Product and process for transformation of thraustochytriales microorganisms

• US 20030166207A1
• Filed: 04/16/2002
• Published: 09/04/2003
• Est. Priority Date: 04/16/2001
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Metz, James G., Matthews, T. Dave, Ramseier, Tom M., Roessler, Paul G.
• Granted Patent: US 7,001,772 B2
• Time in Patent Office: Days
• US Class Current: 435/193
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/82 for plant cells , e.g. plant artificial chromosomes (PACs)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 202/01006 Acetolactate synthase (2.2.1.6)

Abstract

Disclosed are nucleic acid and amino acid sequences for acetolactate synthase, acetolactate synthase regulatory regions, α-tubulin promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and fatty acid desaturase promoter, each from a Thraustochytriales microorganism. Also disclosed are recombinant vectors useful for transformation of Thraustochytriales microorganisms, as well as a method of transformation of Thraustochytriales microorganisms. The recombinant nucleic acid molecules of the present invention can be used for the expression of foreign nucleic acids in a Thraustochytriales microorganism as well as for the deletion, mutation, or inactivation of genes in Thraustochytriales microorganisms.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

a. a nucleic acid sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO;

15, SEQ ID NO;

19, SEQ ID NO;

22 and SEQ ID NO;

24, wherein said protein is an acetolactate synthase;

b. a nucleic acid sequence encoding a protein having an amino acid sequence that is at least about 75% identical to an amino acid sequence of (a), wherein said protein is an acetolactate synthase; and

,

c. a nucleic acid sequence that is fully complementary to said nucleic acid sequence of (a) or (b).

2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a protein having an amino acid sequence that is at least about 85% identical to an amino acid sequence of (a), and wherein said protein is an acetolactate synthase.

3. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a protein having an amino acid sequence that is at least about 95% identical to an amino acid sequence of (a), and wherein said protein is an acetolactate synthase.

4. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a protein having an amino acid sequence that differs from SEQ ID NO:

15 by an amino acid deletion, insertion, or substitution at an amino acid position selected from the group consisting of;

116G, 117A, 192P, 200A, 251K, 358M, 383D, 592V, 595W, and 599F.

5. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:

15, SEQ ID NO;

19, SEQ ID NO;

22 and SEQ ID NO;

24, and wherein said protein is an acetolactate synthase.

6. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence is selected from the group consisting of nucleotides 1260-3314 of SEQ ID NO:

14, nucleotides 1260-3314 of SEQ ID NO;

18, nucleotides 1260-3314 of SEQ ID NO;

21, and nucleotides 1260-3314 of SEQ ID NO;

23.

7. The isolated nucleic acid molecule of claim 1, wherein expression of said protein of (a) or (b) confers reduced sensitivity to compounds selected from the group consisting of:

sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, onto a microorganism of the Order Thraustochytriales that is transformed with said nucleic acid molecule.

8. The isolated nucleic acid molecule of claim 7, wherein said nucleic acid sequence encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:

19, SEQ ID NO;

22 and SEQ ID NO;

24.

9. The isolated nucleic acid molecule of claim 7, wherein said nucleic acid sequence is selected from the group consisting of:

nucleotides 1260-3314 of SEQ ID NO;

14, nucleotides 1260-3314 of SEQ ID NO;

18, nucleotides 1260-3314 of SEQ ID NO;

21, and nucleotides 1260-3314 of SEQ ID NO;

23.

10. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a Schizochytrium acetolactate synthase.

11. The isolated nucleic acid molecule of claim 10, wherein expression of said Schizochytrium acetolactate synthase confers reduced sensitivity to compounds selected from the group consisting of:

sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, onto a microorganism of the Order Thraustochytriales that is transformed with said nucleic acid molecule.

12. A recombinant nucleic acid molecule comprising the isolated nucleic acid molecule of claim 1, operatively linked to a transcription control sequence.

13. A recombinant microorganism of the order Thraustochytriales that is transformed with the recombinant nucleic acid molecule of claim 12.

