Recombinant Beta-Glucosidase Variants for Production of Soluble Sugars from Cellulosic Biomass

Abstract

The invention relates to recombinant expression of a variant form of a fungal C1 strain β-glucosidase. The invention also relates to the generation of fermentable sugars from biomass and the production of biofuels by fermentation of the sugars using genetically modified organisms expressing the β-glucosidase variant. The invention provides methods for producing a fermentable sugar, such as glucose, from cellobiose by contacting cellobiose with a recombinant β-glucosidase variant protein, such as a variant protein secreted by a recombinant host cell into culture medium. Methods of the invention may be used for conversion of a biomass substrate to a fermentable sugar, and ultimately to ethanol or other biofuel.

What is claimed: 
     
         1 . A β-glucosidase (Bgl1) variant that has at least 70% identity to amino acid residues 20-870 of SEQ ID NO:2 and comprises an amino acid substiution at position Q291, an amino acid substitution at position D369, or an amino acid substitution at position E402, as determined with reference to SEQ ID NO:2. 
     
     
         2 . The Bgl1 variant of  claim 1 , wherein the substitution at Q291 is W, A, or F; the substitution at D369 is L, Y, V, A H, R, F, E, M, I, K, C, P, or Q; or the substitution at E402 is N. 
     
     
         3 . A Bgl1 variant of  claim 1  that has, relative to a native C1 Bgl1 comprising amino acids 20-870 of SEQ ID NO:2,
 a) at least 5-fold greater thermoactivity at about pH 5 and about 65° C., or 
 b) at least 3-fold greater thermostability at about pH 5 and about 65° C., or 
 c) both (a) and (b). 
 
     
     
         4 . The Bgl1 variant of  claim 1 , further comprising at least one amino acid substitution at a position selected from the group consisting of Q258, Q313, S434, A475, K495, and G628. 
     
     
         5 . A Bgl1 variant of  claim 4  that has, relative to C1 Bgl1 Variant 3 (SEQ ID NO:5),
 a) at least β-fold greater thermoactivity at about pH 5 and about 70° C., or 
 b) at least 3-fold greater thermostability at about pH 5 and about 65° C., or 
 c) both (a) and (b). 
 
     
     
         6 . The Bgl1 variant of  claim 4 , wherein the variant comprises at least one substitution at a position selected from the group consisting of A689 and Y715. 
     
     
         7 . A Bgl1 variant of  claim 6  that has, relative to C1 Bgl1 Variant 269 (SEQ ID NO:7),
 a) at least 4-fold greater thermoactivity at about pH 4.5 and about 70° C., or 
 b) at least 2-fold greater thermostability at about pH 4.5 and about 70° C., or 
 c) both (a) and (b). 
 
     
     
         8 . The Bgl1 variant of  claim 6 , further comprising at least one amino acid substitution at a position selected from the group consisting of D47, A343, and T687. 
     
     
         9 . A Bgl1 variant of  claim 8  that has, relative to Bgl1 Variant 481 (SEQ ID NO:9),
 a) at least 4-fold greater thermoactivity at about pH 4.2 and about 70° C., or 
 b) at least 4-fold greater thermostability at about pH 4.5 and about 70° C., or 
 c) both (a) and (b). 
 
     
     
         10 . The Bgl1 variant of  claim 6 , further comprising at least one amino acid substitution at a position selected from the group consisting of I106, V260, F314, and A732. 
     
     
         11 . The Bgl1 variant of  claim 10 , further comprising at least one amino acid substitution at position D47, Q85, A109, or A343. 
     
     
         12 . The Bgl1 variant of  claim 11 , further comprising at least one amino acid substitution at position A79. 
     
     
         13 . A composition comprising a Bgl1 variant of  claim 1  and at least one additional normaturally occurring recombinant cellulase polypeptide, selected from
 a) a recombinant endoglucanase (EG) polypeptide 
 b) a recombinant cellobiohydrolase polypeptide (CBH) 
 c) at least one recombinant EG polypeptide and at least one recombinant CBH polypeptide. 
 
     
     
         14 . A recombinant nucleic acid encoding a Bgl1 variant of  claim 1 . 
     
     
         15 . An expression vector comprising the recombinant nucleic acid of  claim 14 . 
     
     
         16 . A host cell that comprises the recombinant nucleic acid of  claim 14 , wherein said nucleic acid is optionally operably linked to a promoter other than the C1 Bgl1 promoter. 
     
     
         17 . The host cell of  claim 16  that also expresses at least one additional normaturally occurring recombinant cellulase polypeptide, selected from
 a) a recombinant endoglucanase (EG) polypeptide, and/or 
 b) a recombinant cellobiohydrolase polypeptide (CBH). 
 
     
     
         18 . A method of producing a Bgl1 polypeptide comprising culturing a host cell of  claim 16  under conditions in which the Bgl1 variant is expressed. 
     
     
         19 . A method of saccharification of a cellulosic substrate comprising
 (a) providing a cellulosic substrate; and   (b) contacting the substrate with a cellulase mixture comprising:
 i) at least one protein with cellobiohydrolase activity; 
 ii) at least one protein with endoglucanase activity; and 
 iii) a β-glucosidase variant of  claim 1  under conditions in which the cellulase mixture catalyzes hydrolysis of the cellulosic substrate. 
   
     
     
         20 . A method of producing glucose comprising combining cellobiose with a β-glucosidase variant of  claim 1  under conditions in which glucose is produced.