Hyaluronan synthase genes and expression thereof inBacillushosts

• US 7,091,008 B1
• Filed: 11/05/2004
• Issued: 08/15/2006
• Est. Priority Date: 07/01/1994
• Status: Expired due to Fees

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: DeAngelis, Paul L., Weigel, Paul H., Kumari, Kshama
• Primary Examiner(s): Wax, Robert A.
• Application Number:
  US10/981,632
• Time in Patent Office: 648 Days
• Field of Search: None
• US Class Current: 435/84
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

The present invention relates to a recombinant Bacillus host cell containing a recombinant vector including a nucleic acid segment having a coding region segment encoding enzymatically active hyaluronan synthase (HAS). The recombinant Bacillus host cell is utilized in a method for producing hyaluronic acid (HA).

Claims

1. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell comprising a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO:

14 wherein the coding region is under control of a promoter.

2. The recombinant host cell of claim 1, wherein the recombinant host cell is a Bacillus subtilis or a Bacillus licheniformis cell.

3. The recombinant host cell of claim 1, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

13.

4. The recombinant host cell of claim 3, wherein the host cell produces hyaluronic acid.

5. The recombinant host cell of claim 1, wherein the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

6. The recombinant host cell of claim 5, wherein the promoter is a Bacillus compatible promoter.

7. The recombinant host cell of claim 1, wherein the Bacillus subtilis cell further comprises a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active UDP-GlcUA biosynthetic pathway enzyme, wherein the enzymatically active UDP-GlcUA biosynthetic pathway enzyme is selected from the group consisting of UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, and combinations thereof.

8. The recombinant host cell of claim 1, wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

9. The recombinant host cell of claim 8, wherein the coding region encoding enzymatically active HAS and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of at least one copy of at least one promoter.

10. The recombinant host cell of claim 8, wherein the coding region encoding enzymatically active HAS and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

11. A method for producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO;

14 into a Bacillus host wherein the coding region is under control of a promoter;

growing the host organism in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

12. The method of claim 11, wherein the Bacillus host is Bacillus subtilis or Bacillus licheniformis.

13. The method according to claim 11, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

14. The method according to claim 13, further comprising the step of purifying the extracted hyaluronic acid.

15. The method of claim 11, further comprising the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

16. The method of claim 11, wherein the coding region encoding enzymatically active hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of the same promoter.

17. The method of claim 11, wherein the coding region encoding enzymatically active hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

18. The method of claim 11, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase, the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

13.

19. The method of claim 11, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

20. The method of claim 19, wherein the promoter is a Bacillus-compatible promoter.

21. A method for producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO;

14 into a Bacillus host wherein the coding region is under control of a promoter;

introducing a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase into the Bacillus host;

growing the Bacillus host in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

22. The method of claim 21, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase, the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

13.

23. The method of claim 21, wherein the coding region encoding enzymatically active hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of the same promoter.

24. The method of claim 21, wherein the coding region encoding enzymatically active hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

25. The method of claim 21, wherein the Bacillus host is Bacillus subtilis or Bacillis licheniformis.

26. The method according to claim 21, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

27. The method according to claim 26, further comprising the step of purifying the extracted hyaluronic acid.

28. The method of claim 21 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

29. The method of claim 28, wherein the promoter is a Bacillus-compatible promoter.

30. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell comprising:

a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO;

14; and

a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

31. The recombinant host cell of claim 30, wherein the recombinant host cell is a Bacillus subtilis or Bacillus licheniformis cell.

32. The recombinant host cell of claim 30, wherein the host cell produces hyaluronic acid.

33. The recombinant host cell of claim 30, wherein the coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

34. The recombinant host cell of claim 33, wherein the promoter is a Bacillus-compatible promoter.

35. The recombinant host cell of claim 30, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

13.

36. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell having a recombinant vector introduced therein, the recombinant vector comprising:

a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO;

14; and

a coding region encoding enzymatically active UDP-glucose dehydrogenase.

37. The recombinant host cell of claim 36, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

13.

38. The recombinant host cell of claim 36, wherein the coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of the same promoter.

