Novel elongase gene, and process for the preparation of polyunsaturated fatty acids

• US 20080160054A1
• Filed: 03/05/2007
• Published: 07/03/2008
• Est. Priority Date: 02/09/2000
• Status: Active Grant

• Current Assignee: Andreas Renz, Ernst Heinz, Jens Lerchl, Thorsten Zank, Ulrich Zahringer
• Original Assignee: Andreas Renz, Ernst Heinz, Jens Lerchl, Thorsten Zank, Ulrich Zahringer
• Inventors: Zank, Thorsten, Lerchl, Jens, Renz, Andreas, Heinz, Ernst, Zahringer, Ulrich
• Granted Patent: US 8,933,300 B2
• Time in Patent Office: Days
• US Class Current: 424/401
• 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 3/06 Antihyperlipidemics
• 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: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The invention relates to a novel elongase gene with the sequences stated in sequence SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or their homologs, derivatives or analogs, to a gene construct comprising this gene or its homologs, derivatives and analogs, and to its use. The invention also relates to vectors or transgenic organisms comprising an elongase gene with the sequence SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or its homologs, derivatives and analogs. The invention furthermore relates to the use of the elongase gene sequences alone or in combination with further elongases and/or further fatty acid biosynthesis genes. The present invention relates to a novel elongase gene with the sequence SEQ ID NO:1 or its homologs, derivatives and analogs.

Furthermore, the invention relates to a process for the preparation of polyunsaturated fatty acids and to a process for introducing DNA into organisms which produce large amounts of oils and, in particular, oils with a high content of unsaturated fatty acids. Moreover, the invention relates to an oil and/or a fatty acid preparation with a higher content of polyunsaturated fatty acids with at least two double bonds and/or a triacylglycerol preparation with a higher content of polyunsaturated fatty acids with at least two double bonds.

Claims

1-28. -28. (canceled)

29. An isolated nucleic acid sequence encoding a polypeptide which elongates C₁₈-fatty acids with at least two double bonds in the fatty acid molecule, said isolated nucleic acid sequence comprising a derivative of SEQ ID NO:

1 which encodes a polypeptide with at least 70% homology at the amino acid level with SEQ ID NO;

2 and wherein said polypeptide has C₁₈-elongase activity.

30. The isolated nucleic acid sequence as claimed in claim 29 derived from Physcomitrella.

31. A gene construct comprising the isolated nucleic acid sequence as claimed in claim 29, wherein the isolated nucleic acid sequence is functionally linked to one or more regulatory signals.

32. The gene construct as claimed in claim 31 further comprising a nucleic acid sequence which encodes a fatty acid biosynthesis gene.

33. The gene construct as claimed in claim 32, wherein the nucleic acid which encodes a fatty acid biosynthesis gene is selected from the group consisting of:

Δ

19-, Δ

17-, Δ

15-, Δ

12-, Δ

9-, Δ

8-, Δ

6-, Δ

5-, Δ

4-desaturase, hydroxylases, Δ

12-acetylenase, acyl-ACP thioesterases, β

-ketoacyl-ACP synthases and β

-ketoacyl-ACP reductases.

34. An amino acid sequence which is encoded by the isolated nucleic acid sequence as claimed in claim 29.

35. A vector comprising the isolated nucleic acid sequence as claimed in claim 29.

36. An organism comprising the isolated nucleic acid sequence of claim 29.

37. The organism as claimed in claim 36, wherein the organism is a microorganism, an animal or a plant.

38. The organism as claimed in claim 36, wherein the organism is a transgenic plant.

39. An antibody which specifically binds the polypeptide which is encoded by the isolated nucleic acid sequence as claimed in claim 29.

40. An antisense nucleic acid molecule which comprises a sequence complementary to the isolated nucleic acid sequence as claimed in claim 29.

41. A process for the preparation of a PUFA, which comprises culturing an organism comprising the isolated nucleic acid sequence of claim 29 under conditions under which PUFAs are formed in the organism.

42. The process as claimed in claim 41, wherein the PUFA is a C₁₈-fatty acid molecule with at least two double bonds in the fatty acid molecule.

43. The process as claimed in claim 42, wherein the C₁₈-fatty acid molecule is isolated from the organism in the form of an oil, lipid or a free fatty acid.

44. The process as claimed in claim 41, wherein the organism is a microorganism, an animal or a plant.

45. The process as claimed in claim 41, wherein the organism is a transgenic plant.

46. The process as claimed in claim 41, wherein the C₁₈-fatty acid is a fatty acid with three double bonds in the molecule.

47. An oil, lipid or fatty acid composition which comprises the PUFA of claim 41 and which is derived from a transgenic plant.

48. An oil, lipid or fatty acid composition which comprises a PUFA and which is derived from a transgenic plant, wherein the transgenic plant comprises at least one of:

the isolated nucleic acid sequence of claim 29, a gene construct comprising said isolated nucleic acid sequence, an antisense nucleic acid molecule which comprises a complementary sequence of said isolated nucleic acid sequence, or a vector comprising said isolated nucleic acid sequence or said gene construct.

49. A composition, comprising at least one of an antibody which specifically binds a polypeptide which is encoded by the isolated nucleic acid sequence of claim 29 and an antisense nucleic acid construct which comprises the complementary sequence of the isolated nucleic acid sequence.

50. A kit comprising the isolated nucleic acid sequence as claimed in claim 29, a gene construct comprising said isolated nucleic acid sequence, an antisense nucleic acid molecule which comprises the complementary sequence of said isolated nucleic acid sequence, an antibody which specifically binds a polypeptide which is encoded by said isolated nucleic acid sequence, an amino acid sequence which is encoded by said isolated nucleic acid sequence or by a gene construct comprising said isolated nucleic acid sequence.

51. A process for identifying an antagonist or agonist of elongases, comprising

a) bringing cells which express the polypeptide of claim 29 into contact with a candidate substance;

b) testing PSE activity;

c) comparing the PSE activity with a standard activity in the absence of the candidate substance, where a PSE activity that is higher than that of the standard indicates that the candidate substance is an agonist and where a PSE activity that is lower than that of the standard indicates that the candidate substance is an antagonist.

52. A composition, comprising at least one of an antagonist or agonist of the elongase of claim 51.

53. An oil, lipid or fatty acid or a fraction thereof, prepared by the process as claimed in claim 41.

54. A feeding stuff, foodstuff, cosmetic or pharmaceutical comprising the oil, lipid or fatty acid composition as claimed in claim 48.

55. The isolated nucleic acid as claimed in claim 29 wherein the sequence is derived from a plant.

56. A gene construct of claim 31, wherein gene expression is enhanced by said regulatory signals.

57. The organism of claim 36 wherein the isolated nucleic acid is within a vector or a gene construct.

58. The process of claim 41, wherein the isolated nucleic acid is within a vector or a gene construct.