Method of producing polyunsaturated fatty acids, novel biosynthesis genes, and novel plant expression constructs

• US 20040053379A1
• Filed: 07/02/2003
• Published: 03/18/2004
• Est. Priority Date: 01/19/2001
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Domergue, Frederic, Lerchl, Jens, Renz, Andreas, Heinz, Ernst, Zahringer, Ulrich
• Granted Patent: US 7,615,679 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• 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 7/6434 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 method for the production of unsaturated fatty acids with at least two double bonds and/or a method for the production of triglycerides with an increased content of polyunsaturated fatty acids with at least two double bonds. The invention furthermore relates to the advantageous use of the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 18 in the method and for generating a transgenic organism, preferably a transgenic plant or a transgenic microorganism, with an increased content of fatty acids, oils or lipids with unsaturated C₁₈-, C₂₀-, or C₂₂-fatty acids.

The invention furthermore relates to novel desaturases with the [lacuna] in the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 11 or its homologs, derivatives and analogs, and to gene constructs encompassing these genes or their homologs, derivatives or analogs, and to their use alone or in combination with biosynthesis genes of polyunsaturated fatty acids as shown advantageously in SEQ ID NO: 7 and SEQ ID NO: 9.

In addition, the invention relates to isolated nucleic acid sequences; expression cassettes comprising the nucleic acid sequences, vectors and transgenic organisms comprising at least one nucleic acid sequence or one expression cassette. In addition, the invention relates to unsaturated fatty acids with at least two double bonds and to triglycerides with an increased content of unsaturated fatty acids with at least two double bonds, and to their use.

Moreover, the invention relates to multiexpression cassettes for seed-specific expression, and to vectors or organisms which encompass a desaturase gene alone or in combination with further desaturases with the sequence SEQ ID NO:7 and/or elongase genes with the sequence SEQ ID NO: 9 or its homologs, derivatives or analogs using said expression cassettes.

Claims

1. A method for the production of fatty acid esters with an increased content of polyunsaturated fatty acids with at least two double bonds, which comprises introducing, into a fatty-acid-ester-producing organism, at least one nucleic acid sequence selected from the group consisting of

a) a nucleic acid sequence with the sequence shown in SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

11,

b) nucleic acid sequences which, owing to the degeneracy of the genetic code, are obtained by backtranslating the amino acid sequences shown in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

12,

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

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

11, which encode polypeptides with the amino acid sequences shown in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

12 and have at least 50% homology at the amino acid level, without essentially reducing the enzymatic action of the polypeptides,

growing the organism, and isolating the fatty acid esters present in the organism.

2. A method as claimed in claim 1, wherein the fatty acid esters produced by the method comprise polyunsaturated C₁₈-, C₂₀- or C₂₂-fatty acid molecules with at least two double bonds in the fatty acid ester.

3. A method as claimed in claim 1 or 2, wherein the C₁₈-, C₂₀- or C₂₂-fatty acid molecules are isolated from the organism in the form of an oil or lipid.

4. A method as claimed in any of claims 1 to 3, wherein the organism is a microorganism, an animal or a plant.

5. A method as claimed in any of claims 1 to 4, wherein the organism is a transgenic plant.

6. A method as claimed in any of claims 1 to 5, wherein the fatty acid esters contain C₁₈-, C₂₀- or C₂₂-fatty acids with three, four or five double bonds in the fatty acid ester.

7. A method as claimed in any of claims 1 to 6, wherein the polyunsaturated fatty acids contained in the fatty acid esters are liberated.

21. An oil, lipid or fatty acid, or a fraction thereof, produced by the method as claimed in any of claims 1 to 7.

22. An oil, lipid or fatty acid composition, comprising an oil, lipid or fatty acid, or a fraction thereof, as claimed in claim 21.

23. The use of an oil, lipid or fatty acid, or a fraction thereof, as claimed in claim 21 or of an oil, lipid or fatty acid composition as claimed in claim 22 in feed, foodstuffs, cosmetics or pharmaceuticals.

26. A kit as claimed in claim 25 comprising an oil, lipid or fatty acid, or a fraction thereof, as claimed in claim 21, or an oil, lipid or fatty acid composition as claimed in claim 22.

8. An isolated nucleic acid sequence encoding a polypeptide with desaturase activity, selected from the group consisting of:

a) a nucleic acid sequence with the sequence shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

11,

b) nucleic acid sequences which, owing to the degeneracy of the genetic code, are obtained by backtranslating the amino acid sequences shown in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

12,

c) derivatives of the nucleic acid sequences shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

11, which encode polypeptides with the amino acid sequences shown in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

12 and have at least 50% homology at the amino acid level, without essentially reducing the enzymatic action of the polypeptides.

9. An amino acid sequence encoded by a nucleic acid sequence as claimed in claim 8.

10. An amino acid sequence as claimed in claim 9 encoded by the sequence shown in SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

11.

11. A nucleic acid construct comprising a nucleic acid sequence as claimed in claim 8, wherein the nucleic acid sequence is linked to one or more regulatory signals.

12. A nucleic acid construct as claimed in claim 11, wherein additional biosynthesis genes of the fatty acid or lipid metabolism are present in the nucleic acid construct.

13. A nucleic acid construct as claimed in claim 11 or 12, wherein the biosynthesis gene of the fatty acid or lipid metabolism which is present in the nucleic acid construct is a gene 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, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s).

14. A vector comprising a nucleic acid sequence as claimed in claim 8 or a nucleic acid construct as claimed in claim 11.

15. An organism comprising at least one nucleic acid sequence as claimed in claim 8, at least one nucleic acid construct as claimed in claim 11 or at least one vector as claimed in claim 14.

16. An organism as claimed in claim 15, wherein the organism is a plant, a microorganism or an animal.

17. A transgenic plant comprising a functional or nonfunctional nucleic acid sequence as claimed in claim 8, a functional or nonfunctional nucleic acid construct as claimed in claim 11 or a functional or nonfunctional vector as claimed in claim 14.

18. The use of a nucleic acid sequence as claimed in claim 8 or of a nucleic acid construct as claimed in claim 11 for generating transgenic plants.

19. An antibody which specifically binds to a polypeptide encoded by one of the nucleic acids as claimed in claim 8.

20. An antisense nucleic acid molecule comprising the complementary sequence of the nucleic acid as claimed in claim 8.

25. A kit encompassing the nucleic acid as claimed in claim 8, the nucleic acid construct as claimed in any of claims 11 to 13, the antibody as claimed in claim 19, the antisense nucleic acid molecule as claimed in claim 20, an antagonist or agonist identified as claimed in claim 24, the composition as claimed in claim 27, the amino acid sequence as claimed in claim 9.

27. A composition comprising the antibody as claimed in claim 19, the antisense nucleic acid construct as claimed in claim 20 or an antagonist or agonist identified as claimed in claim 24.

24. A method of identifying an antagonist or agonist of desaturases, comprising

a) contacting the cells which express the polypeptide of the present invention with a candidate substance;

b) testing the desaturate activity;

c) comparing the desaturase activity with a standard activity in the absence of the candidate material, where an increase in the desaturase activity beyond the standard indicates that the candidate material is an agonist and a reduction in the desaturase activity indicates that the candidate material is an antagonist.