Desaturases and methods of using them for synthesis of polyunsaturated fatty acids

• US 6,825,017 B1
• Filed: 08/17/2001
• Issued: 11/30/2004
• Est. Priority Date: 12/07/1998
• Status: Expired due to Term

• Current Assignee: Washington State University
• Original Assignee: Washington State University
• Inventors: Browse, John A., Watts, Jennifer L., Wallis, James G.
• Primary Examiner(s): Rao, Manjunath N.
• Application Number:
  US09/857,583
• Time in Patent Office: 1,201 Days
• Field of Search: 435/4, 435/6, 435/69.1, 435/183-234, 435/252.34, 435/320.1, 435/325, 435/252.3, 536/23-- 2, 536/23.5, 536/23.6
• US Class Current: 435/190
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• 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 amino acid and nucleic acid sequences of a Δ⁵-desaturase enzyme and a Δ⁸-desaturase enzyme are disclosed. The nucleic acid sequences can be used to design recombinant DNA constructs and vectors. These vectors can then be used to transform various organisms, including for example, plants and yeast. The transformed organisms will then produce polyunsaturated fatty acids. The amino acid sequences are useful for generating enzyme-specific antibodies that are useful for identifying the desaturases.

Claims

1. A purified protein having Δ

⁸ fatty acid desaturase activity, and comprising an amino acid sequence selected from the group consisting of;

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

4;

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

(c) an amino acid sequences having at least 95% sequence identity to the sequences specified in (a) or (b).

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

3. The isolated nucleic acid molecule of claim 2, comprising a sequence as shown in SEQ ID NO:

3.

4. A recombinant nucleic acid molecule, comprising a control sequence operably linked to the nucleic acid sequence of claim 2.

5. A cell transformed with the recombinant nucleic acid molecule of claim 4.

6. A cell transformed with the recombinant nucleic acid molecule of claim 4 and a nucleic acid molecule that encodes a protein having Δ

⁵ fatty acid desaturase activity, selected from the group consisting of;

(a) a nucleic acid molecule as shown in SEQ ID NO;

1; and

(b) a nucleic acid molecule that has at least 95% sequence identity to the nucleic acid molecule shown in (a).

7. The cell of claim 5, wherein the cell is a plant cell.

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

19. A cell transformed with the recombinant nucleic acid molecule of claim 18.

20. A cell transformed with the recombinant nucleic acid molecule of claim 18 and a nucleic acid molecule that encodes a protein having Δ

⁵ fatty acid desaturase activity, selected from the group consisting of;

(a) a nucleic acid molecule as shown in SEQ ID NO;

1; and

(b) a nucleic acid molecule that has at least 95% sequence identity to the nucleic acid molecule shown in (a).

21. The cell of claim 20, wherein the cell is a plant cell.

22. The cell of claim 21, wherein the cell is a plant cell.

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

26. The cell of claim 21, wherein the cell is a plant cell.

8. An isolated nucleic acid molecule that:

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

3; and

(b) encodes a protein having Δ

⁸ fatty acid desaturase activity.

9. An isolated or recombinant Δ

⁸ fatty acid desaturase encoded by the nucleic acid molecule of claim 8.

10. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 8.

11. A cell transformed with the recombinant nucleic acid molecule of claim 10.

13. The isolated nucleic acid molecule of claim 8, wherein the nucleic acid molecule hybridizes under very high-stringency conditions with said nucleic acid probe.

14. An isolated or recombinant Δ

⁸ fatty acid desaturase encoded by the nucleic acid molecule of claim 13.

15. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 13.

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

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

24. A cell transformed with the recombinant nucleic acid molecule of claim 10 and a nucleic acid molecule that encodes a protein having Δ

⁵ fatty acid desaturase activity, selected from the group consisting of;

(a) a nucleic acid molecule as shown in SEQ ID NO;

1; and

(b) a nucleic acid molecule that has at least 95% sequence identity to the nucleic acid molecule shown in (a).

25. The cell of claim 11, wherein the cell is a plant cell.

12. An isolated nucleic acid molecule that:

(a) has at least 95% sequence identity with a nucleic acid sequence as shown in SEQ ID NO;

3; and

(b) encodes a protein having Δ

⁸ fatty acid desaturase activity.