Morphological mutants of filamentous fungi

• US 6,066,493 A
• Filed: 03/13/1997
• Issued: 05/23/2000
• Est. Priority Date: 01/19/1996
• Status: Expired due to Term

• Current Assignee: Novozymes
• Original Assignee: NOVO Nordisk Biotech Incorporated (Novo Nordisk A/S)
• Inventors: Shuster, Jeffrey R., Royer, John C.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Assistant Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US08/816,239
• Time in Patent Office: 1,167 Days
• Field of Search: 435/69.1, 435/254.1, 435/254.11, 435/254.3, 435/254.8, 435/173.1
• US Class Current: 435/254.3
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12R 2001/69 Aspergillus oryzae
• CPC Class Codes: C12R 2001/77 Fusarium

Abstract

The present invention relates to methods of obtaining a mutant cell from a filamentous fungal parent cell, comprising: (a) obtaining mutant cells of the parent cell; (b) identifying the mutant cell which exhibits a more restricted colonial phenotype and/or a more extensive hyphal branching than the parent cell; and (c) identifying the mutant cell which has an improved property for production of a heterologous polypeptide than the parent cell, when the mutant and parent cells are cultured under the same conditions.

Claims

1. A method for obtaining a mutant cell which produces a heterologous protein, comprising:

(a) producing a first population of presumptive mutant cells from an Aspergillus or a Fusarium parent cell;

(b) identifying from the first population a second population of presumptive mutant cells having a more restricted colonial phenotype or a more extensive hyphal branching than the parent cell; and

(c) identifying from the second population the mutant cell, comprising a nucleic acid sequence encoding a heterologous polypeptide, which has a radial extension rate which is at least 10% less than the parent cell, has a hyphal growth unit length that is at least 10% less than the parent cell, and has one or more improved properties selected from the group consisting of (i) produces at least about 10% more heterologous polypeptide than the parent cell, (ii) exhibits about 90% or less of the viscosity of the parent cell, and (iii) secretes more heterologous protein than the parent cell, when cultivated under the same conditions; and

(d) obtaining the mutant cell.

2. The method of claim 1, wherein in step (b), the second population of presumptive mutant cells has a more restricted colonial phenotype and a more extensive hyphal branching than the parent cell.

3. The method of claim 1, wherein the first population of presumptive mutant cells are obtained by irradiation of the parent cell.

4. The method of claim 1, wherein the first population of presumptive mutant cells are obtained by chemical mutagenesis of the parent cell.

5. The method of claim 1, wherein the mutant cell is a spontaneous mutant of the parent cell.

6. The method of claim 1, wherein the radial extension rate is at least 20% less than the parent cell.

7. The method of claim 6, wherein the radial extension rate is at least 30% less than the parent cell.

8. The method of claim 1, wherein the hyphal growth unit length is at least 20% less than the parent cell.

9. The method of claim 8, wherein the hyphal growth unit length is at least 30% less than the parent cell.

10. The method of claim 1, wherein the mutant cell produces at least about 10% more heterologous polypeptide than the parent cell.

11. The method of claim 10, wherein the mutant cell produces at least about 20% more heterologous polypeptide than the parent cell.

12. The method of claim 11, wherein the mutant cell produces at least about 30% more heterologous polypeptide than the parent cell.

13. The method of claim 1, wherein the mutant cell exhibits about 90% or less of the viscosity of the parent cell.

14. The method of claim 13, wherein the mutant cell exhibits about 80% or less of the viscosity of the parent cell.

15. The method of claim 14, wherein the mutant cell exhibits about 50% or less of the viscosity of the parent cell.

16. The method of claim 1, wherein the mutant cell has a further improved property of an improved growth.

17. The method of claim 1, wherein the Aspergillus parent cell is an Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell.

18. The method of claim 1, wherein the Aspergillus parent cell is an Aspergillus oryzae cell.

19. The method of claim 1, wherein the Aspergillus parent cell is an Aspergillus niger cell.

20. The method of claim 1, wherein the Fusarium parent cell is a Fusarium cell of the Section Discolor.

21. The method of claim 20, wherein the Fusarium cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum cell.

22. The method of claim 20, wherein the Fusarium parent cell is Fusarium strain ATCC 20334.

23. The method of claim 1, wherein the Fusarium parent cell is a Fusarium cell of the Section Elegans.

24. The method of claim 23, wherein the Fusarium cell is a Fusarium oxysporum cell.

26. The method of claim 1, wherein the mutant cell is obtained by irradiation of the parent cell.

25. A method for producing a heterologous polypeptide, comprising:

(a) cultivating a mutant cell of aaspergillus or Fusarium parent cell under conditions suitable for production of the heterologous ploypeptide, wherein the mutant cell comprises a nucleic acid sequence encoding the heterologous polypeptide, and wherein the mutant cell has a radial extension rate which is at least 10% less than the parent cell, has ahyphal growth unit length that is at least 10% less than the parent cell, and has one or more improved properties selected from the group consisting of (i) produces at least about 10% more heterologous polypeptide than the parent cell, (ii) exhibits about 90% or less of the viscosity of the parent cell, and (iii) secretes more heterologous protein than the parent cell, when cultivated under the same conditions; and

(b) recovering the heterologous polypeptide.

