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( 12 ) United States Patent US010731168B2 (12 ) United States Patent ( 10 ) Patent No.: US 10,731,168 B2 Landowski et al. (45 ) Date of Patent: * Aug. 4 , 2020 ( 54 ) PROTEASE DEFICIENT FILAMENTOUS ( 2013.01) ; CO7K 2317/622 (2013.01 ) ; COOK FUNGAL CELLS AND METHODS OF USE 2317/94 (2013.01 ) ; YO2P 20/52 (2015.11 ) THEREOF (58 ) Field of Classification Search ( 71) Applicant: GLYKOS FINLAND OY , Helsinki None (FI ) See application file for complete search history . ( 72 ) Inventors : Christopher Landowski, Helsinki (FI ) ; Anne Huuskonen , Helsinki (FI ) ; ( 56 ) References Cited Juhani Saarinen , Helsinki (FI ) ; Ann U.S. PATENT DOCUMENTS Westerholm - Parvinen , Kirkkonummi (FI ) ; Anne Kanerva , Helsinki (FI ) ; 4,758,512 A 7/1988 Goldberg et al. 5,674,728 A 10/1997 Buxton et al . Jari Natunen , Vantaa (FI ) ; Anna - Liisa 5,693,520 A 12/1997 Branner et al. Hänninen , Helsinki (FI ) ; Noora 5,756,338 A 5/1998 Buxton et al. Salovuori, Helsinki ( FI) ; Merja 5,776,730 A 7/1998 Stuart Penttilä , Helsinki (FI ) ; Markku 5,821,104 A 10/1998 Holm et al . Saloheimo, Helsinki (FI ) 5,840,570 A 11/1998 Berka et al . 5,846,802 A 12/1998 Buxton et al . 5,968,774 A 10/1999 Lehmbeck ( 73 ) Assignee : GLYKOS FINLAND OY , Helsinki 5,989,889 A 11/1999 Rey et al . (FI ) 6,013,452 A 1/2000 Christensen et al. 6,013,489 A 1/2000 Musters et al . ( * ) Notice : Subject to any disclaimer , the term of this 6,025,185 A 2/2000 Christensen et al. patent is extended or adjusted under 35 6,291,209 B1 9/2001 Lehmbeck 6,352,841 B1 3/2002 Lehmbeck U.S.C. 154 (b ) by 6 days . 6,509,171 B1 1/2003 Berka et al . This patent is subject to a terminal dis 6,806,062 B1 10/2004 Hjort et al. claimer . 7,122,330 B2 10/2006 Emalfarb et al. 7,163,804 B1 1/2007 Royer et al. 7,198,938 B2 4/2007 Shuster et al . ( 21) Appl. No .: 16 /251,194 7,303,877 B2 12/2007 Connelly et al. 7,323,327 B2 1/2008 Edens et al. ( 22 ) Filed : Jan. 18 , 2019 7,691,621 B2 4/2010 Wang 7,771,971 B2 8/2010 Connelly et al. (65 ) Prior Publication Data 7,794,974 B2 9/2010 Peij et al. 7,858,360 B2 12/2010 Hansen et al . US 2019/0233831 A1 Aug. 1 , 2019 (Continued ) Related U.S. Application Data FOREIGN PATENT DOCUMENTS (63 ) Continuation of application No. 15/ 375,635 , filed on Dec. 12 , 2016 , now Pat. No. 10,240,159 , which is a EP 1 266 011 B1 5/2010 continuation of application No. 14 /370,255 , filed as WO 1997/012045 A1 4/1997 application No. PCT/ EP2013 / 050126 on Jan. 4 , 2013 , (Continued ) now Pat . No. 9,567,596 . OTHER PUBLICATIONS (60 ) Provisional application No. 61/ 583,559, filed on Jan. 5 , 2012 . Mäntylä et al . Cloning of the aspartic protease gene of Trichoderma reesei . Proc. Second Eur. Conf. on Fungal Genetics, Apr. 29 -May 1 , (51 ) Int. Cl. 1994 , Book of Abstracts ( 1994 ) . C12N 15/80 ( 2006.01 ) CO7K 16/28 (2006.01 ) (Continued ) COZK 16/00 ( 2006.01) COZK 14/65 ( 2006.01 ) Primary Examiner Suzanne M Noakes CO7K 14/61 ( 2006.01 ) CO7K 14/56 ( 2006.01 ) Assistant Examiner Jae W Lee C12N 9/58 (2006.01 ) ( 74 ) Attorney , Agent, or Firm — Patrick J. Halloran C12P 21/02 ( 2006.01 ) C12P 21/00 ( 2006.01 ) (52 ) U.S. CI. (57 ) ABSTRACT CPC C12N 15/80 (2013.01 ) ; C07K 14/56 ( 2013.01 ) ; CO7K 14/61 (2013.01 ) ; CO7K 14/65 The present disclosure relates to compositions and methods (2013.01 ) ; COOK 16/00 (2013.01 ) ; CO7K useful for the production of heterologous proteins in fila 16/2887 ( 2013.01 ) ; C12N 9/58 ( 2013.01) ; mentous fungal cells . C12P 21/005 (2013.01 ) ; C12P 21/02 (2013.01 ) ; CO7K 2317/14 ( 2013.01) ; CO7K 2317/24 (2013.01 ) ; CO7K 2317/41 (2013.01 ) ; 16 Claims, 46 Drawing Sheets CO7K 2317/55 (2013.01 ) ; CO7K 2317/569 Specification includes a Sequence Listing . US 10,731,168 B2 Page 2 ( 56 ) References Cited Kubicek , et al . UniProt G9P169 ( 2012 ) . Kubicek , et al. UniProt G9N7YO ( 2012 ) . U.S. PATENT DOCUMENTS Kubicek , et al . UniProt G9MY25 ( 2012 ) . Kubicek , et al. UniProt G9NLJ5 (2012 ) . 7,968,312 B2 6/2011 Sagt et al. LONG , et al . UniProt 12EC22 (2012 ) . 7,977,067 B2 7/2011 Power et al. 8,017,341 B2 9/2011 Nikolaev et al . Suarez , et al. UniProt A4V8W6 ( 2007 ) . 8,119,171 B2 2/2012 Lopez et al . Viterbo , et al. UniProt Q64HWO ( 2004 ) . 8,288,517 B2 10/2012 Clarkson et al. Roberts et al. , “ Heterologous Gene Expression in Aspergillus Niger: 8,389,269 B2 3/2013 Sagt et al. a Glucoamylase - Porcine Pancreatic Prophospholipase A2 Fusion 8,426,164 B2 4/2013 Hjort et al . Protein is Secreted and Processed to Yield Mature Enzyme” , Gene. , 8,450,098 B2 5/2013 Kim et al . 122 , 1992 , pp . 155-161 . 8,633,010 B2 1/2014 Lehmbeck et al. Sharma et al. , “ Approaches for Refining Heterologous Protein 8,647,856 B2 2/2014 Shasky et al . Production in Filamentous Fungi” , World J Microbiol Biotechnol, 8,680,252 B2 3/2014 Emalfarb et al. 25 , 2009 , pp . 2083-2094 . 8,716,004 B2 5/2014 Wang Sharon et al ., “ Transcription Factor PrtT Controls Expression of 8,741,654 B2 6/2014 Bodie et al . Multiple Secreted Proteases in the Human Pathogenic Mold Aspergil 8,812,247 B2 8/2014 Roubos et al. lus Fumigatus” , Infection and Immunity , vol. 77, No. 9 , Sep. 2009 , 8,916,363 B2 12/2014 Gusakov et al. 9,175,296 B2 11/2015 Punt et al. pp . 4051-4060 . 9,862,956 B2 * 1/2018 Emalfarb C12N 15/80 Simkovic et al. , “ Induction of Secretion of Extracellular Proteases 2004/0018573 Al 1/2004 Power et al . from Trichoderma Viride ” , Acta Chimica Slovaca , vol. 1 , No. 1 , 2008/0206816 A1 8/2008 Idiris et al. 2008 , pp . 250-264 . 2009/0176219 Al 7/2009 Parenicova et al . Sims et al. , “ Glutamic Protease Distribution is Limited to Filamen 2009/0221030 A1 9/2009 Bao et al . tous Fungi ” , FEMS Microbiology Letters , 239 , 2004 , pp . 95-101. 2009/0253173 A1 10/2009 Wang Sriranganadane et al. , “ Secreted Glutamic Protease Rescues Aspartic 2009/0275079 Al 11/2009 Edens et al. Protease Pep Deficiency in Aspergillus Fumigatus During Growth 2011/0111977 Al 5/2011 Retallack in Acidic Protein Medium ” , Microbiology, 157 , 2011, pp . 1541 2011/0283422 A1 11/2011 Nelson et al. 1550 . 