Gh61 glycoside hydrolase protein variants and cofactors that enhance gh61 activity

Abstract

The present invention provides various GH61 protein variants comprising various amino acid substitutions. The GH61 protein variants have an improved ability to synergize with cellulase enzymes, thereby increasing the yield of fermentable sugars obtained by saccharification of biomass. In some embodiments, sugars obtained from saccharification are fermented to produce numerous end-products, including but not limited to alcohol.

We claim: 
     
         1 . A non-naturally occurring polynucleotide sequence encoding a glycoside hydrolase 61 (GH61) variant protein that is at least about 90% identical to SEQ ID NO:4. 
     
     
         2 . A non-naturally occurring polynucleotide sequence of  claim 1 , wherein said non-naturally occurring polynucleotide encodes a GH61 variant protein, wherein said GH61 variant protein is at least 90% identical to SEQ ID NO:5. 
     
     
         3 . A recombinant nucleic acid construct comprising the non-naturally occurring polynucleotide sequence of  claim 1 . 
     
     
         4 . The recombinant nucleic acid construct of  claim 3 , wherein said non-naturally occurring polynucleotide sequence encoding a glycoside hydrolase (GH61) variant protein is operably linked to a promoter. 
     
     
         5 . The recombinant nucleic acid construct of  claim 3 , wherein said construct further encodes at least one enzyme in addition to said GH61 variant protein. 
     
     
         6 . The nucleic acid construct of  claim 5 , wherein said at least one additional enzyme is selected from wild-type GH61 enzymes, endoglucanases (EG), beta-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), Type 2 cellobiohydrolases (CBH2), cellulases, hemicellulases, xylanases, xylosidases, amylases, glucoamylases, proteases, esterases, and lipases. 
     
     
         7 . A host cell comprising the nucleic acid construct of  claim 3 . 
     
     
         8 . The host cell of  claim 7 , wherein said host cell further produces at least one enzyme selected from wild-type GH61 enzymes, endoglucanases (EG), beta-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), Type 2 cellobiohydrolases (CBH2), cellulases, hemicellulases, xylanases, xylosidases, amylases, glucoamylases, proteases, esterases, and lipases. 
     
     
         9 . The host cell of  claim 7 , wherein said host cell is a yeast or filamentous fungal cell. 
     
     
         10 . The host cell of  claim 9 , wherein said host cell is  Myceliophthora thermophila.    
     
     
         11 . A method of producing a GH61 variant protein comprising culturing the host cell set forth in  claim 7 , under conditions such that said host cell produces said GH61 variant proteins. 
     
     
         12 . The method of  claim 11 , wherein said host cell further produces at least one additional enzyme selected from wild-type GH61 enzymes, endoglucanases (EG), beta-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), Type 2 cellobiohydrolases (CBH2), cellulases, hemicellulases, xylanases, xylosidases, amylases, glucoamylases, proteases, esterases, and lipases.