PUFA polyketide synthase systems and uses thereof

• US 7,217,856 B2
• Filed: 10/13/2004
• Issued: 05/15/2007
• Est. Priority Date: 01/14/1999
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Weaver, Craig A., Zirkle, Ross, Metz, James G.
• Primary Examiner(s): Nashed; Nashaat T.
• Application Number:
  US10/965,017
• Publication Number:
  US 20050100995A1
• Time in Patent Office: 944 Days
• Field of Search: None
• US Class Current: 800/281
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/62 Carboxylic acid esters
• 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

Disclosed are the complete polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from the bacterial microorganisms Shewanella japonica and Shewanella olleyana, and biologically active fragments and homologues thereof. More particularly, this invention relates to nucleic acids encoding such PUFA PKS systems, to proteins and domains thereof that comprise such PUFA PKS systems, to genetically modified organisms (plants and microorganisms) comprising such PUFA PKS systems, and to methods of making and using the PUFA PKS systems disclosed herein. This invention also relates to genetically modified plants and microorganisms and methods to efficiently produce lipids enriched in various polyunsaturated fatty acids (PUFAs) as well as other bioactive molecules by manipulation of a PUFA polyketide synthase (PKS) system.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to amino acid positions 5–

483 of SEQ ID NO;

4, wherein the amino acid sequence has β

-ketoacyl-ACP synthase (KS) activity.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence that is at least 96% identical to amino acid positions 5–

483 of SEQ ID NO;

4.

3. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence that is at least 97% identical to amino acid positions 5–

483 of SEQ ID NO;

4.

4. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence that is at least 98% identical to amino acid positions 5–

483 of SEQ ID NO;

4.

5. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence that is at least 99% identical to amino acid positions 5–

483 of SEQ ID NO;

4.

6. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence comprising amino acid positions 5–

483 of SEQ ID NO;

4.

7. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of a nucleic acid sequence encoding amino acid positions 5–

483 of SEQ ID NO;

4.

8. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO:

4.

9. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises nucleotides 21139–

22575 of SEQ ID NO;

1.

10. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises nucleotides 21127–

27186 of SEQ ID NO;

1.

11. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:

1.

12. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is from Shewanella japonica.

13. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 1 and an expression control sequence.

14. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 6 and an expression control sequence.

15. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 8 and an expression control sequence.

16. A recombinant plant cell that expresses the recombinant nucleic acid molecule of claim 13.

17. A recombinant plant cell that expresses the recombinant nucleic acid molecule of claim 14.

18. A recombinant plant cell that expresses the recombinant nucleic acid molecule of claim 15.

19. An isolated recombinant cell that expresses the recombinant nucleic acid molecule of claim 13.

20. An isolated recombinant cell that expresses the recombinant nucleic acid molecule of claim 14.

21. An isolated recombinant cell that expresses the recombinant nucleic acid molecule of claim 15.

22. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, an isolated animal cell or a plant cell that expresses a PKS system for production of PUFAs, wherein the isolated animal cell or the plant cell expresses the recombinant nucleic acid molecule of claim 13.

23. The method of claim 22, wherein the isolated animal cell or the plant cell produces a polyunsaturated fatty acid (PUFA) profile that differs from an isolated animal cell or a plant cell that does not express the recombinant nucleic acid molecule.

24. The method of claim 22, wherein the cell or plant is a plant cell.

25. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, an isolated animal cell or a plant cell that expresses a PKS system for production of PUFAs, wherein the isolated animal cell or the plant cell expresses the recombinant nucleic acid molecule of claim 14.

26. The method of claim 25, wherein the isolated animal cell or the plant cell produces a polyunsaturated fatty acid (PUFA) profile that differs from an isolated recombinant cell or a plant cell that does not express the recombinant nucleic acid molecule.

27. The method of claim 25, wherein the cell is a plant cell.

28. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, an isolated animal cell or a plant cell that expresses a PKS system for production of PUFAs, wherein the isolated animal cell or the plant cell expresses the recombinant nucleic acid molecule of claim 15.

29. The method of claim 28, wherein the isolated recombinant cell or the plant cell produces a polyunsaturated fatty acid (PUFA) profile that differs from an isolated recombinant cell or a plant cell that does not express the recombinant nucleic acid molecule.

30. The method of claim 28, wherein the cell is a plant cell.

31. A method to produce a genetically modified plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the plant in the absence of the genetic modification, comprising genetically modifying cells of the plant to express a PKS system for production of PUFAs comprising the recombinant nucleic acid molecule of claim 13.

32. A method to produce a genetically modified plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the plant in the absence of the genetic modification, comprising genetically modifying cells of the plant to express a PKS system for production of PUFAs comprising the recombinant nucleic acid molecule of claim 14.

