Method for producing multiple unsaturated fatty acids in plants

• US 20070028326A1
• Filed: 04/25/2003
• Published: 02/01/2007
• Est. Priority Date: 04/29/2002
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Cirpus, Petra, Lerchl, Jens, Renz, Andreas, Kuijpers, Anne-Marie
• Granted Patent: US 7,893,320 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: 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6481 Phosphoglycerides phosphoglycerides having carboxylic acids with less than seven carbon atoms C12P7/62

Abstract

The present invention relates to a method for the production of fatty acid esters which comprise unsaturated fatty acids with at least three double bonds, and to free unsaturated fatty acids with a content of at least 1% by weight based on the total fatty acids present in the plants, by expressing at least one nucleic acid sequence which encodes a polypeptide with Δ6-desaturase activity and at least one nucleic acid sequence which encodes a polypeptide with Δ6-elongase activity. Advantageously, these nucleic acid sequences can, if appropriate, be expressed in the transgenic plant together with a third nucleic acid sequence which encodes a polypeptide with Δ5-desaturase activity. The invention furthermore relates to the use of defined nucleic acid sequences which encode polypeptides with a Δ6-desaturase activity, Δ6-elongase activity or Δ5-desaturase activity selected from a group of nucleic acid sequences, and/or to the use of nucleic acid constructs comprising the abovementioned nucleic acid sequences.

Claims

1. A process for the production of compounds in transgenic plants, wherein said compounds are comprised of general Formula I:

2. The process of claim 1, wherein R² and R³ independently of one another are C₁₀-C₂₂-alkylcarbonyl-.

3. The process of claim 1, wherein R² and R³ independently of one another are C₁₆-, C₁₈-, C₂₀- or C₂₂-alkylcarbonyl-.

4. The process of claim 1, wherein R² and R³ independently of one another are unsaturated C₁₆-, C₁₈-, C₂₀- or C₂₂-alkylcarbonyl- with one, two, three, four or five double bonds.

5. The process of claim 1, wherein the plant is an oil crop.

6. The process of claim 1, wherein the plant is selected from the group consisting of soya, peanut, oilseed rape, canola, linseed, evening primrose, verbascum, thistle, hazelnut, almond, macadamia, avocado, bay, wild roses, pumpkin/squash, pistachios, sesame, sunflower, safflower, borage, maize, poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica, oil palm, walnut and coconut.

7. The process of claim 1, wherein the compounds of general Formula I are obtained from the transgenic plants by pressing or extraction, and said compounds are in the form of oils, fats, lipids or free fatty acids.

8. The process of claim 7, wherein the oils, fats, lipids or free fatty acids are refined.

9. The process of claim 1, wherein saturated or unsaturated fatty acids present in the compounds are liberated.

10. The process of claim 9, wherein the saturated or unsaturated fatty acids are liberated by alkaline hydrolysis or enzymatic cleavage.

11. The process of claim 1, wherein the compounds comprise at least 5% by weight, of the total fatty acid content of the transgenic plants.

12. The process of claim 1, wherein the nucleic acid sequence that encodes the polypeptide with Δ

6-desaturase activity, Δ

6-elongase activity or Δ

5-desaturase activity is selected from the group consisting of;

a) a nucleic acid sequence that contains the sequence 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, SEQ ID NO;

25, SEQ ID NO;

27, SEQ ID NO;

29 or SEQ ID NO;

31,

b) nucleic acid sequences which, owing to the degeneracy of the genetic code, are obtained by back translation of an amino acid sequence contains the sequence of SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

26, SEQ ID NO;

28, SEQ ID NO;

30 or SEQ ID NO;

32,

c) derivatives of a nucleic acid sequence that contains the sequence 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, SEQ ID NO;

25, SEQ ID NO;

27, SEQ ID NO;

29 or SEQ ID NO;

31, which encode a derivative polypeptide that contains the amino acid sequence of SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

26, SEQ ID NO;

28, SEQ ID NO;

30 or SEQ ID NO;

32, and which has at least 50% homology at the amino acid level, wherein enzymatic activity of the derivative polypeptide is not substantially reduced as compared to said polypeptide.

13. The process of claim 1, wherein one or more of the first, second, and optionally third nucleic acid sequences are linked with one or more regulatory signals in a nucleic acid construct.

14. The process of claim 13, wherein the nucleic acid construct comprises additional biosynthetic genes of fatty acid or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(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, allene oxide synthases, hydroperoxide lyases, fatty acid elongase(s) and combinations thereof.

15. The process of claim 1, wherein the first, second and optionally third nucleic acid sequences are stably integrated in the plant.

16. The process of claim 1, wherein the plant is selected from the group consisting of plant cells, plant tissues, plant organs, plant leaves, plant roots, plant stems, intact plants, plant tubors, plant seeds, and cellular parts of any of the preceding.

17. The process of claim 7, wherein the pressing or the extraction is performed without supplying heat.

18. The process of claim 12, wherein two or more of the first, second and third nucleic acid sequences are selected from said group.

19. The process of claim 12, wherein the first, second and third nucleic acid sequences are selected from said group.

20. The process of claim 13, wherein the first, second and third nucleic acid sequences are linked to with one or more regulatory signals in said nucleic acid construct.