IP Library › Granted Patent US 9,249,430
Granted Patent B2
US 9,249,430 · App. 14/232,011 · Granted Feb 2, 2016

Recombinant microorganisms having phosphoketolase activity and diminished phosphofructokinase activity and/or glucose-6 phosphate dehydrogenase activity

Inventor: Philippe Marliere (Mouscron, BE)
Assignee: Scientist of Fortune, S.A.
C12P5/026C12N9/16C12N9/88C12N15/63C12P7/28C12P19/32C12Y301/03011C12Y401/02009C12Y401/02022Y02P20/52
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Quick Facts
Patent No.
US 9,249,430
App. No.
14/232,011
Granted
Feb 2, 2016
Kind
B2
Abstract

Described are recombinant microorganisms characterized by having phosphoketolase activity, having a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene(s) encoding phosphofructokinase or by reducing phosphofructokinase activity as compared to a non-modified microorganism and having a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or by reducing glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism. These microorganisms can be used for the production of useful metabolites such as acetone, isobutene or propene.

Claims (99)

1. A genetically modified prokaryotic microorganism comprising:

(a) increased phosphoketolase activity as compared to a non-genetically modified microorganism; and

(b) (i) diminished or inactivated phosphofructokinase activity as compared to a non-genetically modified microorganism; or

(ii) not possessing phosphofructokinase activity; and

(c) (i) diminished or inactivated glucose-6-phosphate dehydrogenase activity as compared to a non-genetically modified microorganism; or

(ii) not possessing glucose-6-phosphate dehydrogenase activity; and either

(d) (i) reduced glyceraldehyde 3-phosphate dehydrogenase activity as compared to a non-genetically modified microorganism; or

(ii) increased fructose-1,6-bisphosphate phosphatase activity when grown on glucose as compared to a non-genetically modified microorganism.

2. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to reduce phosphofructokinase activity as compared to a non-genetically modified microorganism.

3. The genetically modified prokaryotic microorganism of claim 2 , wherein a gene encoding a phosphofructokinase is genetically modified so as to reduce phosphofructokinase activity as compared to the non-genetically modified microorganism.

4. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to inactivate phosphofructokinase activity as compared to the non-genetically modified microorganism.

5. The genetically modified prokaryotic microorganism of claim 4 , wherein a gene encoding a phosphofructokinase is inactivated.

6. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to reduce glucose-6-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

7. The genetically modified prokaryotic microorganism of claim 6 , wherein a gene encoding a glucose-6-phosphate dehydrogenase is genetically modified so as to reduce glucose-6-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

8. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to inactivate glucose-6-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

9. The genetically modified prokaryotic microorganism of claim 8 , wherein a gene encoding a glucose-6-phosphate dehydrogenase is inactivated.

10. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to reduce glyceraldehyde 3-phosphate dehydrogenase activity as compared to a non-genetically modified microorganism.

11. The genetically modified prokaryotic microorganism of claim 10 , wherein a gene encoding a glyceraldehyde 3-phosphate dehydrogenase is genetically modified so as to reduce glyceraldehyde 3-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

12. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to inactivate glyceraldehyde 3-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

13. The genetically modified prokaryotic microorganism of claim 12 , wherein a gene encoding a glyceraldehyde 3-phosphate dehydrogenase is inactivated.

14. The genetically modified prokaryotic microorganism of claim 1 , wherein said genetically modified prokaryotic microorganism has increased fructose-1,6-bisphosphate phosphatase activity as compared to a non-genetically modified microorganism when grown on glucose.

15. The genetically modified prokaryotic microorganism of claim 14 , which has been genetically modified to have increased fructose-1,6-bisphosphate phosphatase activity as compared to a non-genetically modified microorganism when grown on glucose.

16. The genetically modified prokaryotic microorganism of claim 15 , wherein the genetically modified prokaryotic microorganism is further genetically modified so as to reduce glyceraldehyde 3-phosphate dehydrogenase activity as compared to a non-genetically modified microorganism.

