, wherein the total fatty acid of the seed comprises at least 10.2% C20 fatty acids (w/w).

11. The process of claim 10, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

12. The process of claim 10, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

13. The process of claim 1, wherein the docosapentaenoic acid is present at a level based on a conversion ratio of eicosapentaenoic acid to docosapentaenoic acid of at least 7% (w/w).

14. The process of claim 13, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

15. The process of claim 13, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

16. The process of claim 13, wherein the docosapentaenoic acid is present at a level based on a conversion ratio of eicosapentaenoic acid to docosapentaenoic acid of at least 10% (w/w).

17. The process of claim 16, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

18. The process of claim 16, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

19. The process of claim 13, wherein the extracted oil is purified after extraction.

20. The process of claim 16, wherein the extracted oil is purified after extraction.

21. The process of claim 1, wherein the triacylglycerols in the extracted oil are hydrolysed after extraction.

22. The process of claim 1, wherein the oilseed plant is a Brassica plant.

23. The process of claim 13, wherein the oilseed plant is a Brassica plant.

24. The process of claim 16, wherein the oilseed plant is a Brassica plant.

25. The process of claim 23, wherein the Brassica plant is a canola plant.

26. A process for producing a composition comprising eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, comprising the steps of (i) obtaining a transgenic seed of an oilseed plant which comprises eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid in an esterified form as part of triglycerides, wherein the total fatty acid of the transgenic seed comprises at least 2.5% ω

3 C20 fatty acids (w/w) and wherein the docosapentaenoic acid is present at a level based on a conversion ratio of eicosapentaenoic acid to docosapentaenoic acid of at least 5% (w/w), (ii) extracting oil from the transgenic seed, and (iii) processing the oil, thereby producing the composition.

27. The process of claim 26, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

28. The process of claim 26, wherein the total fatty acid of the seed comprises at least 2.1% eicosapentaenoic acid and less than 0.1% eicosatrienoic acid (w/w).

29. The process of claim 26, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

30. The process of claim 26, wherein the extracted oil comprises at least 50% triacylglycerols.

31. The process of claim 26, wherein the total fatty acid of the seed comprises at least 7.9% C20 fatty acids (w/w).

32. The process of claim 31, wherein the total fatty acid of the seed comprises at least 10.2% C20 fatty acids (w/w).

33. The process of claim 32, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

34. The process of claim 32, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

35. The process of claim 26, wherein the docosapentaenoic acid is present at a level based on a conversion ratio of eicosapentaenoic acid to docosapentaenoic acid of at least 7% (w/w).

36. The process of claim 35, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

37. The process of claim 35, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

38. The process of claim 35, wherein the docosapentaenoic acid is present at a level based on a conversion ratio of eicosapentaenoic acid to docosapentaenoic acid of at least 10% (w/w).

39. The process of claim 38, wherein the total fatty acid of the seed comprises at least 1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).

40. The process of claim 38, wherein at least 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of the seed is incorporated into triacylglycerols in the seed.

41. The process of claim 35, wherein the extracted oil is purified after extraction.

42. The process of claim 38, wherein the extracted oil is purified after extraction.

43. The process of claim 26, wherein the triacylglycerols in the extracted oil are hydrolysed after extraction.

44. The process of claim 26, wherein the oilseed plant is a Brassica plant.

45. The process of claim 35, wherein the oilseed plant is a Brassica plant.

46. The process of claim 38, wherein the oilseed plant is a Brassica plant.

47. The process of claim 45, wherein the Brassica plant is a canola plant.