14. A recombinant vector for transformation of microorganisms of the Order Thraustochytriales, comprising a nucleic acid sequence encoding an acetolactate synthase that confers reduced sensitivity to compounds selected from the group consisting of:

sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, onto a microorganism of the order Thraustochytriales, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;

a. an amino acid sequence selected from the group consisting of SEQ ID NO;

19, SEQ ID NO;

22 and SEQ ID NO;

24; and

,

b. an amino acid sequence that is at least about 75% identical to an amino acid sequence of (a);

wherein said nucleic acid sequence encoding an acetolactate synthase is operatively linked to a transcription control sequence.

15. The recombinant vector of claim 14, wherein said recombinant vector is an expression vector.

16. The recombinant vector of claim 14, wherein said recombinant vector is a targeting vector.

17. The recombinant vector of claim 14, wherein said nucleic acid sequence encodes an acetolactate synthase having an amino acid sequence that is at least about 85% identical to an amino acid sequence of (a).

18. The recombinant vector of claim 14, wherein said nucleic acid sequence encodes an acetolactate synthase having an amino acid sequence that is at least about 95% identical to an amino acid sequence of (a).

19. The recombinant vector of claim 14, wherein said nucleic acid sequence encodes a protein having an amino acid sequence that differs from SEQ ID NO:

15 by an amino acid deletion, insertion, or substitution at an amino acid position selected from the group consisting of;

116G, 117A, 192P, 200A, 251K, 358M, 383D, 592V, 595W, and 599F.

20. The recombinant vector of claim 14, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of SEQ ID NO:

19, SEQ ID NO;

22 and SEQ ID NO;

24.

21. The recombinant vector of claim 14, wherein said nucleic acid sequence is selected from the group consisting of:

nucleotides 1260-3314 of SEQ ID NO;

18, nucleotides 1260-3314 of SEQ ID NO;

21, and nucleotides 1260-3314 of SEQ ID NO;

23.

22. The recombinant vector of claim 14, wherein said transcription control sequence is selected from the group consisting of a Thraustochytriales α

-tubulin promoter, a Thraustochytriales acetolactate synthase promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and a Thraustochytriales fatty acid desaturase promoter.

23. The recombinant vector of claim 14, wherein said vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

18, SEQ ID NO;

21, and SEQ ID NO;

23.

24. A method for transformation of cells of a microorganism of the Order Thraustochytriales, said method comprising:

a. introducing into cells of a microorganism of the Order Thraustochytriales a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an acetolactate synthase that confers onto said cells reduced sensitivity to compounds selected from the group consisting of;

sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;

i. an amino acid sequence selected from the group consisting of SEQ ID NO;

19, SEQ ID NO;

22 and SEQ ID NO;

24; and

,

ii. an amino acid sequence that is at least about 75% identical to an amino acid sequence of (i); and

,

b. selecting cells that have been successfully transformed with said recombinant nucleic acid molecule by culturing said cells of (a) in a medium containing at least one compound that is inhibitory to untransformed cells, said compound being selected from the group consisting of;

a sulfonylurea compound, an imidazolinone-class inhibitor, and pyrimidinyl oxybenzoates.

25. The method of claim 24, wherein said nucleic acid sequence encodes an acetolactate synthase having an amino acid sequence that is at least about 85% identical to an amino acid sequence of (i).

26. The method of claim 24, wherein said nucleic acid sequence encodes an acetolactate synthase having an amino acid sequence that is at least about 95% identical to an amino acid sequence of (i).

27. The method of claim 24, wherein said nucleic acid sequence encodes a protein having an amino acid sequence that differs from SEQ ID NO:

15 by an amino acid deletion, insertion, or substitution at an amino acid position selected from the group consisting of;

116G, 117A, 192P, 200A, 251K, 358M, 383D, 592V, 595W, and 599F.

28. The method of claim 24, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of SEQ ID NO:

19, SEQ ID NO;

22 and SEQ ID NO;

24.

29. The method of claim 24, wherein said nucleic acid sequence is selected from the group consisting of:

nucleotides 1260-3314 of SEQ ID NO;

18, nucleotides 1260-3314 of SEQ ID NO;

21, and nucleotides 1260-3314 of SEQ ID NO;

23.

30. The method of claim 24, wherein said nucleic acid sequence is operatively linked to a transcription control sequence selected from the group consisting of a Thraustochytriales α

-tubulin promoter, a Thraustochytriales acetolactate synthase promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and a Thraustochytriales fatty acid desaturase promoter.

