Process and materials for production of glucosamine and N-acetylglucosamine

• US 8,124,381 B2
• Filed: 12/19/2007
• Issued: 02/28/2012
• Est. Priority Date: 07/01/2002
• Status: Active Grant

• Current Assignee: Arkion Life Sciences
• Original Assignee: Arkion Life Sciences
• Inventors: Deng, Ming-De, Angerer, J. David, Cyron, Don, Grund, Alan D., Jerrell, Thomas A. Jr., Leanna, Candice, Mathre, Owen, Rosson, Reinhardt, Running, Jeff, Severson, Dave, Song, Linsheng, Wassink, Sarah
• Primary Examiner(s): Ramirez, Delia
• Application Number:
  US11/960,516
• Publication Number:
  US 20120009627A1
• Time in Patent Office: 1,532 Days
• Field of Search: 435/84, 435/15, 435/193, 435/183, 435/325, 435/252.3, 435/252.33, 435/69.1, 435/320.1, 530/350, 536/23.2
• US Class Current: 435/84
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

A biosynthetic method for producing glucosamine and N-acetylglucosamine is disclosed. Such a method includes the fermentation of a genetically modified microorganism to produce glucosamine and/or N-acetylglucosamine. Also disclosed are genetically modified microorganisms that are useful for producing glucosamine and N-acetylglucosamine. In addition, methods of recovering N-acetylglucosamine that has been produced by a fermentation process, including methods that result in N-acetylglucosamine of high purity, are described. Also disclosed is a method to produce glucosamine from N-acetylglucosamine.

Claims

1. A method to produce N-acetylglucosamine-6-phosphate or N-acetylglucosamine by fermentation, comprising:

a) culturing in a fermentation medium a microorganism that is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate acetyltransferase that has an amino acid sequence that is at least 95% identical to SEQ ID NO;

32; and

b) collecting a product produced from the step of culturing which is selected from the group consisting of N-acetylglucosamine-6-phosphate and N-acetylglucosamine.

2. The method of claim 1, wherein the recombinant nucleic acid molecule further comprises a non-native promoter.

3. The method of claim 1, wherein the glucosamine-6-phosphate acetyltransferase has the amino acid sequence of SEQ ID NO:

32.

4. The method of claim 1, wherein expression of the recombinant nucleic acid molecule is inducible.

5. The method of claim 4, wherein expression of the recombinant nucleic acid molecule is inducible by lactose.

6. The method of claim 5, wherein the microorganism further comprises a partial or complete deletion or inactivation of a gene encoding a Lacl repressor protein.

7. The method of claim 1, wherein the microorganism further comprises a partial or complete deletion of an endogenous gene encoding a glucosamine-6-phosphate deaminase.

8. The method of claim 1, wherein the step of culturing includes the step of maintaining the carbon source at a concentration of from about 0.5% to about 5% in the fermentation medium.

9. The method of claim 1, wherein the step of culturing is performed in a fermentation medium comprising yeast extract.

10. The method of claim 1, wherein the step of culturing is performed in a fermentation medium comprising a carbon source selected from the group consisting of glucose, fructose, a pentose sugar, lactose and gluconic acid.

11. The method of claim 10, wherein the pentose sugar is selected from the group consisting of ribose, xylose, and arabinose.

12. The method of claim 1, wherein the step of culturing is performed in a fermentation medium comprising glucose and ribose.

13. The method of claim 1, wherein the step of culturing is performed in a fermentation medium comprising glucose and gluconic acid.

14. The method of claim 1, wherein the step of culturing is performed at a temperature of from about 25°

C. to about 45°

C.

15. The method of claim 1, wherein the step of culturing is performed at about 37°

C.

16. The method of claim 1, wherein the step of culturing is performed at a pH of from about pH 4 to about pH 7.5.

17. The method of claim 1, wherein the step of culturing is performed at a pH of from about pH 6.7 to about pH 7.5.

18. The method of claim 1, wherein the step of culturing is performed at a pH of from about pH 4.5 to about pH 5.

19. The method of claim 1, wherein the microorganism is a bacterium or yeast.

20. The method of claim 1, wherein the microorganism is a bacterium.

21. The method of claim 20, wherein the bacterium is a bacterium from a genus selected from the group consisting of:

Escherichia, Bacillus, Lactobacillus, Pseudomonas and Streptomyces.

22. The method of claim 20, wherein the bacterium is a bacterium from a species selected from the group consisting of:

Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa and Streptomyces lividans.

23. The method of claim 1, wherein the microorganism is a yeast.

24. The method of claim 23, wherein the yeast is a yeast from a genus selected from the group consisting of:

Saccharomyces, Candida, Hansenula, Pichia, Kluveromyces, and Phaffia.

25. The method of claim 23, wherein the yeast is a yeast from a species selected from the group consisting of:

Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Pichia pastoris, P. canadensis, Kluyveromyces marxianus and Phaffia rhodozyma.

26. The method of claim 1, wherein the microorganism is transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bacterial or yeast phosphoglucoisomerase.

27. The method of claim 26, wherein the phosphoglucoisomerase comprises the amino acid sequence of SEQ ID NO:

105.

28. The method of claim 1, wherein the microorganism further comprises a partial or complete deletion of an endogenous gene encoding a phosphofructokinase.

29. The method of claim 1, wherein the microorganism has been transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bacterial or yeast glutamine synthetase.

30. The method of claim 29, wherein the glutamine synthetase comprises the amino acid sequence of SEQ ID NO:

89.

31. The method of claim 1, wherein the microorganism has been transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a bacterial or yeast glucose-6-phosphate dehydrogenase.

