Engineered Light-Emitting Reporter Genes

• US 20090081715A1
• Filed: 09/05/2008
• Published: 03/26/2009
• Est. Priority Date: 09/07/2007
• Status: Abandoned Application

• Current Assignee: Cobalt Technologies, Inc.
• Original Assignee: Cobalt Technologies, Inc.
• Inventors: Contag, Pamela Reilly, Burns-Guydish, Stacy M.
• Application Number:
  US12/205,845
• Publication Number:
  US 20090081715A1
• Time in Patent Office: Days
• US Class Current: 435/8
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Compositions and methods are provided for enhanced expression of light emitting reporters. Such reporters are used in methods for monitoring cultures for production of target compounds.

Claims

1. An isolated non-natural nucleic acid molecule comprising a nucleotide sequence encoding a light-emitting reporter, wherein the nucleotide sequence has an A/T content between about 62% and about 75%.

2. The nucleic acid molecule of claim 1 wherein the A/T content is between 65% and 75%.

3. The nucleic acid molecule of claim 1 wherein the light-emitting report is luciferase.

4. The nucleic acid molecule of claim 1 wherein the light-emitting report is self-contained.

5. The nucleic acid molecule of claim 1 wherein the nucleotide sequence encodes a Lux A polypeptide and/or a Lux B polypeptide.

6. The nucleic acid molecule of claim 1 wherein the nucleotide sequence encodes a Lux A polypeptide having the amino acid sequence of SEQ ID NO:

2.

7. The nucleic acid molecule of claim 6 wherein the nucleotide sequence comprises SEQ ID NO:

1.

8. The nucleic acid molecule of claim 1 wherein the nucleotide sequence encodes a Lux B polypeptide having the amino acid sequence of SEQ ID NO:

4.

9. The nucleic acid molecule of claim 8 wherein the nucleotide sequence comprises SEQ ID NO:

3.

10. The nucleic acid molecule of claim 1 wherein the nucleotide sequence is a polycistronic sequence.

11. The nucleic acid molecule of claim 10 wherein the polycistronic sequence encodes a lux A polypeptide and a lux B polypeptide.

12. The nucleic acid molecule of claim 11 wherein the lux A polypeptide and the lux B polypeptide are from Photorhabdus luminescens, or Vibrio fischeri.

13. The nucleic acid molecule of claim 11 wherein the lux A polypeptide and the lux B polypeptide are from a genus of organisms selected from a group consisting of Photorhabdus, Kenorhabdus, or Vibrio.

14. The nucleic acid molecule of claim 10 wherein the polycistronic sequence encodes a lux A polypeptide, a lux B polypeptide, a lux C polypeptide, a lux D polypeptide and a lux E polypeptide.

15. The nucleic acid molecule of claim 14 wherein the lux polypeptides are from Photorhabdus luminescens.

16. The nucleic acid molecule of claim 12 wherein the lux C polypeptide has the amino acid sequence of SEQ ID NO:

6, the lux D polypeptide has the amino acid sequence of SEQ ID NO;

5 and the lux E polypeptide has the amino acid sequence of SEQ ID NO;

10.

17. The nucleic acid molecule of claim 16 wherein the nucleotide sequence encoding the lux C polypeptide comprises SEQ ID NO:

6, the nucleotide sequence encoding the lux D polypeptide comprises SEQ ID NO;

8 and the nucleotide sequence encoding the lux E polypeptide comprises SEQ ID NO;

10.

18. The nucleic acid molecule of claim 1 wherein the nucleotide sequence encodes a lux A polypeptide and/or a lux B polypeptide having a non-natural amino acid sequence.

19. A recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%.

20. The recombinant nucleic acid molecule of claim 19 wherein the A/T content is between 65% and 75%.

21. The recombinant nucleic acid molecule of claim 19 wherein the light emitting report is luciferase.

22. The recombinant nucleic acid molecule of claim 19 which is a plasmid.

23. The recombinant nucleic acid molecule of claim 19 which is a transposon.

24. The recombinant nucleic acid molecule of claim 19 wherein the expression control sequence functions in a gram positive bacterium.

25. The recombinant nucleic acid molecule of claim 19 wherein the expression control sequence comprises a promoter that is functional in Clostridium.

26. The recombinant nucleic acid molecule of claim 24 wherein the promoter selected from the group consisting of promoters of genes for butanol dehydrogenase, butyraldehyde dehydrogenase, ethanol dehydrogenase, acid aldehyde dehydrogenase, acetoacetate decarboxylase, butyrate kinase, phosphobutyryltransferase, phosphotransacetylase, acetate kinase, acyl CoA transferase, lactate dehydrogenase and butryl CoA transferase.

27. The recombinant nucleic acid molecule of claim 19 comprising an operon wherein the coding nucleotide sequence is a polycistronic sequence.

28. The recombinant nucleic acid molecule of claim 27 wherein the polycistronic sequence encodes a lux A polypeptide and a lux B polypeptide.

29. The recombinant nucleic acid molecule of claim 28 wherein the polycistronic sequence further encodes a lux C polypeptide, a lux D polypeptide and a lux E polypeptide.

30. The recombinant nucleic acid molecule of claim 29 wherein the polycistronic sequence comprises SEQ ID NO:

12.

31. The recombinant nucleic acid molecule of claim 29 wherein the polycistronic sequence further encodes a lux R polypeptide and a lux I polypeptide.

32. The recombinant nucleic acid molecule of claim 27 wherein the expression control sequence comprises a Shine-Dalgarno sequence (AGGAGG) operatively linked with each cistron.

