Novel delta-12 desaturase and methods of using it for synthesis of polyunsaturated fatty acids

• US 20030172398A1
• Filed: 12/20/2000
• Published: 09/11/2003
• Est. Priority Date: 12/21/1999
• Status: Abandoned Application

• Current Assignee: Washington State University
• Original Assignee: Washington State University
• Inventors: Browse, John A.
• Application Number:
  US09/748,516
• Publication Number:
  US 20030172398A1
• Time in Patent Office: Days
• US Class Current: 800/281
• 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

Desaturase enzymes, and especially animal Δ¹²-desaturases, and the use of such enzymes to alter fatty acid saturation, especially fatty acid saturation in oilseeds, are disclosed. Also disclosed are nucleic acid sequences encoding animal Δ¹²-desaturase enzymes.

Claims

1. A purified desaturase protein, comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence as shown in SEQ. ID NO. 2;

(b) an amino acid sequence that differs from that specified in (a) by one or more conservative amino acid substitutions;

(c) an amino acid sequences having at least 60% sequence identity to the sequences specified in (a) or (b); and

(d) fragments of (a), (b), or (c),

wherein the purified protein has desaturase activity.

2. The desaturase protein of claim 1, wherein the desaturase protein is a Δ

¹²-desaturase.

3. An isolated nucleic acid molecule, encoding a protein according to claim 1.

4. The isolated nucleic acid molecule of claim 3, comprising a sequence as shown in SEQ ID NO:

1.

5. A recombinant nucleic acid molecule, comprising a control sequence operably linked to the nucleic acid molecule of claim 3.

6. A cell, transformed with the recombinant nucleic acid molecule of claim 5.

7. The cell of claim 6, wherein the cell is a plant cell.

8. A transgenic organism, comprising a recombinant nucleic acid molecule according to claim 5, wherein the transgenic organism is selected from the group consisting of plants, bacteria, insects, fungi, and animals.

9. The transgenic organism of claim 8, wherein the organism is a plant.

10. The transgenic organism of claim 9, wherein the plant is selected from the group consisting of oil palm, sunflower, safflower, rapeseed, canola, soy, peanut, cotton, corn, rice, Arabidopsis, mustard, wheat, barley, potato, tomato, yam, apple, and pear plants.

29. A method for creating a double bond between two carbons in a fatty acid, comprising:

contacting a fatty acid with a purified desaturase protein according to claim 1;

and

allowing the desaturase protein to form a double-bond between two carbons in the fatty acid.

30. The method of claim 29, wherein the desaturase protein is expressed in a transgenic organism and the double-bond formation occurs in vivo.

31. The method of claim 30, wherein the desaturase is expressed in an organism selected from the group consisting of eukaryotes and prokaryotes.

32. The method of claim 29, wherein the desaturase is expressed in vitro, and the double-bond formation occurs in vitro.

11. An isolated nucleic acid molecule that:

(a) hybridizes under low-stringency conditions with a nucleic acid probe, the probe comprising a sequence as shown in SEQ ID NO;

1, and fragments thereof, and

(b) encodes a protein having desaturase activity.

12. A desaturase protein encoded by the nucleic acid molecule of claim 11.

13. The desaturase protein of claim 12, wherein the desaturase protein is a Δ

¹²-desaturase.

14. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 11.

15. A cell transformed with the recombinant nucleic acid molecule of claim 14.

16. The cell of claim 15, wherein the cell is a plant cell.

17. A transgenic organism, comprising the transformed cell of claim 15, wherein the transgenic organism is selected from the group consisting of plants, bacteria, insects, fungi, and animals.

18. The transgenic organism of claim 17, that is a plant.

19. The transgenic organism of claim 18, wherein the plant is selected from the group consisting of oil palm, sunflower, safflower, rapeseed, canola, soy, peanut, cotton, corn, rice, Arabidopsis, mustard, wheat, barley, potato, tomato, yam, apple, and pear plants.

20. A specific binding agent that binds to the desaturase protein of claim 12.

21. An isolated nucleic acid molecule that:

(a) has at least 60% sequence identity with a nucleic acid sequence as shown in SEQ ID NO;

1; and

(b) encodes a protein having desaturase activity.

22. A method for identifying a nucleic acid sequence, comprising:

(a) hybridizing the nucleic acid sequence to at least 10 contiguous nucleotides of a sequence as shown in SEQ ID NO;

1; and

(b) identifying the nucleic acid sequence as corresponding to a nucleic acid encoding a desaturase.

23. The method of claim 22, wherein hybridizing the nucleic acid sequence is performed under low-stringency conditions.

24. A nucleic acid molecule identified by the method of claim 22.

25. A desaturase encoded by the nucleic acid molecule of claim 24.

26. A specific binding agent that binds the desaturase of claim 25.

27. The method of claim 22, wherein step (a) occurs in a PCR reaction.

28. The method of claim 22, wherein step (a) occurs during a library screening.