Desaturase genes and uses thereof

• US 6,635,451 B2
• Filed: 01/25/2001
• Issued: 10/21/2003
• Est. Priority Date: 01/25/2001
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Das, Tapas, Huang, Yung-Sheng, Mukerji, Pradip, Thurmond, Jennifer, Pereira, Suzette L.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SULLIVAN, DANIEL M
• Application Number:
  US09/769,863
• Publication Number:
  US 20030157144A1
• Time in Patent Office: 999 Days
• Field of Search: 536/23.1, 536/23.2, 435/320.1, 435/71.1, 435/189, 424/93.7, 424/93.21
• US Class Current: 435/71.1
• 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 nucleic acid molecule having a nucleotide sequence comprising or complementary to the nucleotide sequence of SEQ ID NO:

28.

2. The isolated nucleic acid molecule of claim 1 wherein said molecule encodes a functionally active desaturase which utilizes a polyunsaturated fatty acid as a substrate.

3. The isolated nucleic acid molecule of claim 1 wherein said molecule is derived from a fungus.

4. The isolated nucleic acid molecule of claim 3 wherein said fungus is Thraustochytrium aureum.

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

a) isolating a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO;

28;

b) constructing a vector comprising;

i) said isolated nucleotide acid molecule operably linked to ii) a regulatory sequence;

c) introducing said vector into a host cell for a time and conditions sufficient for expression of said desaturase, wherein said host cell is a prokaryotic or a eukaryotic cell, wherein said eukaryotic cell is selected from the group consisting of an insect cell, a plant cell and a fungal cell.

6. The method of claim 5 wherein said prokaryotic cell is selected from the group consisting of E. coli, cyanobacteria and B. subtilis.

7. The method of claim 5 wherein said fungal cell is a yeast cell.

8. The method of claim 7 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.

11. The host cell of claim 7, wherein said host cell is a prokaryotic cell or a eukaryotic cell, wherein said eukaryotic cell is selected from the group consisting of an insect cell, a plant cell and a fungal cell.

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

13. The host cell of claim 11 wherein said fungal cell is a yeast cell.

14. The host cell of claim 13 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.

15. The host cell of claim 14 wherein said yeast cell is Saccharomyces cerevisiae.

9. A vector comprising:

a) an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO;

28, operably linked to b) a regulatory sequence.

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

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

a) isolating a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO;

28;

b) constructing a vector comprising said isolated nucleic acid molecule;

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

5-desaturase enzyme, wherein said host cell is a prokaryotic cell or a eukaryotic cell, wherein said eukaryotic cell is selected from the group consisting of an insect cell, a plant cell and a fungal cell; 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.

17. The method according to claim 16, wherein said substrate polyunsaturated fatty acid is DGLA or 20-4:

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

18. The method according to claim 16 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.

19. The method according to claim 18 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.

20. The method of claim 18 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.

21. The method of claim 20 wherein said final polyunsaturated fatty acid is (n-6)-docosapentaenoic acid or docosahexaenoic (DHA) acid.

22. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:

29.