2011/0294191 A1 12/2011 Wang 2012/0030839 A1 2/2012 Emalfarb et al . Suárez et al. , “ Characterization of Genes Encoding Novel Peptidases 2012/0107856 A1 5/2012 Punt et al. in the Biocontrol Fungus Trichoderma Harzianum CECT 2413 2012/0149064 Al 6/2012 Wang et al. Using the TrichoEST FunctionalGenomics Approach ” , Curr Genet , 2012/0213728 A1 8/2012 Meehl et al . 51, 2007 , pp . 331-342 . 2012/0231502 A1 9/2012 Hamilton et al. Uusitalo et al. , Enzyme Production by Recombinant Trichoderma 2012/0232007 A1 9/2012 Bobrowicz et al. Reesei Strains. Journal of Biotechnology, 17 , 1991 , pp . 35-50 . 2012/0276075 A1 11/2012 Monod et al. Van Den Hombergh et al. , “New Protease Mutants in Aspergillus 2012/0288892 Al 11/2012 Maiyuran et al . Niger Result in Strongly Reduced in Vitro Degradation of Target 2012/0328626 A1 12/2012 Sethuraman et al. Proteins ; Genetical and Biochemical Characterization of Seven 2013/0011875 Al 1/2013 Meehl et al. Complementation Groups” , Curr Genet, 28 , 1995 , pp . 299-308 . 2013/0084608 Al 4/2013 Szabo et al . Van Den Hombergh et al. , “ Improve the Efficiency of Protein 2014/0212977 Al 7/2014 Yaver et al. Expression in Fungi ” , Cherntech 26 , Feb. 1996 , pp . 30-37 . 2015/0337279 Al 11/2015 Zhang et al. Van Den Hombergh et al ., “ Aspergillus as a Host for Heterologous Protein Production : The Problem of Proteases ” , Tibtech , vol. 15 , FOREIGN PATENT DOCUMENTS Jul . 1997 , pp . 256-263 . Van Den Hombergh et al ., “ Production of the Homologous Pectin WO 1997/046689 A1 12/1997 WO 2000/046375 A2 8/2000 Lyase B Protein in Six Genetically Defined Protease -Deficient WO 2004/067709 A2 8/2004 Aspergillus Niger Mutant Stains” , Curr Genet , vol. 32 , Jul. 1997 , WO 2005/087922 A1 9/2005 pp . 73-81 . WO 2011075677 A2 6/2011 Van Kuyk et al ., " Analysis of Two Aspergillus Nidulans Genes WO 2012021883 A2 2/2012 Encoding Extracellular Proteases ” , Fungal Genetics and Biology , WO 2012024662 A2 2/2012 vol . 29 , Apr. 2000 , pp . 201-210 . WO 2012048334 A2 4/2012 Vázquez -Laslop et al. , " Characterization of a Vacuolar Protease in WO 2012061382 A2 5/2012 Neurospora Crassa and the Use ofGene Riping to Generate Protease WO 2013048661 A2 4/2013 Deficient Strains” , The Journal of Biological Chemistry , vol. 271, No. 36 , Sep. 1996 , pp . 21944-21949 . Vinterová et al . , “ Evidence for the Presence of Proteolytically OTHER PUBLICATIONS Active Secreted Aspartic Proteinase of Candida Parapsilosis in the Cell Wall ” , Protein Science , vol. 20 , Dec. 2011 , pp . 2004-2012 . Berka et al. Comparative genomic analysis of the thermophilic Wang et al. , “ Bioprocessing Strategies to Improve Heterologous biomass- degrading fungi Myceliophthora thermophila and Thielavia Protein Production in Filamentous Fungal Fermentations” , Biotech terrestris . Nat Biotechnol. Oct. 2 , 2011 ; 29 ( 10 ) : 922-7 . nology Advances , vol . 23, Mar. 2005 , pp . 115-129 . Berka et al . ( 2011) UniProt G2Q4X9 . Wang et al. , “ Isolation of Four Pepsin - Like Protease Genes from Berka et al .
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