Methods and compositions for synthesis of long chain poly-unsaturated fatty acids in plants

• US 6,075,183 A
• Filed: 04/11/1997
• Issued: 06/13/2000
• Est. Priority Date: 04/11/1997
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated, Calgene Incorporated (Pharmacia Corp.)
• Original Assignee: Abbott Laboratories Incorporated, Calgene Incorporated (Pharmacia Corp.)
• Inventors: Mukerji, Pradip, Huang, Yung-Sheng, Thurmond, Jennifer, Chaudhary, Sunita, Knutzon, Deborah
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US08/834,033
• Time in Patent Office: 1,159 Days
• Field of Search: 536/23.2, 435/134, 435/69.1, 435/419, 435/468, 435/471, 435/430, 435/252.3, 800/281, 800/298
• US Class Current: 800/281
• 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: 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 compositions and methods for preparing poly-unsaturated long chain fatty acids in plants, plant parts and plant cells, such as leaves, roots, fruits and seeds. Nucleic acid sequences and constructs encoding fatty acid desaturases, including Δ5-desaturases, Δ6-desaturases and Δ12-desaturases, are used to generate transgenic plants, plant parts and cells which contain and express one or more transgenes encoding one or more desaturases. Expression of the desaturases with different substrate specificities in the plant system permit the large scale production of poly-unsaturated long chain fatty acids such as docosahexaenoic acid, eicosapentaenoic acid, α-linoleic acid, gamma-linolenic acid, arachidonic acid and the like for modification of the fatty acid profile of plants, plant parts and tissues. Manipulation of the fatty acid profiles allows for the production of commercial quantities of novel plant oils and products.

Claims

1. A nucleic acid construct comprising one or more nucleotide sequences depicted in a SEQ ID NO:

selected from the group consisting of SEQ ID NO;

1 and SEQ ID NO;

3 linked to a heterologous nucleotide sequence.

2. A nucleic acid construct comprising one or more nucleotide sequences depicted in a SEQ ID NO:

selected from the group consisting of SEQ ID NO;

1 and SEQ ID NO;

3 operably associated with an expression control sequence in a plant cell.

3. The nucleic acid construct according to claim 2, wherein said nucleotide sequence is derived from a fungus.

4. The nucleic acid construct according to claim 3, wherein said fungus is of the genus Mortierella.

5. The nucleic acid construct according to claim 4, wherein said fungus is of the species alpina.

6. A nucleic acid construct comprising a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence depicted in SEQ ID NO:

2, wherein said nucleotide sequence is operably associated with a transcription or an expression control sequence functional in a plant cell, wherein said nucleotide sequence encodes a functionally active polypeptide which removes hydrogen atoms from carbons 6 and 7 as numbered from the carboxy terminus of a fatty acid molecule to form an unsaturated bond.

7. A nucleic acid construct comprising a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence depicted in SEQ ID NO:

4, wherein said nucleotide sequence is operably associated with a transcription or an expression control sequence functional in a plant cell, wherein said nucleotide sequence encodes a functionally active polypeptide which removes hydrogen atoms from carbons 5 and 6 as numbered from the carboxy terminus of a fatty acid molecule to form an unsaturated bond.

8. A nucleic acid construct comprising a nucleotide sequence which encodes a functionally active desaturase having the amino acid sequence depicted in a SEQ ID NO:

selected from the group consisting of SEQ ID NO;

4, and SEQ ID NO;

2, wherein said nucleotide sequence is operably associated with a promoter functional in a plant cell.

9. The nucleic acid construct according to claim 8, wherein said plant cell is a seed cell.

10. The nucleic acid construct according to claim 9, wherein said seed cell is an embryo cell.

13. The nucleic acid construct of claim 8, wherein said amino acid sequence is derived from the genus Mortierella.

11. A microbial or plant host cell comprising at least one copy of a DNA sequence which encodes at least one functionally active Mortierella alpina fatty acid desaturase having an amino acid sequence as depicted in SEQ NO:

selected from the group consisting of SEQ ID NO;

2 and SEQ ID NO;

4, wherein said cell or parent of said cell was transformed with a vector comprising said DNA sequence, and wherein said DNA sequence is operably associated with an expression control sequence.

12. The recombinant host cell of claim 11, wherein said host cell is a plant cell.

16. The host cell according to claim 12, wherein said host cell is a plant cell enriched in 18:

1 or 18;

2 fatty acids after said cell has been transformed with said vector.

17. The host cell according to claim 16, wherein said plant cell is from a plant selected from the group consisting of Brassica, soybean, safflower, and sunflower.

18. The host cell according to claim 12, wherein said expression control sequence is endogenous to said plant cell.

22. The host cell according to claim 12, wherein said plant cell which is enriched in 18:

3 or 18;

4 fatty acids after said cell has been transformed with said vector.

14. An isolated or purified polynucleotide comprising the nucleotide sequence depicted in SEQ ID NO:

1 or SEQ ID NO;

3.

15. An isolated or purified nucleotide sequence encoding the polypeptide depicted in SEQ ID NO:

2 or SEQ ID NO;

4.

19. A method for obtaining altered long chain polyunsaturated fatty acid biosynthesis in a plant, said method comprising growing a plant having cells which contain one or more transgenes encoding a transgene expression product having an amino acid sequence depicted in a SEQ ID NO:

selected from the group consisting of SEQ ID NO;

4 and SEQ ID NO;

2 which desaturates a fatty acid molecule at carbon 6 or carbon 12 from the carboxyl end of said fatty acid molecule, wherein said one or more transgenes is operably associated with an expression control sequence, under conditions whereby said one or more transgenes is expressed, whereby long chain polyunsaturated fatty acid biosynthesis in said cells is altered.

20. The method according to claim 19, wherein said one or more transgenes is derived from Mortierella alpina.

21. The method according to claim 19, wherein said long chain polyunsaturated fatty acid is arachidonic acid.