Production of .gamma.-linolenic acid by a .DELTA.6-desaturase

• US 5,614,393 A
• Filed: 12/30/1994
• Issued: 03/25/1997
• 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., Nuccio, Michael, Reddy, Avutu S., Nunberg, Andrew N., Freyssinet, Georges L.
• Primary Examiner(s): RORIES, CHARLES
• Application Number:
  US08/366,779
• Time in Patent Office: 816 Days
• Field of Search: 800/205, 800/250, 800/255, 800/DIG. 14, 800/DIG. 17, 800/DIG. 23, 800/DIG. 26, 800/DIG. 43, 800/DIG. 56, 435/69.1, 435/41, 435/134, 435/172.3, 435/240.4, 435/243, 435/252.3, 435/320.1, 435/70.1, 435/71.1, 435/189, 435/240.1, 435/240.45, 435/254.11, 536/23.6, 536/23.2, 536/24.1, 935/6, 935/14, 935/22, 935/24, 935/29, 935/30, 935/66-70, 935/72
• US Class Current: 435/134
• 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 7/6436 Fatty acid esters
• 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 a borage Δ

6-desaturase.

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

4.

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 promotor 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 which effects 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 promoter, a napin promoter, the 35S promoter from CaMV, or a helianthinin tissue-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.

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

12. A cell comprising the vector of claim 4.

13. A cell comprising the vector of claim 5.

14. A cell comprising the vector of claim 6.

15. The cell of claim 12 wherein said cell is an animal cell, a bacterial cell, a plant cell or a fungal cell.

16. The cell of claim 13 wherein said cell is an animal cell, a bacterial cell, a plant cell or a fungal cell.

17. The cell of claim 14 wherein said cell is an animal cell, a bacterial cell, a plant cell or a fungal cell.

18. A transgenic bacterium or plant comprising the isolated nucleic acid of any one of claims 1-3.

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

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

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

22. A plant or progeny of said plant which has been regenerated from the plant cell of claim 15.

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

24. 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.

25. 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.

26. 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.

27. 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.

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

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

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

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

32. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacterium or plant deficient or lacking in or producing low levels Of GLA which comprises transforming said organism with the isolated nucleic acid of any one of claims 1-3.

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

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

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

39. The method of inducing or increasing production of octadecatetraenoic acid in at least one of a plant deficient or lacking in or producing low levels of octadecatetraenoic acid, a bacterium which produces α

-linolenic acid, or a bacterium which exhibits a Δ

-15 desaturase activity on a GLA substrate which comprises transforming said plant or bacterium with the isolated nucleic acid of any one of claims 1-3.

40. The method of inducing or increasing production of octadecatetraenoic acid in at least one of a plant deficient or lacking in or producing low levels of octadecatetraenoic acid, a bacterium which produces α

-linolenic acid, or a bacterium which exhibits a Δ

-15 desaturase activity on a GLA substrate which comprises transforming said plant or bacterium with the vector of claim 4.

41. The method of inducing or increasing production of octadecatetraenoic acid in at least one of a plant deficient or lacking in or producing low levels of octadecatetraenoic acid, a bacterium which produces α

-linolenic acid, or a bacterium which exhibits a Δ

-15 desaturase activity on a GLA substrate which comprises transforming said plant or bacterium with the vector of claim 5.

42. The method of inducing or increasing production of octadecatetraenoic acid in at least one of a plant deficient or lacking in or producing low levels of octadecatetraenoic acid, a bacterium which produces α

-linolenic acid, or a bacterium which exhibits a Δ

-15 desaturase activity on a GLA substrate which comprises transforming said plant or bacterium with the vector of claim 6.

43. The method of inducing or increasing production of octadecatetraenoic acid in at least one of a plant deficient or lacking in or producing low levels of octadecatetraeonic acid, a bacterium which produces α

-linolenic acid, or a bacterium which exhibits a Δ

-15 desaturase activity on a GLA substrate which comprises transforming said plant or bacterium with the vector of claim 7.

44. The method of claim 42 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.

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

5.

36. A method of inducing or increasing production of gama linolenic acid (GLA) in a bacterium or plant deficient or lacking in or producing low levels of GLA and linoleic acid (LA) which comprises transforming said organism with an isolated nucleic acid encoding borage Δ

6-desaturase and an isolated nucleic acid encoding Δ

12-desaturase.

38. The method of any one of claims 36 or 37 wherein said isolated nucleic acid encoding Δ

6-desaturase comprises nucleotides 44 to 1390 of SEQ. ID NO;

4.

37. A method of inducing or increasing production of gamma linolenic acid (GLA) in a bacterium or plant deficient or lacking in or producing low levels of GLA and linoleic acid (LA) which comprises transforming said organism with at least one expression vector comprising an isolated nucleic acid encoding borage Δ

6-desaturase and an isolated nucleic acid encoding Δ

12-desaturase.