Fungal proteases

Abstract

The present invention provides fungal proteases and improved fungal strains that are deficient in protease production.

What is claimed is: 
     
         1 . A fungal protease comprising the polypeptide sequence set forth in SEQ ID NOS:3, 6, 9, and/or 12, or a biologically active fragment thereof. 
     
     
         2 . An isolated polynucleotide sequence encoding the fungal protease set forth in  claim 1 . 
     
     
         3 . The isolated polynucleotide sequence set forth in  claim 2 , wherein said sequence is selected from SEQ ID NOS:1, 2, 4, 5, 7, 8, 10, and/or 11, and/or a fragment and/or fusion of said SEQ ID NOS:1, 2, 4, 5, 7, 8, 10, and/or 11. 
     
     
         4 . The isolated polynucleotide sequence set forth in  claim 2 , wherein said polynucleotide hybridizes to the full length complement of SEQ ID NO:1, 2, 4, 5, 7, 8, 10, and/or 11, under stringent hybridization conditions. 
     
     
         5 . The isolated polynucleotide of  claim 4 , obtainable from a filamentous fungus. 
     
     
         6 . The isolated polynucleotide of  claim 5 , wherein said filamentous fungus is  Myceliophthora.    
     
     
         7 . The isolated polynucleotide of  claim 6 , wherein said filamentous fungus is  Myceliophthora thermophila.    
     
     
         8 . A vector comprising the polynucleotide sequence of  claim 2 . 
     
     
         9 . The vector of  claim 8 , wherein said polynucleotide sequence is operably linked to regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell. 
     
     
         10 . A host cell comprising the vector of  claim 9 , wherein said host cell is prokaryotic or eukaryotic cell. 
     
     
         11 . The host cell of  claim 10 , wherein said host cell is a eukaryotic cell. 
     
     
         12 . The host cell of  claim 11 , wherein said host cell is a yeast or filamentous fungal cell. 
     
     
         13 . The host cell of  claim 11 , wherein said eukaryotic cell is  Myceliophthora    
     
     
         14 . An isolated  Myceliophthora  deficient in at least one protease native to said  Myceliophthora , wherein said protease comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence set forth in SEQ ID NO:3, 6, 9, and/or 12. 
     
     
         15 . The  Myceliophthora  of  claim 14 , wherein said  Myceliophthora  is  Myceliophthora thermophila.    
     
     
         16 . The  Myceliophthora  of  claim 14 , wherein said  Myceliophthora  produces at least one enzyme. 
     
     
         17 . The  Myceliophthora  of  claim 16 , wherein said at least one enzyme comprises at least one cellulase. 
     
     
         18 . The  Myceliophthora  of  claim 17 , wherein said at least one cellulase is selected from beta-glucosidases, endoglucanases, cellobiohydrolases, cellobiose dehydrogenases, endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and/or alpha-glucuronyl esterases. 
     
     
         19 . The  Myceliophthora  of  claim 17 , wherein said cellulase is a recombinant cellulase selected from EG1b, EG2, EG3, EG4, EG5, EG6, CBH1a, CBH1b, CBH2a, CBH2b, GH61a, GH61f, GH61p, and/or BGL. 
     
     
         20 . The  Myceliophthora  of  claim 17 , wherein said  Myceliophthora  further produces at least one non-cellulase enzyme. 
     
     
         21 . The  Myceliophthora  of  claim 20 , wherein said at least one non-cellulase enzyme comprises at least one lipase, amylase, glucoamylase, and/or protease. 
     
     
         22 . A composition comprising the  Myceliophthora  of  claim 14 . 
     
     
         23 . A composition comprising at least one of said enzymes produced by said  Myceliophthora  of  claim 16 . 
     
     
         24 . A method for producing the  Myceliophthora  of  claim 14 , comprising providing a  Myceliophthora  having protease activity, wherein said protease comprises at least one amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with at least one polypeptide sequence set forth in SEQ ID NO:3, 6, 9, and/or 12; and mutating said  Myceliophthora  under conditions such that said protease is mutated to produce a protease-deficient  Myceliophthora.    
     