33. The recombinant nucleic acid molecule of claim 19 comprising a first restriction sequence upstream of the expression control sequence, a second restriction sequence between a promoter of the expression control sequence and the coding nucleotide sequence and a third restriction sequence downstream of the coding nucleotide sequence.

34. The recombinant nucleic acid molecule of claim 28 comprising a first restriction sequence and a second restriction sequence upstream and downstream, respectively, of the sequence encoding the lux A polypeptide and the lux B polypeptide.

35. A recombinant cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%.

36. The recombinant cell of claim 35 wherein the cell is a Clostridium cell.

37. The recombinant cell of claim 36 wherein Clostridium is C. acetobutylicum, C. perfringens, C. saccharobutylicum, C. puniceum, C. saccharoperobutylicum or C. beijerinckii.

38. The recombinant cell of claim 35 wherein the cell is and other bacteria with an AT rich DNA.

39. The recombinant cell of claim 35 wherein the recombinant nucleic acid is not integrated into the cell genome.

40. The recombinant cell of claim 35 wherein the recombinant nucleic acid is integrated into the cell genome.

41. The recombinant cell of claim 35 comprising a plurality of different recombinant nucleic acid molecules, wherein the different recombinant nucleic acid molecules comprise different expression control sequences and different coding nucleotide sequences encoding light-emitting reporters that report light of different wavelengths.

42. An isolated polypeptide comprising a light-emitting reporter, wherein the polypeptide is encoded by a nucleotide sequence having an A/T content from between about 62% and about 75%.

43. The polypeptide of claim 42 wherein the A/T content is between 65% and 75%.

44. The polypeptide of claim 42 wherein the light-emitting reporter is luciferase.

45. The polypeptide of claim 42 wherein the light-emitting report is self-contained.

46. The polypeptide of claim 42 further comprising a Lux A polypeptide and/or a Lux B polypeptide.

47. The polypeptide of claim 42 further having the amino acid sequence of SEQ ID NO:

2.

48. The polypeptide of claim 47 encoded by the nucleotide sequence of SEQ ID NO:

1.

49. The polypeptide of claim 42 further having the amino acid sequence of SEQ ID NO:

4.

50. The polypeptide of claim 42 encoded by the nucleotide sequence comprises SEQ ID NO:

3.

51. The polypeptide of claim 42 wherein the nucleotide sequence is a polycistronic sequence encoding luciferase.

52. The polypeptide of claim 51 wherein the polycistronic sequence encodes a lux A polypeptide and a lux B polypeptide.

53. The polypeptide of claim 52 wherein the lux A polypeptide and the lux B polypeptide are from Photorhabdus luminescens, or Vibrio fischeri.

54. The polypeptide of claim 52 wherein the lux A polypeptide and the lux B polypeptide are from a genus of organisms selected from a group consisting of Photorhabdus, Kenorhabdus, and Vibrio.

55. The polypeptide of claim 51 wherein the polycistronic sequence encodes a lux A polypeptide, a lux B polypeptide, a lux C polypeptide, a lux D polypeptide and a lux E polypeptide.

56. The polypeptide of claim 55 wherein the lux polypeptides are from Photorhabdus luminescens.

57. A method comprising:

a) culturing a recombinant cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%; and

b) measuring the light emitted from the reporter in the culture.

58. The method of claim 57 wherein the light-emitting reporter is self-contained.

59. The method of claim 57 wherein the cell is Clostridium and the expression control sequence comprises a Clostridium promoter.

60. The method of claim 57 wherein the expression control sequence is from a low-GC bacteria.

61. The method of claim 60 wherein the light-emitting reporter is from Photorhabdus luminescens.

68. The method of claim 60, wherein said bacteria is Clostridium.

62. A method for regulating fermentation in a bacterial cell culture comprising;

monitoring expression of a light emitting reporter in bacteria in said culture, wherein said light emitting reporter is encoded by a nucleic acid sequence comprising total A/T content of about 62% to about 75%;

wherein said bacteria is low-GC bacteria; and

regulating conditions in said culture based on said monitoring.

63. A method for identifying and/or optimizing fermentation culture conditions comprising:

culturing a plurality of cultures, wherein the bacteria are the same, wherein the culture conditions are different, and wherein one culture condition serves as a control condition;

monitoring the expression of a light emitting reporter in said bacteria in said cultures, wherein said light emitting reporter is encoded by a nucleic acid sequence comprising total A/T content of about 62% to about 75%; and

wherein said bacteria is low-GC bacteria; and

identifying said cultures that have a higher expression of the light emitting reporter compared to a control culture.

64. The method of claim 63, wherein said cultures with a higher expression of the light emitting report compared to the control culture indicate culture conditions that will result in higher productivity than a control culture condition.

65. The method of claim 63, wherein the culture conditions vary by nutrient, vitamin, mineral, salt, or cofactor composition.

66. The method of claim 63, wherein the culture conditions vary by a physical parameter selected from temperature, pH, oxygen partial pressure, osmotic pressure, or dilution rate of said culture.

67. A method for identifying mutants with higher productivity comprising:

mutagenizing a plurality of bacteria that express a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%;

isolating pure cultures derived from individual mutants;

culturing the pure cultures of mutants;

measuring the light emitted from the reporter in the cultures; and

selecting mutants that have a higher emission of light than an unmutagenized parent strain.

69. A kit comprising:

a) a first container containing a first nucleic acid molecule comprising an expression control sequence and

b) a second container containing a second nucleic acid molecule comprising a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 65% and about 75%;

c) wherein the first and second nucleic acid molecules comprise compatible restriction sequences which, when the first and second nucleic acid molecules are ligated together, put the expression control sequence in operative linkage with the coding nucleotide sequence and create a restriction sequence.

70. A kit containing:

a) a Clostridium cell; and

b) recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%.