Fatty acid desaturases from fungi

• US 20060156435A1
• Filed: 05/21/2003
• Published: 07/13/2006
• Est. Priority Date: 05/22/2002
• Status: Active Grant

• Current Assignee: Adeka Corporation, Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Froman, Byron, Voelker, Toni, Ursin, Virginia M.
• Granted Patent: US 7,622,632 B2
• Time in Patent Office: Days
• US Class Current: 800/281
• 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)

Abstract

The invention relates generally to methods and compositions concerning fungal desaturase enzymes that modulate the number and location of double bonds in long chain poly-unsaturated fatty acids (LC-PUFA'"'"'s). In Particular, the invention relates to methods and compositions for improving omega-3 fatty acid profiles in plant products and parts using desaturase enzymes and nucleic acids encoding for such enzymes. In particular embodiments, the desaturase enzymes are fungal −15 desaturases. Also provided are improved canola oil compositions having SDA and maintaining beneficial oleic acid content.

Claims

1. An isolated polynucleotide comprising a nucleic acid sequence that encodes a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 15, wherein the polynucleotide is selected from the group consisting of:

(a) a polynucleotide encoding the polypeptide of SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

34;

(b) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

4 or SEQ ID NO;

33;

(c) a polynucleotide hybridizing to one or more of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

4 or SEQ ID NO;

33, or a complement thereof, under conditions of 5×

SSC, 50% formamide and 42°

C.; and

(d) a fungal polynucleotide encoding a polypeptide having at least one of the amino acid motifs;

TrpIleLeuAlaHisGluCysGlyHisGlyAlaSerPhe (WILAHECGHGASF) (SEQ ID NO;

6);

LeuAlaHisGluCysGlyHis (LAHECGH) (SEQ ID NO;

7);

HisSerPheLeuLeuValProTyrPheSerTrpLys (HSFLLVPYFSWK) (SEQ ID NO;

8);

LeuLeuValProTyrPheSerTrpLys (LLVPYFSWK) (SEQ ID NO;

9);

His(His/Ala)ArgHisHisArg(Phe/Tyr)ThrThr (H(H/A)RHHR(F/Y)TT) (SEQ ID NO;

10, SEQ ID NO;

19, SEQ ID NO;

20, or SEQ ID NO;

21);

TrpValHisHisTrpLeuValAlaIleThrTyrLeu(His/Gln)HisThrHis (WVHHWLVAITYL(H/Q)HTH) (SEQ ID NO;

11);

AlaIleThrTyrLeu(His/Gln)HisThr (AITYL(H/Q)HT) (SEQ ID NO;

12);

GlyAlaLeuAlaThrValAspArg (GALATVDR) (SEQ ID NO;

13) or HisValValHisHisLeuPheXaaArgIleProPheTyr (HVVHHLFXRIPFY) (SEQ ID NO;

14 or SEQ ID NO;

22).

2. The isolated polynucleotide of claim 1, wherein said polynucleotide is from a phyla selected from the group consisting of zygomycota, basidiomycota, and ascomycota.

3. The isolated polynucleotide of claim 1, wherein the polynucleotide is from a species selected from the group consisting of Neurospora crassa, Aspergillus nidulans and Botrytis cinerea.

4. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having at least one of the amino acid motifs:

TrpIleLeuAlaHisGluCysGlyHisGlyAlaSerPhe (WILAHECGHGASF) (SEQ ID NO;

6);

LeuAlaHisGluCysGlyHis (LAHECGH) (SEQ ID NO;

7);

HisSerPheLeuLeuValProTyrPheSerTrpLys (HSFLLVPYFSWK) (SEQ ID NO;

8);

LeuLeuValProTyrPheSerTrpLys (LLVPYFSWK) (SEQ ID NO;

9);

His(His/Ala)ArgHisHisArg(Phe/Tyr)ThrThr (H(H/A)RHHR(FY)TT) (SEQ ID NO;

10, SEQ ID NO;

