human desaturase gene and uses thereof

• US 20030104596A1
• Filed: 07/09/2002
• Published: 06/05/2003
• Est. Priority Date: 04/11/1997
• Status: Active Grant

• Current Assignee: Amanda Eun-Yeong Leonard, Pradip Mukerji, Tapas Das, Yung-Sheng Huang
• Original Assignee: Amanda Eun-Yeong Leonard, Pradip Mukerji, Tapas Das, Yung-Sheng Huang
• Inventors: Das, Tapas, Mukerji, Pradip, Huang, Yung-Sheng, Leonard, Amanda Eun-Yeong
• Granted Patent: US 6,858,416 B2
• Time in Patent Office: Days
• US Class Current: 435/190
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23L 33/10 using additives addition of substantially indigestible substances A23L33/21
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23L 33/13 Nucleic acids or derivatives thereof A23L33/145 takes precedence
• CPC Class Codes: A23L 33/17 Amino acids, peptides or proteins
• CPC Class Codes: A23L 33/40 Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
• CPC Class Codes: A23L 7/126 Snacks or the like obtained by binding, shaping or compacting together cereal grains or cereal pieces, e.g. cereal bars
• CPC Class Codes: A23L 9/10 Puddings; Dry powder puddings
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2250/1862 Arachidonic acid
• CPC Class Codes: A23V 2250/1882 Polyunsaturated fatty acids
• CPC Class Codes: A23V 2250/19 Genetically modified oil
• CPC Class Codes: A61K 31/202 having three or more double bonds, e.g. linolenic eicosanoids, e.g. leukotrienes A61K31/557
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 8/361 Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
• CPC Class Codes: A61K 8/67 Vitamins
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin
• CPC Class Codes: C12N 15/81 : for yeasts
• 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 7/6432 : Eicosapentaenoic acids [EPA]
• 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 a gene involved in the desaturation of polyunsaturated fatty acids at carbon 5 (i.e., “human Δ5-desaturase”) and to uses thereof. In particular, human 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). 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 represented by SEQ ID NO:

1 (FIG.

     12).

2. The isolated nucleotide sequence of claim 1 wherein said sequence is represented by SEQ ID NO:

1.

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 mammal.

5. The nucleotide sequence of claim 4 wherein said Sequence is derived from a human.

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

7. 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 of said purified protein of claim 6.

46. 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 31, said another polyunsaturated fatty acid produced according to the method of claim 33, and said final polyunsaturated fatty acid produced according to the method of claim 35 and 2) a pharmaceutically acceptable carrier.

47. The pharmaceutical composition of claim 46 wherein said pharmaceutical composition is administered to a human or an animal.

48. The pharmaceutical composition of claim 46 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 method of producing a human Δ

5-desaturase comprising the steps of;

a) isolating said nucleotide sequence represented by SEQ ID NO;

1 (FIG.

     12);

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 human Δ

5-desaturase.

9. The method of claim 8 wherein said host cell is a eukaryotic cell or a prokaryotic cell.

10. The method of claim 9 wherein said prokaryotic cell is selected from the group consisting of E. coli, cyanobacteria, and B. subtilis.

11. The method of claim 9 wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.

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

13. The method of claim 12 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.

14. The method of claim 13 wherein said yeast cell is Saccharomyces cerevisiae.

15. A vector comprising:

a) a nucleotide sequence as represented by SEQ ID NO;

1 (FIG.

     12) operably linked to b) a promoter.

16. A host cell comprising said vector of claim 15.

17. The host cell of claim 16, wherein said host cell is a eukaryotic cell or a prokaryotic cell.

18. The host cell of claim 17 wherein said prokaryotic cell is selected from the group consisting of E. coli, Cyanobacteria, and B. subtilis.

19. The host cell of claim 17 wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.

20. The host cell of claim 19 wherein said fungal cell is a yeast cell.

21. The host cell of claim 20 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.

22. The host cell of claim 21 wherein said host cell is Saccharomyces cerevisiae.

23. A plant cell, plant or plant tissue comprising said vector of claim 15, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid by said plant cell or tissue.

24. The plant cell, plant or plant tissue of claim 23 wherein said polyunsaturated fatty acid is AA or EPA.

25. One or more plant oils or acids expressed by said plant cell, plant or plant tissue of claim 23.

26. A transgenic plant comprising said vector of claim 15, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid in seeds of said transgenic plant.

27. A mammalian cell comprising said vector of claim 15, wherein expression of said nucleotide sequence of said vector results in production of altered levels of AA or EPA when said cell is grown in a culture media comprising a fatty acid selected from the group consisting of an essential fatty acid, LA and ALA.

28. A transgenic, non-human mammal whose genome comprises a DNA sequence encoding a human Δ

5-desaturase operably linked to a promoter.

29. The transgenic, non-human mammal of claim 28, wherein said DNA sequence is represented by SEQ ID NO:

1 (FIG.

     12).

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

5-desaturase.

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

a) isolating said nucleotide sequence represented by SEQ ID NO;

1 (FIG.

     12);

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 said human Δ

5-desaturase enzyme; and

d) exposing said expressed human Δ

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

32. The method according to claim 31, wherein said substrate polyunsaturated fatty acid is DGLA or 20:

4n−

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

33. The method according to claim 31 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.

34. The method according to claim 33 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.

35. The method of claim 33 further comprising the steps 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.

36. The method of claim 35 wherein said final polyunsaturated fatty acid is (n−

6)-docosapentaenoic acid or docosahexaenoic (DHA) acid.

37. 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 31, said another polyunsaturated fatty acid produced according to the method of claim 33, and said final polyunsaturated fatty acid produced according to the method of claim 35.

38. The nutritional composition of claim 37 wherein said product polyunsaturated fatty acid is AA or EPA.

39. The nutritional composition of claim 37 wherein said another polyunsaturated fatty acid is adrenic acid or (n−

3)-docosapentaenoic acid.

40. The nutritional composition of claim 37 wherein said final polyunsaturated fatty acid is DHA.

41. The nutritional composition of claim 37 wherein said nutritional composition is selected from the group consisting of an infant formula, a dietary supplement and a dietary substitute.

42. The nutritional composition of claim 41 wherein said nutritional composition is administered to a human or an animal.

43. The nutritional composition of claim 42 wherein said nutritional composition is administered enterally or parenterally.

44. The nutritional composition of claim 41 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.

45. The nutritional composition of claim 44 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.

49. 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 31, said another polyunsaturated fatty acid produced according to the method of claim 33 and said final polyunsaturated fatty acid produced according to the method of claim 35.

50. The animal feed of claim 49 wherein said product polyunsaturated fatty acid is AA or EPA.

51. The animal feed of claim 49 wherein said another polyunsaturated fatty acid is adrenic acid or (n−

3)-docosapentaenoic acid.

52. The animal feed of claim 49 wherein said final polyunsaturated fatty acid is (n−

6)-docosapentaenoic acid or DHA.

53. 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 31, said another polyunsaturated fatty acid produced according to the method of claim 33 and said final polyunsaturated fatty acid produced according to the method of claim 35.

54. 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 37 in an amount sufficient to effect said treatment.