NUCLEIC ACIDS AND VECTORS FOR USE WITH METHANOTROPHIC BACTERIA

• US 20170121718A1
• Filed: 06/18/2015
• Published: 05/04/2017
• Est. Priority Date: 06/18/2014
• Status: Active Grant

• Current Assignee: Calysta Incorporated
• Original Assignee: Calysta Incorporated
• Inventors: Saville, Renee M., Silverman, Joshua, Minshull, Jeremy, Ness, Jon Edward, Leonard, Effendi, Welch, Mark, Stumpe, Jana
• Granted Patent: US 10,443,060 B2
• Time in Patent Office: Days
• 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 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Y 101/01244 Methanol dehydrogenase (1.1.1.244)

Abstract

The present disclosure provides nucleic acids and vectors for use with methanotrophic bacteria. Related host cells and methods for using such nucleic acids and vectors for expressing polypeptides or other genetic manipulation of methanotrophic bacteria are also provided. In one aspect, the present disclosure is directed to a non-naturally occurring nucleic acid molecule, comprising (1) a promoter that is functional in a methanotrophic bacterium, and (2) a native or altered methanol dehydrogenase (MDH) ribosomal binding sequence, provided that when the promoter is an MDH gene promoter, the nucleic acid comprises an altered MDH ribosomal binding sequence.

Claims

1. A non-naturally occurring nucleic acid molecule, comprising:

(1) a promoter that is functional in a methanotrophic bacterium, and

(2) a native or altered methanol dehydrogenase (MDH) ribosomal binding sequence,

provided that when the promoter is an MDH gene promoter, the nucleic acid comprises an altered MDH ribosomal binding sequence.

2. The nucleic acid of claim 1, wherein the promoter is a constitutive promoter.

3. The nucleic acid of claim 2, wherein the constitutive promoter is an MDH promoter, 30S ribosomal protein S16 promoter, 30S ribosomal protein S12 promoter, 50S ribosomal protein L13 promoter, hexulose 6-phosphate synthase promoter, T5 promoter, or Trc promoter.

4. The nucleic acid of claim 3, wherein the MDH promoter comprises a promoter portion of SEQ ID NO.:

14, the 30S ribosomal protein S16 promoter comprises a promoter portion of SEQ ID NO.;

15, the 30S ribosomal protein S12 promoter comprises a promoter portion of SEQ ID NO.;

43, the 50S ribosomal protein L13 promoter comprises a promoter portion of SEQ ID NO.;

44, the hexulose 6-phosphate synthase promoter comprises a promoter portion of SEQ ID NO.;

16, the T5 promoter comprises a promoter portion of SEQ ID NO.;

17, and the Trc promoter comprises a promoter portion of SEQ ID NO.;

18.

5. The nucleic acid of claim 1, wherein the promoter is an inducible promoter.

6. The nucleic acid of any one of claims 1 to 5, comprising a native MDH ribosomal binding sequence.

7. The nucleic acid of claim 6, wherein the native MDH ribosomal binding sequence comprises SEQ ID NO.:

40.

8. The nucleic acid of any one of claims 1 to 5, comprising an altered MDH ribosomal binding sequence.

9. The nucleic acid of claim 8, wherein the altered MDH ribosomal binding sequence encodes (1) a native MDH ribosomal binding site and a variant spacer, (2) a variant MDH ribosomal binding site and a native spacer, or (3) a variant MDH ribosomal binding site and a variant spacer.

10. The nucleic acid of claim 9, wherein the altered MDH ribosomal binding sequence comprises SEQ ID NO.:

20, 22, 24, 26, 27, 31, 32, or 41.

15. The nucleic acid of any one of claims 1-14, further comprising a nucleic acid molecule encoding a target polypeptide operably linked to the promoter sequence and the ribosomal binding sequence.

16. The nucleic acid of claim 15, wherein the target polypeptide is a marker or reporter protein.

17. The nucleic acid of claim 15, wherein the target polypeptide is a protein capable of improving production of a desired chemical or metabolite.

18. The nucleic acid of claim 17, wherein the target polypeptide is an isoprene synthase or lactate dehydrogenase.

19. The nucleic acid of claim 15, wherein the target polypeptide is Lad repressor protein or a BenR activator protein.

20. The nucleic acid of claim 19, wherein the nucleic acid further comprises a second promoter, a lac operator to which Lad repressor protein is capable of binding, and a sequence encoding a second target polypeptide, wherein the second promoter and the lac operator are operably linked to the nucleic acid molecule encoding the second target polypeptide.

21. The nucleic acid of claim 20, wherein the second target polypeptide is a marker or reporter protein.

22. The nucleic acid of claim 20, wherein the second target polypeptide is a protein capable of improving production of a desired chemical or metabolite.

23. The nucleic acid of claim 22, wherein the second target polypeptide is an isoprene synthase or lactate dehydrogenase.

