Methods and compositions for synthesis of long chain polyunsaturated fatty acids

• US 6,136,574 A
• Filed: 07/29/1999
• Issued: 10/24/2000
• Est. Priority Date: 04/11/1997
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories, Calgene LLC (Bayer AG)
• Original Assignee: Abbott Laboratories, Calgene LLC (Bayer AG)
• Inventors: Mukerji, Pradip, Huang, Yung-Sheng, Thurmond, Jennifer, Chaudhary, Sunita, Knutzon, Deborah
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US09/363,574
• Time in Patent Office: 453 Days
• Field of Search: 435/189, 435/134, 435/136, 435/254.2, 435/254.21, 536/23.2, 536/23.1, 536/23.7, 536/23.74
• US Class Current: 435/134
• 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: 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 17/00 Drugs for dermatological disorders
• 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 7/6431 Linoleic acids [18:2[n-6]]
• 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 fatty acid desaturases able to catalyze the conversion of oleic acid to linoleic acid, linoleic acid to gamma-linolenic acid, or of alpha-linolenic acid to stearidonic acid. Nucleic acid sequences encoding desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising a desaturase gene, and recombinant host microorganism or animal expressing increased levels of a desaturase are described. Methods for desaturating a fatty acid and for producing a desaturated fatty acid by expressing increased levels of a desaturase are disclosed. Fatty acids, and oils containing them, which have been desaturated by a 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 desaturase produced by a recombinant host microorganism or animal also are described.

Claims

1. A method for production of γ

-linolenic acid in a yeast culture, said method comprising;

growing a yeast culture having a plurality of recombinant yeast cells, wherein said yeast cells or an ancestor of said yeast cells were transformed with a vector comprising Mortierella alpina DNA encoding a Δ

6 desaturase polypeptide which converts linoleic acid to γ

-linolenic acid, wherein said DNA is operably associated with an expression control sequence functional in said yeast cells, under conditions whereby said DNA is expressed, whereby γ

-linolenic acid is produced from linoleic acid in said yeast culture.

2. The method according to claim 1, wherein said linolenic acid is exogenously supplied.

3. The method according to claim 1 wherein said conditions are inducible.

4. The method according to claim 1 wherein said fungal DNA comprises the DNA sequence depicted in SEQ ID NO:

1.

5. The method of according to claim 1 wherein said polypeptide comprises the polypeptide sequence depicted in SEQ ID NO:

2.

6. A method for production of stearidonic acid in a yeast culture, said method comprising:

growing a yeast culture having a plurality of recombinant yeast cells, wherein said yeast cells or an ancestor of said yeast cells were transformed with a vector comprising Mortierella alpina DNA encoding a Δ

6 desaturase polypeptide which converts α

-linolenic acid to stearidonic acid, wherein said DNA is operably associated with an expression control sequence functional in said yeast cells, under conditions whereby said DNA is expressed, whereby stearidonic acid is produced from α

-linolenic acid in said yeast culture.

7. The method according to claim 6 wherein said α

-linolenic acid is exogenously supplied.

8. The method according to claim 6 wherein said conditions are inducible.

9. The method according to claim 6 wherein said Mortierella alpina DNA comprises the DNA sequence depicted in SEQ ID NO:

1.

10. The method according to claim 6 wherein the polypeptide comprises the polypeptide sequence depicted in SEQ ID NO:

2.

11. A method for production of linoleic acid in a yeast culture, said method comprising:

growing a yeast culture having a plurality of recombinant yeast cells, wherein said yeast cells or an ancestor of said yeast cells were transformed with a vector comprising Mortierella alpina DNA encoding a Δ

12 desaturase polypeptide which converts oleic acid to linoleic acid, wherein said DNA is operably associated with an expression control sequence functional in said yeast cells, under conditions whereby said DNA is expressed, whereby linoleic acid is produced oleic acid in said yeast culture.

12. The method according to claim 11 wherein said conditions are inducible.

13. The method according to claim 11 wherein said Mortierella alpina DNA comprises the DNA sequence depicted in SEQ ID NO:

3.

14. The method according to claim 11 wherein the polypeptide comprises the polypeptide sequence depicted in SEQ ID NO:

4.

15. A method for production of stearidonic acid in a eukaryotic cell culture, said method comprising:

growing a eukaryotic cell culture having a plurality of recombinant eukaryotic cells, wherein said recombinant eukaryotic cells or ancestors of said recombinant eukaryotic cells were transformed with a vector comprising Mortierella alpina DNA encoding a polypeptide which converts α

-linolenic to stearidonic acid, wherein said DNA is operably associated with an expression control sequence functional in said recombinant eukaryotic cells, under conditions whereby said DNA is expressed, whereby stearidonic acid is produced from α

-linolenic in said eukaryotic cell culture.

17. The method according to claim 15, wherein said eukaryotic cells are selected from the group consisting of mammalian cells, fungal cells, avian cells and algal cells.

19. The method according to claim 17, wherein said fungal cells are yeast cells of the genus Saccharomyces.

16. A method for production of linoleic acid in a eukaryotic cell culture, said method comprising:

growing a eukaryotic cell culture having a plurality of recombinant eukaryotic cells, wherein said recombinant eukaryotic cells or ancestors of said recombinant eukaryotic cells were transformed with a vector comprising Mortierella alpina DNA encoding a polypeptide which converts oleic acid to linoleic acid, wherein said DNA is operably associated with an expression control sequence functional in said recombinant eukaryotic cells, under conditions whereby said DNA is expressed, whereby linoleic acid is produced from oleic acid in said eukaryotic cell culture.

18. The method according to claim 16 wherein said eukaryotic cells are selected from the group of mammalian cells, fungal cells, avian cells and algal cells.

20. The method according to claim 18, wherein said fungal cells are yeast cells of the genus Saccharomyces.

21. A method of making α

-linolenic acid, said method comprising;

growing a recombinant yeast cell comprising a nucleotide sequence which encodes a functionally active Δ

6 desaturase having the amino acid sequence depicted in SEQ ID NO;

2, and at least one nucleic acid construct comprising a nucleotide sequence which encodes a functionally active Δ

12 desaturase having an amino acid sequence depicted in SEQ ID NO;

4, wherein said nucleic acid constructs are operably associated with transcription control sequences functional in a yeast cell under conditions whereby said nucleotide sequences are expressed, whereby γ

-linolenic acid is produced in said yeast cell.

22. The method according to claim 21 wherein said conditions are inducible.