31. The method of claim 24, wherein said recombinant nucleic acid molecule further comprises a nucleic acid sequence encoding a protein to be produced by said cell, wherein said nucleic acid sequence encoding said protein is operatively linked to a transcription control sequence.

32. The method of claim 31, wherein said protein is associated with the synthesis of a fatty acid selected from the group consisting of docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA) and arachadonic acid (ARA).

33. The method of claim 31, wherein said protein is selected from the group consisting of:

a fatty acid synthase, a fatty acid desaturase, a fatty acid elongase, a protein associated with a polyketide synthase complex and a protein associated with incorporation of fatty acids into phospholipids or into triacylglycerol molecules.

34. The method of claim 31, wherein said protein is an ω

-3 fatty acid desaturase is encoded by a nucleic acid sequence SEQ ID NO;

29.

35. The method of claim 31, wherein said protein is a polyenoic fatty acid isomerase.

36. The method of claim 31, wherein said protein is selected from the group consisting of HMG-CoA synthase, HMG-CoA reductase, squalene synthase, phytoene synthase, phytoene desaturase, a carotenoid cyclase, a carotenoid hyroxylase, a carotenoid ketolase, vitamin E and lipoic acid.

37. The method of claim 24, wherein said recombinant nucleic acid molecule in step (a) further comprises a nucleic acid sequence that hybridizes with a target nucleic acid sequence in said microorganism such that a gene comprising said target nucleic acid sequence is mutated or inactivated by homologous recombination with said second nucleic acid sequence.

38. The method of claim 37, wherein said target nucleic acid sequence encodes a protein selected from the group consisting of lipases, fatty acid oxidation enzymes, proteins involved in carbohydrate synthesis, proteins involved in synthesis of products of isoprenoid pathways, proteins involved in synthesis of cell wall components, proteins involved in the saturated fatty acid synthesis pathways, proteins involved in the polyunsaturated fatty acid synthesis pathways, proteins associated with a polyketide synthase complex, and proteins associated with incorporation of fatty acids into phospholipids or triacylglycerol molecules.

39. The method of claim 24, further comprising the step of introducing into said cell at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a protein to be expressed, said nucleic acid sequence being operatively linked to a transcription control sequence.

40. The method of claim 24, further comprising the step of introducing into said cell at least one additional recombinant nucleic acid molecule comprising a second nucleic acid sequence that hybridizes with a target nucleic acid sequence in said microorganism such that a gene comprising said target nucleic acid sequence is mutated or inactivated by homologous recombination with said second nucleic acid sequence.

41. The method of claim 24, further comprising the step of introducing into said cell a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bleomycin-binding protein.

42. The method of claim 41, wherein said recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bleomycin-binding protein further comprises a nucleic acid sequence encoding a second protein to be expressed by said cell, wherein said nucleic acid sequence encoding said second protein is operatively linked to a transcription control sequence.

43. The method of claim 42, wherein said transcription control sequence is selected from the group consisting of a Thraustochytriales α

-tubulin promoter, a Thraustochytriales acetolactate synthase promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and a Thraustochytriales fatty acid desaturase promoter.

44. The method of claim 41, wherein said recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bleomycin-binding protein further comprises a second nucleic acid sequence that hybridizes with a target nucleic acid sequence in said microorganism such that a gene comprising said target nucleic acid sequence is mutated or inactivated by homologous recombination with said second nucleic acid sequence.

45. The method of claim 41, wherein said recombinant nucleic acid molecule comprises a nucleic acid sequence SEQ ID NO:

9.

46. The method of claim 24, wherein said microorganism is from a genus selected from the group consisting of Thraustochytrium, Labyrinthuloides, Japonochytrium, and Schizochytrium.

47. The method of claim 24, wherein said microorganism is from a species selected from the group consisting of Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium minutum, Thraustochytrium sp., Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum, and Japonochytrium sp.

48. The method of claim 24, wherein said step of introducing is performed by a method selected from the group consisting of particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.

49. A recombinant microorganism of the order Thraustochytriales, transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an acetolactate synthase that confers onto said microorganism reduced sensitivity to compounds selected from the group consisting of:

sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;

a. an amino acid sequence selected from the group consisting of SEQ ID NO;

19, SEQ ID NO;

22 and SEQ ID NO;

24; and

,

b. an amino acid sequence that is at least about 75% identical to an amino acid sequence of (a).

