Fad4, Fad5, Fad5-2 and Fad6, novel fatty acid desaturase family members and uses thereof

• US 20020156254A1
• Filed: 09/28/2001
• Published: 10/24/2002
• Est. Priority Date: 09/28/2000
• Status: Active Grant

• Current Assignee: Bioriginal Food & Science Corp. (Omega Protein Corporation)
• Original Assignee: Bioriginal Food & Science Corp. (Omega Protein Corporation)
• Inventors: Hong, Haiping, Qiu, Xiao
• Granted Patent: US 7,087,432 B2
• Time in Patent Office: Days
• US Class Current: 536/23.1
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• 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: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/24 Antidepressants
• 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 27/02 Ophthalmic agents
• 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 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• 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 9/10 : 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 9/0071 : acting on paired donors with incorporation of molecular oxygen (1.14)
• 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
• CPC Class Codes: C12Y 114/19003 : Linoleoyl-CoA desaturase (1.14.19.3)
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturase family members. The invention also provides recombinant expression vectors containing desaturase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g., DHA.

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:

(a) the nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5, or SEQ ID NO;

7;

(b) the nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, or SEQ ID NO;

8; and

(c) the nucleic acid molecule which encodes a naturally-occurring allelic variant of a polypeptide comprising the amino acid sequence s set forth in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, or SEQ ID NO;

8.

3. An isolated nucleic acid molecule which hybridizes to a complement of the nucleic acid molecule of claim 1 or 2 under stringent conditions.

4. A vector comprising the nucleic acid molecule of claim 1 or 2.

5. The vector of claim 4, which is an expression vector.

6. A host cell transfected with the expression vector of claim 5.

7. The host cell of claim 6, wherein the cell is selected from the group consisting of a plant cell, a microbial cell, and an animal cell.

8. The host cell of claim 7, wherein the plant cell is a cell obtained from an oilseed crop selected from the group consisting of flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycine and Soja sp.), sunflower (Helianthus sp.), corron (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), and peanut (Arachis sp.).

9. The host cell of claim 8, wherein the microbial cell is selected from the group consisting of Thraustochytrium, Pythium irregulare, Schizochytrium, and Crythecodinium.

10. A method of producing a polypeptide comprising culturing the host cell of claim 6 to produce the polypeptide.

13. A method of producing an unsaturated fatty acid comprising transfecting or transforming a cell with the isolated nucleic acid molecule of claim 1 or 2 and culturing or cultivating the cell under conditions such that the unsaturated fatty acid is produced.

22. A method of producing a cell capable of generating an unsaturated fatty acid comprising, introducing into said cell the nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule encodes a desaturase having an activity of catalyzing the formation of a double bond in a fatty acyl chain.

23. The method of claim 22, wherein the cell is selected from the group consisting of a plant cell, an animal cell, and a microbial cell.

24. The method of claim 23, wherein the plant cell is a cell from an oilseed plant.

25. The method of claim 24, wherein the oilseed plant is flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycine and Soja sp.), sunflower (Helianthus sp.), corron (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), and peanut (Arachis sp.).

26. The method of claim 22, wherein the unsaturated fatty acid is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,7,10,13,16,19).

27. A method for modulating the production of an unsaturated fatty acid comprising, culturing the cell of claim 6, such that modulation of the production of an unsaturated fatty acid occurs.

28. The method of claim 27, wherein the production of unsaturated fatty acid is enhanced.

29. The method of claim 27, wherein the unsaturated fatty acid is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,77,10,13,16,19).

30. The method of claim 27, further comprising recovering the unsaturated fatty acid produced.

31. A method for large scale production of unsaturated fatty acids, comprising culturing the cell of claim 6, such that an unsaturated fatty acid is produced.

32. The method of claim 31, wherein the production of unsaturated fatty acids is enhanced.

33. The method of claim 31, wherein the unsaturated fatty acid produced is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,7,10,13,16,19).

34. The method of claim 29, further comprising recovering the unsaturated fatty acid produced.

38. A composition comprising the cell produced by the method of claim 22.

39. A composition of claim 35, wherein the composition is used in animal feed.

40. A composition of claim 36, wherein the composition is used in animal feed.

41. A composition of claim 36, wherein the composition is used as a dietary supplement.

42. A method for supplementing the diet of a human or an animal, comprising administering the composition of claim 35.

43. A method of treating a patient having a disorder, comprising administering the composition of claim 35 such that the patient is treated.

44. The method of claim 43, wherein the disorder is selected from the group consisting of stress, diabetes, cancer, inflammatory disorders, and cardiovascular disorders.

2. An isolated nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid molecule comprising a nucleotide sequence which is at least 70% identical to the nucleotide sequence of SEQ ID NO;

1, 3, 5, 7, or a complement thereof;

(b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO;

1, 3, 5, 7, or a complement thereof;

(c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 50% identical to the amino acid sequence of SEQ ID NO;

2, 4, 6, or 8; and

(d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO;

2, 4, 6, or 8, wherein the fragment comprises at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO;

2, 4, 6, or 8.

11. An isolated polypeptide selected from the group consisting of:

(a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO;

2, 4, 6, or 8, wherein the fragment comprises at least 10 contiguous amino acids of SEQ ID NO;

2, 4, 6, or 8;

(b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO;

2, 4, 6, or 8, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to complement of a nucleic acid molecule consisting of SEQ ID NO;

1, 3, 5, or 7 under stringent conditions;

(c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 70% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO;

1, 3, 5, or 7; and

(d) a polypeptide comprising an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO;

2, 4, 6, or 8.

12. The isolated polypeptide of claim 11 comprising the amino acid sequence of SEQ ID NO:

2, 4, 6, or 8.

14. The method of claim 13, wherein the cell is selected from the group consisting of a plant cell, an animal cell, and a microbial cell.

15. The method of claim 14, wherein the plant is selected from an oilseed crop.

16. The method of claim 15, wherein the oilseed crop is selected from the group consisting of flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycine and Soja sp.), sunflower (Helianthus sp.), corron (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), and peanut (Arachis sp.).

17. The method of claim 13, further comprising the step of recovering the unsaturated fatty acid.

18. The method of claim 13, wherein the unsaturated fatty acid is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,7,10,13,16,19).

19. A method of producing an unsaturated fatty acid comprising contacting a composition comprising at least one desaturase target molecule with at least one isolated polypeptide of claim 11 or 12 under conditions such that the unsaturated fatty acid is produced.

20. The method of claim 19, further comprising the step of recovering the unsaturated fatty acid.

21. The method of claim 19, wherein the unsaturated fatty acid is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,7,10,13,16,19).

35. A composition comprising the polypeptide of claim 11 or 12.

36. A composition comprising the polypeptide produced by the method of claim 13.

37. A composition comprising the polypeptide produced by the method of claim 19.