32. The method of claim 21, wherein the glucose-6-phosphate dehydrogenase comprises the amino acid sequence of SEQ ID NO:

95.

33. The method of claim 1, wherein the microorganism further comprises a partial or complete deletion of at least one endogenous gene encoding an enzyme involved in glycogen synthesis selected from the group consisting of:

ADP-glucose pyrophosphorylase, glycogen synthase and a branching enzyme.

34. The method of claim 1, further comprising recovering an intracellular product from the microorganism selected from the group consisting of:

intracellular glucosamine-6-phosphate, glucosamine-1-phosphate, N-acetylglucosamine-1-phosphate, and glucosamine.

35. The method of claim 1, wherein N-acetylglucosamine produced by the fermentation method is recovered by precipitating N-acetylglucosamine-containing solids from the fermentation broth.

36. The method of claim 1, wherein N-acetylglucosamine produced by the fermentation method is recovered by crystallizing N-acetylglucosamine-containing solids from the fermentation broth.

37. The method of claim 1, further comprising the step of contacting the fermentation medium with at least one ion exchange resin.

38. A method to produce N-acetylglucosamine-6-phosphate, N-acetylglucosamine, glucosamine or glucosamine HCl by fermentation, comprising:

culturing in a fermentation medium a microorganism that is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate acetyltransferase that has an amino acid sequence that is at least 95% identical to SEQ ID NO;

32; and

collecting a product produced from the step of culturing which is selected from the group consisting of N-acetylglucosamine-6-phosphate and N-acetylglucosamine;

and wherein the method further comprises a step selected from the group consisting of;

a) purifying N-acetylglucosamine from the fermentation medium;

b) dephosphorylating N-acetylglucosamine-6-phosphate to produce N-acetylglucosamine; and

c) treating N-acetylglucosamine to produce a glucosamine product selected from the group consisting of;

glucosamine and glucosamine HCl.

39. The method of claim 38, wherein step (c) comprises hydrolyzing N-acetylglucosamine under acid and heat conditions or by enzymatic deacetylation.

40. A method to produce N-acetylglucosamine-6-phosphate or N-acetylglucosamine by fermentation, comprising:

a) culturing in a fermentation medium a microorganism that expresses;

i) a recombinant nucleic acid molecule encoding a glucosamine-6-phosphate acetyltransferase that has an amino acid sequence that is at least 95% identical to SEQ ID NO;

32; and

ii) a recombinant nucleic acid molecule encoding a glucosamine-6-phosphate synthase that has an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of;

SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, and SEQ ID NO;

20; and

b) collecting a product produced from the step of culturing which is selected from the group consisting of N-acetylglucosamine-6-phosphate and N-acetylglucosamine.

41. The method of claim 40, wherein the glucosamine-6-phosphate acetyltransferase has the amino acid sequence of SEQ ID NO:

32.

42. The method of claim 40, wherein the glucosamine-6-phosphate synthase has the amino acid sequence of SEQ ID NO:

6.

43. The method of claim 40, wherein the microorganism further comprises a partial or complete deletion of an endogenous gene encoding a phosphofructokinase.

44. The method of claim 43, wherein the microorganism further comprises a partial or complete deletion of endogenous genes encoding N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, and N-acetyl-glucosamine-specific enzyme II^Nag.

45. The method of claim 43, wherein the microorganism further comprises a partial or complete deletion of an endogenous gene encoding mannose transporter EIIM,P/III^Man.

46. The method of claim 43, wherein the recombinant nucleic acid molecules of (a)(i) and (a)(ii) are inducible by lactose or galactose.

47. The method of claim 43, wherein the step of culturing is performed in a fermentation medium comprising glucose and fructose.

48. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

4.

49. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

6.

50. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

8.

51. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

10.

52. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

12.

53. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

14.

54. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

16.

55. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

18.

56. The method of claim 40, wherein the glucosamine-6-phosphate synthase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

20.

57. The method of claim 40, wherein the microorganism is a bacterium or yeast.

58. The method of claim 40, wherein the microorganism is a bacterium.

59. The method of claim 40, wherein the microorganism is a yeast.

60. A method to produce N-acetylglucosamine-6-phosphate or N-acetylglucosamine by fermentation, comprising:

a) culturing in a fermentation medium an E. coli that expresses;

i) a recombinant nucleic acid molecule encoding a glucosamine-6-phosphate acetyltransferase that is at least 95% identical to the amino acid sequence of SEQ ID NO;

32; and

ii) a recombinant nucleic acid molecule encoding a glucosamine-6-phosphate synthase that is at least 95% identical to the amino acid sequence of SEQ ID NO;

6; and

b) collecting a product produced from the step of culturing which is selected from the group consisting of N-acetylglucosamine-6-phosphate and N-acetylglucosamine.

61. The method of claim 60, wherein the E. coli further comprises a partial or complete deletion of the pfkA gene.

62. The method of claim 61, wherein the E. coli further comprises a partial or complete deletion of the nagΔ

, nagB, and nagE genes.

63. The method of claim 61, wherein the E. coli further comprises a partial or complete deletion of the manXYZ gene.

64. The method of claim 61, wherein the recombinant nucleic acid molecules of (a)(i) and (a)(ii) are inducible by lactose or galactose.

65. The method of claim 61, wherein the step of culturing is performed in a fermentation medium comprising glucose and fructose.

66. The method of claim 60, wherein the glucosamine-6-phosphate acetyltransferase has the amino acid sequence of SEQ ID NO:

32.

67. The method of claim 60, wherein the glucosamine-6-phosphate synthase has the amino acid sequence of SEQ ID NO:

6.