Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same

• US 5,578,463 A
• Filed: 08/02/1994
• Issued: 11/26/1996
• Est. Priority Date: 08/29/1985
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Berka, Randy M., Hayenga, Kirk J., Lawlis, Virgil B., Gray, Gregory L., Cullen, Daniel
• Primary Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US08/284,942
• Time in Patent Office: 847 Days
• Field of Search: 435/69.1, 435/69.7, 435/69.8, 435/172.3, 435/183, 435/205, 435/254.3, 435/320.1, 536/23.1, 536/23.2, 536/23.74, 536/24.1, 935/27, 935/47, 935/48, 935/68
• US Class Current: 435/69.1
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12N 9/58 derived from fungi
• CPC Class Codes: C12N 9/6481 Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)

Abstract

Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3'"'"' terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.

Claims

1. A DNA construct for use in transforming an Aspergillus host to enable secretion an of a heterologous polypeptide, said DNA construct comprising promoter DNA from a fungal gene operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and said heterologous polypeptide.

2. The DNA construct of claim 1 wherein said fungal gene is from a filamentous fungus.

3. The DNA construct of claim 2 wherein said filamentous fungus is Mucor or Humicola.

4. The DNA construct of claim 3 wherein said gene from said Humicola comprises a glucoamylase gene.

5. The DNA construct of claim 3 wherein said gene from said Mucor comprises a carboxyl protease gene.

6. A DNA construct according to claims 1 or 2 wherein said heterologous polypeptide is biochemically active.

7. A DNA construct according to claims 1 or 2 wherein said heterologous polypeptide comprises a mammalian polypeptide.

8. A DNA construct according to claims 1 or 2 wherein said mammalian polypeptide comprises chymosin or prochymosin.

9. A DNA construct according to claims 1 or 2 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Aspergillus host.

10. A DNA construct according to claims 1 or 2 wherein said heterologous polypeptide comprises an enzyme.

11. A DNA construct according to claim 10 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

12. A DNA construct according to claims 1 or 2 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

13. A DNA construct according to claim 12 wherein said signal peptide is from a secreted polypeptide selected from the group consisting of Aspergillus glucoamylase, Humicola glucoamylase, and Mucor carboxyl protease.

14. A DNA construct according to claims 1 or 2 wherein said signal peptide is from a source other than a filamentous fungus.

15. A DNA construct according to claim 14 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide.

16. A DNA construct according to claim 15 wherein said mammalian polypeptide comprises preprochymosin.

17. A DNA construct according to claims 1 or 2 further comprising DNA encoding a selection characteristic expressible in said Aspergillus host.

18. A DNA construct according to claim 17 wherein said selection characteristic is selected from the group consisting of the acetamidase gene, pyr4, argB and trpC.

19. A transformed Aspergillus expression host capable of secreting a heterologous polypeptide, said host being transformed with a vector comprising promoter DNA from a fungal gene operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and said heterologous polypeptide.

20. The Aspergillus host of claim 19 wherein said fungal gene is from a filamentous fungus.

21. The Aspergillus host of claim 20 wherein said filamentous fungus is selected from the group consisting of Mucor and Humicola.

22. The Aspergillus host of claim 21 wherein said gene from said Humicola comprises a glucoamylase gene.

23. The Aspergillus host of claim 21 wherein said gene from said Mucor comprises a carboxyl protease gene.

24. The Aspergillus host of claim 19 or 20 wherein said heterologous polypeptide is biochemically active.

25. The Aspergillus host of claim 19 or 20 wherein said heterologous polypeptide comprises a mammalian polypeptide.

26. The Aspergillus host of claim 25 wherein said mammalian polypeptide comprises chymosin or prochymosin.

27. The Aspergillus host of claim 19 or 20 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Aspergillus host.

28. The Aspergillus host of claim 19 or 20 wherein said heterologous polypeptide comprises an enzyme.

29. The Aspergillus host of claim 28 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

30. The Aspergillus host of claim 19 or 20 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

31. The Aspergillus host of claim 30 wherein said signal peptide is from a secreted polypeptide selected from the group consisting of Aspergillus glucoamylase, Humicola glucoamylase, and Mucor carboxyl protease.

32. The Aspergillus host of claim 19 or 20 wherein said signal peptide is from a source other than a filamentous fungus.

33. The Aspergillus host of claim 32 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide.

34. The Aspergillus host of claim 33 wherein said mammalian polypeptide comprises preprochymosin.

35. The Aspergillus host of claim 19 or 20 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Aspergillus host.

36. The Aspergillus host of claim 35 wherein said selection characteristic is selected from the group consisting of acetamidase, pyr4, argB and trpC.

37. The Aspergillus host of claim 19 or 20 wherein said host Aspergillus is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus awamori and Aspergillus nidulans.

