Heterologous polypeptides expressed in aspergillus

• US 5,364,770 A
• Filed: 09/25/1989
• Issued: 11/15/1994
• Est. Priority Date: 08/29/1985
• Status: Expired due to Term

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Berka, Randy M., Hayenga, Kirk J., Lawlis, Virgil B., Cullen, Daniel, Gray, Gregory L.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US07/413,010
• Time in Patent Office: 1,877 Days
• Field of Search: 435/320.1, 435/254, 435/69.7, 435/69.8, 435/69.1, 435/71.1, 435/183, 435/205, 435/172.3, 435/913, 435/254.3, 935/47, 935/48, 935/27, 935/68, 536/23.1, 536/23.2, 536/23.74
• 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 of a heterologous polypeptide, said DNA construct comprising promoter DNA from an Aspergillus gene operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and said heterologous polypeptide.

2. A DNA construct according to claim 1 wherein said Aspergillus gene comprises a glucoamylase gene.

3. A DNA construct according to claim 2 wherein said glucoamylase gene comprises the glucoamylase gene from Aspergillus niger.

4. A DNA construct according to claim 1 wherein said heterologous polypeptide is biochemically active.

5. A DNA construct according to claim 1 wherein said heterologous polypeptide comprises a mammalian polypeptide.

6. A DNA construct according to claim 5 wherein said mammalian polypeptide comprises chymosin or prochymosin.

7. A DNA construct according to claim 1 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Aspergillus host.

8. A DNA construct according to claim 1 wherein said heterologous polypeptide comprises an enzyme.

9. A DNA construct according to claim 8 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

10. A DNA construct according to claim 1 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

11. A DNA construct according to claim 10 wherein said signal peptide is from a secreted polypeptide selected from the group consisting of Aspergillus glucoamylase, Humicola glucoamylase, and Mucor carboxyl protease.

12. A DNA construct according to claim 1 wherein said signal peptide is from a source other than a filamentous fungus.

13. A DNA construct according to claim 12 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide.

14. A DNA construct according to claim 13 wherein said mammalian polypeptide comprises preprochymosin.

15. A DNA construct according to claim 1 further comprising DNA encoding a selection characteristic expressible in said Aspergillus host.

16. A DNA construct according to claim 15 wherein said selection characteristic is selected from the group consisting of the acetamidase gene, pyr4, argB and trpC.

17. A DNA construct for use in transforming an Aspergillus host to enable secretion therefrom of a polypeptide foreign thereto, said DNA construct comprising promoter DNA operably linked to coding DNA to enable expression thereof, said coding DNA comprising DNA coding for said foreign polypeptide operably linked to DNA coding for a signal peptide enabling said polypeptide to be secreted when said coding DNA is expressed, said promoter DNA and the signal peptide-encoding DNA corresponding in sequence to the glucoamylase gene of Aspergillus.

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

19. The Aspergillus host of claim 18 wherein said Aspergillus gene comprises a glucoamylase gene.

20. The Aspergillus host of claim 19 wherein said glucoamylase gene comprises the glucoamylase gene from Aspergillus niger.

21. The Aspergillus host of claim 18 wherein said heterologous polypeptide is biochemically active.

22. The Aspergillus host of claim 18 wherein said heterologous polypeptide comprises a mammalian polypeptide.

23. The Aspergillus host of claim 22 wherein said mammalian polypeptide comprises chymosin or prochymosin.

24. The Aspergillus host of claim 18 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Aspergillus host.

25. The Aspergillus host of claim 18 wherein said heterologous polypeptide comprises an enzyme.

26. The Aspergillus host of claim 25 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

27. The Aspergillus host of claim 18 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

28. The Aspergillus host of claim 27 wherein said signal peptide is from a secreted polypeptide selected from the group consisting of Aspergillus glucoamylase, Humicola glucoamylase, and Mucor carboxyl protease.

29. The Aspergillus host of claim 18 wherein said signal peptide is from a source other than a filamentous fungus.

30. The Aspergillus host of claim 29 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide.

31. The Aspergillus host of claim 30 wherein said mammalian polypeptide comprises preprochymosin.

32. The Aspergillus host of claim 18 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Aspergillus host.

33. The Aspergillus host of claim 32 wherein said selection characteristic is selected from the group consisting of the acetamidase gene, pyr4, argB and trpC.

34. The Aspergillus host of claim 18 wherein said host Aspergillus is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus awamori and Aspergillus nidulans.

35. A process for producing a polypeptide comprising:

culturing an Aspergillus host transformed with a vector comprising promoter DNA from an Aspergillus 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.

36. The process of claim 35 wherein said culturing is carried out in a culture medium comprising utilizable carbon, nitrogen and phosphate sources, surfactant and trace elements.

37. The process of claim 35 further comprising the step of isolating said secreted heterologous polypeptide.

38. The process of claim 35 wherein said Aspergillus gene comprises a glucoamylase gene.

39. The process of claim 38 wherein said glucoamylase gene comprises the glucoamylase gene from Aspergillus niger.

40. The process of claim 35 wherein said heterologous polypeptide is biochemically active.

41. The process of claim 35 wherein said heterologous polypeptide comprises a mammalian polypeptide.

42. The process of claim 44 wherein said mammalian polypeptide comprises chymosin or prochymosin.

43. The process of claim 35 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Aspergillus host.

44. The process of claim 35 wherein said heterologous polypeptide comprises an enzyme.

45. The process of claim 44 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor miehei carboxyl protease.

46. The process of claim 35 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus.

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

48. The process of claim 35 wherein said signal peptide is from a source other than a filamentous fungus.

49. The process of claim 48 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide.

50. The process of claim 49 wherein said mammalian polypeptide comprises preprochymosin.

51. The process of claim 35 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Aspergillus host.

52. The process of claim 51 wherein said election characteristic is selected from the group consisting of the acetamidase gene, pyr4, argB and trpC.

53. The process of claim 35 wherein said Aspergillus host is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus awamori and Aspergillus nidulans.