Method for Increasing the Content of Polyunsaturated Long-Chained Fatty Acids in Transgenic Organisms

• US 20080076164A1
• Filed: 07/15/2005
• Published: 03/27/2008
• Est. Priority Date: 07/16/2004
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Qiu, Xiao, Vrinten, Patricia, Cirpus, Petra, Bauer, Jorg
• Granted Patent: US 7,842,852 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/88 Lyases (4.)

Abstract

The present invention relates to a method for increasing the content of polyunsaturated long-chain fatty acids in an organism by introducing into said organism nucleic acids coding for polypeptides or proteins exhibiting a phospholipase, ketoacyl-CoA reductase and/or dehydratase activity. Advantageously, said enzymes originate from Ostreococcus or Thraustochytrium. The present invention further relates to the nucleic acid sequences, nucleic acid constructs, vectors and organisms containing the nucleic acid sequences according to the present invention, vectors containing the nucleic acid sequences and/or the nucleic acid constructs as well as to transgenic organisms containing the above mentioned nucleic acid sequences, nucleic acid constructs and/or vectors.

Advantageously, said previously mentioned nucleic acid sequences, nucleic acid constructs, vectors can optionally be expressed in the organism together with further nucleic acid sequences coding for polypeptides or proteins of the biosynthesis of the fatty acid or lipid metabolism. Particularly advantageous herein are those nucleic acid sequences of the fatty acid or lipid metabolism that code for a Δ-9 elongase, Δ-8 desaturase, Δ-6 desaturase, a Δ-5 desaturase, Δ-4 desaturase, Δ-12 desaturase, Δ-5 elongase and/or Δ-6 elongase activity. Advantageously, said desaturases and elongases originate from organisms like Thalassiosira, Euglena, Isochrysis, Physcomitrella, Thraustochytrium, Borago, Phytophthora, Crypthecodinium, Oncorhynchus, Primula, Xenopus, Ciona, Arabidopsis, Mortierella, Caenorhabditis, Phaeodactylum, Ceratodon or Ostreococcus. A further part of the present invention relates to oils, lipids and/or fatty acids produced according to the method of the present invention and the use thereof. Furthermore, the present invention relates to unsaturated fatty acids and triglycerides having an increased content of unsaturated fatty acids and the use thereof.

Claims

1-26. -26. (canceled)

27. A method for producing oils and/or lipids having a high content of polyunsaturated fatty acids containing at least two double bonds in a transgenic organism comprising:

a) introducing at least one nucleic acid sequence coding for a phospholipase A2 activity into the organism, or

b) introducing at least one nucleic acid sequence coding for a ketoacyl-CoA reductase activity into the organism, or

c) introducing at least one nucleic acid sequence coding for a dehydratase activity into the organism, and

d) cultivating the transgenic organism.

28. The method according to claim 27, characterized in that the oil and/or lipid is isolated from the transgenic organism.

29. The method according to claim 27, characterized in that the nucleic acid sequence coding for a polypeptide or a protein exhibiting phospholipase A2, ketoacyl-CoA reductase or dehydratase activity is selected from the group consisting of:

a) a nucleic acid sequence having the sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7,

b) a nucleic acid sequence which encodes one of the amino acid sequences depicted in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8, and

c) a derivative of the nucleic acid sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7 coding for a polypeptide or a protein having at least 40% identity at the amino acid level to SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and exhibiting phospholipase A2, ketoacyl-CoA reductase or dehydratase activity.

30. The method according to claim 27, characterized in that at least one nucleic acid sequence coding for a polypeptide or a protein exhibiting Δ

-12 desaturase, Δ

-9 elongase, Δ

-8 desaturase, Δ

-6 desaturase, Δ

-6 elongase, Δ

-5 desaturase, Δ

-5 elongase, ω

-3 desaturase, Δ

-4 desaturase activity is additionally introduced into the organism.

31. The method according to claim 27, characterized in that the oil and/or lipid is selected from the group consisting of linoleic acid, γ

-linolenic acid, stearidonic acid, dihomo-γ

-linolenic acid, ω

-3-eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, ω

-6-docosapentaenoic acid, ω

-6-docosatetraenoic acid, ω

-3-docosapentaenoic acid and docosahexaenoic acid.

32. The method according to claim 27, characterized in that the polyunsaturated fatty acids are isolated from the oil and/or lipid in the form of free fatty acids.

33. The method according to claim 27, characterized in that the transgenic organism is a transgenic microorganism or a transgenic plant.

34. The method according to claim 27, characterized in that the transgenic organism is an oil-producing plant, a vegetable plant, or an ornamental plant.

35. The method according to claim 27, characterized in that the transgenic organism is a transgenic plant selected from the group consisting of plant classes or families:

Adelotheciaceae, Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae, Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae and Prasinophyceae.

