Morphological mutants of filamentous fungi

• US 6,184,026 B1
• Filed: 09/24/1999
• Issued: 02/06/2001
• 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): TATE, CHRISTOPHER ROBIN
• Application Number:
  US09/405,564
• Time in Patent Office: 501 Days
• Field of Search: 435/69.1, 435/254.1, 435/254.11, 435/254.6, 435/173.1
• US Class Current: 435/254.6
• 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 a Trichoderma 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 radial extension rate is at least 20% less than the parent cell.

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

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

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

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

8. The method of claim 1, wherein the Trichoderma parent cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

14. The method of claim 1, wherein the Trichoderma parent cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

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

(a) cultivating a mutant cell of a Trichoderma parent cell under conditions suitable for production of the heterologous polypeptide, 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 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

(b) recovering the heterologous polypeptide.

10. The method of claim 9, wherein the radial extension rate is at least 20% less than the parent cell.

11. The method of claim 9, wherein the hyphal growth unit length is at least 20% less than the parent cell.

12. The method of claim 9, wherein the mutant cell produces at least about 10% more heterologous polypeptide than the parent cell.

13. The method of claim 9, wherein the mutant cell exhibits about 90% or less of the viscosity of the parent cell.

15. A mutant cell of a Trichoderma 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 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.

16. The mutant cell of claim 15, wherein the radial extension rate is at least 20% less than the parent cell.

17. The mutant cell of claim 15, wherein the hyphal growth unit length is at least 20% less than the parent cell.

18. The mutant cell of claim 15, wherein the mutant cell produces at least about 10% more heterologous polypeptide than the parent cell.

19. The mutant cell of claim 15, wherein the mutant cell exhibits about 90% or less of the viscosity of the parent cell.

20. The mutant cell of claim 15, wherein the Trichoderma parent cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.