Process for making DNA libraries in filamentous fungal cells using a novel cloned gene involved in the mismatch repair system of filamentous fungal cells

• US 6,518,042 B1
• Filed: 02/24/2000
• Issued: 02/11/2003
• Est. Priority Date: 02/24/1999
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Christiansen, Lars, Vind, Jesper, Borchert, Torben Vedel
• Primary Examiner(s): Yucel, Remy
• Assistant Examiner(s): Davis, Katharine F
• Application Number:
  US09/512,250
• Time in Patent Office: 1,083 Days
• Field of Search: 435/254.11, 435/254.3, 435/254.7, 435/6, 435/69.1, 435/471, 435/23.1, 536/23.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/38 from Aspergillus
• CPC Class Codes: C12N 15/80 for fungi

Abstract

A process for making DNA libraries in filamentous fungal cells using a novel cloned gene involved in the mismatch repair system of filamentous fungal cells.

Claims

1. A filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises a DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:

2.

3. The filamentous fungal cell of claims 1 or 2, wherein the gene involved in the mismatch repair is defective.

4. The filamentous fungal cell of claims 1 or 2, wherein the gene involved in the mismatch repair has been inactivated transitorily.

5. The filamentous fungal cell of claims 1 or 2, wherein the filamentous fungal cell is a strain of Fusarium.

6. The filamentous fungal cell of claims 1 or 2, wherein the filamentous fungal cell is a strain of Aspergillus.

7. A process for preparing a filamentous fungal cell population wherein individual cells in the population comprise individually different DNA sequences of interest representing a DNA library of interest, the process comprising the following steps:

(a) placing individually different DNA sequences of interest in a filamentous fungal cell population comprising a filamentous fungal cell of claims 1 or 2, and

(b) growing the population of (a) for a period of time allowing an individual DNA sequence of interest in the population to be duplicated at least once under conditions wherein the mismatch repair system gene of claims 1 or 2 has been inactivated.

8. A process for production of a polypeptide of interest comprising:

(a) placing individually different DNA sequences of interest in a filamentous fungal cell population comprising a filamentous fungal cell of claims 1 or 2, wherein the DNA sequences of interest encode a polypeptide of interest;

(b) growing the population of (a) for a period of time allowing an individual DNA sequence of interest in the population to be duplicated at least once under conditions wherein the mismatch repair system gene of claims 1 or 2 has been inactivated; and

(c) selecting from the resultant population of filamentous fungal cells of step (b) a desired polypeptide of interest.

9. A process for production of a polypeptide of interest, comprising:

(a) placing a DNA sequence encoding a polypeptide of interest into the filamentous fungal cell of claims 1 or 2,

(b) cultivating the filamentous fungal cell of said (a) for a period of time allowing the DNA sequence encoding the polypeptide of interest to be duplicated at least once under conditions wherein the mismatch repair system gene has been inactivated;

(c) isolating the polypeptide of interest.

10. The process of claim 7, wherein the mismatch repair system gene under step (b) is defective.

11. the process of claim 7, wherein the mismatch repair system gene under step (b) has been inactivated transitorily.

12. The process of claim 7, wherein the filamentous fungal cell is a strain of Fusarium.

13. The process of claim 7, wherein the filamentous fungal cell is a strain of Aspergillus.

14. The process of claim 7, further comprising the step of recombining homologous DNA sequences of interest.

15. The process of claim 8, which further comprises the following steps:

(d) placing the DNA sequence encoding the polypeptide of interest of step (c) of claim 13 or the modified polypeptide of interest of step (d) into a filamentous fungal cell, which is suitable for large scale production of the polypeptide of interest;

(e) cultivating the filamentous fungal cell of step (a) in a fermentor of at least 10000 m3 under conditions permitting expression of the polypeptide of interest; and

(f) isolating the polypeptide of interest.

16. The process of claim 8, wherein the polypeptide of interest is a polypeptide derived from a filamentous fungal cell.

17. The process of claim 8, wherein the polypeptide of interest is an enzyme selected from an amylase, a protease, a cellulase, a lipase, a xylanase and a phospholipase.

18. The process of claim 15, wherein the filamentous fungal cell which is suitable for large scale production of the polypeptide of interest of step (d) of claim 14 is another filamentous fungal cell as compared to the filamentous fungal cell of step (a) of claim 13.

2. A filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises a DNA sequence encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO:

2.