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 recombinant Aspergillus cell comprising an inactivated dpp5 (SEQ ID NO: 15) chromosomal gene or functionally homologous sequences thereto, wherein the functionally homoloqous sequences of dpp5 (SEQ ID NO: 15) have at least 99% sequence identity thereto.
2. The recombinant filamentous fungal cell of claim 1 further comprising a second inactivated chromosomal gene.
3. The recombinant filamentous fungal cell of claim 2 , wherein the two inactivated chromosomal genes are dpp4 (SEQ ID NO:13) and dpp5 (SEQ ID NO:15) or homologous sequences thereto having at least 95% sequence identity thereto.
4. The recombinant filamentous fungal cell of claim 1 , further comprising an inactivated chromosomal gene selected from 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); and functionally homologous sequences thereto having at least 95% sequence identity.
5. The recombinant filamentous fungal cell of claim 1 , further comprising an inactivated pepA.
6. The recombinant filamentous fungal cell of claim 1 , wherein the inactivated gene has been deleted.
7. The recombinant fungal cell of claim 1 ,wherein the inactivated gene has been disrupted.
8. The recombinant filamentous fungal cell of claim 1 , further comprising an inactivated gene selected from pepB (SEQ ID NO:25), pepC (SEQ ID NO:27), pepD (SEQ ID NO:29) and homologous sequences and combinations thereof.
9. The recombinant filamentous fungal cell of claim 1 further comprising an introduced nucleic acid which encodes a protein of interest.
10. The recombinant filamentous fungal cell of claim 9 , wherein the protein of interest is an enzyme.
11. The recombinant filamentous fungal cell of claim 8 , wherein the protein of interest is a protease inhibitor.
12. The recombinant filamentous fungal cell of claim 9 , wherein the protein of interest is an antibody or fragment thereof.
13. A method for obtaining a protein of interest from an Aspergillus strain comprising
a) obtaining a recombinant Aspergillus cell according to claim 1 ;
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.
14. The method according to claim 13 , wherein the protein is a protease inhibitor.
15. The method according to claim 13 , wherein the protein is an antibody or fragment thereof.
16. The method according to claim 13 , wherein the protein is an enzyme.
17. The method according to claim 13 , wherein the Aspergillus is A. niger.