Desaturase genes and uses thereof

• US 20030190733A1
• Filed: 05/08/2003
• Published: 10/09/2003
• Est. Priority Date: 01/25/2001
• Status: Active Grant

• Current Assignee: Jennifer Thurmond, Pradip Mukerji, Suzette L. Pereira, Tapas Das, Yung-Sheng Huang
• Original Assignee: Jennifer Thurmond, Pradip Mukerji, Suzette L. Pereira, Tapas Das, Yung-Sheng Huang
• Inventors: Das, Tapas, Mukerji, Pradip, Huang, Yung-Sheng, Thurmond, Jennifer, Pereira, Suzette L.
• Granted Patent: US 7,241,619 B2
• Time in Patent Office: Days
• US Class Current: 435/190
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/04 for urolithiasis
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 15/12 for climacteric disorders
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/04 Antipruritics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/14 for baldness or alopecia
• CPC Class Codes: A61P 17/16 Emollients or protectives, e.g. against radiation
• 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 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 3/00 : 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: A61P 3/06 : Antihyperlipidemics
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 3/14 : for calcium homeostasis vitamin D A61P3/02; parathyroid hormones A61P5/18; calcitonin A61P5/22; osteoporosis A61P19/10; bone metastasis A61P35/04
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/08 : Vasodilators for multiple indications
• 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: 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: C12P 7/6427 : Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6431 : Linoleic acids [18:2[n-6]]
• 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

Abstract

The subject invention relates to the identification of genes involved in the desaturation of polyunsaturated fatty acids at carbon 5 (i.e., “Δ5-desaturase”) and at carbon 6 (i.e., “Δ6-desaturase”) and to uses thereof. In particular, Δ5-desaturase may be utilized, for example, in the conversion of dihomo-γ -linolenic acid (DGLA) to arachidonic acid (AA) and in the conversion of 20:4n-3 to eicosapentaenoic acid (EPA). Delta-6 desaturase may be used, for example, in the conversion of linoleic (LA) to γ-linolenic acid (GLA). AA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

Claims

1. An isolated nucleotide sequence corresponding to or complementary to at least about 50% of the nucleotide sequence selected from the group consisting of SEQ ID NO:

13, SEQ ID NO;

19 and SEQ ID NO;

28.

2. The isolated nucleotide sequence of claim 1 wherein said sequence is selected from the group consisting of SEQ ID NO:

13, SEQ ID NO;

19 and SEQ ID NO;

28.

3. The isolated nucleotide sequence of claims 1 or 2 wherein said sequence encodes a functionally active desaturase which utilizes a polyunsaturated fatty acid as a substrate.

4. The nucleotide sequence of claim 1 wherein said sequence is derived from a fungus.

5. The nucleotide sequence of claim 4 wherein said fungus from which SEQ ID NO:

13 and SEQ ID NO;

19 are derived is Saprolegnia diclina.

6. The nucleotide sequence of claim 4 wherein said fungus from which SEQ ID NO:

28 is derived is Thraustochytrium aureum.

7. A purified protein encoded by said nucleotide sequence of claims 1 or 2.

53. A pharmaceutical composition comprising 1) at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of claim 32 or 38, said another polyunsaturated fatty acid produced according to the method of claim 34 or 40, and said final polyunsaturated fatty acid produced according to the method of claim 36 or 42 and 2) a pharmaceutically acceptable carrier.

54. The pharmaceutical composition of claim 53 wherein said pharmaceutical composition is administered to a human or an animal.

55. The pharmaceutical composition of claim 53 wherein said pharmaceutical composition further comprises an element selected from the group consisting of a vitamin, a mineral, a carbohydrate, an amino acid, a free fatty acid, a phospholipid, an antioxidant, and a phenolic compound.

8. A purified polypeptide which desaturates polyunsaturated fatty acids at carbon 5 and has at least about 50% amino acid similarity to the amino acid sequence comprising SEQ ID NO:

20 or SEQ ID NO;

29.