36. The method according to claim 32, characterized in that the free fatty acids are isolated at a concentration of at least 5% by weight, based on the total lipid content of the transgenic organism.

37. Oils, lipids or fatty acids or a fraction thereof, produced by the method according to claim 27.

38. An oil, lipid or fatty acid composition comprising the polyunsaturated fatty acids produced by the method according to claim 27 and originated from a transgenic plant.

39. A method for producing an oil, lipid or fatty acid composition by mixing oil, lipids or fatty acids according to claim 37 with animal oil, lipids or fatty acids.

40. Feed, food, cosmetics or pharmaceuticals comprising oil, lipids or fatty acids according to claim 37.

41. An isolated nucleic acid sequence coding for a polypeptide or a protein exhibiting phospholipase A2 activity, characterized in that the nucleic acid sequence is selected from the group consisting of:

a) a nucleic acid sequence having the sequence depicted in SEQ ID NO;

1,

b) a nucleic acid sequence which encodes the amino acid sequence depicted in SEQ ID NO;

2, and

c) a derivative of the nucleic acid sequence depicted in SEQ ID NO;

1, which codes for a polypeptide or a protein having at least 40% homology at the amino acid level to SEQ ID NO;

2 and exhibiting a phospholipase A2 activity.

44. The isolated nucleic acid sequence according to claim 41, wherein the sequence is originated from an alga, a fungus, a microorganism, a plant, or a non-human animal.

45. The isolated nucleic acid sequence according to claim 41, wherein the sequence is originated from the order Salmoniformes, the diatoms genera Thalassiosira or Crypthecodinium, the classes Prasinophyceae or Phycomycota, or the families Euglenaceae or Pythiaceae.

46. An amino acid sequence encoded by the isolated nucleic acid sequence according to claim 41.

47. A gene construct containing the isolated nucleic acid according to claim 41, wherein the nucleic acid is functionally linked to one or more regulatory signals.

48. The Gene construct according to claim 47, characterized in that the nucleic acid construct contains at least one additional biosynthesis gene of the fatty acid or lipid metabolism selected from the group consisting of:

acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA;

lysophospholipid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-Coenzyme A carboxylase(s), acyl-Coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allene oxide synthase(s), hydroperoxide lyases and fatty acid elongase(s).

49. The gene construct according to claim 47, characterized in that the nucleic acid construct contains at least one additional biosynthesis gene of the fatty acid or lipid metabolism selected from the group consisting of Δ

-4 desaturase, Δ

-5 desaturase, Δ

-6 desaturase, Δ

-8 desaturase, Δ

-9 desaturase, Δ

-12 desaturase, Δ

-6 elongase, Δ

-5 elongase and Δ

-9 elongase.

50. A vector containing the nucleic acid according to claim 41.

51. A transgenic non-human organism containing at least one nucleic acid according to claim 41.

52. The transgenic non-human organism according to claim 51, wherein the organism is a plant.

42. An isolated nucleic acid sequence coding for a polypeptide or a protein exhibiting ketoacyl-CoA reductase activity, selected from the group consisting of:

a) a nucleic acid sequence having the sequence depicted in SEQ ID NO;

3,

b) a nucleic acid sequence which encodes the amino acid sequence depicted in SEQ ID NO;

4, and

c) a derivative of the nucleic acid sequence depicted in SEQ ID NO;

3, which codes for a polypeptide or a protein having at least 40% homology at the amino acid level to SEQ ID NO;

4 and exhibiting a ketoacyl-CoA reductase activity.

53. An amino acid sequence encoded by the isolated nucleic acid sequence according to claim 42.

54. A gene construct containing the isolated nucleic acid according to claim 42, wherein the nucleic acid is functionally linked to one or more regulatory signals.

55. A vector containing the nucleic acid according to claim 42.

56. A transgenic non-human organism containing at least one nucleic acid according to claim 42.

43. An isolated nucleic acid sequence coding for a polypeptide or a protein exhibiting dehydratase activity, selected from the group consisting of:

a) a nucleic acid sequence having the sequence depicted in SEQ ID NO;

5 or SEQ ID NO;

7,

b) a nucleic acid sequence which encodes one of the amino acid sequences depicted in SEQ ID NO;

6 or SEQ ID NO;

8, and

c) a derivative of the nucleic acid sequence depicted in SEQ ID NO;

5 or SEQ ID NO;

7, which codes for a polypeptide or a protein having at least 40% identity at the amino acid level to SEQ ID NO;

6 or SEQ ID NO;

8 and exhibiting a dehydratase activity.

57. An amino acid sequence encoded by the isolated nucleic acid sequence according to claim 43.

58. A gene construct containing the isolated nucleic acid according to claim 43, wherein the nucleic acid is functionally linked to one or more regulatory signals.

59. A vector containing the nucleic acid according to claim 43.

60. A transgenic non-human organism containing at least one nucleic acid according to claim 43.