33. A method to produce a genetically modified plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the plant in the absence of the genetic modification, comprising genetically modifying cells of the plant to express a PKS system for production of PUFAs comprising the recombinant nucleic acid molecule of claim 15.

34. A method to produce lipids enriched for a polyunsaturated fatty acid (PUFA) selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), comprising growing under conditions effective to produce the lipids, a plant that expresses the recombinant nucleic acid molecule of claim 13 and that produces the PUFA, wherein the production of the PUFA is enriched in the plant as compared to in the absence of the expression of the recombinant nucleic acid molecule.

35. A method to produce lipids enriched for a polyunsaturated fatty acid (PUFA) selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), comprising growing under conditions effective to produce the lipids, a plant that expresses the recombinant nucleic acid molecule of claim 14 and that produces the PUFA, wherein the production of the PUFA is enriched in the or plant as compared to in the absence of the expression of the recombinant nucleic acid molecule.

36. A method to produce lipids enriched for a polyunsaturated fatty acid (PUFA) selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), comprising growing under conditions effective to produce the lipids, a plant that expresses the recombinant nucleic acid molecule of claim 15 and that produces the PUFA, wherein the production of the PUFA is enriched in the plant as compared to in the absence of the expression of the recombinant nucleic acid molecule.

37. A recombinant microbial cell that expresses a recombinant vector comprising the nucleic acid molecule of claim 1 and an expression control sequence.

38. The recombinant microbial cell of claim 37, wherein the microbial cell is a bacterium.

39. The recombinant microbial cell of claim 37, wherein the microbial cell is from a Thraustochytrid.

40. The recombinant cell of claim 39, wherein the Thraustochytrid is a Schizochytrium or a Thraustochytrium.

41. A recombinant microbial cell that expresses a recombinant vector comprising the nucleic acid molecule of claim 6 and an expression control sequence.

42. The recombinant microbial cell of claim 41, wherein the microbial cell is a bacterium.

43. The recombinant microbial cell of claim 41, wherein the microbial cell is from a Thraustochytrid.

44. The recombinant cell of claim 43, wherein the Thraustochytrid is a Schizochytrium or a Thraustochytrium.

45. A recombinant microbial cell that expresses a recombinant vector comprising the nucleic acid molecule of claim 8 and an expression control sequence.

46. The recombinant microbial cell of claim 45, wherein the microbial cell is a bacterium.

47. The recombinant microbial cell of claim 45, wherein the microbial cell is from a Thraustochytrid.

48. The recombinant cell of claim 47, wherein the Thraustochytrid is a Schizochytrium or a Thraustochytrium.

49. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, a microbial cell that expresses a PKS system for production of PUFAs, wherein the microbial cell expresses a recombinant vector comprising the nucleic acid molecule of claim 1 and an expression control sequence.

50. The method of claim 49, wherein the microbial cell produces a polyunsaturated fatty acid (PUFA) profile that differs from a microbial cell that does not express the recombinant nucleic acid molecule.

51. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, a microbial cell that expresses a PKS system for production of PUFAs, wherein the microbial cell expresses a recombinant vector comprising the nucleic acid molecule of claim 6 and an expression control sequence.

52. The method of claim 51, wherein the microbial cell produces a polyunsaturated fatty acid (PUFA) profile that differs from a microbial cell that does not express the recombinant nucleic acid molecule.

53. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, a microbial cell that expresses a PKS system for production of PUFAs, wherein the microbial cell expresses a recombinant vector comprising the nucleic acid molecule of claim 8 and an expression control sequence.

54. The method of claim 53, wherein the microbial cell produces a polyunsaturated fatty acid (PUFA) profile that differs from a microbial cell that does not express the recombinant nucleic acid molecule.

55. A method to produce lipids enriched for a polyunsaturated fatty acid (PUFA) selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), comprising culturing under conditions effective to produce the lipids, a microorganism that expresses a recombinant vector comprising the nucleic acid molecule of claim 1 and an expression control sequence, and that produces the PUFA, wherein the production of the PUFA is enriched in the microorganism as compared to in the absence of the expression of the recombinant nucleic acid molecule.

56. A method to produce lipids enriched for a polyunsaturated fatty acid (PUFA) selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), comprising culturing under conditions effective to produce the lipids, a microorganism that expresses a recombinant vector comprising the nucleic acid molecule of claim 6 and an expression control sequence, and that produces the PUFA, wherein the production of the PUFA is enriched in the microorganism as compared to in the absence of the expression of the recombinant nucleic acid molecule.

57. A method to produce lipids enriched for a polyunsaturated fatty acid (PUFA) selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), comprising culturing under conditions effective to produce the lipids, a microorganism that expresses a recombinant vector comprising the nucleic acid molecule of claim 8 and an expression control sequence, and that produces the PUFA, wherein the production of the PUFA is enriched in the microorganism as compared to in the absence of the expression of the recombinant nucleic acid molecule.