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

• US 7,402,735 B2
• Filed: 10/26/2004
• Issued: 07/22/2008
• Est. Priority Date: 12/07/1998
• Status: Expired due to Fees

• Current Assignee: Washington State University
• Original Assignee: Washington State University
• Inventors: Browse, John A., Watts, Jennifer L., Wallis, James G.
• Primary Examiner(s): McElwain; Elizabeth F
• Application Number:
  US10/975,692
• Publication Number:
  US 20060090221A1
• Time in Patent Office: 1,365 Days
• Field of Search: None
• US Class Current: 800/298
• 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 transgenic organism, comprising a cell comprising a first recombinant nucleic acid molecule comprising a promoter sequence operably linked to a first heterologous 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, wherein the transgenic organism is a plant or a yeast.

3. The transgenic organism of claim 1, wherein the first recombinant nucleic acid molecule is contained in a first transformation vector.

4. The transgenic organism according to claim 1, wherein the heterologous nucleic acid molecule hybridizes under very high-stringency conditions with the nucleic acid probe.

5. The transgenic organism according to claim 1, wherein the cell further comprises a second recombinant nucleic acid molecule that comprises a promoter sequence operably linked to a second heterologous nucleic acid molecule, wherein the second heterologous nucleic acid molecule encodes a protein having Δ

⁵ fatty acid desaturase activity and is 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).

6. The transgenic organism according to claim 5, wherein the transgenic organism is a plant.

7. The transgenic organism according to claim 5, wherein the second recombinant nucleic acid molecule is contained in a transformation vector.

8. The transgenic organism according to claim 5, wherein the first recombinant nucleic acid molecule and the second nucleic acid molecule are contained in a single transformation vector.

2. A transgenic plant, comprising a cell comprising a first recombinant nucleic acid molecule comprising a promoter sequence operably linked to a first heterologous 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.

18. The transgenic organism of claim 2, wherein the first recombinant nucleic acid molecule is contained in a first transformation vector.

19. The transgenic organism according to claim 2, wherein the heterologous nucleic acid molecule hybridizes under very high-stringency conditions with the nucleic acid probe.

9. A transgenic organism, comprising a cell comprising a first recombinant nucleic acid molecule that comprises a promoter sequence operably linked to a first heterologous 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, wherein the transgenic organism is a plant or a yeast.

11. The transgenic organism of claim 9, wherein the cell further comprises a second recombinant nucleic acid molecule that comprises a promoter sequence operably linked to a second heterologous nucleic acid molecule, wherein the second heterologous nucleic acid molecule encodes a protein having Δ

⁵ fatty acid desaturase activity and is 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).

12. The transgenic organism according to claim 11, wherein the transgenic organism is a plant.

10. A transgenic plant, comprising a cell comprising a first recombinant nucleic acid molecule that comprises a promoter sequence operably linked to a first heterologous 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.

13. A transgenic organism, comprising a first recombinant nucleic acid molecule comprising a promoter sequence operably linked to a first heterologous nucleic acid molecule that encodes a protein having Δ

⁸ fatty acid desaturase activity, wherein the protein comprises 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 sequence having at least 95% sequence identity to the sequences specified in (a) or (b),

wherein the transgenic organism is a plant or a yeast.

15. The transgenic organism according to claim 13, wherein the first heterologous nucleic acid molecule comprises the sequence shown in SEQ ID NO:

3.

16. The transgenic organism according to claim 13, further comprising a second recombinant nucleic acid molecule that comprises a promoter sequence operably linked to a second heterologous nucleic acid molecule, wherein the second heterologous nucleic acid molecule encodes a protein having Δ

⁵ fatty acid desaturase activity and is 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).

17. A transgenic organism according to claim 16, wherein the transgenic organism is a plant.

14. A transgenic plant, comprising a first recombinant nucleic acid molecule comprising a promoter sequence operably linked to a first heterologous nucleic acid molecule that encodes a protein having Δ

⁸ fatty acid desaturase activity, wherein the protein comprises 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 sequence having at least 95% sequence identity to the sequences specified in (a) or (b).

20. The transgenic organism according to claim 14, wherein the first heterologous nucleic acid molecule comprises the sequence shown in SEQ ID NO:

3.