19, SEQ ID NO;

20, or SEQ ID NO;

21);

TrpValHisHisTrpLeuValAlaIleThrTyrLeu(His/Gln)HisThrHis (WVHHWLVAITYL(H/Q)HTH (SEQ ID NO;

11);

AlaIleThrTyrLeu(His/Gln)HisThr (AITYL(H/Q)HT) (SEQ ID NO;

12);

GlyAlaLeuAlaThrValAspArg (GALATVDR) (SEQ ID NO;

13) or HisValValHisHisLeuPheXaaArgIleProPheTyr (HVVHHLFXRIPFY) (SEQ ID NO;

14 or SEQ ID NO;

22).

5. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes the polypeptide of SEQ ID NO:

3, SEQ ID NO;

5 or SEQ ID NO;

34.

6. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

4 or SEQ ID NO;

33.

7. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes to one or more of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

4 or SEQ ID NO;

33 under conditions of 5×

SSC, 50% formamide and 42°

C.

8. A recombinant vector comprising the isolated polynucleotide of claim 1.

9. The recombinant vector of claim 8, further comprising at least one additional sequence chosen from the group consisting of:

(a) regulatory sequences operatively linked to the polynucleotide;

(b) selection markers operatively linked to the polynucleotide;

(c) marker sequences operatively linked to the polynucleotide;

(d) a purification moiety operatively linked to the polynucleotide; and

(e) a targeting sequence operatively linked to the polynucleotide.

10. The recombinant vector of claim 8, further defined as comprising a promoter operably linked to said isolated polynucleotide.

11. The recombinant vector of claim 10, wherein the promoter is a developmentally-regulated, organelle-specific, tissue-specific, constitutive or cell-specific promoter.

12. The recombinant vector of claim 10, wherein said promoter is selected from the group consisting of 35S CaMV, 34S FMV, Napin, 7S, Glob, and Lee.

13. The recombinant vector of claim 8, defined as an isolated expression cassette.

14. The recombinant vector of claim 8, further defined as comprising a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 6 and/or a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 12.

24. A transgenic plant transformed with the recombinant vector of claim 8.

25. The transgenic plant of claim 24, further defined as transformed with a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 6.

26. A host cell transformed with the recombinant vector of claim 8.

27. The host cell of claim 26, wherein said host cell expresses a protein encoded by said vector.

28. The host cell of claim 26, wherein the cell has inherited said recombinant vector from a progenitor of the cell.

29. The host cell of claim 26, wherein the cell has been transformed with said recombinant vector.

30. The host cell of claim 26, wherein said host cell is a plant cell.

38. A method of producing seed oil containing omega-3 fatty acids from plant seeds, comprising the steps of:

(a) obtaining seeds of a plant according to claim 24;

and

(b) extracting the oil from said seeds.

39. A method of producing a plant comprising seed oil containing altered levels of omega-3 fatty acids comprising introducing the recombinant vector of claim 8 into an oil-producing plant.

40. The method of claim 39, wherein introducing the recombinant vector comprises plant breeding.

41. The method of claim 39, wherein introducing the recombinant vector comprises the steps of:

(a) transforming a plant cell with the recombinant vector of claim 8;

and

(b) regenerating said plant from the plant cell, wherein the plant has altered levels of omega-3 fatty acids.

42. The method of claim 39, wherein the plant is a plant selected from the group consisting of Arabidopsis thaliana, oilseed Brassica, rapeseed, sunflower, safflower, canola, corn, soybean, cotton, flax, jojoba, Chinese tallow tree, tobacco, cocoa, peanut, fruit plants, citrus plants, and plants producing nuts and berries.

43. The method of claim 39, wherein the plant is further defined as transformed with a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 6.

44. The method of claim 43, wherein stearidonic acid is increased.

45. The method of claim 39, further defined as comprising introducing the recombinant vector of claim 8 into a plurality of oil-producing plants and screening said plants or progeny thereof having inherited the recombinant vector for a plant having a desired profile of omega-3 fatty acids.

