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 polynucleotide comprising a nucleic acid sequence encoding a GH61 variant protein that is at least about 90% identical to SEQ ID NO:2 or a polynucleotide that hybridizes under stringent hybridization conditions to the polynucleotide and/or a complement of a polynucleotide encoding said GH61 variant protein. 
     
     
         2 . A polynucleotide sequence encoding a GH61 variant protein, wherein said polynucleotide sequence is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of SEQ ID NOS:1, 4, 7, and/or 10, or a polynucleotide that hybridizes under stringent hybridization conditions to the polynucleotide and/or a complement of any of SEQ ID NOS:1, 4, 7, and/or 10. 
     
     
         3 . A recombinant nucleic acid construct comprising at least one polynucleotide sequence encoding at least one GH61 protein, wherein the polynucleotide is selected from: (a) a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2, 3, 5, 6, 8, and/or 9, wherein the amino acid sequence comprises at least one substitution and/or substitution set provided herein; (b) a polynucleotide that hybridizes under stringent hybridization conditions to at least a fragment of a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, 3, 5, 6, 8, and/or 9, and wherein said amino acid sequence comprises at least one substitution and/or at least one substitution set provided herein; and/or (c) a polynucleotide that hybridizes under stringent hybridization conditions to the complement of at least a fragment of a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, 3, 5, 6, 8, and/or 9, and wherein said amino acid sequence comprises at least one substitution and/or at least one substitution set provided herein. 
     
     
         4 . A recombinant nucleic acid construct comprising at least one polynucleotide sequence encoding at least one GH61 protein, wherein the polynucleotide is selected from: (a) a polynucleotide that encodes a polypeptide comprising 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 to SEQ ID NO:2, wherein the amino acid sequence comprises at least one substitution and/or substitution set provided herein; (b) a polynucleotide that hybridizes under stringent hybridization conditions to a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, and wherein said amino acid sequence comprises at least one substitution and/or at least one substitution set provided herein; and/or (c) a polynucleotide that hybridizes under stringent hybridization conditions to the complement of a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, and wherein said amino acid sequence comprises at least one substitution and/or at least one substitution set provided herein. 
     
     
         5 . The recombinant nucleic acid construct of  claim 3 , wherein the polynucleotide sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of SEQ ID NOS:1, 4, 7, and/or 10, and wherein said polynucleotide sequence comprises at least one mutation and/or at least one mutation set provided herein. 
     
     
         6 . The recombinant nucleic acid construct of  claim 3 , wherein the polynucleotide sequence comprises at least one mutation or mutation set selected from t60c/c573g, t60c/c573g/g1026a, c573g, t60c/c291a/c573g, t60c/c291a, t60c/c876t, a312g, t60c, t379a/c380g/g381c, c300t, t204c/t379a/c380g/g381c/c385t, g1026a, c246t, c597g, c72t, c732g/c843t/c882t, c909t, c912g, g921a, c792t, g972t, g921a, t379a/c380g/g381c/c454a/c456a/c732t/c843t/c849t, c520a/c522g; t60c/c573g; t60c/c288t/c573g; t60c/c198t/c573g; and/or t60c/g399a/c573g. 
     
     
         7 . The recombinant nucleic acid construct of  claim 3 , wherein said nucleic acid sequence is operably linked to a promoter. 
     
     
         8 . The nucleic acid construct of  claim 3 , wherein said construct further encodes at least one enzyme in addition to said GH61 variant protein. 
     
     
         9 . The nucleic acid construct of  claim 8 , 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. 
     
     
         10 . An expression construct comprising at least one nucleic acid construct of  claim 3 . 
     
     
         11 . A host cell comprising the nucleic acid construct of  claim 3 . 
     
     
         12 . The host cell of  claim 11 , 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. 
     
     
         13 . The host cell of  claim 11 , wherein said host cell is a yeast or filamentous fungal cell. 
     
     
         14 . A method of producing at least one GH61 variant protein comprising culturing the host cell set forth in  claim 3 , under conditions such that said host cell produces at least one of GH61 variant proteins. 
     
     
         15 . The method of  claim 14 , 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.