Compositions and methods for producing isoprene

• US 8,288,148 B2
• Filed: 12/15/2008
• Issued: 10/16/2012
• Est. Priority Date: 12/13/2007
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Cervin, Marguerite A., Chotani, Gopal K., Feher, Frank J., McAuliffe, Joseph C., Miasnikov, Andrei, Peres, Caroline M., Puhala, Aaron S., Sanford, Karl J., Valle, Fernando, Whited, Gregory M., La Duca, Richard
• Primary Examiner(s): Desai, Anand
• Assistant Examiner(s): CHOWDHURY, IQBAL HOSSAIN
• Application Number:
  US12/335,071
• Publication Number:
  US 20090203102A1
• Time in Patent Office: 1,401 Days
• Field of Search: None
• US Class Current: 435/252.33
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/1229 Phosphotransferases with a phosphate group as acceptor (2.7.4)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12Y 203/01009 Acetyl-CoA C-acetyltransferase (2.3.1.9)
• CPC Class Codes: C12Y 207/01036 Mevalonate kinase (2.7.1.36)
• CPC Class Codes: C12Y 401/01033 Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
• CPC Class Codes: C12Y 402/03027 Isoprene synthase (4.2.3.27)
• CPC Class Codes: C12Y 503/03002 Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

The invention features methods for producing isoprene from cultured cells. The invention also provides compositions that include these cultured cells.

Claims

1. Recombinant bacterial cells capable of increased production of isoprene, the cells comprising nucleic acids encoding:

(a) an isoprene synthase polypeptide, wherein the isoprene synthase polypeptide is encoded by a heterologous nucleic acid,

(b) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or

one or more mevalonate (MVA) pathway polypeptides,

wherein the cells produce greater than 400 nmole/g_wcm/hr of isoprene.

2. The cells of claim 1, wherein the cells produce at least 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, or 12,500 nmole/g_wcm/hr of isoprene.

3. The cells of claim 1, wherein the cells produce between 400 nmole/gwcm/hr to 2.0×

105 nmole/g_wcm/hr of isoprene.

4. The cells of claim 1, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

5. The cells of claim 4, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

6. The cells of claim 4, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

7. The cells of claim 1, wherein the nucleic acid encoding an IDI polypeptide of (b) is a heterologous nucleic acid encoding an IDI polypeptide.

8. The cells of claim 7, wherein the IDI polypeptide is a yeast IDI polypeptide.

9. The cells of claim 1, wherein the nucleic acid encoding an IDI polypeptide of (b) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

10. The cells of claim 1, wherein the nucleic acid encoding a DXS polypeptide of (c) is a heterologous nucleic acid encoding a DXS polypeptide.

11. The cells of claim 1, wherein the nucleic acid encoding a DXS polypeptide of (c) is a copy of an endogenous nucleic acid encoding a DXS polypeptide.

12. The cells of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid.

13. The cells of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a copy of an endogenous nucleic acid.

14. The cells of claim 1, wherein the cells comprise two or more polypeptides of the MVA pathway.

15. The cells of claim 1, wherein the cells comprise three or more polypeptides of the MVA pathway.

16. The cells of claim 1, wherein the cells comprise four or more polypeptides of the MVA pathway.

17. The cells of claim 1, wherein the cells comprise polypeptides of the entire MVA pathway.

18. The cells of claim 1, wherein the cells comprise polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

19. The cells of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is over-expressed.

20. The cells of claim 19, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

21. The cells of claim 19, wherein the over-expressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

22. The cells of claim 1, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

23. The cells of claim 1, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

24. The cells of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is in a vector.

25. The cells of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is integrated into a chromosome of the cells.

26. The cells of claim 1, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and one or more MVA pathway polypeptides.

27. The cells of claim 26, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and polypeptides of the entire MVA pathway.

28. The cells of claim 1, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

81. The cells of claim 1 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one or more vectors.

82. The cells of claim 1 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one vector.

29. Recombinant bacterial cells capable of increased production of isoprene, the cells comprising nucleic acids encoding:

(a) an isoprene synthase polypeptide, wherein the isoprene synthase polypeptide is encoded by a heterologous nucleic acid,

(b) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or

one or more mevalonate (MVA) pathway polypeptides,

wherein the cells have an average volumetric productivity of isoprene greater than 0.1 mg/L_broth/hr.