9. A purified polypeptide which desaturates polyunsaturated fatty acids at carbon 6 and has at least about 50% amino acid similarity to the amino acid sequence comprising SEQ ID NO:

14.

10. A method of producing a desaturase comprising the steps of:

a) isolating said nucleotide sequence represented selected from the group consisting of SEQ ID NO;

13, SEQ ID NO;

19 and SEQ ID NO;

28;

b) constructing a vector comprising;

i) said isolated nucleotide sequence operably linked to ii) a promoter;

c) introducing said vector into a host cell under time and conditions sufficient for expression of said desaturase.

11. The method of claim 10 wherein said host cell is a eukaryotic cell or a prokaryotic cell.

12. The method of claim 11 wherein said prokaryotic cell is selected from the group consisting of E. coli, cyanobacteria, and B. subtilis.

13. The method of claim 11 wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.

14. The method of claim 11 wherein said fungal cell is a yeast cell.

15. The method of claim 14 wherein said yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp., Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. and Pichia spp.

16. A vector comprising:

a) a nucleotide sequence selected from the group consisting of SEQ ID NO;

13, SEQ ID NO;

19 and SEQ ID NO;

28, operably linked to b) a promoter.

17. A host cell comprising said vector of claim 16.

18. The host cell of claim 17, wherein said host cell is a eukaryotic cell or a prokaryotic cell.

19. The host cell of claim 18 wherein said prokaryotic cell is selected from the group consisting of E. coli, Cyanobacteria, andy B. subtilis.

20. The host cell of claim 19 wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.

21. The host cell of claim 20 wherein said fungal cell is a yeast cell.

22. The host cell of claim 21 wherein said yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp., Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. and Pichia spp.

23. The host cell of claim 22 wherein said host cell is Saccharomyces cerevisiae.

24. A plant cell, plant or plant tissue comprising said vector of claim 16, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid by said plant cell or tissue.

25. The plant cell, plant or plant tissue of claim 24 wherein said polyunsaturated fatty acid is selected from the group consisting of AA, EPA, GLA and STA.

26. One or more plant oils or acids expressed by said plant cell, plant or plant tissue of claim 25.

27. A transgenic plant comprising said vector of claim 16, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid in seeds of said transgenic plant.

62. A mammalian cell comprising said vector of claim 16, wherein expression of said nucleotide sequence of said vector results in production of altered levels of AA, EPA, GLA or STA, when said cell is grown in a culture media comprising at least one fatty acid selected from the group consisting of LA, ALA, DGLA and ESP.

28. A transgenic, non-human mammal whose genome comprises a DNA sequence encoding an enzyme selected from the group consisting of Δ

5-desaturase and Δ

6-desaturase, operably linked to a promoter.

29. The transgenic, non-human mammal of claim 28, wherein said DNA sequence is selected from the group consisting of SEQ ID NO:

13, SEQ ID NO;

19 and SEQ ID NO;

28.

30. A fluid produced by said transgenic, non-human mammal of claim 29 wherein said fluid comprises a detectable level of at least Δ

5-desaturase when said DNA sequence comprises either SEQ ID NO;

19 or SEQ ID NO;

28.

31. A fluid produced by said transgenic, non-human mammal of claim 29 wherein said fluid comprises a detectable level of at least Δ

6-desaturase when said DNA sequence comprises SEQ ID NO;

13.

32. A method for producing a polyunsaturated fatty acid comprising the steps of:

a) isolating a nucleotide sequence selected from the group consisting of SEQ ID NO;

19 and SEQ ID NO;

28;

b) constructing a vector comprising said isolated nucleotide sequence;

c) introducing said vector into a host cell under time and conditions sufficient for expression of Δ

5-desaturase enzyme; and

d) exposing said expressed Δ

5-desaturase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.

33. The method according to claim 32, wherein said substrate polyunsaturated fatty acid is DGLA or 20:

4n-3 and said product polyunsaturated fatty acid is AA or EPA, respectively.