15. An isolated polynucleotide comprising a nucleic acid sequence that encodes a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 12, wherein the polynucleotide is selected from the group consisting of:

(a) a polynucleotide encoding the polypeptide of SEQ ID NO;

32 or SEQ ID NO;

40;

(b) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO;

31 or SEQ ID NO;

39; and

(c) a polynucleotide hybridizing to one or both of SEQ ID NO;

31 or SEQ ID NO;

39, or a complement thereof, under conditions of 5×

SSC, 50% formamide and 42°

C.; and

16. The isolated polynucleotide of claim 15, wherein said polynucleotide is from a phyla selected from the group consisting of zygomycota, basidiomycota, and ascomycota.

17. The isolated polynucleotide of claim 15, wherein the polynucleotide is from a species selected from the group consisting of Neurospora crassa and Botrytis cinerea.

18. A recombinant vector comprising the isolated polynucleotide of claim 15.

19. The recombinant vector of claim 18, further defined as comprising a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 6 and/or a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 15.

31. A transgenic plant transformed with the recombinant vector of claim 15.

32. The transgenic plant of claim 31, further defined as transformed with a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 6 and/or a nucleic acid sequence encoding a polypeptide having desaturase activity that desaturates a fatty acid molecule at carbon 15.

33. A host cell transformed with the recombinant vector of claim 15.

34. The host cell of claim 33, wherein said host cell expresses a protein encoded by said vector.

35. The host cell of claim 33, wherein the cell has inherited said recombinant vector from a progenitor of the cell.

36. The host cell of claim 33, wherein the cell has been transformed with said recombinant vector.

37. The host cell of claim 33, wherein said host cell is a plant cell.

46. A method of producing a plant comprising seed oil containing altered levels of omega-3 fatty acids comprising introducing the recombinant vector of claim 15 into an oil-producing plant.

47. The method of claim 46, wherein introducing the recombinant vector comprises plant breeding.

48. The method of claim 46, wherein introducing the recombinant vector comprises the steps of:

(a) transforming a plant cell with the recombinant vector of claim 15;

and

(b) regenerating said plant from the plant cell, wherein the plant has altered levels of omega-3 fatty acids.

49. The method of claim 46, wherein the plant is a plant selected from the group consisting of Arabidopsis thaliana, oilseed Brassica, rapeseed, sunflower, safflower, canola, corn, soybean, cotton, flax, jojoba, Chinese tallow tree, tobacco, cocoa, peanut, fruit plants, citrus plants, and plants producing nuts and berries.

20. A fungal polypeptide or fragment thereof having desaturase activity that desaturates a fatty acid molecule at carbon 15 and comprises at least one of the amino acid motifs:

TrpIleLeuAlaHisGluCysGlyHisGlyAlaSerPhe (WILAHECGHGASF) (SEQ ID NO;

6);

LeuAlaHisGluCysGlyHis (LAHECGH) (SEQ ID NO;

7);

HisSerPheLeuLeuValProTyrPheSerTrpLys (HSFLLVPYFSWK) (SEQ ID NO;

8);

LeuLeuValProTyrPheSerTrpLys (LLVPYFSWK) (SEQ ID NO;

9);

His(His/Ala)ArgHisHisArg(Phe/Tyr)ThrThr (H(H/A)RHHR(F/Y)TT) (SEQ ID NO;

10, SEQ ID NO;

19, SEQ ID NO;

20, or SEQ ID NO;

21);

TrpValHisHisTrpLeuValAlaIleThrTyrLeu(His/Gln)HisThrHis (WVHHWLVAITYL(H/Q)HTH) (SEQ ID NO;

11);

AlaIleThrTyrLeu(His/Gln)HisThr (AITYL(H/Q)HT) (SEQ ID NO;

12);

GlyAlaLeuAlaThrValAspArg (GALATVDR) (SEQ ID NO;

     13) or HisValValHisHisLeuPheXaaArgIleProPheTyr (HVVHHLFXRIPFY) (SEQ ID NO;

14 or SEQ ID NO;

22).

