Production of gamma linolenic acid by a Δ6-desaturase

• US 6,355,861 B1
• Filed: 09/19/1997
• Issued: 03/12/2002
• Est. Priority Date: 10/10/1991
• Status: Expired due to Term

• Current Assignee: Bayer SAS (Bayer AG)
• Original Assignee: Rhone-Poulenc Agrochimie
• Inventors: Thomas, Terry L.
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US08/934,254
• Time in Patent Office: 1,635 Days
• Field of Search: 536/23.2, 800/281, 800/298, 435/69.1, 435/71.1, 435/468, 435/471, 435/252.3, 435/440, 435/410, 435/325
• US Class Current: 800/281
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 114/19003 Linoleoyl-CoA desaturase (1.14.19.3)

Abstract

Linoleic acid is converted into γ-linolenic acid by the enzyme Δ6-desaturase. The present invention is directed to isolated nucleic acids comprising the Δ6-desaturase gene. More particularly, the isolated nucleic acid comprises the promoter, coding region and termination regions of the Δ6-desaturase gene. The present invention provides recombinant constructions comprising the Δ6-desaturase coding region in functional combination with heterologous regulatory sequences. The nucleic acids and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms.

Claims

1. An isolated nucleic acid encoding an evening primrose Δ

6-desaturase.

2. The isolated nucleic acid of claim 1 comprising at least one of the nucleotide sequence of SEQ ID NO:

26 or nucleotides 49 to 1401 of SEQ ID NO;

26.

4. A vector comprising the nucleic acid of any one claims 1-3.

5. An expression vector comprising the isolated nucleic acid of any one of claims 1-3 operably linked to a promoter which effects expression of the gene product of said isolated nucleic acid.

6. An expression vector comprising the isolated nucleic acid of any one of claims 1-3 operably linked to a promoter and a termination signal capable of effecting expression of the gene product of said isolated nucleic acid.

7. The expression vector of claim 5 wherein said promoter is a Δ

6- desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycinin in promoter, a napin promoter, the 35S promoter from CaMV, a helianthinin tissue-specific promoter, an oleosin seed-specific promoter, or an albumin seed-specific promoter.

8. The expression vector of claim 6 wherein said promoter is a Δ

6-desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycinin promoter, a napin promoter, the 35S promoter from CaMV, a helianthinin tissue-specific promoter, an oleosin seed-specific promoter, or an albumin seed-specific promoter.

9. An expression vector comprising the isolated nucleic acid of any one of claims 1-3 operably linked to a constitutive promoter.

10. An expression vector comprising the isolated nucleic acid of any one of claims 1-3 operably linked to a tissue specific promoter.

11. The expression vector of claim 6 wherein said termination signal is a Synechocystis termination signal, a nopaline synthase termination signal, or a seed-specific termination signal.

12. A cell comprising the vector of claim 4 wherein said cell is a bacterial cell, a plant cell, or a fungal cell.

13. A cell comprising the vector of claim 5 wherein said cell is a bacterial cell, a plant cell, or a fungal cell.

14. A cell comprising the vector of claim 6 wherein said cell is a bacterial cell, a plant cell, or a fungal cell.

15. A transgenic bacterium or plant comprising the isolated nucleic acid of any one of claims 1-3, wherein said nucleic acid is integrated into the genome of said bacterium or plant by genetic transformation.

16. A transgenic bacterium or plant comprising the vector of claim 4.

17. A transgenic bacterium or plant comprising the vector of claim 5.

18. A transgenic bacterium or plant comprising the vector of claim 6.

19. A plant which has been regenerated from the plant cell of claim 12, wherein said plant comprises said vector.

20. The plant of claim 19 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.

21. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises:

(a) transforming a plant cell with the isolated nucleic acid of any one of claims 1-3; and

(b) regenerating a plant with increased GLA content from said plant cell.

22. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises:

(a) transforming a plant cell with the vector of claim 4; and

(b) regenerating a plant with increased GLA content from said plant cell.

23. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises:

(a) transforming a plant cell with the vector of claim 5; and

(b) regenerating a plant with increased GLA content from said plant cell.

24. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises:

(a) transforming a plant cell with the vector of claim 6; and

(b) regenerating a plant with increased GLA content from said plant cell.

25. The method of claim 21 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.

26. The method of claim 22 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.

27. The method of claim 23 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.

28. The method of claim 24 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.

29. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant deficient in GLA which comprises transforming said bacteria or plant with the isolated nucleic acid of any one of claims 1-3.

30. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant deficient in GLA which comprises transforming said bacteria or plant with the vector of claim 4.

31. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant deficient in GLA which comprises transforming said bacteria or plant with the vector of claim 5.

32. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant deficient in GLA which comprises transforming said bacteria or plant with the vector of claim 6.

33. Progeny of the plant of claim 19, wherein said progeny comprises said vector.

34. A plant which has been regenerated from the plant cell of claim 13, wherein said plant comprises said vector.

35. A plant which has been regenerated from the plant cell of claim 14, wherein said plant comprises said vector.

36. Progeny of the plant of claim 34, wherein said progeny comprises said vector.

37. Progeny of the plant of claim 35, wherein said progeny comprises said vector.

38. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant lacking in or producing low levels of GLA which comprises transforming said bacteria or plant with the vector of claim 4.

39. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant lacking in or producing low levels of GLA which comprises transforming said bacteria or plant with the vector of claim 5.

40. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacteria or plant lacking in or producing low levels of GLA which comprises transforming said bacteria or plant with the vector of claim 6.

3. An isolated nucleic acid that codes for the amino acid sequence of SEQ ID NO:

27.