Method for the production of polyunsaturated fatty acids

• US 20060168687A1
• Filed: 01/29/2004
• Published: 07/27/2006
• Est. Priority Date: 02/27/2003
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Renz, Andreas, Zank, Thorsten, Abbadi, Amine, Heinz, Ernst, Domergue, Frederic
• Granted Patent: US 7,537,920 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 9/1029 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 producing polyunsaturated fatty acids in an organism by introducing nucleic acids into said organism which code for polypeptides having acyl-CoA:lysophospholipid acyltransferase activity. Advantageously, these nucleic acid sequences may, if appropriate together with further nucleic acid sequences coding for biosynthesis polypeptides of the fatty acid or lipid metabolism, be expressed in the transgenic organism. The invention furthermore relates to the nucleic acid sequences, to nucleic acid constructs comprising the nucleic acid sequences of the invention, to vectors comprising said nucleic acid sequences and/or said nucleic acid constructs and to transgenic organisms comprising the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors. A further part of the invention relates to oils, lipids and/or fatty acids produced by the process of the invention and to their use.

Claims

1. An isolated nucleic acid sequence coding for a polypeptide having acyl-CoA:

lysophospholipid-acyltransferase activity, wherein the acyl-CoA;

lysophospholipid acyltransferase encoded by said nucleic acid sequence specifically converts C₁₆, C₁₈-, C₂₀- or C₂₂-fatty acids having at least one double bond in the fatty acid molecule.

2. The isolated nucleic acid sequence according to claim 1, selected from the group consisting of:

a) a nucleic acid sequence having the sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7,

b) nucleic acid sequences which can be derived from the coding sequence comprised in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7 as a result of the degenerated genetic code,

c) derivatives of the nucleic acid sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7 which code for polypeptides having the amino acid sequence depicted in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and are at least 40% homologous at the amino acid level to SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and have an acyl-CoA;

lysophospholipid-acyltransferase activity.

3. The isolated nucleic acid sequence according to claim 1, which is derived from a eukaryote.

4. An amino acid sequence encoded by an isolated nucleic acid sequence according to claim 1.

5. A gene construct comprising an isolated nucleic acid according to claim 1, wherein said nucleic acid is functionally linked to one or more regulatory signals.

6. The gene construct according to claim 5, wherein the nucleic acid construct comprises additional biosynthetic genes of the 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 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).

7. The gene construct according to claim 5, wherein the nucleic acid construct comprises additional biosynthetic genes of the fatty acid or lipid metabolism, selected from the group consisting of Δ

4-desaturase, Δ

5-desaturase, Δ

6-desaturase, Δ

8-desaturase, Δ

9-desaturase, Δ

12-desaturase, Δ

5-elongase, Δ

6-elongase or Δ

9-elongase.

8. A vector comprising a nucleic acid according to claim 1.

9. A transgenic nonhuman organism comprising at least one nucleic acid according to claim 1.

10. The transgenic nonhuman organism according to claim 9, which organism is a microorganism, a nonhuman animal or a plant.

11. The transgenic nonhuman organism according to claim 9, which organism is a plant.

26. A vector comprising the gene construct according to claim 5.

27. A transgenic nonhuman organism comprising the gene construct according to claim 5.

28. A transgenic nonhuman organism comprising the vector according to claim 8.

12. A process for producing polyunsaturated fatty acids in an organism, wherein said process comprises the following steps:

a) introducing into the organism at least one nucleic acid sequence having the sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7, which sequence codes for a polypeptide having an acyl-CoA;

lysophospholipid-acyltransferase activity;

or

b) introducing into said organism at least one nucleic acid sequence which can be derived, as a result of the degenerated genetic code, from the coding sequence comprised in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7, or

c) introducing into said organism at least one derivative of the nucleic acid sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7, which code for polypeptides having the amino acid sequence depicted in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and which are at least 40% homologous at the amino acid level to SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and have an equivalent acyl-CoA;

lysophospholipid-acyltransferase activity, and

d) culturing and harvesting said organism.

13. A process for producing polyunsaturated fatty acids according to claim 12, wherein, in addition to the nucleic acid sequences mentioned under (a) to (c), further nucleic acid sequences have been introduced into said organism, which code for polypeptides of the 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 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).

14. The process for producing polyunsaturated fatty acids according to claim 12, wherein, in addition to the nucleic acid sequences mentioned under (a) to (c), further nucleic acid sequences have been introduced into the organism, which code for polypeptides selected from the group consisting of Δ

4-desaturase, Δ

5-desaturase, Δ

6-desaturase, A8-desaturase, Δ

9-desaturase, Δ

12-desaturase, Δ

5-elongase, Δ

6-elongase or Δ

9-elongase activity.

15. The process for producing polyunsaturated fatty acids according to claim 12, wherein C₁₆-, C₁₈-, C₂₀- or C₂₂-fatty acids are used as substrate of the acyl-CoA:

lysophospholipid acyltransferases.

16. The process for producing polyunsaturated fatty acids according to claim 12, wherein the polyunsaturated fatty acids are isolated from the organism in the form of an oil, lipid or a free fatty acid.

17. The process for producing polyunsaturated fatty acids according to claim 12, wherein the polyunsaturated fatty acid produced in said process is a C₁₈-, C₂₀- or C₂₂-fatty acids having at least two double bonds in the molecule.

18. The process for producing polyunsaturated fatty acids according to claim 12, wherein a polyunsaturated fatty acid selected from the group consisting of dihomo-γ

-linolenic acid, arachidonic acid, eisosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid is produced in said process.

19. The process for producing polyunsaturated fatty acids according to claim 12, wherein the organism is a microorganism, a nonhuman animal or a plant.

20. The process for producing polyunsaturated fatty acids according to claim 12, wherein the organism is a transgenic plant.

21. The process for producing polyunsaturated fatty acids according to claim 20, wherein the transgenic plant is an oil crop plant.

22. An oil, a lipid or a fatty acid or a fraction thereof, prepared by the process according to claim 12.

23. An oil composition, a lipid composition or a fatty acid composition which comprises polyunsaturated fatty acids prepared by a process according to claim 12 and is derived from transgenic plants.

25. A method of making feed, foodstuffs, cosmetics or pharmaceuticals comprising incorporating the fatty acids prepared according to claim 12 in said feed, foodstuffs, cosmetics or pharmaceuticals.

29. A method of making feed, foodstuffs, cosmetics or pharmaceuticals comprising incorporating the oil composition, lipid composition or fatty acid composition according to claim 23 in said feed, foodstuffs, cosmetics or pharmaceuticals.

24. (canceled)