# US 11,299,522 B2 · Method of producing proteins in filamentous fungi with decreased CLR2 activity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,522 B2 - Document ID: US11299522B2 - Status: Granted - Kind: B2 - Application number: 15/779,595 - Filed: 2016-12-02 - Published: 2020-10-01 - Granted: 2022-04-12 - Earliest claimed: 2015-12-02 - Assignees: BASF SE - Applicants: BASF SE - Inventors: Stefan Haefner; Andreas Thywissen; Holger Hartmann; Nico Boehmer - Web page: https://patentgrep.com/patent/US11299522B2 ## CPC - C07K 14/37: from fungi - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12N 9/16: acting on ester bonds (3.1) - C12N 9/2491: {Beta-mannosidase (3.2.1.25), i.e. mannanase} - C12N 15/80: for fungi - C12Y 301/03008: 3-Phytase (3.1.3.8) - C12Y 302/01025: Beta-mannosidase (3.2.1.25), i.e. mannanase - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - 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) - C12Y: ENZYMES ## Abstract The present invention relates to a method of producing a recombinant polypeptide a filamentous fungus which is genetically modified to decrease or eliminate the activity of cellulase regulator 2 (CLR2) and to express said recombinant polypeptide. The method further relates to a filamentous fungus Myceliophthora thermophila, which is genetically modified to decrease or eliminate the activity of CLR2 and the use of this filamentous fungus in the production of a recombinant polypeptide. ## Claims ### Claim 1 A method of producing a recombinant polypeptide in a filamentous fungus which is genetically modified by disruption of the clr2 gene to decrease or eliminate the activity of cellulase regulator 2 (CLR2) and disruption of the xyr1 gene to decrease or eliminate the activity of xylanase regulator 1 (XYR1) compared to the corresponding filamentous fungus not having the genetic modification which is cultured under the same conditions as the genetically modified filamentous fungus, wherein the filamentous fungus is Myceliophthora thermophila, wherein the filamentous fungus genetically modified to decrease or eliminate the activity of cellulase regulator 2 (CLR2) has been further genetically modified to express said recombinant polypeptide, wherein the recombinant polypeptide is expressed under the control of a promoter which is not activatable by CLR2, wherein the recombinant polypeptide is a polypeptide which is heterologous to the filamentous fungus, wherein the filamentous fungus genetically modified to decrease or eliminate the activity of cellulose regulator 2 (CLR2) has further been genetically modified by disruption of the alp1 gene to decrease or eliminate the activity of alkaline protease 1 (ALP1), said method comprising: (i) growing said genetically modified filamentous fungus in a culture medium which does not contain cellulose or any derivative thereof which is capable of inducing CLR2 activity; and (ii) isolating the recombinant polypeptide from the culture medium. ### Claim 2 (depends on 1) The method of claim 1, wherein the recombinant polypeptide is a hydrolase. ### Claim 3 (depends on 1) The method of claim 1, wherein said genetically modified filamentous fungus is capable of accumulating the recombinant polypeptide in a higher purity than the corresponding filamentous fungus not having the genetic modification which is cultured under the same conditions as the genetically modified filamentous fungus. ### Claim 4 (depends on 1) The method of claim 1, wherein said clr2 gene comprises a nucleic acid sequence selected from the group consisting of: (a) the nucleic acid sequence of nucleotides 3001 to 5570 of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2 or a part of said nucleic acid sequence encoding a part of the polypeptide of SEQ ID NO: 3 required for CLR2 activity; (b) a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 3 or a part of said polypeptide having the same CLR2 activity as the polypeptide of SEQ ID NO: 3; and (c) a nucleic acid sequence encoding a polypeptide having CLR2 activity and having at least 70% sequence identity to the nucleic acid sequence of nucleotides 3001 to 5570 of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2. ### Claim 5 (depends on 1) The method of claim 1, wherein said filamentous fungus which is genetically modified to decrease or eliminate the activity of cellulose regulator 2 (CLR2) further comprises at least one additional genetic modification. ### Claim 6 (depends on 5) The method of claim 5, wherein the at least one additional genetic modification decreases or eliminates the activity of a transcription factor. ### Claim 7 (depends on 1) The method of claim 1, wherein the xyr1 gene is deleted.