Novel method for the production of polyunsaturated fatty acids

• US 20060246556A1
• Filed: 12/11/2003
• Published: 11/02/2006
• Est. Priority Date: 12/19/2002
• Status: Active Grant

• Current Assignee: University of Bristol
• Original Assignee: University of Bristol
• Inventors: Napier, Johnathan A, Lazarus, Colin, Zank, Thorsten, Heinz, Ernst, Sayanova, Olga, Zahringer, Ulrich, Qi, Baoxiu
• Granted Patent: US 7,714,185 B2
• Time in Patent Office: Days
• US Class Current: 435/128
• 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: 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 an improved process for the specific production of poly-unsaturated omega-3 and omega-6 fatty acids and a process for the production of triglycerides having an increased content of unsaturated fatty acids, in particular omega-3 and omega-6 fatty acids having at least two double bonds and a 20 or 22 carbon atom chain length. The invention relates to the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism, hav-ing an increased content of fatty acids, oils or lipids containing C20- or C22-fatty acids with a delta-5, 7, 8, 10 double bond, respectively due to the expression of a delta-8-desaturase and a delta-9-elon-gase from organisms such as plants preferably Algae like Isochrysis galbana or Euglena gracilis. In addition the invention relates to a process for the production of poly unsaturated fatty acids such as Eicosapentaenoic, Arachidonic, Docosapentaenoic or Doosahexaenoic acid through the co-expression of a delta-8-desaturase, a delta-9-elongase and a delta-5 desaturase in organisms such as microorganisms or plants. The invention additionally relates to the use of specific nucleic acid sequences encoding for the aforementioned proteins with delta-8-desaturase-, delta-9-elongase- or delta-5-desaturase-activity, nucleic acid constructs, vectors and organisms containing said nucleic acid sequences. The invention further relates to unsaturated fatty acids and triglycerides having an increased content of at least 1% by weight of unsaturated fatty acids and use thereof.

Claims

1. A process for the production of compounds of the following general formula I

2. The process as claimed in claim 1, wherein the nucleic acid sequences which encode polypeptides with Δ

-8-desaturase, Δ

-9-elongase or Δ

-5-desaturase are selected from the group consisting of

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

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7 or SEQ ID NO;

9

b) a nucleic acid sequence which is derived from the sequence depicted in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7 or SEQ ID NO;

9 according to the degeneracy of the genetic code,

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

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7 or SEQ ID NO;

9 which encodes polypeptides having at least 50% homology to the sequence encoding amino acid sequences depicted in SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8 or SEQ ID NO;

10 and which sequences function as a Δ

-8-desaturase, Δ

-9-elongase or Δ

-5-desaturase.

3. The process as claimed in claim 1, wherein the substituents R² and R³ are independent of each other saturated or unsaturated C₁₀-C₂₂-alkylcarbonyl-.

4. The process as claimed in claim 1, wherein the substituents R² and R³ are independent of each other saturated or unsaturated C₁₆-, C₁₈-, C₂₀- or C₂₂-alkylcarbonyl-.

5. The process as claimed in claim 1, wherein the substituents R² and R³ are independent of each other unsaturated C₁₆-, C₁₈-, C₂₀- or C₂₂-alkylcarbonyl- with at least three double bonds.

6. The process as claimed in claim 1, wherein the transgenic organism is an oil producing plant.

7. The process as claimed in claim 1, wherein the transgenic plant is selected from the group consisting of rapeseed, poppy, mustard, hemp, castor bean, sesame, olive, calendula, punica, hazel nut, almond, macadamia, avocado, pumpkin, walnut, laurel, pistachio, primrose, canola, peanut, linseed, soybean, safflower, sunflower and borage.

8. The process as claimed in claim 1, wherein the compounds of the general formula I are isolated in the form of their oils, lipids of free fatty acids.

9. The process as claimed in claim 1, wherein the compounds of the general formula I are isolated in a concentration of at least 5% by weight referred to the total lipid content.

10. An isolated nucleic acid sequence comprising a nucleotide sequence which encodes a Δ

-8-desaturase selected from the group consisting of

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

1,

b) a nucleic acid sequence which is derived from the sequence depicted in SEQ ID NO;

1 according to the degeneracy of the genetic code and which sequences function as a Δ

-8-desaturase.

12. An amino-acid sequence encoded by an isolated nucleic acid sequence as claimed in claims 10.

13. A gene construct comprising an isolated nucleic acid having the sequence SEQ ID NO:

1 as claimed in claim 10, where the nucleic acid is functionally linked to one or more regulatory signals.

14. A gene construct as claimed in claim 13, whose gene expression is increased by the regulatory signals.

15. A vector comprising a nucleic acid as claimed in claim 10.

16. An organism comprising at least one nucleic acid as claimed in claim 10.

17. The organism as claimed in claim 16, wherein the organism is a microorganism, a non-human animal or a plant.

18. The organism as claimed in claim 16, wherein the organism is a transgenic plant.

11. An isolated nucleic acid sequence comprising a nucleotide sequence which encodes a Δ

-5-desaturase selected from the group consisting of

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

5,

b) a nucleic acid sequence which is derived from the sequence depicted in SEQ ID NO;

5 according to the degeneracy of the genetic code,

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

5 which encodes polypeptides having at least 50% homology to the sequence encoding amino acid sequences depicted in SEQ ID NO;

6 and which sequences function as a Δ

-5-desaturase.

19. An amino-acid sequence encoded by an isolated nucleic acid sequence as claimed in claim 11.

20. A gene construct comprising an isolated nucleic acid having the sequence SEQ ID NO:

5 as claimed in claim 11, where the nucleic acid is functionally linked to one or more regulatory signals.

21. A gene construct as claimed in claim 20, whose gene expression is increased by the regulatory signals.

22. A vector comprising a nucleic acid as claimed in claim 11.

23. An organism comprising at least one nucleic acid as claimed in claim 11.

24. The organism as claimed in claim 23, wherein the organism is a microorganism, a non-human animal or a plant.

25. The organism as claimed in claim 23, wherein the organism is a transgenic plant.