50. The recombinant microorganism of claim 49, wherein said nucleic acid sequence encodes an acetolactate synthase having an amino acid sequence that is at least about 85% identical to an amino acid sequence of (a).

51. The recombinant microorganism of claim 49, wherein said nucleic acid sequence encodes an acetolactate synthase having an amino acid sequence that is at least about 95% identical to an amino acid sequence of (a).

52. The recombinant microorganism of claim 49, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of SEQ ID NO:

19, SEQ ID NO;

22 and SEQ ID NO;

24.

53. The recombinant microorganism of claim 49, wherein said nucleic acid sequence is selected from the group consisting of:

nucleotides 1260-3314 of SEQ ID NO;

18, nucleotides 1260-3314 of SEQ ID NO;

21, and nucleotides 1260-3314 of SEQ ID NO;

23.

54. The recombinant microorganism of claim 49, wherein said recombinant nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

18, SEQ ID NO;

21 and SEQ ID NO;

23.

55. The recombinant microorganism of claim 49, wherein said nucleic acid sequence encoding an acetolactate synthase is operatively linked to a promoter that functions in a Thraustochytriales microorganism.

56. The recombinant microorganism of claim 49, wherein said nucleic acid sequence encoding an acetolactate synthase is operatively linked to a transcription control sequence selected from the group consisting of a Thraustochytriales α

-tubulin promoter, a Thraustochytriales acetolactate synthase promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and a Thraustochytriales fatty acid desaturase promoter.

57. The recombinant microorganism of claim 49, wherein said recombinant nucleic acid molecule further comprises a nucleic acid sequence encoding a first protein for production by said microorganism, wherein said nucleic acid sequence encoding said first protein is operatively linked to a transcription control sequence.

58. The recombinant microorganism of claim 49, wherein said recombinant cell is further transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bleomycin-binding protein.

59. The recombinant microorganism of claim 58, wherein said recombinant nucleic acid molecule comprises a nucleic acid sequence SEQ ID NO:

9.

60. The recombinant microorganism of claim 58, wherein said recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bleomycin-binding protein further comprises a nucleic acid sequence encoding a second protein for production by said cell, wherein said nucleic acid sequence encoding said second protein is operatively linked to a transcription control sequence.

61. The recombinant microorganism of claim 49, further comprising at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a protein for production by said cell.

62. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

a. nucleotides 441-894 of SEQ ID NO;

9;

b. a nucleic acid sequence that is at least about 95% identical to nucleotides 441-894 of SEQ ID NO;

9 over the full length of nucleotides 441-894 of SEQ ID NO;

9, wherein said nucleic acid sequence has at least basal α

-tubulin promoter transcriptional activity; and

c. an isolated polynucleotide comprising a nucleic acid sequence that is fully complementary to said polynucleotide of (a) or (b).

63. The isolated nucleic acid molecule of claim 62, wherein said isolated nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

4 and nucleotides 441-894 of SEQ ID NO;

9.

64. A recombinant vector for transformation of microorganisms of the Order Thraustochytriales, comprising a nucleic acid sequence encoding a bleomycin binding protein operatively linked to a promoter selected from the group consisting of a Thraustochytriales α

-tubulin promoter, a Thraustochytriales acetolactate synthase promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and a Thraustochytriales fatty acid desaturase promoter.

65. The recombinant vector of claim 64, wherein said Thraustochytriales acetolactate synthase promoter comprises nucleotides 1-1259 of SEQ ID NO:

14.

66. The recombinant vector of claim 64, wherein said α

-tubulin promoter comprises a nucleic acid sequence selected from the group consisting of nucleotides 441-894 of SEQ ID NO;

9, and a nucleic acid sequence that is at least about 95% identical to nucleotides 441-894 of SEQ ID NO;

9 over the full length of nucleotides 441-894 of SEQ ID NO;

9, wherein said nucleic acid sequence has at least basal α

-tubulin promoter transcriptional activity.

67. The recombinant vector of claim 64, wherein said recombinant vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

8 and SEQ ID NO;

9.

68. The recombinant vector of claim 64, wherein said promoter from a Thraustochytriales polyketide synthase (PKS) system comprises SEQ ID NO:

34 or a fragment thereof that has at least basal PKS promoter transcriptional activity.