27. The method of claim 25, wherein the mutant cell is obtained by chemical mutagenesis of the parent cell.

28. The method of claim 25, wherein the mutant cell is a spontaneous mutant of the parent cell.

29. The method of claim 25, wherein the radial extension rate is at least 20% less than the parent cell.

30. The method of claim 29, wherein the radial extension rate is at least 30% less than the parent cell.

31. The method of claim 25, wherein the hyphal growth unit length is at least 20% less than the parent cell.

32. The method of claim 31, wherein the hyphal growth unit length is at least 30% less than the parent cell.

33. The method of claim 25, wherein the mutant cell produces at least about 10% more heterologous polypeptide than the parent cell.

34. The method of claim 33, wherein the mutant cell produces at least about 20% more heterologous polypeptide than the parent cell.

35. The method of claim 34, wherein the mutant cell produces at least about 30% more heterologous polypeptide than the parent cell.

36. The method of claim 25, wherein the mutant cell exhibits about 90% or less of the viscosity of the parent cell.

37. The method of claim 36, wherein the mutant cell exhibits about 80% or less of the viscosity of the parent cell.

38. The method of claim 37, wherein the mutant cell exhibits about 50% or less of the viscosity of the parent cell.

39. The method of claim 25, wherein the mutant cell has a further improved property of improved growth.

40. The method of claim 25, wherein the Aspergillus parent cell is an Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell.

41. The method of claim 25, wherein the Aspergillus parent cell is an Aspergillus oryzae cell.

42. The method of claim 25, wherein the Aspergillus parent cell is an Aspergillus niger cell.

43. The method of claim 25, wherein the Fusarium parent cell is a Fusarium cell of the Section Discolor.

44. The method of claim 43, wherein the Fusarium cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum cell.

45. The method of claim 43, wherein the Fusarium parent cell is Fusarium strain ATCC 20334.

46. The method of claim 25, wherein the Fusarium parent cell is a Fusarium cell of the Section Elegans.

47. The method of claim 46, wherein the Fusarium cell is a Fusarium oxysporum cell.

48. A mutant cell of an Aspergillus or a Fusarium parent cell comprising a nucleic acid sequence encoding a heterologous polypeptide, wherein the mutant cell has a radial extension rate which is at least 10% less than the parent cell, has ahyphal growth unit length that is at least 10% less than the parent cell, and has one or more improved properties selected from the group consisting of (i) produces at least about 10% more heterologous polypeptide than the parent cell, (ii) exhibits about 90% or less of the viscosity of the parent cell, and (iii) secretes more heterologous protein than the parent cell, when cultivated under the same conditions.

49. The mutant cell of claim 48, wherein the mutant cell produces at least about 10% more heterologous polypeptide than the parent cell.

50. The mutant cell of claim 49, wherein the mutant cell produces at least about 20% more heterologous polypeptide than the parent cell.

51. The mutant cell of claim 50, wherein the mutant cell produces at least about 30% more heterologous polypeptide than the parent cell.

52. The mutant cell of claim 48, wherein the mutant cell exhibits about 90% or less of the viscosity of the parent cell.

53. The mutant cell of claim 52, wherein the mutant cell exhibits about 80% or less of the viscosity of the parent cell.

54. The mutant cell of claim 53, wherein the mutant cell exhibits about 50% or less of the viscosity of the parent cell.

55. The mutant cell of claim 48, which has a further improved property of improved growth.

56. The mutant cell of claim 48, wherein the Aspergillus parent cell is an Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell.

57. The mutant cell of claim 48, wherein the Fusarium parent cell is a Fusarium cell of the Section Discolor.

58. The mutant cell of claim 57, wherein the Fusarium cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum cell.

59. The mutant cell of claim 57, wherein the Fusarium parent cell is Fusarium strain ATCC 20334.

60. The mutant cell of claim 48, wherein the Fusarium parent cell is a Fusarium cell of the Section Elegans.

61. The mutant cell of claim 60, wherein the Fusarium cell is a Fusarium oxysporurm cell.

62. The mutant cell of claim 48, wherein the radial extension rate is at least 20% less than the parent cell.

63. The mutant cell of claim 48, wherein the hyphal growth unit length is at least 20% less than the parent cell.

64. The mutant cell of claim 63, wherein the hyphal growth unit length is at least 30% less than the parent cell.