Gene inactivated mutants with altered protein production
A recombinant filamentous fungal cell (e.g. Aspergillus ) having one or more inactivated chromosomal genes is provided. The chromosomal genes in some embodiments correspond to derA, derB, htmA, mnn9, mnn10, ochA, dpp4, dpp5, pepAa, pepAb, pepAc, pepAd, pepF and combinations thereof. The recombinant fungal cells may include further inactivated chromosomal genes which correspond to pepA, pepB, pepC and pepD. The recombinant filamentous fungal cells may include a heterologous nucleic acid encoding a protein of interest. Also provided are methods of producing a protein of interest in said recombinant filamentous fungal cell.
1. A method for producing a protein of interest in a filamentous fungal cell comprising
a) obtaining a recombinant filamentous fungal cell comprising one or more inactivated chromosomal genes selected from derB (SEQ ID NO:3), mnn9 (SEQ ID NO:7), mnn10 (SEQ ID NO:9), ochA (SEQ ID NO:11), dpp4 (SEQ ID NO:13), pepAa (SEQ ID NO:17), pepAb (SEQ ID NO:19), pepAc (SEQ ID NO:31), pepAd (SEQ ID NO:21), pepF (SEQ ID NO:23), functionally homologous sequences thereto, wherein the functionally homologous sequences have at least 95% sequence identity thereto, and combinations thereof,
b) introducing a nucleic acid sequence which encodes a protein of interest into said recombinant fungal cell, and
c) growing the recombinant fungal cell under suitable culture conditions for the expression and production of said protein of interest.
2. The method according to claim 1 further comprising recovering the protein of interest.
3. The method according to claim 2 , wherein the expression of the protein of interest is enhanced compared to the expression of said protein of interest in a corresponding parent cell.
4. The method according to claim 3 , wherein the filamentous fungal cell is Aspergillus cell.
5. A method for obtaining a protein of interest from an Aspergillus strain comprising
a) obtaining a recombinant Aspergillus cell comprising one or more inactivated chromosomal genes selected from derB (SEQ ID NO:3), mnn9 (SEQ ID NO:7), mnn10 (SEQ ID NO:9), ochA (SEQ ID NO:11), dpp4 (SEQ ID NO:13), pepAa (SEQ ID NO:17), pepAb (SEQ ID NO:19), pepAc (SEQ ID NO:31), pepAd (SEQ ID NO:21), pepF (SEQ ID NO:23), functionally homologous sequences thereto, wherein the functionally homologous sequences have at least 95% sequence identity thereto, and combinations thereof;
b) transforming the recombinant Aspergillus cell with a nucleic acid sequence encoding a protein of interest;
c) growing the transformed Aspergillus cell under suitable growth conditions to allow expression of the protein of interest, and
d) recovering said protein.
6. The method according to claim 5 , wherein the protein is a protease inhibitor.
7. The method according to claim 5 , wherein the protein is an antibody or fragment thereof.
8. The method according to claim 5 , wherein the protein is an enzyme.
9. The method according to claim 5 , wherein the Aspergillus is A. niger.
10. The method according to claim 1 , wherein the recombinant filamentous fungal cell comprises two inactivated chromosomal genes.
11. The method according to claim 10 , wherein the two inactivated chromosomal genes are mnn9 (SEQ ID NO:7) and ochA (SEQ ID NO:11), or homologous sequences thereto having at least 95% sequence identity thereto.
12. The method according to claim 1 , wherein the inactivated gene has been deleted.
13. The method according to claim 1 , wherein the inactivated gene has been disrupted.