Delta4-desaturase genes and uses thereof

• US 20030134400A1
• Filed: 04/11/2002
• Published: 07/17/2003
• Est. Priority Date: 05/04/2001
• Status: Active Grant

• Current Assignee: Abbott Laboratories
• Original Assignee: Abbott Laboratories
• Inventors: Das, Tapas, Mukerji, Pradip, Huang, Yung-Sheng, Thurmond, Jennifer, Pereira, Suzette L., Leonard, Amanda Eun-Yeong
• Granted Patent: US 7,045,683 B2
• 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: A61P 3/02 Nutrients, e.g. vitamins, minerals
• 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 7/6434 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 or fragment thereof comprising or complementary to a nucleotide sequence encoding a polypeptide having desaturase activity, wherein the amino acid sequence of said polypeptide has at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

37, SEQ ID NO;

46 and SEQ ID NO;

56.

3. The isolated nucleotide sequence of claim 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 or 2 wherein said sequence is derived from an organism selected from the group consisting of a fungus and an algae.

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, SEQ ID NO;

17 and SEQ ID NO;

45 is derived from the fungus Thraustochytrium aureum, said sequence comprising SEQ ID NO;

36 is derived from the fungus Schizochytrium aggregatum, and said sequence comprising SEQ ID NO;

55 is derived from the algae Isochrysis galbana.

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

2. An isolated nucleotide sequence or fragment thereof comprising or complementary to a nucleotide sequence having at least 50% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

15 and SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

36, SEQ ID NO;

45 and SEQ ID NO;

55.

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

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

37, SEQ ID NO;

46 and SEQ ID NO;

56.

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

a) isolating a nucleotide sequence comprising or complementary to a nucleotide sequence;

i) encoding a polypeptide having an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

37, SEQ ID NO;

46 and SEQ ID NO;

56 or

ii) having at least 50% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

36, SEQ ID NO;

45 and SEQ ID NO;

55;

b) constructing a vector comprising;

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

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

9. A vector comprising:

a) an isolated nucleotide sequence comprising or complementary to a nucleotide sequence;

i) encoding a polypeptide having an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

37, SEQ ID NO;

46 and SEQ ID NO;

56 or

ii) having at least 50% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

36, SEQ ID NO;

45 and SEQ ID NO;

55, operably linked to

b) a promoter.

10. A host cell comprising said vector of claim 9.

11. A plant cell, plant or plant tissue comprising said vector of claim 9, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid by said plant cell, plant or plant tissue.

12. The plant cell, plant or plant tissue of claim 11 wherein said polyunsaturated fatty acid is selected from the group consisting of ω

6-docosapentaenoic acid and docosahexaenoic acid.

13. One or more plant oils or acids expressed by said plant cell, plant or plant tissue of claim 11.

14. A transgenic plant comprising said vector of claim 9, wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid in seeds of said transgenic plant.

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

a) isolating a nucleotide sequence comprising or complementary to a nucleotide sequence;

i) encoding a polypeptide having an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

37, SEQ ID NO;

46 and SEQ ID NO;

56 or

ii) having at least 50% identity to 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, SEQ ID NO;

36, SEQ ID NO;

45, and SEQ ID NO;

55;

b) constructing a vector comprising said isolated nucleotide sequence;

c) introducing said vector into a host cell for a time and under 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.

16. The method according to claim 15, 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.

17. The method according to claim 15 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.

18. The method according to claim 17 wherein said product polyunsaturated fatty acid is ω

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

22. A 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 15 and said another polyunsaturated fatty acid produced according to the method of claim 17.

23. The composition of claim 22 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.

24. The composition of claim 22 wherein said another polyunsaturated fatty acid is docosahexaenoic acid.

25. The composition of claim 22 wherein said composition is selected from the group consisting of an infant formula, a dietary supplement and a dietary substitute.

26. The composition of claim 22 wherein said composition is administered to a human or an animal.

27. The composition of claim 26 wherein said composition is administered enterally or parenterally.

28. A method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to said patient said composition of claim 22 in an amount sufficient to effect said prevention or treatment.

19. 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, SEQ ID NO;

21, SEQ ID NO;

37, SEQ ID NO;

46 and SEQ ID NO;

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

20. The method of claim 19 wherein said substrate polyunsaturated fatty acid is selected from the group consisting of linoleic acid, γ

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

-linolenic acid, eicosatetraenoic acid, adrenic acid, elcosapentaenoic acid.

21. The method of claim 19 wherein said final product polyunsaturated fatty acid is selected from the group consisting of ω

6-docosapentaenoic acid and docosahexaenoic acid.

29. An isolated nucleotide sequence or fragment thereof comprising or complementary to a nucleotide sequence encoding a polypeptide having desaturase activity, wherein the amino acid sequence of said polypeptide has at least 30% identity to the amino acid sequence of SEQ ID NO:

56.

30. An isolated nucleotide sequence or fragment thereof comprising or complementary to a nucleotide sequence having at least 40% identity to the nucleotide sequence of SEQ ID NO:

55.

31. A purified polypeptide which desaturates polyunsaturated fatty acids at carbon 4 and has an amino acid sequence having at least 30% identity to the amino acid sequence of SEQ ID NO:

56.