PUFA polyketide synthase systems and uses thereof

• US 7,211,418 B2
• Filed: 03/26/2004
• Issued: 05/01/2007
• 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., Barclay, William R., Metz, James G., Flatt, James H.
• Primary Examiner(s): Nashed; Nashaat T.
• Application Number:
  US10/810,352
• Publication Number:
  US 20040235127A1
• Time in Patent Office: 1,131 Days
• Field of Search: None
• US Class Current: 435/134
• CPC Class Codes: A23D 7/001 Spread compositions characterised by ingredients other than fatty acid triglycerides A23D7/0056
• CPC Class Codes: A23L 33/115 Fatty acids or derivatives thereof; Fats or oils
• CPC Class Codes: A23L 33/135 Bacteria or derivatives thereof, e.g. probiotics
• CPC Class Codes: C07K 14/28 from Vibrionaceae (F)
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• 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 7/6434 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 invention generally relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems, to homologues thereof, to isolated nucleic acid molecules and recombinant nucleic acid molecules encoding biologically active domains of such a PUFA PKS system, to genetically modified organisms comprising PUFA PKS systems, to methods of making and using such systems for the production of bioactive molecules of interest, and to novel methods for identifying new bacterial and non-bacterial microorganisms having such a PUFA 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 SEQ ID NO:

66, wherein the amino acid sequence has β

-hydroxyacyl-ACP dehydrase (DH) activity.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence that is at least 97% identical to SEQ ID NO:

66.

3. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence that is at least 99% identical to SEQ ID NO:

66.

4. The isolated nucleic acid molecule of claim 1, wherein the nucleic sequence encodes an amino acid sequence of SEQ ID NO:

66.

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

66.

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

65.

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

62.

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

61.

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

10. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 4, and an expression control sequence.

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

12. A recombinant microbial or plant cell that expresses the recombinant nucleic acid molecule of claim 9.

13. The recombinant cell of claim 12, wherein the recombinant cell is a plant cell.

14. The recombinant cell of claim 12, wherein the recombinant cell is a microbial cell.

15. The recombinant cell of claim 14, wherein the microbial cell is from a Thraustochytrid.

16. The recombinant cell of claim 15, wherein the Thraustochytrid is a Schizochytrium.

17. A recombinant microbial or plant cell that expresses the recombinant nucleic acid molecule of claim 10.

18. The recombinant cell of claim 17, wherein the recombinant cell is a plant cell.

19. The recombinant cell of claim 17, wherein the recombinant cell is a microbial cell.

20. The recombinant cell of claim 19, wherein the microbial cell is from a Thraustochytrid.

21. The recombinant cell of claim 20, wherein the Thraustochytrid is a Schizochytrium.

22. A recombinant microbial or plant cell hat expresses the recombinant nucleic acid molecule of claim 11.

23. The recombinant cell of claim 22, wherein the recombinant cell is a plant cell.

24. The recombinant cell of claim 22, wherein the recombinant cell is a microbial cell.

25. The recombinant cell of claim 24, wherein the recombinant cell is a Thraustochytrid microorganism.

26. The recombinant cell of claim 25, wherein the recombinant cell is a Schizochytrium.

27. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, a microorganism or a plant that expresses a PKS system for production of PUFAs, wherein the microorganism or plant an organism expresses the recombinant nucleic acid molecule of claim 9.

28. The method of claim 27, wherein the microorganism or a plant produces a polyunsaturated fatty acid (PUFA) profile that differs from a microorganism or plant that does not express the recombinant nucleic acid molecule.

29. The method of claim 28, wherein the microorganism or plant produces docosahexaenoic acid (DHA), and wherein the production of DHA is increased in the microorganism or a plant as compared to a microorganism or plant that does not express the recombinant nucleic acid molecule.

30. The method of claim 27, wherein the microorganism or a plant is a microorganism.

31. The method of claim 27, wherein the microorganism or a plant is a plant.

32. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, a microorganism or a plant that expresses a PKS system for production of PUFAs, wherein the microorganism or plant expresses the recombinant nucleic acid molecule of claim 10.

33. The method of claim 32, wherein the microorganism or a plant is a microorganism.

34. The method of claim 32, wherein the microorganism or a plant is a plant.

35. A method to produce at least one polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the PUFA, a microorganism or a plant that expresses a PKS system for production of PUFAs, wherein the microorganism or plant expresses the recombinant nucleic acid molecule of claim 11.

36. The method of claim 35, wherein the microorganism or a plant is a microorganism.

37. The method of claim 35, wherein the microorganism or a plant is a plant.

38. 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 9.

39. 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 10.

40. 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 11.

41. A method to produce lipids enriched for docosahexaenoic acid (DHA), comprising culturing under conditions effective to produce the lipids, a Thraustochytrid microorganism that expresses the recombinant nucleic acid molecule of claim 9 and that produces DHA, wherein the production of DHA is enriched in the Thraustochytrid microorganism as compared to in the absence of the expression of the recombinant nucleic acid molecule.

42. A method to produce lipids enriched for docosahexaenoic acid (DHA), comprising culturing under conditions effective to produce the lipids, a Thraustochytrid microorganism that expresses the recombinant nucleic acid molecule of claim 10 and that produces DHA, wherein the production of DHA is enriched in the Thraustochytrid microorganism as compared to in the absence of the expression of the recombinant nucleic acid molecule.

43. A method to produce lipids enriched for docosahexaenoic acid (DHA), comprising culturing under conditions effective to produce the lipids, a Thraustochytrid microorganism that expresses the recombinant nucleic acid molecule of claim 11 and that produces DHA, wherein the production of DHA is enriched in the Thraustochytrid microorganism as compared to in the absence of the expression of the recombinant nucleic acid molecule.

44. An isolated recombinant cell that expresses the recombinant nucleic acid molecule of claim 9.

45. An isolated recombinant cell that expresses the recombinant nucleic acid molecule of claim 10.

46. An isolated recombinant cell that expresses the recombinant nucleic acid molecule of claim 11.