Methods for producing polypeptides in protease-deficient mutants of Trichoderma

Patent metadata

Field	Value
Publication number	US-11001869-B2
Application number	US-202016995820-A
Country	US
Kind code	B2
Filing date	Aug 18, 2020
Priority date	Dec 18, 2009
Publication date	May 11, 2021
Grant date	May 11, 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. A method of producing a heterologous polypeptide, the method comprising:
(i) cultivating an isolated mutant of a parent Trichoderma strain in a medium for the production of the heterologous polypeptide; and
(ii) recovering the heterologous polypeptide from the cultivation medium;
wherein the mutant strain comprises a polynucleotide encoding the heterologous polypeptide and a first aspartic protease gene, wherein the first aspartic protease gene is modified rendering the mutant strain at least 95% deficient in the production of the first aspartic protease compared to the parent Trichoderma strain when cultivated under identical conditions;
wherein the first aspartic protease is selected from:
(a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4 or at least 95% sequence identity to amino acids 21 to 407 of SEQ ID NO: 4, 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: 3 or at least 95% sequence identity to nucleotides 61 to 1299 of SEQ ID NO: 3, or the cDNA thereof, wherein the nucleotide sequence encodes an amino acid sequence having aspartic protease activity.

2. The method of claim 1 , wherein the first aspartic protease comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 4 or at least 96% sequence identity to amino acids 21 to 407 of SEQ ID NO: 4.

3. The method of claim 1 , wherein the first aspartic protease comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 4 or at least 97% sequence identity to amino acids 21 to 407 of SEQ ID NO: 4.

4. The method of claim 1 , wherein the first aspartic protease comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4 or at least 98% sequence identity to amino acids 21 to 407 of SEQ ID NO: 4.

5. The method of claim 1 , wherein the first aspartic protease comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4 or at least 99% sequence identity to amino acids 21 to 407 of SEQ ID NO: 4.

6. The method of claim 1 , wherein the first aspartic protease comprises SEQ ID NO: 4 or amino acids 21 to 407 of SEQ ID NO: 4.

7. The method of claim 1 , wherein the first aspartic protease is encoded by a polynucleotide comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 3 or at least 96% sequence identity to nucleotides 61 to 1299 of SEQ ID NO: 3, or the cDNA thereof.

8. The method of claim 1 , wherein the first aspartic protease is encoded by a polynucleotide comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 3 or at least 97% sequence identity to nucleotides 61 to 1299 of SEQ ID NO: 3, or the cDNA thereof.

9. The method of claim 1 , wherein the first aspartic protease is encoded by a polynucleotide comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 3 or at least 98% sequence identity to nucleotides 61 to 1299 of SEQ ID NO: 3, or the cDNA thereof.

10. The method of claim 1 , wherein the first aspartic protease is encoded by a polynucleotide comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 3 or at least 99% sequence identity to nucleotides 61 to 1299 of SEQ ID NO: 3, or the cDNA thereof.

11. The method of claim 1 , wherein the first aspartic protease is encoded by a polynucleotide comprising SEQ ID NO: 3 or nucleotides 61 to 1299 of SEQ ID NO: 3, or the cDNA thereof.

12. The method of claim 1 , wherein the mutant strain is completely deficient in the production of the first aspartic protease compared to the parent Trichoderma strain when cultivated under identical conditions.

13. The method of claim 1 , wherein the heterologous polypeptide is an enzyme.

14. The method of claim 1 , wherein the heterologous polypeptide is an acetylmannan esterase, acetyxylan esterase, aminopeptidase, alpha-amylase, arabinanase, arabinofuranosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, coumaric acid esterase, cyclodextrin glycosyltransferase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, feruloyl esterase, GH61 polypeptide having cellulolytic enhancing activity, alpha-galactosidase, beta-galactosidase, glucocerebrosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, glucuronidase, glucuronoyl esterase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, mannanase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme, ribonuclease, alpha-1,6-transglucosidase, transglutaminase, urokinase, xylanase, or beta-xylosidase.

15. The method of claim 1 , wherein the heterologous polypeptide is a phytase.

16. The method of claim 1 , wherein the heterologous polypeptide is a proteolytic enzyme.

17. The method of claim 1 , wherein the heterologous polypeptide is an alpha-glucosidase.

18. The method of claim 1 , wherein the heterologous polypeptide is a cellulase.

19. The method of claim 1 , wherein the heterologous polypeptide is a xylanase.

20. The method of claim 1 , wherein the heterologous polypeptide is a glucoamylase.

21. The method of claim 1 , wherein the heterologous polypeptide is an alpha-amylase.

22. The method of claim 1 , wherein the parent Trichoderma strain is selected from Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , and Trichoderma viride.

23. The method of claim 1 , wherein the parent Trichoderma strain is Trichoderma reesei.

24. The method of claim 1 , wherein the mutant strain produces more biologically active heterologous polypeptide compared to the parent Trichoderma strain when cultured under identical production conditions.

25. The method of claim 1 , wherein the mutant strain further comprises a second protease gene, wherein the second protease gene is modified rendering the mutant strain at least 95% deficient in the production of a second protease compared to the parent Trichoderma strain when cultivated under identical conditions.

26. The method of claim 1 , wherein:
the parent Trichoderma strain is Trichoderma reesei;
the heterologous polypeptide is selected from a phytase, alpha-amylase, glucoamylase, cellulase, alpha-galactosidase, proteolytic enzyme, and xylanase; and
the mutant strain produces more biologically active heterologous polypeptide compared to the parent Trichoderma strain when cultured under identical production conditions.

27. The method of claim 1 , wherein the mutant strain further comprises a subtilisin-like serine protease gene, wherein the subtilisin-like serine protease gene is modified rendering the mutant strain at least 95% deficient in the production of a subtilisin-like serine protease compared to the parent Trichoderma strain when cultivated under identical conditions;
wherein the subtilisin-like serine protease is selected from:
(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

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

• C12P21/02

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

• C12P21/00 Primary

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

• C12N15/04

  Fungi · CPC title

• C12N15/80

  for fungi · CPC title