Methods and compositions for synthesis of long chain polyunsaturated fatty acids

• US 6,589,767 B1
• Filed: 08/19/1999
• Issued: 07/08/2003
• Est. Priority Date: 04/11/1997
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories Incorporated, Monsanto Company (Bayer AG)
• Original Assignee: Abbott Laboratories Incorporated, Calgene Incorporated (Pharmacia Corp.)
• Inventors: Thurmond, Jennifer, Chaudhary, Sunita, Knutzon, Deborah, Mukerji, Pradip, Huang, Yung-Sheng
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): STEADMAN, DAVID J
• Application Number:
  US09/377,452
• Time in Patent Office: 1,419 Days
• Field of Search: 435/189
• US Class Current: 435/189
• 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: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23L 33/40 Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2250/1862 Arachidonic acid
• CPC Class Codes: A23V 2250/1882 Polyunsaturated fatty acids
• CPC Class Codes: A23V 2250/19 Genetically modified oil
• 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 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: C12N 15/81 for yeasts
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The present invention relates to a fatty acid Δ5-desaturase able to catalyze the conversion of dihomo-gamma-linolenic acid to arachidonic acid. Nucleic acid sequences encoding a Δ5-desaturase, nucleic acid sequences which hybridize thereto, DNA constructs comprising a Δ5-desaturase gene, and recombinant host microorganism or animal expressing increased levels of a Δ5-desaturase are described. Methods for desaturating a fatty acid at the Δ5 position and for producing arachidonic acid by expressing increased levels of a Δ5 desaturase are disclosed. Fatty acids, and oils containing them, which have been desaturated by a Δ5-desaturase produced by recombinant host microorganisms or animals are provided. Pharmaceutical compositions, infant formulas or dietary supplements containing fatty acids which have been desaturated by a Δ5-desaturase produced by a recombinant host microorganism or animal also are described.

Claims

1. A purified or isolated polypeptide encoded by the nucleic acid depicted in SEQ ID NO:

1.

3. The polypeptide of claim 1, wherein said polypeptide is produced by expression of said nucleic acid in a host cell, wherein said nucleic acid is operably linked to a promoter functional in the host cell.

4. The polypeptide of claim 3 wherein said host cell is a yeast cell or an algae cell.

5. The polypeptide of claim 4 wherein said host cell is a yeast cell.

6. The polypeptide of claim 4 wherein said host cell is an algae cell.

13. A catalytically active deletion mutant of the polypeptide of claim 1, wherein the deletion mutant lacks at least one amino acid of said polypeptide, wherein said deletion mutant forms a monounsaturated bond between carbons 5 and 6 of a fatty acid as numbered from a caiboxy terminus thereof.

19. The polypeptide of claim 5 wherein said yeast cell is a Saccharomyces cell.

20. The polypeptide of claim 6 wherein said algae cell is a marine algae cell.

2. A purified or isolated polypeptide comprising the amino acid sequence depicted in SEQ ID NO:

2.

14. A catalytically active deletion mutant of the potypeptide of claim 2, wherein the deletion mutant lacks at least one amino acid of said polypeptide, wherein said deletion mutant forms a monounsaturated bond between carbons 5 and 6 of a fatty acid as numbered from a carboxy terminus thereof.

7. A purified or isolated polypeptide encoded by a nucleic acid with at least 80% homology to the sequence depicted in SEQ ID NO:

1, wherein said polypeptide forms a monounsaturated bond between carbons 5 and 6 of a fatty acid as numbered from a carboxy terminus thereof.

8. The polypeptide of claim 7, wherein said polypeptide is encoded by a nucleic acid with at least 90% homology to the sequence depicted in SEQ ID NO:

1.

9. The polypeptide of claim 8, wherein said polypeptide is encoded by a nucleic acid with at least 95% homology to the sequence depicted in SEQ ID NO:

1.

15. A catalytically active deletion mutant of the polypeptide of claim 7, wherein the deletion mutant lacks at least one amino acid of said polypeptide.

16. A catalytically active deletion mutant of the polypeptide of claim 8, wherein the deletion mutant lacks at least one amino acid of said polypeptide.

17. A catalytically active deletion mutant of the polypeptide of claim 9, wherein the deletion mutant lacks at least one amino acid of said polypeptide.

21. The polypeptide of claim 7 wherein the sequence of said polypeptide comprises a sequence selected from the group consisting of residues 30-38, 41-44, 171-175, 203-212 and 387-394 of SEQ ID NO:

2.

10. A purified or isolated polypeptide, wherein said polypeptide has at least 80% homology to the sequence depicted in SEQ ID NO:

2 and said polypeptide forms a monounsaturated bond between carbons 5 and 6 of a fatty acid as numbered from a carboxy terminus thereof.

11. The polypeptide of claim 10, wherein said polypeptide has at least 90% homology to the sequence depicted in SEQ ID NO:

2.

12. The polypeptide of claim 11, wherein said polypeptide has at least 95% homology to the sequence depicted in SEQ ID NO:

2.

18. A catalytically active deletion mutant of the polypeptide of claim 10, wherein the deletion mutant lacks at least one amino acid of said polypeptide.

22. The polyeptide of claim 10 wherein the sequence of said polypeptide comprises a sequence selected from the group consisting of residues 30-38, 41-44, 171-175, 203-212 and