17. The genetically modified prokaryotic microorganism of claim 16 wherein a gene encoding a glyceraldehyde 3-phosphate dehydrogenase is genetically modified so as to reduce glyceraldehyde 3-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

18. The genetically modified prokaryotic microorganism of claim 15 , wherein said genetically modified prokaryotic microorganism has been transformed with a nucleic acid encoding a fructose-1,6-bisphosphate phosphatase so as to have increased fructose-1,6-bisphosphate phosphatase activity when grown on glucose as compared to the non-genetically modified microorganism.

19. The genetically modified prokaryotic microorganism of claim 1 , wherein said genetically modified prokaryotic microorganism is a bacterium.

20. The genetically modified prokaryotic microorganism of claim 1 , wherein a gene encoding a PEP-dependent PTS transporter has been inactivated.

21. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into acetone.

22. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into isobutene.

23. The genetically modified prokaryotic microorganism of claim 1 , wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into propene.

24. The genetically modified prokaryotic microorganism of claim 1 wherein the genetically modified prokaryotic microorganism is capable of converting glucose into acetyl-CoA.

25. A method for producing acetyl-CoA comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 24 in a suitable medium; and

(b) optionally recovering said acetyl-CoA.

26. A method for producing acetone comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 21 in a suitable medium; and

(b) optionally recovering said acetone from the culture medium.

27. A method for producing isobutene comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 22 in a suitable medium; and

(b) optionally recovering said isobutene.

28. A method for producing propene comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 23 in a suitable medium; and

(b) optionally recovering said propene.

29. The genetically modified prokaryotic microorganism of claim 1 , wherein the non-genetically modified microorganism does not have phosphoketolase activity.

30. The genetically modified prokaryotic microorganism of claim 29 , wherein the non-genetically modified microorganism is genetically modified so as to comprise a nucleotide sequence encoding a phosphoketolase.

31. The genetically modified prokaryotic microorganism of claim 30 , wherein the genetically modified prokaryotic microorganism is further genetically modified by mutation and selection for increased phosphoketolase activity as compared to the non-genetically modified microorganism.

32. The genetically modified prokaryotic microorganism of claim 30 , wherein the genetically modified microorganism is E. coli.

33. The genetically modified prokaryotic microorganism of claim 1 , wherein the non-genetically modified microorganism has phosphoketolase activity.

34. The genetically modified prokaryotic microorganism of claim 33 , wherein the non-genetically modified microorganism is genetically modified by mutation and selection for increased phosphoketolase activity as compared to the non-genetically modified microorganism.

35. The genetically modified prokaryotic microorganism of claim 33 , wherein the non-genetically modified microorganism is genetically modified so as to comprise a nucleotide sequence allowing for the increased expression of a phosphoketolase as compared to the non-genetically modified microorganism.

36. The genetically modified prokaryotic microorganism of claim 35 , wherein the nucleotide sequence encodes a phosphoketolase.

37. The genetically modified prokaryotic microorganism of claim 35 , wherein the nucleotide sequence comprises a heterologous expression control sequence.

38. A genetically modified prokaryotic microorganism comprising:

(a) increased phosphoketolase activity as compared to a non-genetically modified microorganism; and

(b) (i) diminished or inactivated phosphofructokinase activity as compared to a non-genetically modified microorganism; or

(ii) not possessing phosphofructokinase activity; and

(c) (i) diminished or inactivated glucose-6-phosphate dehydrogenase activity as compared to a non-genetically modified microorganism; or

(ii) not possessing glucose-6-phosphate dehydrogenase activity; and either

(d) (i) reduced glyceraldehyde 3-phosphate dehydrogenase activity as compared to a non-genetically modified microorganism; or

(ii) increased fructose-1,6-bisphosphate phosphatase activity when grown on glucose as compared to a non-genetically-modified microorganism;

wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into acetone, isobutene, or propene.

39. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to reduce phosphofructokinase activity as compared to a non-genetically modified microorganism.

40. The genetically modified prokaryotic microorganism of claim 39 , wherein a gene encoding a phosphofructokinase is genetically modified so as to reduce phosphofructokinase activity as compared to the non-genetically modified microorganism.

41. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to inactivate phosphofructokinase activity as compared to the non-genetically modified microorganism.

42. The genetically modified prokaryotic microorganism of claim 41 , wherein a gene encoding a phosphofructokinase is inactivated.

43. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to reduce glucose-6-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

44. The genetically modified prokaryotic microorganism of claim 43 , wherein a gene encoding a glucose-6-phosphate dehydrogenase is genetically modified so as to reduce glucose-6-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

45. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is genetically modified so as to inactivate glucose-6-phosphate dehydrogenase activity as compared to the non-genetically modified microorganism.

46. The genetically modified prokaryotic microorganism of claim 45 , wherein a gene encoding a glucose-6-phosphate dehydrogenase is inactivated.

47. The genetically modified prokaryotic microorganism of claim 38 , wherein said genetically modified prokaryotic microorganism has increased fructose-1,6-bisphosphate phosphatase activity as compared to a non-genetically modified microorganism when grown on glucose.

48. The genetically modified prokaryotic microorganism of claim 47 , which has been genetically modified to have increased fructose-1,6-bisphosphate phosphatase activity as compared to a non-genetically modified microorganism when grown on glucose.

49. The genetically modified prokaryotic microorganism of claim 48 , wherein said genetically modified prokaryotic microorganism has been transformed with a nucleic acid encoding a fructose-1,6-bisphosphate phosphatase so as to have fructose-1,6-bisphosphate phosphatase activity when grown on glucose.

50. The genetically modified prokaryotic microorganism of claim 38 , wherein said genetically modified prokaryotic microorganism is a bacterium.

51. The genetically modified prokaryotic microorganism of claim 38 , wherein a gene encoding a PEP-dependent PTS transporter has been inactivated.

52. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into acetone.

53. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into isobutene.

54. The genetically modified prokaryotic microorganism of claim 38 , wherein the genetically modified prokaryotic microorganism is capable of converting acetyl-CoA into propene.

55. The genetically modified prokaryotic microorganism of claim 38 wherein the genetically modified prokaryotic microorganism is capable of converting glucose into acetyl-CoA.

56. A method for producing acetyl-CoA comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 55 in a suitable medium; and

(b) optionally recovering said acetyl-CoA.

57. A method for producing acetone comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 52 in a suitable medium; and

(b) optionally recovering said acetone from the culture medium.

58. A method for producing isobutene comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 52 in a suitable medium; and

(b) optionally recovering said isobutene.

59. A method for producing propene comprising:

(a) culturing the genetically modified prokaryotic microorganism of claim 52 in a suitable medium; and

(b) optionally recovering said propene.

60. The genetically modified prokaryotic microorganism of claim 38 , wherein the non-genetically modified microorganism does not have phosphoketolase activity.

61. The genetically modified prokaryotic microorganism of claim 60 , wherein the non-genetically modified microorganism is genetically modified so as to comprise a nucleotide sequence encoding a phosphoketolase.

62. The genetically modified prokaryotic microorganism of claim 61 , wherein the genetically modified prokaryotic microorganism is further genetically modified by mutation and selection for increased phosphoketolase activity as compared to the non-genetically modified microorganism.

63. The genetically modified prokaryotic microorganism of claim 61 , wherein the genetically modified microorganism is E. coli.

64. The genetically modified prokaryotic microorganism of claim 38 , wherein the non-genetically modified microorganism has phosphoketolase activity.

65. The genetically modified prokaryotic microorganism of claim 64 , wherein the non-genetically modified microorganism is genetically modified by mutation and selection for increased phosphoketolase activity as compared to the non-genetically modified microorganism.

66. The genetically modified prokaryotic microorganism of claim 64 , wherein the non-genetically modified microorganism is genetically modified so as to comprise a nucleotide sequence allowing for the increased expression of a phosphoketolase as compared to the non-genetically modified microorganism.

67. The genetically modified prokaryotic microorganism of claim 66 , wherein the nucleotide sequence encodes a phosphoketolase.

68. The genetically modified prokaryotic microorganism of claim 66 , wherein the nucleotide sequence comprises a heterologous expression control sequence.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 15, 2014
From: MARLIERE, PHILIPPE
To: SCIENTIST OF FORTUNE, S.A.
Reel/Frame 032905/0165 →
Priority Claims (1)
EP 111735635 · Jul 12, 2011 · regional
Continuity (1)
Related Publication 20140248669A1 · Sep 4, 2014