21. The fungal polypeptide of claim 20, further defined as comprising all of said amino acid motifs.

22. A fungal polypeptide comprising the amino acid sequence of SEQ ID NO:

3, SEQ ID NO;

5 or SEQ ID NO;

34;

or a fragment thereof having desaturase activity that desaturates a fatty acid molecule at carbon 15.

23. A fungal polypeptide comprising the amino acid sequence of SEQ ID NO:

32 or SEQ ID NO;

40;

or a fragment thereof having desaturase activity that desaturates a fatty acid molecule at carbon 12.

50. An endogenous canola seed oil having a stearidonic acid content of from about 8% to about 27% and an oleic acid content of from about 40% to about 70%.

51. The canola seed oil of claim 50, further defined as comprising less than 10% combined alpha-linoleic acid, linoleic acid and gamma-linoleic acid.

52. The canola seed oil of claim 50, wherein the stearidonic acid content is further defined as from about 10% to about 20%.

53. The canola seed oil of claim 50, wherein the stearidonic acid content is further defined as from about 12% to about 17%.

54. The canola seed oil of claim 50, wherein the oleic acid content is further defined as from about 45% to about 65%.

55. The canola seed oil of claim 50, wherein the oleic acid content is further defined as from about 55% to about 65%.

56. The canola seed oil of claim 50, wherein the stearidonic acid content is further defined as from about 12% to about 17% and the oleic acid content is further defined as from about 55% to about 65%.

57. The canola seed oil of claim 50, further defined as from Brassica napus seed.

58. The canola seed oil of claim 50, further defined as from Brassica rapa seed.

59. The canola seed oil of claim 50, wherein the ratio of omega-6 to omega-3 fatty acids in the oil is from about 1:

1 to about 1;

4.

60. The canola seed oil of claim 59, wherein the ratio of omega-6 to omega-3 fatty acids in the oil is further defined as from about 1:

2 to about 1;

4.

61. A method of increasing the nutritional value of an edible product for human or animal consumption, comprising adding the canola seed oil of claim 50 to the edible product.

62. The method of claim 61, wherein the edible product is human food.

63. The method of claim 61, wherein the edible product is animal feed.

64. The method of claim 61, wherein the edible product is a food supplement.

65. The method of claim 61, wherein the canola seed oil increases the stearidonic acid content of the edible product.

66. The method of claim 61, wherein the canola seed oil decreases the ratio of omega-6 to omega-3 fatty acids of the edible product.

67. The method of claim 61, wherein the edible product lacks stearidonic acid prior to adding the canola seed oil.

68. A method of manufacturing food or feed, comprising adding the canola seed oil of claim 50 to starting food or feed ingredients to produce the food or feed.

69. The method of claim 68, further defined as a method of manufacturing food.

70. The method of claim 68, further defined as a method of manufacturing feed.

71. Food or feed made by the method of claim 68.

72. A method of providing stearidonic acid to a human or animal, comprising administering the canola seed oil of claim 50 to said human or animal.

73. The method of claim 72, wherein the canola seed oil is administered in an edible composition.

74. The method of claim 73, wherein the edible composition is food or feed.

75. The method of claim 74, wherein the food comprises beverages, infused foods, sauces, condiments, salad dressings, fruit juices, syrups, desserts, icings and fillings, soft frozen products, confections or intermediate food.

76. The method of claims 73, wherein the edible composition is substantially a liquid.

77. The method of claim 73, wherein the edible composition is substantially a solid.

78. The method of claim 73, wherein the edible composition is a food supplement.

79. The method of claim 73, wherein the edible composition is a nutraceutical.

80. The method of claim 72, wherein the canola seed oil is administered to a human.

81. The method of claim 72, wherein the canola seed oil is administered to an animal.

82. The method of claim 81, wherein the canola seed oil is administered to livestock or poultry.