24. The nucleic acid of any one of claims 20-23, wherein the second promoter is an MDH promoter.

25. The nucleic acid of claim 24, wherein the second promoter comprises an MDH promoter portion of SEQ ID NO.:

14.

28. The nucleic acid of claim 15, wherein the target polypeptide is a BenR activator protein.

29. The nucleic acid of claim 28, wherein the nucleic acid further comprises a second promoter and a nucleic acid molecule encoding a second target polypeptide, wherein the second promoter is operably linked to the nucleic acid molecule encoding the second target polypeptide.

30. The nucleic acid of claim 29, wherein the second target polypeptide is a marker or reporter protein.

31. The nucleic acid of claim 29, wherein the second target polypeptide is a protein capable of improving production of a desired chemical or metabolite.

32. The nucleic acid of claim 29, wherein the second target polypeptide is an isoprene synthase or lactate dehydrogenase.

33. The nucleic acid of any one of claims 28-32, wherein the second promoter is a P_ben promoter.

34. The nucleic acid of claim 33, wherein the P_ben promoter comprises a promoter portion of SEQ ID NO.:

48.

38. A vector, comprising the nucleic acid of any one of claims 1 to 37.

39. A host cell, comprising the nucleic acid of any one of claims 1 to 37.

40. The host cell of claim 39, wherein the nucleic acid is integrated into the genome of the host cell.

41. The host cell of claim 39, wherein the nucleic acid is present in a vector.

42. The host cell of claim 39, wherein the host cell is a Methylococcus or Methylosinus.

43. The host cell of claim 39, wherein the host cell is Methylococcus capsulatus or Methylosinus trichosporium.

44. The host cell of claim 39, wherein the host cell is Methylococcus capsulatus Bath or Methylosinus trichosporium OB3b.

45. The host cell of any one of claims 39-44, wherein the host comprises the nucleic acid of any one of claims 15-18.

46. The host cell of any one of claims 39-44, wherein the host comprises the nucleic acid of any one of claim 20, 26, or 27.

47. The host cell of any one of claims 39-44, wherein the host comprises the nucleic acid of any one of claim 33, 35, 36, or 37.

48. A method for producing a target polypeptide, comprising culturing the host cell of claim 45 under conditions and for a time sufficient to express the target polypeptide.

49. The method of claim 48, further comprising purifying the target polypeptide.

50. A method for producing a target polypeptide, comprising culturing the host cell of claim 46 in the presence of an inducer that binds to the lad repressor protein to express the second target polypeptide.

51. A method for producing a target polypeptide, comprising culturing the host cell of claim 47 in the presence of an inducer that binds to the BenR protein to express the second target polypeptide.

64. The vector of any one of claims 52-63, further comprising (6) the nucleic acid of any one of claims 1 to 37.

70. The host cell of any one of claims 66-69, wherein the vector comprises the nucleic acid of any one of claims 15-18.

71. The host cell of any one of claims 66-69, wherein the vector comprises the nucleic acid of claim 20.

72. The host cell of any one of claims 66-69, wherein the vector comprises the nucleic acid of any one of claims 20-27.

73. The host cell of any one of claims 66-69, wherein the vector comprises the nucleic acid of any one of claims 28-37.

74. A method for producing a target polypeptide, comprising culturing the host cell of claim 70 to express the target polypeptide.

75. The method of claim 74, further comprising purifying the target polypeptide.

76. A method for producing a target polypeptide, comprising culturing the host cell of claim 72 in the presence of an inducer that binds to lad repressor protein to express the second target polypeptide.

77. A method for producing a target polypeptide, comprising culturing the host cell of claim 73 in the presence of an inducer that binds to BenR activator protein to express the second target polypeptide.

11. A non-naturally occurring nucleic acid molecule, comprising:

(1) a promoter selected from an MDH promoter, 30S ribosomal protein S16 promoter, 30S ribosomal protein S12 promoter, 50S ribosomal protein L13 promoter, hexulose 6-phosphate synthase promoter, T5 promoter, or Trc promoter, and

(2) a ribosomal binding sequence that is functional in a methanotrophic bacterium,

wherein if the promoter sequence and the ribosomal binding sequence are from the same gene, then at least one of the sequences are not a native sequence.

12. The nucleic acid of claim 11, wherein the MDH promoter comprises a promoter portion of SEQ ID NO.:

14, the 30S ribosomal protein S16 promoter comprises a promoter portion of SEQ ID NO.;

15, the 30S ribosomal protein S12 promoter comprises a promoter portion of SEQ ID NO.;

43, the 50S ribosomal protein L13 promoter comprises a promoter portion of SEQ ID NO.;

44, the hexulose 6-phosphate synthase promoter comprises a promoter portion of SEQ ID NO.;

16, the T5 promoter comprises a promoter portion of SEQ ID NO.;

17, and the Trc promoter comprises a promoter portion of SEQ ID NO.;

18.

