Novel elongase gene and method for producing multiple-unsaturated fatty acids

• US 20040111763A1
• Filed: 07/31/2002
• Published: 06/10/2004
• Est. Priority Date: 02/09/2000
• Status: Active Grant

• Current Assignee: BASF AG (BASF SE)
• Original Assignee: BASF AG (BASF SE)
• Inventors: Lerchl, Jens, Renz, Andreas, Zank, Thorsten, Heinz, Ernst, Zahringer, Ulrich
• Granted Patent: US 7,544,859 B2
• Time in Patent Office: Days
• US Class Current: 800/281
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The invention relates to a novel elongase gene with the sequences stated in sequence SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or their homologs, derivatives or analogs, to a gene construct comprising this gene or its homologs, derivatives and analogs, and to its use. The invention also relates to vectors or transgenic organisms comprising an elongase gene with the sequence SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or its homologs, derivatives and analogs. The invention furthermore relates to the use of the elongase gene sequences alone or in combination with further elongases and/or further fatty acid biosynthesis genes. The present invention relates to a novel elongase gene with the sequence SEQ ID NO:1 or its homologs, derivatives and analogs.

Furthermore, the invention relates to a process for the preparation of polyunsaturated fatty acids and to a process for introducing DNA into organisms which produce large amounts of oils and, in particular, oils with a high content of unsaturated fatty acids. Moreover, the invention relates to an oil and/or a fatty acid preparation with a higher content of polyunsaturated fatty acids with at least two double bonds and/or a triacylglycerol preparation with a higher content of polyunsaturated fatty acids with at least two double bonds.

Claims

1. An isolated nucleic acid derived from a plant or alga and encoding a polypeptide which elongates C₁₆, C₁₈- or C₂₀-fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms.

5. A gene construct encompassing an isolated nucleic acid as claimed in any of claims 1 to 4, wherein the nucleic acid is functionally linked to one or more regulatory signals.

6. A gene construct as claimed in claim 5, whose gene expression is enhanced by the regulatory signals.

7. A gene construct comprising a nucleic acid sequence as claimed in any of claims 1 to 4 or a gene construct as claimed in claim 5 or 6 and at least one further nucleic acid which encodes a fatty acid biosynthesis gene.

8. A gene construct as claimed in claim 7, wherein the nucleic acid which encodes a fatty acid biosynthesis gene is selected from the group consisting of Δ

19-, Δ

17-, Δ

15-, Δ

12-, Δ

9-, Δ

8-, Δ

6-, Δ

5-, Δ

4-desaturase, the various hydroxylases, Δ

12-acetylenase, acyl-ACP thioesterases, β

-ketoacyl-ACP synthases or β

-ketoacyl-ACP reductases.

9. An amino acid sequence which is encoded by the isolated nucleic acid sequence as claimed in any of claims 1 to 4 or the gene construct as claimed in any of claims 5 to 8.

10. A vector encompassing a nucleic acid as claimed in any of claims 1 to 4 or a gene construct as claimed in any of claims 5 to 8.

11. An organism encompassing at least one nucleic acid as claimed in any of claims 1 to 4, a gene construct as claimed in any of claims 5 to 8 or a vector as claimed in claim 10.

12. An organism as claimed in claim 11, wherein the organism is a microorganism, an animal or a plant.

13. An organism as claimed in claim 11 or 12, wherein the organism is a transgenic plant.

14. An antibody which specifically binds a polypeptide which is encoded by one of the nucleic acids as claimed in any of claims 1 to 4.

15. An antisense nucleic acid molecule which encompasses the complementary sequence of the nucleic acid as claimed in any of claims 1 to 4.

16. A process for the preparation of PUFAs, which comprises culturing an organism which encompasses a nucleic acid as claimed in claim 1, a gene construct as claimed in claim 6 or a vector as claimed in claim 10, encoding a polypeptide which elongates C₁₆-, C₁₈- or C₂₀-fatty acids with at least two double bonds in the fatty acid molecule by at least two carbon atoms under conditions under which PUFAs are formed in the organism.

17. A process as claimed in claim 16, wherein the PUFAs prepared by the process are C₁₈-, C₂₀- or C₂₂-fatty acid molecules with at least two double bonds in the fatty acid molecule.

18. A process as claimed in claim 16 or 17, wherein the C₁₈-, C₂₀- or C₂₂-fatty acid molecules are isolated from the organism in the form of an oil, lipid or a free fatty acid.

19. A process as claimed in any of claims 16 to 18, wherein the organism is a microorganism, an animal or a plant.

20. A process as claimed in any of claims 16 to 19, wherein the organism is a transgenic plant.

21. A process as claimed in any of claims 16 to 20, wherein the C₁₆-, C₁₈- or C₂₀-fatty acid is a fatty acid with three double bonds in the molecule.

22. An oil, lipid or fatty acid or a fraction thereof, prepared by the process as claimed in any of claims 16 to 21.

24. An oil, lipid or fatty acid composition as claimed in claim 23, wherein the PUFAs are derived from transgenic plants which encompass a nucleotide sequence as claimed in any of claims 1 to 4, the gene construct as claimed in any of claims 5 to 8, the antisense nucleic acid molecule as claimed in claim 15 or the vector as claimed in claim 10.

25. The use of the oil, lipid or fatty acid composition as claimed in any of claims 22 to 24 in feeding stuffs, foodstuffs, cosmetics or pharmaceuticals.

26. A composition, comprising the antibody as claimed in claim 14, the antisense nucleic acid construct as claimed in claim 15 or an antagonist or agonist identified as claimed in claim 28.

27. A kit, comprising the nucleic acid as claimed in any of claims 1 to 4, the gene construct as claimed in any of claims 5 to 8, the antisense nucleic acid molecule as claimed in claim 15, the antibody as claimed in claim 14, an antagonist or agonist identified as claimed in claim 28, the composition as claimed in claim 26, the amino acid sequence as claimed in claim 9 and/or oil, lipids and/or fatty acids or a fraction thereof as claimed in any of claims 22 to 25.

2. An isolated nucleic acid encompassing a nucleotide sequence encoding a polypeptide which elongates C₁₆-, C₁₈- or C₂₀-fatty acids with at least two double bonds in the fatty acid molecule, selected from the group consisting of

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

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7,

b) a nucleic acid sequence which, in accordance with the degeneracy of the genetic code, is derived from the sequence shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7,

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

1, SEQ ID NO;

3, SEQ ID NO;

5 or SEQ ID NO;

7 which encode polypeptides of the amino acid sequence shown in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6 or SEQ ID NO;

8 and which have at least 50% homology at the amino acid level without the enzymatic action of the polypeptides being substantially reduced.

3. An isolated nucleic acid sequence as claimed in claim 2, wherein the sequence is derived from a plant, alga or fungus.

4. An isolated nucleic acid sequence as claimed in claim 2 or 3, wherein the sequence is derived from Physcomitrella, Thraustochytrium or Crypthecodinium.

23. An oil, lipid or fatty acid composition which comprises PUFAs and which is derived from a transgenic plant.

28. A process for identifying an antagonist or agonist of elongases, comprising

a) bringing the cells which express the polypeptide of the present invention into contact with a candidate substance;

b) testing the PSE activity;

c) comparing the PSE activity with a standard activity in the absence of the candidate substance, where a PSE activity that is higher than that of the standard indicates that the candidate substance is an agonist and where a PSE activity that is lower than that of the standard indicates that the candidate substance is an antagonist.