Elongase genes and uses thereof

• US 20050009140A1
• Filed: 08/05/2004
• Published: 01/13/2005
• Est. Priority Date: 09/02/1998
• Status: Active Grant

• Current Assignee: Amanda Eun-Yeong Leonard, Pradip Mukerji, Suzette L. Pereira, Yung-Sheng Huang
• Original Assignee: Amanda Eun-Yeong Leonard, Pradip Mukerji, Suzette L. Pereira, Yung-Sheng Huang
• Inventors: Mukerji, Pradip, Huang, Yung-Sheng, Pereira, Suzette L., Leonard, Amanda Eun-Yeong
• Granted Patent: US 7,375,205 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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: A61K 2800/86 Products or compounds obtained by genetic engineering
• 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 8/606 Nucleosides; Nucleotides; Nucleic acids
• CPC Class Codes: A61K 8/64 Proteins; Peptides; Derivatives or degradation products thereof
• CPC Class Codes: A61P 11/06 Antiasthmatics
• 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 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 17/06 Antipsoriatics
• 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 25/00 Drugs for disorders of the nervous system
• 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 3/04 : Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 : for HIV
• 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 9/10 : 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: A61Q 19/00 : Preparations for care of the skin
• CPC Class Codes: C12N 15/8247 : involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/1029 : transferring groups other than amino-acyl groups (2.3.1)
• 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 several genes involved in the elongation of polyunsaturated acids (i.e., “elongases”) and to uses thereof. At least two of these genes are also involved in the elongation of monounsaturated fatty acids. In particular, elongase is utilized in the conversion of gamma linolenic acid (GLA) to dihomogamma linolenic acid (DGLA) and in the conversion of AA to adrenic acid (ADA), or eicosapentaenoic acid (EPA) to ω3-docosapentaenoic acid (DPA). DGLA may be utilized in the production of polyunsaturated fatty acids, such as arachidonic acid (AA), docosahexaenoic acid (DHA), EPA, adrenic acid, ω6-docosapentaenoic acid or ω3-docosapentaenoic acid which 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 elongase activity, wherein the amino acid sequence of said polypeptide has at least 50% identity to an amino acid sequence comprising SEQ ID NO:

121.

3. The isolated nucleotide sequence of claims 1 or 2 wherein said sequence encodes a functionally active elongase which utilizes a polyunsaturated fatty acid as a substrate.

4. The isolated nucleotide sequence of claim 1 whrein said sequence is derived from an algae.

5. The isolated nucleotide sequence of claim 4 wherein said algae is Pavlova sp.

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

7. A purified polypeptide which elongates polyunsaturated fatty acids and has at least about 30% amino acid identity to the amino acid sequence of said purified protein of claim 6.

2. An isolated nucleotide sequence or fragment thereof comprising or complementary to a nucleotide sequence having at least 50% nucleotide sequence identity to a nucleotide sequence comprising SEQ ID NO:

119.

40. 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 29, said another polyunsaturated fatty acid produced according to the method of claim 31, and said final polyunsaturated fatty acid produced according to the method of claim 33 and 2) a pharmaceutically acceptable carrier.

8. A method of producing an elongase enzyme comprising the steps of:

a) isolating a nucleotide sequence comprising SEQ ID NO;

119;

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 elongase enzyme.

9. The method of claim 8 wherein said host cell is selected from the group consisting of 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 selected from the group consisting of Saccharomyces spp., Candida spp., Lipomyces starkey, Yarrowia spp., Kluyveromyces spp., Hansenula spp., Aspergillusspp., Penicillium spp., Neurospora spp., Trichoderma spp. and Pichia spp.

13. The method of claim 12 wherein said fungal cell is a yeast cell selected from the group consisting of Saccharomyces spp., Candida spp., Hansenula 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 comprising SEQ ID NO;

119 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 selected from the group consisting of 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 selected from the group consisting of Saccharomyces spp., Candida spp., Lipomyces starkey, Yarrowia spp., Kluyveromyces spp., Hansenula spp., Aspergillusspp., Penicillium spp., Neurospora spp., Trichoderma spp. and Pichia spp.

21. The host cell of claim 20 wherein said fungal cell is a yeast cell selected from the group consisting of Saccharomyces spp., Candida spp., Hansenula spp. and Pichia spp.

22. The host cell of claim 21 wherein said yeast 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, plant or plant tissue.

24. The plant cell, plant or plant tissue of claim 23 wherein said polyunsaturated fatty acid is selected from the group consisting of ADA and ω

3-docosapentaenoic acid.

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 transgenic, non-human mammal whose genome comprises a DNA sequence encoding an elongase, operably linked to a promoter, wherein said DNA sequence comprises SEQ ID NO:

119.

28. A fluid produced by said transgenic, non-human mammal of claim 27 wherein said fluid comprises a detectable level of at least one elongase or products thereof.

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

a) isolating a nucleotide sequence comprising SEQ ID NO;

119;

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 an elongase enzyme encoded by said isolated nucleotide sequence; and

d) exposing said expressed elongase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.

30. The method according to claim 29, wherein said substrate polyunsaturated fatty acid is selected from the group consisting of AA and EPA, and said product polyunsaturated fatty acid is selected from the group consisting of ADA and ω

3-docosapentaenoic acid, respectively.

31. The method according to claim 29 further comprising the step of exposing said expressed elongase enzyme to at least one desaturase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.

32. The method according to claim 31 wherein said product polyunsaturated fatty acid is selected from the group consisting of ADA and ω

3-docosapentaenoic acid, said another polyunsaturated fatty acid is selected from the group consisting of ω

6-docosapentaenoic acid, ω

3-docosapentaenoic acid and docohexaenoic acid, and said at least one desaturase is Δ

4-desaturase with respect to production of ω

6-docosapentaenoic acid or docosahexaenoic acid, and Δ

19-desaturase with respect to production of ω

3-docosapentaenoic acid or docosahexaenoic acid.

33. The method of claim 32 further comprising the step of exposing said another polyunsaturated fatty acid to one or more enzymes selected from the group consisting of at least one elongase and at least one additional desaturase in order to convert said another polyunsaturated fatty acid to a final polyunsaturated fatty acid.

34. The method of claim 33 wherein said final 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 claim 29, said another polyunsaturated fatty acid produced according to the method of claim 31, and said final polyunsaturated fatty acid produced according to the method of claim 33.

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

3-docosapentaenoic.

37. The nutritional composition of claim 35 wherein said another polyunsaturated fatty acid is selected from the group consisting ω

6-docosapentaenoic acid, ω

3-docosapentaenoic acid and docosahexaenoic acid.

38. The nutritional composition of claim 35 wherein said final polyunsaturated fatty acid is docosahexaenoic acid.

39. 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.

41. 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 29, said another polyunsaturated fatty acid produced according to the method of claim 31 and said final polyunsaturated fatty acid produced according to the method of claim 33.

42. 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 35 in an amount sufficient to effect said prevention or treatment.