Method for Producing Polyunsaturated C20 and C22 Fatty Acids with at Least Four Double Bonds in Transgenic Plants

• US 20090158462A1
• Filed: 03/21/2006
• Published: 06/18/2009
• Est. Priority Date: 03/22/2005
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Cirpus, Petra, Bauer, Jörg
• Granted Patent: US 8,049,064 B2
• 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: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• 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 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• 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 a process for the production of polyunsaturated fatty acids in transgenic plants, by introducing, into the plant, the nucleic acids which code for polypeptides with Δ6-desaturase, Δ6-elongase, a Δ5-desaturase, Δ5-elongase, Δ4-desaturase, Δ12-desaturase and/or ω3-desaturase activity. These desaturases and elongases are advantageously derived from Phytophthora sojae.

The invention furthermore relates to the nucleic acid sequences, nucleic acid constructs, vectors and organisms comprising the nucleic acid sequences according to the invention, vectors comprising the nucleic acid sequences and/or the nucleic acid constructs and to transgenic plants comprise the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors.

A further part of the invention relates to fatty acid compositions produced by the process according to the invention and to their use.

Claims

1. A process for the production of polyunsaturated C₂₀- or C₂₂-fatty acids with at least four double bonds in transgenic plants with a content of at least 15% by weight based on the total triglyceride content of the transgenic plants comprising:

a) introducing, into the transgenic plant, a nucleic acid construct which comprises nucleic acid sequences which codes for a Δ

6-desaturase, a Δ

6-elongase and a Δ

5-desaturase, or

b) introducing, into the transgenic plant, a nucleic acid construct which comprises nucleic acid sequences which codes for a Δ

6-desaturase, a Δ

6-elongase, a Δ

5-desaturase, a Δ

12-desaturase and ω

3-desaturase, or

c) introducing, into the transgenic plant, a nucleic acid construct which comprises nucleic acid sequences which codes for a Δ

6-desaturase, a Δ

6-elongase, a Δ

5-desaturase, a Δ

5-elongase and Δ

4-desaturase, or

d) introducing, into the transgenic plant, a nucleic acid construct which comprises nucleic acid sequences which codes for a Δ

6-desaturase, a Δ

6-elongase, a Δ

5-desaturase, a Δ

5-elongase, Δ

4-desaturase, a Δ

12-desaturase and ω

3-desaturase, and

e) obtaining the oils or lipids from the plants.

2. The process according to claim 1, wherein the nucleic acid construct comprises a nucleic acid sequence selected from the group consisting of:

SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

13, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

23, and SEQ ID NO;

25.

3. The process according to claim 1, wherein the polyunsaturated C₂₀- or C₂₂-fatty acids with at least four double bonds is arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid.

4. The process according to claim 1, wherein the polyunsaturated C₂₀- or C₂₂-fatty acids with at least four double bonds is arachidonic acid.

5. The process according to claim 1, wherein the polyunsaturated C₂₀- or C₂₂-fatty acids with at least four double bonds is eicosapentaenoic acid or docosahexaenoic acid.

6. The process according claim 3, wherein the arachidonic acid or eicosapentaenoic acid is present in the transgenic plant with a content of at least 15% by weight based on the total triglyceride content.

7. The process according to claim 3, wherein the docosahexaenoic acid with a content of at least 4% by weight based on the total triglyceride content is present in the transgenic plant.

8. The process according to claim 1, wherein a polyunsaturated fatty acid selected from the group consisting of C22:

4<sup>Δ

7,10,13,16</sup>-, C22;

5<sup>Δ

4,7,10,13,16</sup>- or C22;

5<sup>Δ

7,10,13,16,19</sup>-fatty acid is present in the triglycerides in an amount of less than 0.5% by weight based on the total fatty acid content of the triglycerides.

9. The process according to claim 1, wherein the transgenic plant is an oil crop plant or useful plant.

10. The process according to claim 1, wherein the transgenic plant is selected from the group consisting of peanut, oilseed rape, canola, sunflower, safflower, Carthamus tinctoria, poppy, mustard, hemp, castor-oil plant, olive, sesame, Calendula, Punica, evening primrose, verbascum, thistle, hazelnut, almond, macadamia, avocado, bay, pumpkin, linseed, soya, pistachios, borage, oil palm, coconut, walnut, maize, wheat, rye, oats, triticale, rice, barley, cotton, cassava or pepper.

11. The process according to claim 1, wherein the polyunsaturated C₂₀- or C₂₂-fatty acids with at least four double bonds are isolated from the oils or lipids in the form of the free fatty acids.

12. A process for the preparation of fatty acid compositions comprising mixing oils, lipids or free fatty acids produced by the process according to claim 1 with animal, microbial or vegetable oils, lipids or fatty acids.

13. A process for preparing feedstuffs, foodstuffs, cosmetics or pharmaceuticals comprising mixing the oil, lipids or fatty acids produced by the process according to claim 1 with animal, microbial or vegetable oils, lipids or fatty acids in feedstuffs, foodstuffs, cosmetics or pharmaceuticals.

14. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

a) the nucleotide sequence shown in SEQ ID NO;

1, 3, 5, 7, 9, 23 or 25,

b) a nucleotide sequence which codes for a protein with the polypeptide sequence shown in SEQ ID NO;

2, 4, 6, 8, 10, 24, or 26,

c) a derivative of the nucleotide sequence shown in SEQ ID NO;

1 which codes for a polypeptide having at least 70% identity at the amino acid level with SEQ ID NO;

2 and a Δ

6-desaturase activity,

d) a derivative of the nucleotide sequence shown in SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7 which codes for a polypeptide having at least 70% identity at the amino acid level with SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and which has a Δ

6-elongase activity,

e) a derivative of the nucleotide sequence shown in SEQ ID NO;

9 which codes for a polypeptide having at least 40% identity at the amino acid level with SEQ ID NO;

10 and which has a Δ

5-desaturase activity,

f) a derivative of the nucleotide sequence shown in SEQ ID NO;

23 which codes for a polypeptide with at least 40% identity at the amino acid level with SEQ ID NO;

24 and which has a ω

3-desaturase activity, and

g) a derivative of the nucleotide sequence shown in SEQ ID NO;

25 which codes for a polypeptide with at least 40% identity at the amino acid level with SEQ ID NO;

26 and which has a Δ

12-desaturase activity.

19. The isolated nucleic acid according to claim 14, where the nucleotide sequence is derived from a microorganism or a plant.

20. A protein which is encoded by the isolated nucleic acid according to claim 14.

21. A gene construct comprising the isolated nucleic acid according to claim 14, wherein the nucleic acid is linked operably with one or more regulatory signals.

22. The gene construct according to claim 21, wherein the nucleic acid construct comprises additional biosynthesis genes of the fatty acid or lipid metabolism selected from the group acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:

lysophospholipid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s).

23. The gene construct as claimed in claim 21, wherein the nucleic acid construct comprises additional biosynthesis genes of the fatty acid or lipid metabolism selected from the group of the Δ

4-desaturases, Δ

5-desaturases, Δ

6-desaturases, Δ

8-desaturases, Δ

12-desaturases or Δ

9-elongases.

24. A vector comprising the nucleic acid according to claim 14 or a gene construct comprising the nucleic acid.

25. A transgenic nonhuman organism, comprising at least one nucleic acid according to claim 14, a gene construct comprising the nucleic acid, or a vector comprising the nucleic acid.

26. The transgenic nonhuman organism according to claim 25, which organism is a plant.

15-18. -18. (canceled)