30. The cells of claim 29, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

31. The cells of claim 30, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

32. The cells of claim 30, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

33. The cells of claim 29, wherein the nucleic acid encoding an IDI polypeptide of (b) is a heterologous nucleic acid encoding an IDI polypeptide.

34. The cells of claim 33, wherein the IDI polypeptide is a yeast IDI polypeptide.

35. The cells of claim 29, wherein the nucleic acid encoding an IDI polypeptide of (b) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

36. The cells of claim 29, wherein the nucleic acid encoding the DXS polypeptide is a heterologous nucleic acid encoding the DXS polypeptide.

37. The cells of claim 29, wherein the nucleic acid encoding the DXS polypeptide is a copy of an endogenous nucleic acid encoding the DXS polypeptide.

38. The cells of claim 29, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid.

39. The cells of claim 29, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a copy of an endogenous nucleic acid.

40. The cells of claim 29, wherein the cells comprise two or more polypeptides of the MVA pathway.

41. The cells of claim 29, wherein the cells comprise three or more polypeptides of the MVA pathway.

42. The cells of claim 29, wherein nucleic acids encode the cells comprise four or more polypeptides of the MVA pathway.

43. The cells of claim 29, wherein the cells comprise polypeptides of the entire MVA pathway.

44. The cells of claim 29, wherein the cells comprise polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

45. The cells of claim 29, wherein at least one of the nucleic acids encoding a polypeptide of any of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is over-expressed.

46. The cells of claim 45, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

47. The cells of claim 45, wherein the overexpressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

48. The cells of claim 29, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

49. The cells of claim 29, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

50. The cells of claim 29, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is in a vector.

51. The cells of claim 29, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is integrated into a chromosome of the cells.

52. The cells of claim 29, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and one or more MVA pathway polypeptides.

53. The cells of claim 52, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and (ii) polypeptides of the entire MVA pathway.

54. The cells of claim 29, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and (ii) polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

83. The cells of claim 29 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one or more vectors.

84. The cells of claim 29 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one vector.

55. Recombinant bacterial cells capable of increased production of isoprene, the cells comprising nucleic acids encoding:

(a) an isoprene synthase polypeptide, wherein the isoprene synthase polypeptide is encoded by a heterologous nucleic acid,

(b) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or

one or more mevalonate (MVA) pathway polypeptides,

wherein the cells produce at least 0.0037 grams of isoprene per gram of dry cell mass (g_dcm).

56. The cells of claim 55, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

57. The cells of claim 56, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

58. The cells of claim 56, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

59. The cells of claim 55, wherein the nucleic acid encoding an IDI polypeptide of (b) is a heterologous nucleic acid encoding an IDI polypeptide.

60. The cells of claim 59, wherein the IDI polypeptide is a yeast IDI polypeptide.

61. The cells of claim 55, wherein the nucleic acid encoding an IDI polypeptide of (b) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

62. The cells of claim 55, wherein the nucleic acid encoding the DXS polypeptide is a heterologous nucleic acid encoding the DXS polypeptide.

63. The cells of claim 55, wherein the nucleic acid encoding the DXS polypeptide is a copy of an endogenous nucleic acid encoding the DXS polypeptide.

64. The cells of claim 55, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid.

65. The cells of claim 55, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a copy of an endogenous nucleic acid.

66. The cells of claim 55, wherein the cells comprise two or more polypeptides of the MVA pathway.

67. The cells of claim 55, wherein the cells comprise three or more polypeptides of the MVA pathway.

68. The cells of claim 55, wherein the cells comprise four or more polypeptides of the MVA pathway.

69. The cells of claim 55, wherein the cells comprise polypeptides of the entire MVA pathway.

70. The cells of claim 55, wherein the cells comprise polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

71. The cells of claim 55, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is over-expressed.

72. The cells of claim 71, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

73. The cells of claim 71, wherein the overexpressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

74. The cells of claim 55, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

75. The cells of claim 55, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

76. The cells of claim 55, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is in a vector.

77. The cells of claim 55, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is integrated into a chromosome of the cells.

78. The cells of claim 55, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and one or more MVA pathway polypeptides.

79. The cells of claim 78, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and polypeptides of the entire MVA pathway.

80. The cells of claim 55, wherein the cells comprise nucleic acids encoding:

a DXS polypeptide and polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

85. The cells of claim 55 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one or more vectors.

86. The cells of claim 55 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one vector.