38. A process for producing a polypeptide comprising:

transforming an Aspergillus host, with a vector comprising a DNA construct comprising promoter DNA from a fungal gene operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and a heterologous polypeptide, and

culturing said transformed filamentous fungus under conditions which permit the expression of said coding DNA and secretion of said heterologous polypeptide.

39. The process of claim 38 wherein said culturing is carried out in a culture medium comprising utilizable carbon, nitrogen and phosphate sources, surfactant and trace elements.

40. The process of claim 38 further comprising the step of isolating said secreted heterologous polypeptide.

41. The process of claim 38 wherein said fungal gene is from a filamentous fungus.

42. The process of claim 44 wherein said filamentous fungus is Mucor or Humicola.

43. The process of claim 44 wherein said gene from said Humicola comprises a glucoamylase gene.

44. The process of claim 41 wherein said gene from said Mucor comprises a carboxyl protease gene.

66. The process of claims 38 or 41 wherein said culturing is carried out in a culture medium comprising utilizable carbon, nitrogen and phosphate sources, surfactant and trace elements.

67. The process of claims 38 or 41 further comprising the step of isolating said secreted heterologous polypeptide.

68. The process of claims 38 or 41 wherein said heterologous polypeptide is biochemically active.

69. The process of claims 38 or 41 wherein said heterologous polypeptide comprises a mammalian polypeptide.

70. The process of claim 69 wherein said mammalian polypeptide comprises chymosin or prochymosin.

71. The process of claims 38 or 41 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Aspergillus host.

72. The process of claims 38 or 41 wherein said heterologous polypeptide comprises an enzyme.

73. The process of claims 38 or 41 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

74. The process of claims 38 or 41 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

75. The process of claims 38 or 41 wherein said signal peptide is from a secreted polypeptide selected from the group consisting of Aspergillus glucoamylase, Humicola glucoamylast, and Mucor carboxyl protease.

76. The process of claims 38 or 41 wherein said signal peptide is from a source other than a filamentous fungus.

77. The process of claims 38 or 41 wherein said signal peptide comprises the signal peptide from secreted mammalian polypeptide.

78. The process of claims 38 or 41 where said mammalian polypeptide comprises preprochymosin.

79. The process of claims 38 or 41 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Aspergillus host.

80. The process of claims 38 or 41 wherein said election characteristic is selected from the group consisting of the acetamidase gene, pyr4 argB and trpC.

81. The process of claims 38 or 41 wherein said Aspergillus host is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus awamori and Aspergillus nidulans.

45. A process for producing a polypeptide comprising:

culturing a member of the genus Aspergillus transformed with a vector comprising promoter DNA from a fungal gene operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and a heterologous polypeptide, wherein said culturing is under conditions which permit the expression of said coding DNA and secretion of said heterologous polypeptide.

46. The process of claim 45 wherein said fungal gene is from a filamentous fungus.

47. The process of claim 46 wherein said filamentous fungus is Mucor or Humicola.

48. The process of claim 47 wherein said gene from said Humicola comprises a glucoamylase gene.

49. The process of claim 47 wherein said gene from said Mucor comprises a carboxyl protease gene.

50. The process of claims 45 or 46 wherein said culturing is carried out in a culture medium comprising utilizable carbon, nitrogen and phosphate sources, surfactant and trace elements.

51. The process of claims 45 or 46 further comprising the step of isolating said secreted heterologous polypeptide.

52. The process of claims 45 or 46 wherein said heterologous polypeptide is biochemically active.

53. The process of claims 45 or 46 wherein said heterologous polypeptide comprises a mammalian polypeptide.

54. The process of claim 53 wherein said mammalian polypeptide comprises chymosin or prochymosin.

55. The process of claims 45 or 46 wherein said heterologous polypeptide comprises a polypetide from a filamentous fungus other than said Aspergillus host.

57. The process of claims 45 or 46 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

58. The process of claims 45 or 46 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

59. The process of claims 45 or 46 wherein said signal peptide is from a secreted polypeptide selected from the group consisting of Aspergillus glucoamylase, Humicola glucoamylase, and Mucor carboxyl protease.

60. The process of claims 45 or 46 wherein said signal peptide is from a source other than a filamentous fungus.

61. The process of claims 45 or 46 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide.

62. The process of claims 45 or 46 where said mammalian polypeptide comprises preprochymosin.

63. The process of claims 45 or 46 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Aspergillus host.

64. The process of claims 45 or 46 wherein said election characteristic is selected from the group consisting of the acetamidase gene, pgr4, argB and trpC.

65. The process of claims 45 or 46 wherein said Aspergillus host is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus awamori and Aspergillus nidulans.

56. The process of claims or 46 wherein said heterologous polypeptide comprises an enzyme.