39. The recombinant host cell of claim 36, wherein the coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

40. The recombinant host cell of claim 36, wherein the recombinant host cell is a Bacillus subtilis or Bacillus licheniformis cell.

41. The recombinant host cell of claim 36, wherein the host cell produces hyaluronic acid.

42. The recombinant host cell of claim 36, wherein the coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

43. The recombinant host cell of claim 42, wherein the promoter is a Bacillus-compatible promoter.

44. A recombinant host cell wherein the recombinant host cell is a Bacillus cell having enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc, the recombinant host cell further having a recombinant vector comprising a purified nucleic acid segment having a coding region encoding an enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 introduced therein.

45. The recombinant host cell of claim 44, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

13.

46. The recombinant host cell of claim 44, wherein the recombinant host cell is a Bacillus subtilis or Bacillus licheniformis cell.

47. The recombinant host cell of claim 44, wherein the recombinant host cell further includes at least one modified RNA polymerase promoter wherein, when the modified RNA polymerase promoter is recognized by an RNA polymerase, the RNA polymerase is capable of expressing RNA in an amount greater than an endogenous RNA polymerase promoter.

48. The recombinant host cell of claim 47, wherein the modification is a mutation.

49. The recombinant host cell of claim 47, wherein the modification is tandem promoter elements.

50. The recombinant host cell of claim 44, wherein the recombinant host cell is transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding a functionally active enzyme in a UDP-sugar precursor biosynthesis pathway.

51. The recombinant host cell of claim 50, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose dehydrogenase.

52. The recombinant host cell of claim 50, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose pyrophosphorylase.

53. The recombinant host cell of claim 44, wherein the recombinant host cell further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus cell.

54. The recombinant host cell of claim 44, wherein the recombinant host cell further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus cell.

55. The recombinant host cell of claim 44, wherein the recombinant host cell further includes at least one nucleic acid segment having a coding region encoding a functionally active enzyme in a UDP-sugar precursor biosynthesis pathway such that the recombinant host cell has an activity greater than a native host cell expressing an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

56. The recombinant host cell of claim 44, wherein the recombinant host cell further includes at least one mutated UDP-sugar precursor biosynthesis gene, wherein the mutated UDP-sugar precursor gene increases a half-life of a transcribed messenger RNA.

57. The recombinant host cell of claim 44, wherein the recombinant host cell further includes at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency.

58. The recombinant host cell of claim 44, wherein the mutation in the UDP-sugar precursor biosynthesis gene occurs in a ribosome binding site in the UDP-sugar precursor biosynthesis gene such that a ribosome has an increased binding affinity for the ribosome binding site.

59. A method for producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO;

14 into a Bacillus host, wherein the Bacillus host has an enhanced production of at least one of UDP-GlcUA and U DP-GlcNAc;

growing the Bacillus host in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

60. The method of claim 59, wherein in the step of introducing a purified nucleic acid segment encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14, the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO;

13.

61. The method of claim 59, wherein the Bacillus host is Bacillus subtilis or Bacillus licheniformis.

62. The method according to claim 59, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host further includes at least one modified RNA polymerase promoter having an increased promoter activity.

63. The method of claim 62, wherein the modification of the RNA polymerase promoter is a mutation.

64. The method of claim 62, wherein the modification of the RNA polymerase promoter is tandem promoter elements.

65. The method according to claim 59, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host is transformed with a vector comprising a purified nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme.

66. The method according to claim 65, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose dehydrogenase.

67. The method according to claim 65, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose pyrophosphorylase.

68. The method according to claim 59, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus host.

69. The method according to claim 59, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus host.

70. The method according to claim 59, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host further includes at least one nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme such that the recombinant host cell has an activity greater than a native host cell expressing an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

71. The method according to claim 59, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host comprises at least one mutated endogenous UDP-sugar precursor biosynthesis gene wherein the mutation results in an increase of a half-life of a messenger RNA transcribed from the mutated UDP-sugar precursor gene.

72. The method according to claim 59, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus pyogenes hyaluronan synthase of SEQ ID NO:

14 into a Bacillus host, the Bacillus host comprises at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency.

73. The method according to claim 72, wherein, the mutation in the UDP-sugar precursor biosynthesis gene occurs in a ribosome binding site in the UDP-sugar precusor biosynthesis gene such that a ribosome has an increased binding affinity for the ribosome binding site.

