Protease deficient filamentous fungal cells and methods of use thereof

Patent metadata

Field	Value
Publication number	US-9567596-B2
Application number	US-201314370255-A
Country	US
Kind code	B2
Filing date	Jan 4, 2013
Priority date	Jan 5, 2012
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

Abstract

The present disclosure relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells.

First claim

We claim:

1. A Trichoderma cell comprising at least three endogenous proteases having reduced or eliminated activity wherein nucleic acids encoding the proteases are modified to reduce or eliminate expression of the proteases and further comprising a recombinant polynucleotide encoding a heterologous polypeptide, wherein the heterologous polypeptide is produced at a level of at least twofold higher than the production level of the polypeptide in a corresponding parental Trichoderma cell, and wherein the Trichoderma cell has reduced or eliminated activity in at least
(i) a gap1 protease of SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, or homologues thereof having at least 90% sequence identity thereto,
(ii) a gap2 protease of SEQ ID NO: 129, SEQ ID NO:130, SEQ ID NO:131, or homologues thereof having at least 90% sequence identity thereto, and
(iii) a pep3 protease of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or homologues thereof having at least 90% sequence identity thereto.

2. The Trichoderma cell of claim 1 , wherein at least one of the nucleic acids is a gene comprising a mutation within the coding sequence of the gene that reduces or eliminates the protease activity.

3. The Trichoderma cell of claim 1 , wherein the Trichoderma cell is further modified to reduce or eliminate expression of the protease activity of at least one other Trichoderma protease selected from the group consisting of
a pep1 protease of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or homologues thereof having at least 90% sequence identity thereto,
a pep2 protease of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, or homologues thereof having at least 90% sequence identity thereto,
a pep4 protease of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or homologues thereof having at least 90% sequence identity thereto,
a pep5 protease of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or homologues thereof having at least 90% sequence identity thereto,
a pep8 protease of SEQ ID NO: 507, SEQ ID NO: 508, SEQ ID NO: 509 or homologues thereof having at least 90% sequence identity thereto,
a pep11 protease of SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, or homologues thereof having at least 90% sequence identity thereto,
a pep12 protease of SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, or homologues thereof having at least 90% sequence identity thereto,
a tsp1 (protease of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or homologues thereof having at least 90% sequence identity thereto,
a slp1 protease of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:86, or homologues thereof having at least 90% sequence identity thereto,
a slp2 protease of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, or homologues thereof having at least 90% sequence identity thereto,
a slp3 protease of SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:168, SEQ ID NO: 169, or homologues thereof having at least 90% sequence identity thereto, and
a slp7 protease of SEQ ID NO: 231, SEQ ID NO:232, SEQ ID NO:233, or homologues thereof having at least 90% 4 sequence identity thereto.

4. The Trichoderma cell of claim 1 , wherein the heterologous polypeptide is a mammalian polypeptide that is either a naturally non-glycosylated polypeptide or a naturally glycosylated polypeptide.

5. The Trichoderma cell of claim 4 , wherein the mammalian polypeptide is a glycosylated mammalian polypeptide.

6. The Trichoderma cell of claim 4 , wherein the mammalian polypeptide is selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, a growth factor, an interferon, a cytokine, and an interleukin.

7. The Trichoderma cell of claim 1 , wherein the Trichoderma cell further comprises a modification to reduce or eliminate the activity of an ALG3 gene encoding dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase.

8. The Trichoderma cell of claim 7 , wherein the gene encoding ALG3 is deleted from the Trichoderma cell.

9. The Trichoderma cell of claim 1 , wherein the Trichoderma cell further comprises a polynucleotide encoding an α-1,2-mannosidase.

10. The Trichoderma cell of claim 1 , further comprising a polynucleotide encoding an N-acetylglucosaminyltransferase I catalytic domain.

11. The Trichoderma cell of claim 10 , further comprising a polynucleotide encoding an N-acetylglucosaminyltransferase II catalytic domain.

12. The Trichoderma cell of claim 1 , further comprising a polynucleotide encoding a mannosidase II and/or a galactosyl transferase.

13. A method of improving the extracellular stability of a polypeptide, comprising
a) providing the Trichoderma cell of claim 1 , and
b) culturing the Trichoderma cell such that the heterologous polypeptide is expressed, wherein the heterologous polypeptide exhibits increased extracellular stability compared to the heterologous polypeptide when produced in a corresponding parental Trichoderma cell.

14. A method of making a heterologous polypeptide, comprising
a) providing the Trichoderma cell of claim 1 ,
b) culturing the Trichoderma cell such that the heterologous polypeptide is expressed, and
c) purifying the heterologous polypeptide.

Assignees

• Novartis Ag
• Glykos Finland Oy

Inventors

• Landowski Christopher
• Huuskonen Anne
• Saarinen Juhani
• Westerholm-Parvinen Ann
• Kanerva Anne
• Natunen Jari
• Hanninen Anna-Liisa
• Salovuori Noora
• Penttila Merja
• Saloheimo Markku

Classifications

• C12P21/005 Primary

  Glycopeptides, glycoproteins · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/58 Primary

  derived from fungi · CPC title

• C07K14/65

  Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2 · CPC title