34. The method according to claim 32 further comprising the step of exposing said product polyunsaturated fatty acid to an elongase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.

35. The method according to claim 34 wherein said product polyunsaturated fatty acid is AA or EPA and said another polyunsaturated fatty acid is adrenic acid or (n-3)-docosapentaenoic acid, respectively.

36. The method of claim 34 further comprising the step of exposing said another polyunsaturated fatty acid to an additional desaturase in order to convert said another polyunsaturated fatty acid to a final polyunsaturated fatty acid.

37. The method of claim 36 wherein said final polyunsaturated fatty acid is (n-6)-docosapentaenoic acid or docosahexaenoic (DHA) acid.

44. A nutritional composition comprising at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of claim 32 or 38, said another polyunsaturated fatty acid produced according to the method of claim 34 or 40, and said final polyunsaturated fatty acid produced according to the method of claim 36 or 42.

45. The nutritional composition of claim 44 wherein said product polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of AA, EPA, GLA and STA.

46. The nutritional composition of claim 44 wherein said another polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of adrenic acid, (n-3)-docosapentaenoic acid, DGLA and EPA.

47. The nutritional composition of claim 44 wherein said final polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of (n-6)-docosapentaenoic acid, docosahexaenoic (DHA) acid, AA and EPA.

48. The nutritional composition of claim 44 wherein said nutritional composition is selected from the group consisting of an infant formula, a dietary supplement and a dietary substitute.

49. The nutritional composition of claim 48 wherein said nutritional composition is administered to a human or an animal.

50. The nutritional composition of claim 49 wherein said nutritional composition is administered enterally or parenterally.

51. The nutritional composition of claim 44 wherein said nutritional composition further comprises at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, monoglycerides, diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and protein hydrolysates.

52. The nutritional composition of claim 51 wherein said nutritional composition further comprises at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex and at least one mineral selected from the group consisting of calcium magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium and iron.

56. An animal feed comprising at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of claim 32 or 38, said another polyunsaturated fatty acid produced according to the method of claim 34 or 36 and said final polyunsaturated fatty acid produced according to the method of claim 36 or 42.

57. The animal feed of claim 56 wherein said product polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of AA, EPA, GLA and STA.

58. The animal feed of claim 56 wherein said another polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of adrenic acid, (n-3)-docosapentaenoic acid, DGLA and EPA.

59. The animal feed of claim 56 wherein said final polyunsaturated fatty acid is at least one polyunsaturated fatty acid is selected from the group consisting of (n-6)-docosapentaenoic acid, DHA, AA and EPA.

60. A cosmetic comprising a polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of claim 32 or 38, said another polyunsaturated fatty acid produced according to the method of claim 34 or 40 and said final polyunsaturated fatty acid produced according to the method of claim 36 or 42.

61. A method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to said patient said nutritional composition of claim 44 in an amount sufficient to effect said treatment.

38. A method for producing a polyunsaturated fatty acid comprising the steps of:

a) isolating a nucleotide sequence comprising SEQ ID NO;

13;

b) constructing a vector comprising said isolated nucleotide sequence;

c) introducing said vector into a host cell under time and conditions sufficient for expression of Δ

6-desaturase enzyme; and

d) exposing said expressed Δ

6-desaturase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.

39. The method according to claim 38, wherein said substrate polyunsaturated fatty acid is LA or ALA and said product polyunsaturated fatty acid is GLA or STA, respectively.

40. The method according to claim 38 further comprising the step of exposing said product polyunsaturated fatty acid to an elongase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.

41. The method according to claim 40 wherein said product polyunsaturated fatty acid is GLA or STA and said another polyunsaturated fatty acid is DGLA or ETA, respectively.

42. The method of claim 40 further comprising the step of exposing said another polyunsaturated fatty acid to an additional desaturase in order to convert said another polyunsaturated fatty acid to a final polyunsaturated fatty acid.

43. The method of claim 42 wherein said final polyunsaturated fatty acid is AA or EPA.