Methods for producing polypeptides in protease-deficient mutants of Trichoderma

Patent metadata

Field	Value
Publication number	US-11078509-B2
Application number	US-202016996159-A
Country	US
Kind code	B2
Filing date	Aug 18, 2020
Priority date	Dec 18, 2009
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

The present invention relates to mutants of a parent Trichoderma strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of a first subtilisin-like serine protease gene, a first aspartic protease gene, a trypsin-like serine protease gene, a second subtilisin-like serine protease gene, and a second aspartic protease gene, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of a first subtilisin-like serine protease, a first aspartic protease, a trypsin-like serine protease, a second subtilisin-like serine protease, and a second aspartic protease, respectively, compared to the parent Trichoderma strain when cultivated under identical conditions. The present invention also relates to methods of producing a polypeptide in such mutants and methods for producing such mutants.

First claim

What is claimed is:

1. An isolated mutant of a parent Trichoderma strain, said parent strain comprising and a trypsin-like serine protease gene encoding a trypsin-like serine protease, wherein the mutant strain is at least 95% deficient in the production of the trypsin-like serine protease compared to the parent Trichoderma strain when cultivated under identical conditions;
wherein the trypsin-like serine protease is selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6 or at least 95% sequence identity to amino acids 20 to 259 of SEQ ID NO: 6, wherein said amino acid sequence has trypsin-like serine protease activity; and
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5 or at least 95% sequence identity to nucleotides 58 to 930 of SEQ ID NO: 5, or the cDNA thereof, wherein the nucleotide sequence encodes an amino acid sequence having trypsin-like serine protease activity; and
wherein the mutant further comprises a polynucleotide encoding a polypeptide that is heterologous to the parent Trichoderma strain.

2. The isolated mutant of claim 1 , wherein the parent Trichoderma strain is selected from the group consisting of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , and Trichoderma viride.

3. The isolated mutant of claim 1 , wherein the parent Trichoderma strain is Trichoderma reesei.

4. The isolated mutant of claim 1 , which is completely deficient in the trypsin-like serine protease compared to the parent Trichoderma strain when cultivated under identical conditions.

5. The isolated mutant of claim 1 , wherein the amino acid sequence has at least 97% sequence identity to SEQ ID NO: 6 or at least 97% sequence identity to amino acids 20 to 259 of SEQ ID NO: 6.

6. The isolated mutant of claim 1 , wherein the trypsin-like serine protease comprises SEQ ID NO: 6 or amino acids 20 to 259 of SEQ ID NO: 6.

7. The isolated mutant of claim 1 , wherein the parent strain further comprises an aspartic protease gene encoding an aspartic protease, a first subtilisin-like serine protease gene encoding a first subtilisin-like serine protease, and a second subtilisin-like serine protease gene encoding a second subtilisin-like serine protease, wherein the mutant strain is at least 95% deficient in the production of one or more of the aspartic protease, the first subtilisin-like serine protease, and the second subtilisin-like serine protease compared to the parent Trichoderma strain when cultivated under identical conditions;
wherein the aspartic protease is selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 108 or at least 95% sequence identity to amino acids 18 to 395 of SEQ ID NO: 108, wherein said amino acid sequence has aspartic protease activity; and
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 107 or at least 95% sequence identity to nucleotides 52 to 1339 of SEQ ID NO: 107, or the cDNA thereof, wherein the nucleotide sequence encodes an amino acid sequence having aspartic protease activity;

wherein the first subtilisin-like serine protease is selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or at least 95% sequence identity to amino acids 20 to 882 of SEQ ID NO: 2, wherein said amino acid sequence has subtilisin-like serine protease activity; and
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 1 or at least 95% sequence identity to nucleotides 58 to 2774 of SEQ ID NO: 1, or the cDNA thereof, wherein the nucleotide sequence encodes an amino acid sequence having subtilisin-like serine protease activity; and

wherein the second subtilisin-like serine protease is selected from the group consisting of:
(a) a polypeptide comprising of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 100 or at least 95% sequence identity to amino acids 16 to 540 of SEQ ID NO: 100, wherein said amino acid sequence has subtilisin-like serine protease activity; and
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 99 or at least 95% sequence identity to nucleotides 46 to 1681 of SEQ ID NO: 99, or the cDNA thereof, wherein the nucleotide sequence encodes an amino acid sequence having subtilisin-like serine protease activity.

8. The isolated mutant of claim 7 , wherein the aspartic protease comprises SEQ ID NO: 108 or amino acids 18 to 395 of SEQ ID NO: 108.

9. The isolated mutant of claim 7 , wherein the first subtilisin-like serine protease comprises SEQ ID NO: 2 or amino acids 20 to 882 of SEQ ID NO: 2.

10. The isolated mutant of claim 7 , wherein the second subtilisin-like serine protease comprises SEQ ID NO: 100 or amino acids 16 to 540 of SEQ ID NO: 100.

11. A method of producing a polypeptide, comprising:
(a) cultivating the isolated mutant of claim 1 in a medium for the production of the heterologous polypeptide; and
(b) recovering the heterologous polypeptide from the cultivation medium.

12. The method of claim 11 , wherein the parent Trichoderma strain is selected from the group consisting of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , and Trichoderma viride.

13. The method of claim 11 , wherein the parent Trichoderma strain is Trichoderma reesei.

14. The method of claim 11 , wherein the mutant is completely deficient in the trypsin-like serine protease compared to the parent Trichoderma strain when cultivated under identical conditions.

15. The method of claim 11 , wherein the amino acid sequence has at least 97% sequence identity to SEQ ID NO: 6 or at least 97% sequence identity to amino acids 20 to 259 of SEQ ID NO: 6.

16. The method of claim 11 , wherein the trypsin-like serine protease comprises SEQ ID NO: 6 or amino acids 20 to 259 of SEQ ID NO: 6.

17. The method of claim 11 , wherein the parent strain further comprises an aspartic protease gene encoding an aspartic protease, a first subtilisin-like serine protease gene encoding a first subtilisin-like serine protease, and a second subtilisin-like serine protease gene encoding a second subtilisin-like serine protease, wherein the mutant strain at least 95% deficient in the production of one or more of the aspartic protease, the first subtilisin-like serine protease, and the second subtilisin-like serine protease compared to the parent Trichoderma strain when cultivated under identical conditions;
wherein the aspartic protease is selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 108 or at least 95% sequence identity to amino acids 18 to 395 of SEQ ID NO: 108, wherein said amino acid sequence has aspartic protease activity; and
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 107 or at least 95% sequence identity to nucleotides 52 to 1339 of SEQ ID NO: 107, or the cDNA thereof, wherein the nucleotide sequence encodes an amino acid sequence having aspartic protease activity;

wherein the first subtilisin-l

Assignees

• Novozymes Inc

Inventors

• Maiyuran Suchindra
• Jang Abigail
• Brown Kimberly
• Merino Sandra
• Shasky Jeffrey
• Abbate Eric

Classifications

• C12N9/58 Primary

  derived from fungi · CPC title

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12P21/02

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

• C12N15/04

  Fungi · CPC title

• C12N15/80

  for fungi · CPC title