13. The nucleic acid of claim 11 or claim 12, wherein the ribosomal binding sequence is a native ribosomal binding sequence.

14. The nucleic acid of claim 11 or claim 12, wherein the ribosomal binding sequence is an altered ribosomal binding sequence.

26. A non-naturally occurring nucleic acid, comprising:

(1) an inducible promoter that comprises an operator of the lac operon and is functional in a methanotrophic bacterium;

(2) a first ribosomal binding sequence;

(3) a nucleic acid molecule encoding a target protein;

(4) a constitutive promoter that is functional in a methanotrophic bacterium;

(5) a second ribosomal binding sequence; and

(6) a nucleic acid molecule encoding a Lad protein;

wherein the nucleic acid sequence encoding the target protein (3) is operably linked to the inducible promoter (1) and the ribosomal biding sequence (2), and the nucleic acid molecule encoding a repressor or activator protein (6) is operably linked to the constitutive promoter (4) and second ribosomal binding sequence (5).

27. A non-naturally occurring nucleic acid, comprising:

(1) an MDH promoter flanked by two or more operators of the lac operon;

(2) a first MDH ribosomal binding sequence;

(3) a nucleic acid molecule encoding a target polypeptide operably linked to sequences (1) and (2);

(4) a 30S ribosomal protein S16 promoter;

(5) a second MDH ribosomal binding sequence; and

(6) a nucleic acid molecule encoding a repressor or activator protein operably linked to promoter (4) and ribosomal binding sequence (5).

35. A non-naturally occurring nucleic acid, comprising:

(1) an inducible promoter that comprises a promoter region of the benzoate metabolic operon and is functional in a methanotrophic bacterium;

(2) a first ribosomal binding sequence;

(3) a nucleic acid molecule encoding a target protein;

(4) a constitutive promoter that is functional in a methanotrophic bacterium;

(5) a second ribosomal binding sequence; and

(6) a nucleic acid molecule encoding a BenR protein;

wherein the nucleic acid molecule encoding the target protein (3) is operably linked to the inducible promoter (1) and the first ribosomal binding sequence (2), and the nucleic acid molecule encoding the BenR protein (6) is operably linked to the constitutive promoter (4) and the second ribosomal binding sequence (5).

36. A non-naturally occurring nucleic acid, comprising:

(1) a ribosomal protein promoter;

(2) an MDH ribosomal binding sequence;

(3) a nucleic acid molecule encoding BenR, operably linked to promoter (1) and ribosomal binding sequence (2);

(4) a P_ben promoter;

(5) a benA ribosomal binding sequence; and

(6) a nucleic acid molecule encoding a target protein operably linked to promoter (4) and ribosomal binding sequence (5).

37. A non-naturally occurring nucleic acid of 36, wherein the target protein is an enzyme.

52. A vector, comprising:

(1) a beta-lactamase (bla) or neomycin phosphotransferase (neo) promoter sequence operably linked to a sequence encoding a TrfA protein, and

(2) an origin of vegetative replication (oriV) that is functional in a methanotrophic bacterium.

53. The vector of claim 52, wherein the vector comprises the bla promoter sequence as set forth in SEQ ID NO.:

6.

54. The vector of claim 52, wherein the vector comprises the neo promoter sequence as set forth in SEQ ID NO.:

7.

55. The vector of claim 52, wherein the sequence encoding the TrfA protein comprises SEQ ID NO.:

3.

56. The vector of any one of claims 52-55, wherein the oriV comprises SEQ ID NO.:

1 or SEQ ID NO.;

2.

57. The vector of any one of claims 52-56, further comprises (3) an origin of transfer (oriT).

58. The vector of claim 57, wherein the oriT comprises SEQ ID NO.:

8.

59. The vector of any one of claims 52-58, wherein the vector further comprises (4) a kanamycin resistance cassette.

60. The vector of claim 59, wherein the kanamycin resistance cassette comprises a promoter sequence as set forth in SEQ ID NO.:

9 or 11 and a sequence encoding kanamycin resistant protein as set forth in SEQ ID NO.;

10 or 12.

61. The vector of any one of claims 52-60, further comprises (5) a second origin of replication that is functional in a second bacterium, wherein the second origin of replication is different from the oriV or oriT, and the second bacterium is not a methanotrophic bacterium.

62. The vector of claim 61, wherein the second bacterium is E. coli.

63. The vector of claim 61, wherein the origin of replication comprises SEQ ID NO.:

13.

65. The vector of any one of claims 52-64, wherein the total length of components (1) to (5), if present, is no more than 8 kb.

66. A host cell, comprising the vector of any one of claims 52-65.

67. The host cell of claim 66, wherein the host cell is a Methylococcus or Methylosinus.

68. The host cell of claim 66, wherein the host cell is Methylococcus capsulatus or Methylosinus trichosporium.

69. The host cell of claim 66, wherein the host cell is Methylococcus capsulatus Bath or Methylosinus trichosporium OB3b.