Delta4 - desaturase genes and uses thereof

• US 20030082754A1
• Filed: 05/04/2001
• Published: 05/01/2003
• Est. Priority Date: 05/04/2001
• Status: Abandoned Application

• Current Assignee: Abbott Laboratories
• Original Assignee: Abbott Laboratories
• Inventors: Das, Tapas, Mukerji, Pradip, Huang, Yung-Sheng, Thurmond, Jennifer
• Application Number:
  US09/849,199
• Publication Number:
  US 20030082754A1
• Time in Patent Office: Days
• US Class Current: 435/134
• 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 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 9/12 Ready-to-eat liquid or semi-liquid desserts, e.g. puddings, not to be mixed with liquids, e.g. water, milk
• 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: 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 7/6434 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 4 (i.e., “Δ4-desaturase”). In particular, Δ4-desaturase may be utilized, for example, in the conversion of adrenic acid to ω6-docosapentaenoic acid and in the conversion of ω3-docosapentaenoic acid to docosahexaenoic acid. The polyunsaturated fatty acids produced by use of the enzyme 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:

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17.

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

14, SEQ ID NO;

15 and SEQ I6 NO;

17.

3. The isolated nucleotide sequence of claims 1 or 2 wherein said sequence encodes a functionally active desaturase which utilizes a monounsaturated or 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 sequence selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17 is derived from the fungus Thraustochytrium aureum.

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

43. 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 28 and said another polyunsaturated fatty acid produced according to the method of claim 30 and 2) a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 43 wherein said pharmaceutical composition is administered to a human or an animal.

45. The pharmaceutical composition of claim 44 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.

7. A purified polypeptide which desaturates polyunsaturated fatty acids at carbon 4 and has at least about 50% amino acid similarity to an amino acid sequence selected from the group consisting of SEQ ID NO:

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21.

8. 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;

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17;

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.

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 Escherichia coli, Cyanobacteria, and Bacillus 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. A vector comprising:

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

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17, operably linked to b) a promoter.

15. A host cell comprising said vector of claim 14.

16. The host cell of claim 15, wherein said host cell is a eukaryotic cell or a prokaryotic cell.

17. The host cell of claim 16 wherein said prokaryotic cell is selected from the group consisting of Escherichia coli, Cyanobacteria, and Bacillus subtilis.

18. The host cell of claim 16 wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.

19. The host cell of claim 18 wherein said fungal cell is a yeast cell.

20. The host cell of claim 19 wherein said yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carisbergensis, Candida spp., Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. and Pichia spp.

21. A plant cell, plant or plant tissue comprising said vector of claim 14, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid by said plant cell or tissue.

22. The plant cell, plant or plant tissue of claim 21 wherein said polyunsaturated fatty acid is selected from the group consisting of ω

6-docosapentaenoic acid and docosahexaenoic acid.

23. One or more plant oils or acids expressed by said plant cell, plant or plant tissue of claim 22.

24. A transgenic plant comprising said vector of claim 14, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid in seeds of said transgenic plant.

25. A transgenic, non-human mammal whose genome comprises a nucleotide sequence encoding Δ

4-desaturase, operably linked to a promoter.

26. The transgenic, non-human mammal of claim 25, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17.

27. A fluid produced by or tissue of said transgenic, non-human mammal of claim 26 wherein said fluid or tissue comprises a detectable level of at least Δ

4-desaturase when said nucleotide sequence is selected from the group consisting of SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17.

28. 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;

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17;

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

4-desaturase; and

d) exposing said expressed Δ

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

29. The method according to claim 28, wherein said substrate polyunsaturated fatty acid is adrenic acid or ω

3-docosapentaenoic acid and said product polyunsaturated fatty acid is ω

6-docosapentaenoic acid or docosahexaenoic acid, respectively.

30. The method according to claim 29 further comprising the step of exposing said product polyunsaturated fatty acid to a desaturase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.

31. The method according to claim 30 wherein said product polyunsaturated fatty acid is ω

6-docosapentaenoic acid and said another polyunsaturated fatty acid is docosahexaenoic acid.

35. 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 28 and said another polyunsaturated fatty acid produced according to the method of claim 30.

36. The nutritional composition of claim 35 wherein said product polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of ω

6-docosapentaenoic acid and docosahexaenoic acid.

37. The nutritional composition of claim 35 wherein said another polyunsaturated fatty acid is docosahexaenoic acid.

38. The nutritional composition of claim 35 wherein said nutritional composition is selected from the group consisting of an infant formula, a dietary supplement and a dietary substitute.

39. The nutritional composition of claim 35 wherein said nutritional composition is administered to a human or an animal.

40. The nutritional composition of claim 36 wherein said nutritional composition is administered enterally or parenterally.

41. The nutritional composition of claim 35 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.

42. The nutritional composition of claim 41 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.

46. 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 28 and said another polyunsaturated fatty acid produced according to the method of claim 30.

47. The animal feed of claim 46 wherein said product polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of ω

6-docosapentaenoic acid and docosahexaenoic acid.

48. The animal feed of claim 46 wherein said another polyunsaturated fatty acid is docosahexaenoic acid.

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

a) exposing a substrate polyunsaturated fatty acid to one or more enzymes selected from the group consisting of a desaturase and an elongase in order to convert said substrate to a product polyunsaturated fatty acid; and

b) exposing said product polyunsaturated fatty acid of step (a) to a Δ

4-desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21, in order to convert said product polyunsaturated fatty acid to a final product polyunsaturated fatty acid.

33. The method of claim 32 wherein said substrate polyunsaturated fatty acid is selected from the group consisting of linoleic acid, α

-linolenic acid, stearidonic acid, arachidonic acid, dihomo-γ

-linolenic acid, ω

6-docosapentaenoic acid and eicosapentaenoic acid.

34. The method of claim 32 wherein said final product polyunsaturated fatty acid is selected from the group consisting of eicosapentaenoic acid, ω

6-docosapentaenoic acid and docosahexaenoic acid.

49. 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 32 in an amount sufficient to effect said treatment.