# US 11,078,510 B2 · Methods for producing polypeptides in protease-deficient mutants of Trichoderma > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,510 B2 - Document ID: US11078510B2 - Status: Granted - Kind: B2 - Application number: 16/996,168 - Filed: 2020-08-18 - Published: 2020-12-10 - Granted: 2021-08-03 - Earliest claimed: 2009-12-18 - Assignees: Novozymes, Inc. - Applicants: Novozymes, Inc. - Inventors: Suchindra Maiyuran; Abigail Jang; Kimberly Brown; Sandra Merino; Jeffrey Shasky; Eric Abbate - Web page: https://patentgrep.com/patent/US11078510B2 ## CPC - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C12N 9/58: derived from fungi - C12N 15/04: Fungi - C12N 15/80: for fungi - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## 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. ## Claims ### Claim 1 An isolated mutant of a parent Trichoderma strain, said parent strain comprising an aspartic protease gene encoding an aspartic protease, wherein the mutant strain is at least 95% deficient in the production of the aspartic 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; and wherein the mutant further comprises a polynucleotide encoding a polypeptide that is heterologous to the parent Trichoderma strain. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The isolated mutant of claim 1, wherein the parent Trichoderma strain is Trichoderma reesei. ### Claim 4 (depends on 1) The isolated mutant of claim 1, which is completely deficient in the aspartic protease compared to the parent Trichoderma strain when cultivated under identical conditions. ### Claim 5 (depends on 1) The isolated mutant of claim 1, wherein the amino acid sequence has at least 97% sequence identity to SEQ ID NO: 108 or at least 97% sequence identity to amino acids 18 to 395 of SEQ ID NO: 108. ### Claim 6 (depends on 1) The isolated mutant of claim 1, wherein the aspartic protease comprises SEQ ID NO: 108 or amino acids 18 to 395 of SEQ ID NO: 108. ### Claim 7 (depends on 1) The isolated mutant of claim 1, wherein the parent strain further comprises a trypsin-like serine protease gene encoding a trypsin-like serine 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 trypsin-like serine 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 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; 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. ### Claim 8 (depends on 7) The isolated mutant of claim 7, wherein the trypsin-like serine protease comprises SEQ ID NO: 6 or amino acids 20 to 259 of SEQ ID NO: 6. ### Claim 9 (depends on 7) 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. ### Claim 10 (depends on 7) 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. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 11) 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. ### Claim 13 (depends on 11) The method of claim 11, wherein the parent Trichoderma strain is Trichoderma reesei. ### Claim 14 (depends on 11) The method of claim 11, wherein the mutant is completely deficient in the aspartic protease compared to the parent Trichoderma strain when cultivated under identical conditions. ### Claim 15 (depends on 11) The method of claim 11, wherein the amino acid sequence has at least 97% sequence identity to SEQ ID NO: 108 or at least 97% sequence identity to amino acids 18 to 395 of SEQ ID NO: 108. ### Claim 16 (depends on 11) The method of claim 11, wherein the aspartic protease comprises SEQ ID NO: 108 or amino acids 18 to 395 of SEQ ID NO: 108. ### Claim 17 (depends on 11) The method of claim 11, wherein the parent strain further comprises a trypsin-like serine protease gene encoding a trypsin-like serine 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 trypsin-like serine 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 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; wherein the first subtilisin-like serine protease gene 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. ### Claim 18 (depends on 17) The method of claim 17, wherein the trypsin-like serine protease comprises SEQ ID NO: 6 or amino acids 20 to 259 of SEQ ID NO: 6. ### Claim 19 (depends on 17) The method of claim 17, wherein the first subtilisin-like serine protease comprises SEQ ID NO: 2 or amino acids 20 to 882 of SEQ ID NO: 2. ### Claim 20 (depends on 17) The method of claim 17, wherein the second subtilisin-like serine protease comprises SEQ ID NO: 100 or amino acids 16 to 540 of SEQ ID NO: 100.