Synthesis of long-chain polyunsaturated fatty acids by recombinant cell

• US 9,951,357 B2
• Filed: 09/23/2016
• Issued: 04/24/2018
• 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): FRONDA, CHRISTIAN L
• Application Number:
  US15/274,454
• Publication Number:
  US 20170073712A1
• Time in Patent Office: 578 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 recombinant plant cell which synthesises eicosapentaenoic acid (EPA), comprising more than one heterologous polynucleotide, wherein said polynucleotides encode:

a) a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase;

or

b) a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase;

wherein the more than one polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in the cell, wherein the enzymes encoded by said polynucleotides comprise at least one desaturase which is able to act on an acyl-CoA substrate, and wherein the synthesis of EPA requires the sequential action of said enzymes.

2. The cell of claim 1, wherein the enzymes encoded by the polynucleotides comprise at least one Δ

5 elongase which catalyses the conversion of EPA to docosapentaenoic acid (DPA) in the cell.

3. The cell of claim 1 which is a plant cell from an angiosperm, an oilseed plant cell or a cell in a seed.

4. The cell of claim 1, wherein at least one promoter is a seed specific promoter.

5. The cell of claim 1, wherein said cell is capable of synthesising DPA.

6. The cell of claim 5, wherein said cell is capable of synthesising docosahexaenoic acid (DHA).

7. The cell of claim 1, wherein said cell

i) is capable of producing EPA from a fatty acid which is α

-linolenic acid (ALA), linoleic acid (LA), γ

-linolenic acid (GLA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), or any combination or mixture of these,

ii) is capable of producing DHA from a fatty acid which is ALA, LA, GLA, arachidonic acid (ARA), SDA, ETA, EPA, DPA, or any combination or mixture of these, and/or

iii) is capable of producing DPA from a fatty acid which is ALA, LA, GLA, ARA, SDA, ETA, EPA, or any combination or mixture of these.

9. A transgenic plant or plant part comprising at least one cell according to claim 1.

10. The plant or plant part of claim 9 wherein said plant is an angiosperm.

11. The plant of claim 9 which comprises

at least one plant part synthesizing DHA.

12. A transgenic seed comprising a cell according to claim 1.

13. The seed of claim 12 which further comprises

i) DPA, or

ii) DNA, and DPA.

14. The seed of claim 13, wherein the seed is derived from an oilseed plant.

15. The seed of claim 14, wherein the oilseed plant is oilseed rape, maize, sunflower, soybean, sorghum, flax, sugar beet, cotton, peanut, poppy, mustard, castor bean, sesame, or safflower.

16. The seed of claim 12, wherein the seed has a germination rate which is substantially the same as that of an isogenic non-transgenic seed.

17. The seed of claim 16 which comprises DHA which is synthesized in the seed.

18. The seed of claim 12 which is homozygous for the heterologous polynucleotides.

19. A transgenic plant which comprises the transgenic seed of claim 12.

20. A method of producing a long-chain polyunsaturated fatty acid (LC-PUFA), the method comprising culturing, under suitable conditions, a recombinant cell according to claim 1, wherein said LC-PUFA comprises EPA.

21. A method of producing oil comprising LC-PUFA, wherein said LC-PUFA comprises EPA, comprising obtaining the seed according to claim 12, and extracting oil from said seed.

22. The method according to claim 21, wherein said oil is further purified.

23. The method according to claim 21, wherein said oil is further treated by hydrolysis with a strong base to release the free fatty acids.

24. A feedstuff comprising the recombinant cell of claim 1.

25. The plant of claim 9 which is a Brassica napus plant or Arabidopsis thaliana plant, comprising seed which synthesizes EPA, wherein the total fatty acid of the seed comprises 1.5% EPA.

26. The plant of claim 9 which is a Brassica napus plant or Arabidopsis thaliana plant, comprising seed which synthesizes DPA, wherein the total fatty acid of the seed comprises 0.13% DPA.

27. The plant of claim 9 which is a Brassica napus plant or Arabidopsis thaliana plant, comprising seed which synthesizes at least one ω

3 C20 LC-PUFA, wherein the total fatty acid of the seed comprises 4.1% ω

3 C20 LC-PUFA.

28. The plant of claim 9 which is a Brassica napus plant or Arabidopsis thaliana plant, comprising seed which synthesizes EPA, wherein the efficiency of conversion of ALA to EPA in the seed is at least 14.6%.