74. A method for producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO;

10 into a Bacillus host, wherein the Bacillus host has an enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc;

growing the Bacillus host in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

75. The method of claim 74, wherein in the step of introducing a purified nucleic acid segment encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10, the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO;

9.

76. The method of claim 74, wherein the Bacillus host is Bacillus subtilis or Bacillus licheniformis.

77. The method according to claim 74, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host further includes at least one modified RNA polymerase promoter having an increased promoter activity.

78. The method of claim 77, wherein the modification of the RNA polymerase promoter is a mutation.

79. The method of claim 77, wherein the modification of the RNA polymerase promoter is tandem promoter elements.

80. The method according to claim 74, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host is transformed with a vector comprising a purified nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme.

81. The method according to claim 80, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose dehydrogenase.

82. The method according to claim 80, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose pyrophosphorylase.

83. The method according to claim 74, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus host.

84. The method according to claim 74, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus host.

85. The method according to claim 74, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host further includes at least one nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme such that the recombinant host cell has an activity greater than a native host cell expressing an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

86. The method according to claim 74, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host comprises at least one mutated endogenous UDP-sugar precursor biosynthesis gene wherein the mutation results in an increase of a half-life of a messenger RNA transcribed from the mutated UDP-sugar precursor gene.

87. The method according to claim 74, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 into a Bacillus host, the Bacillus host comprises at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency.

88. The method according to claim 87, wherein, the mutation in the UDP-sugar precursor biosynthesis gene occurs in a ribosome binding site in the UDP-sugar precusor biosynthesis gene such that a ribosome has an increased binding affinity for the ribosome binding site.

89. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell comprising a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO:

10 wherein the coding region is under control of a promoter.

90. The recombinant host cell of claim 89, wherein the recombinant host cell is a Bacillus subtilis or a Bacillus licheniformis cell.

91. The recombinant host cell of claim 89, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

9.

92. The recombinant host cell of claim 91, wherein the host cell produces hyaluronic acid.

93. The recombinant host cell of claim 89, wherein the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

94. The recombinant host cell of claim 93, wherein the promoter is a Bacillus compatible promoter.

95. The recombinant host cell of claim 89, wherein the Bacillus subtilis cell further comprises a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active UDP-GlcUA biosynthetic pathway enzyme, wherein the enzymatically active UDP-GlcUA biosynthetic pathway enzyme is selected from the group consisting of UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, and combinations thereof.

96. The recombinant host cell of claim 89, wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

97. The recombinant host cell of claim 96, wherein the coding region encoding enzymatically active HAS and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of at least one copy of at least one promoter.

98. The recombinant host cell of claim 96, wherein the coding region encoding enzymatically active HAS and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

99. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell comprising:

a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO;

10; and

a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

100. The recombinant host cell of claim 99, wherein the recombinant host cell is a Bacillus subtilis or Bacillus licheniformis cell.

101. The recombinant host cell of claim 99, wherein the host cell produces hyaluronic acid.

102. The recombinant host cell of claim 99, wherein the coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

103. The recombinant host cell of claim 102, wherein the promoter is a Bacillus-compatible promoter.

104. The recombinant host cell of claim 99, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

9.

105. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell having a recombinant vector introduced therein, the recombinant vector comprising:

a purified nucleic acid segment having a coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO;

10; and

a coding region encoding enzymatically active UDP-glucose dehydrogenase.

106. The recombinant host cell of claim 105, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

9.

107. The recombinant host cell of claim 105, wherein the coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of the same promoter.

108. The recombinant host cell of claim 105, wherein the coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

109. The recombinant host cell of claim 105, wherein the recombinant host cell is a Bacillus subtilis or Bacillus licheniformis cell.

110. The recombinant host cell of claim 105, wherein the host cell produces hyaluronic acid.

111. The recombinant host cell of claim 105, wherein the coding region encoding enzymatically active Pasteurella multocida hyaluronan synthase of SEQ ID NO:

10 of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

112. The recombinant host cell of claim 111, wherein the promoter is a Bacillus-compatible promoter.