Process for the production of unsaturated fatty acids

• US 20060078973A1
• Filed: 02/27/2003
• Published: 04/13/2006
• Est. Priority Date: 03/01/2002
• Status: Active Grant

• Current Assignee: Cornell University
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Renz, Andreas, Rosenfield, Claire-Lise, Feussner, Ivo, Gipmans, Martijn, Knipple, Douglas C.
• Granted Patent: US 7,700,833 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The invention relates to processes for the production of unsaturated fatty acids, preferably of conjugated polyunsaturated fatty acids such as conjugated linoleic acid (CLA), by the recombinant expression of desaturases from insects of the order Lepidoptera. Expression preferably takes place in an organism selected from the group of the plant organisms, yeasts, fungi and algae. Also according to the invention are recombinant expression cassettes for the recombinant expression of desaturases from insects of the order Lepidoptera, and the transgenic organisms transformed with these.

Claims

1. A process for production of triglycerides comprising unsaturated fatty acids, which comprises recombinantly expressing at least one fatty-acid desaturase from insects of the order Lepidoptera in an organism selected from the group consisting of plant organisms, yeasts, fungi and algae.

2. The process of claim 1, wherein the fatty-acid desaturase is capable of generating a double bond at position C8, C9, C10, C11 or C12 in fatty acids, fatty-acid CoA esters, or fatty-acid derivatives.

3. The process of claim 1, wherein the fatty-acid desaturase generates a cis or trans double bond in fatty acids, fatty-acid CoA esters, or fatty acid derivatives with a fatty acid chain length of 16 or 18 C atoms.

4. The process of claim 1, wherein the fatty-acid desaturase has at least 65% homology with a fatty-acid destuarse containing the sequence of SEQ ID NO:

2, 4, 6, 8, 10, 12, 14 or 22.

5. The process of claim 1, wherein the nucleic acid sequence of the fatty-acid desaturase comprises:

a) a sense strand comprising the sequence of SEQ ID NO;

15 or 17;

or

b) an antisense strand comprising the sequence of SEQ ID NO;

16 or 18.

6. The process of claim 1, wherein the unsaturated fatty acids comprise a conjugated linoleic acid.

7. The process of claim 1, wherein the organism is selected from the group consisting of Mucor circinelloides, Mortierella alpina, Pythium insidiosum, Cryptococcus curvatus, linseed, soya, oilseed rape, coconut, oil palm, safflower, castor-oil plant, peanut, cacao tree and sunflower.

8. Triglycerides produced by the process of claim 1.

9. A method for preparation of foodstuffs, feedstuffs, cosmetics or fine chemicals containing the triglycerides of claim 8.

21. The process of claim 1, wherein the fatty-acid desaturase generates a trans double bond in the unsaturated fatty acids.

22. The process of claim 21, wherein greater than 60% of the unsaturated fatty acids contain the trans double bond.

23. The process of claim 21, wherein greater than 90% of the unsaturated fatty acids contain the trans double bond.

10. A polypeptide with E11-desaturase activity, wherein the polypeptide converts C16 or C18 acyl-CoA fatty acids and comprises at least one sequence selected from the group consisting of:

a) amino acid sequences comprised of the sequence of SEQ ID NO;

2, 4, 6 or 22;

b) amino acid sequences with at least 65% homology with one of the amino acid sequences of SEQ ID NO;

2, 4, 6 or 22; and

c) amino acid sequences which encompass a fragment of at least 20 contiguous amino acid residues of a the sequence of SEQ ID NO;

2, 4, 6 or 22.

11. The polypeptide of claim 10, which comprises the amino acid sequence of SEQ ID NO:

22.

12. A nucleic acid sequence that encodes the polypeptide of claim 11.

13. A recombinant expression cassette comprising at least one nucleic acid sequence encoding a fatty-acid desaturase, which is derived from insects of the order Lepidoptera, and that is under the control of a promoter which is functional in plants, plant organisms, algae, yeasts or fungi.

14. The recombinant expression cassette of claim 13, wherein the promoter is functional in at least one organism selected from the group consisting of Mucor circinelloides, Mortierella alpina, Pythium insidiosum, Cryptococcus curvatus, soya, linseed, oilseed rape, coconut, oil palm, safflower, castor-oil plant, peanut, cocoa bean and sunflower.

15. The recombinant expression cassette of claim 13, wherein the fatty-acid desaturase contains a polypeptide which comprises at least one sequence selected from the group consisting of:

a) amino acid sequences comprised of the sequence of SEQ ID NO;

2, 4, 6, 8, 10, 12, 14 or 22;

b) amino acid sequences with at least 65% homology with one of the amino acid sequences of SEQ ID NO;

2, 4, 6, 8, 10, 12, 14 or 22; and

c) amino acid sequences which encompass a fragment of at least 20 contiguous amino acid residues of the sequence of SEQ ID NO;

2, 4, 6, 8, 10, 12, 14 or 22.

16. The recombinant expression cassette of claim 13, wherein the fatty-acid desaturase contains the amino acid sequence of SEQ ID NO:

22.

17. A recombinant expression vector comprising at least one expression cassette of claim 13.

18. A transgenic organism comprising at least one recombinant expression cassette of claim 13.

19. The transgenic organism of claim 18, which is a plant organism.

20. The transgenic organism of claim 18, which is selected from the group consisting of Mucor circinelloides, Mortierella alpina, Pythium insidiosum, Cryptococcus curvatus, soya, linseed, oilseed rape, coconut, oil palm, safflower, castor-oil plant, peanut, cocoa tree and sunflower.

24. A transgenic organism comprising at least one recombinant expression vector of claim 17.