     
         25 . The method of  claim 24 , wherein said  Myceliophthora  is  Myceliophthora thermophila.    
     
     
         26 . A method for producing at least one enzyme, comprising providing said  Myceliophthora  of  claim 14 , under conditions such that said at least one enzyme is produced by said  Myceliophthora.    
     
     
         27 . The method of  claim 26 , wherein said enzyme comprises at least one cellulase is selected from beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), glycoside hydrolase 61s (GH61s), and/or endoglucanases (EGs). 
     
     
         28 . The method of  claim 27 , wherein said cellulase is a  Myceliophthora  cellulase selected from beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), glycoside hydrolase 61s (GH61s), and/or endoglucanases (EGs). 
     
     
         29 . The method of  claim 28 , wherein said cellulase is selected from EG1b, EG2, EG3, EG4, EG5, EG6, CBH1a, CBH1b, CBH2a, CBH2b, GH61a, GH61f, GH61p, and/or BGL. 
     
     
         30 . The method of  claim 27 , wherein said  Myceliophthora  further produces at least one non-cellulase enzyme. 
     
     
         31 . A composition comprising at least one enzyme produced using the method of  claim 26 . 
     
     
         32 . The composition of  claim 31 , further comprising said  Myceliophthora.    
     
     
         33 . The composition of  claim 31 , wherein said at least one enzyme is selected from beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), glycoside hydrolase 61s (GH61s), and/or endoglucanases (EGs). 
     
     
         34 . The composition of  claim 33 , wherein said cellulase is selected from EG1b, EG2, EG3, EG4, EG5, EG6, CBH1a, CBH1b, CBH2a, CBH2b, GH61a, and/or BGL. 
     
     
         35 . The composition of  claim 31 , further comprising at least one non-cellulase enzyme. 
     
     
         36 . The composition of  claim 35 , wherein said at least one non-cellulase enzyme is a recombinant non-cellulase enzyme. 
     
     
         37 . The composition of  claim 35 , wherein said at least one non-cellulase enzyme comprises at least one lipase, amylase, glucoamylase, protease, oxidase and/or reductase. 
     
     
         38 . A saccharification method comprising (a) providing biomass and protease-deficient  Myceliophthora  in a culture broth, (b) culturing said protease-deficient  Myceliophthora  under conditions in which said at least one enzyme is secreted by said protease-deficient  Myceliophthora  into said culture broth to provide an enzyme-containing broth, and (c) combining the enzyme-containing broth and said biomass under conditions such that saccharification occurs, where (b) may take place before or simultaneously with (c). 
     
     
         39 . The saccharification method of  claim 38 , wherein fermentable sugars are produced during said saccharification. 
     
     
         40 . A method of producing at least one fermentable sugar from at least one cellulosic substrate, comprising contacting the cellulosic substrate with at least one enzyme selected from beta-glucosidase (Bgl), at least one endoglucanase (EG), at least one type 2b cellobiohydrolase (CBH2b), at least one glycoside hydrolase 61(GH61), and/or at least one type 1a cellobiohydrolase (CBH1a) produced by a protease-deficient  Myceliophthora , under conditions in which the fermentable sugar is produced. 
     
     
         41 . The method of  claim 40 , further comprising the step of producing an end-product from said at least one cellulosic substrate, the method further comprising the step of contacting the fermentable sugars with a microorganism in a fermentation to produce the end-product. 
     
     
         42 . The method of  claim 41 , wherein the end-product is a fermentation end product. 
     
     
         43 . The method of  claim 42 , wherein the fermentation end product is selected from alcohols, organic acids, diols, fatty acids, lactic acid, acetic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric acid, amino acids, 1,3-propanediol, ethylene, glycerol, fatty alcohols, butadiene, and beta-lactams. 
     
     
         44 . The method of  claim 43 , wherein said fermentation end product is at least one alcohol selected from ethanol and butanol.