29. The plant of claim 9 which is a Brassica napus plant or Arabidopsis thaliana plant, comprising seed which synthesizes ω

3 polyunsaturated fatty acids that are the products of Δ

6-desaturation of ALA and/or the products of Δ

9 elongation of ALA, wherein the efficiency of conversion of ALA to said products in the seed is at least 22%.

30. The plant of claim 9 which is a Brassica napus plant or Arabidopsis thaliana plant, comprising seed which synthesizes DPA from EPA, and wherein the efficiency of conversion of EPA to DPA in the seed is at least 7%.

31. The plant cell of claim 1 which is a Brassica napus cell or Arabidopsis thaliana cell, wherein the heterologous polynucleotides encode a Δ

6 desaturase, a 46 elongase and a Δ

5 desaturase.

32. The cell of claim 31, comprising a heterologous polynucleotide encoding a Δ

5 elongase which catalyses the conversion of EPA to DPA in the cell.

33. The cell of claim 32, wherein said cell is capable of synthesising DHA.

34. The plant cell of claim 1 which is a Brassica napus cell or Arabidopsis thaliana cell, wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase.

35. The cell of claim 34, wherein said cell is capable of synthesising DHA.

38. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising at least one ω

3 C20 long-chain polyunsaturated fatty acid (LC-PUFA) which is synthesized in the seed, wherein the total fatty acid of the seed comprises 2.5% ω

3 C20 LC-PUFA, wherein the heterologous polynucleotides encode a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

39. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising ALA and EPA, wherein the efficiency of conversion of ALA to EPA in the seed is at least 14.6%, wherein the heterologous polynucleotides encode a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

40. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising ω

3 polyunsaturated fatty acids that are the products of Δ

6-desaturation of ALA and/or the products of Δ

9 elongation of ALA, which products are synthesized in the seed, wherein the efficiency of conversion of ALA to said products in the seed is at least 22%, wherein the heterologous polynucleotides encode a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

41. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising DPA which is synthesized from EPA in the seed, wherein the efficiency of conversion of EPA to DPA in the seed is at least 7%, wherein the heterologous polynucleotides encode a 46 desaturase, a Δ

6 elongase and a Δ

5 desaturase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

42. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising at least one ω

3 C20 LC-PUFA which is synthesized in the seed, wherein the total fatty acid of the seed comprises 2.5% ω

3 C20 LC-PUFA, wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

43. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising ALA and EPA, wherein the efficiency of conversion of ALA to EPA in the seed is at least 14.6%, wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

44. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising n3 polyunsaturated fatty acids that are the products of Δ

6-desaturation of ALA and/or the products of Δ

9 elongation of ALA, which products are synthesized in the seed, wherein the efficiency of conversion of ALA to said products in the seed is at least 22%, wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

45. The seed of claim 12 which is a Brassica napus seed or Arabidopsis thaliana seed comprising DPA which is synthesized from EPA in the seed, wherein the efficiency of conversion of EPA to DPA in the seed is at least 7%, wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase, and wherein the polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica napus seed or Arabidopsis thaliana seed.

8. A method of producing a recombinant plant cell which synthesises one or more long-chain polyunsaturated fatty acids (LC-PUFA(s)), the method comprising introducing into the cell more than one heterologous polynucleotide, wherein said polynucleotides encode:

a) a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase;

or

b) a Δ

5/Δ

6 bifunctional′

desaturase and a Δ

5/Δ

6 bifunctional elongase;

wherein the more than one polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in the cell,

wherein said one or more LC-PUFAs comprise EPA,

wherein the enzymes encoded by said polynucleotides comprise at least one desaturase which is able to act on an acyl-CoA substrate, and

wherein the synthesis of EPA requires the sequential action of said enzymes.

36. The method of claim 8, wherein the plant cell is a Brassica napus cell or Arabidopsis thaliana cell, and wherein the heterologous polynucleotides encode a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase.

37. The method of claim 8, wherein the plant cell is a Brassica napus cell or Arabidopsis thaliana cell, and wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase.