Variant cbh i polypeptides with reduced product inhibition

Abstract

The present disclosure relates to variant CBH I polypeptides that have reduced product inhibition, and compositions, e.g., cellulase compositions, comprising variant CBH I polypeptides. The variant CBH I polypeptides and related compositions can be used in variety of agricultural and industrial applications. The present disclosure further relates to nucleic acids encoding variant CBH I polypeptides and host cells that recombinantly express the variant CBH I polypeptides.

What is claimed is: 
     
         1 . A polypeptide comprising a variant cellobiohydrolase I (“CBH I”) catalytic domain as compared to a reference CBH I catalytic domain, comprising:
 (a) a substitution at the amino acid position corresponding to R268 of  T. reesei  CBH I (“R268 substitution”); 
 (b) a substitution at the amino acid position corresponding to R411 of  T. reesei  CBH I (“R411 substitution”); or 
 (c) both an R268 substitution and an R411 substitution, 
 
       wherein substitution (a), (b) or (c) decreases product inhibition as compared to the reference CBH I catalytic domain. 
     
     
         2 . A method for producing ethanol, comprising:
 (a) treating biomass with a composition according to any one of claims  37  to  43  or with a fermentation broth according to  claim 1 , thereby producing monosaccharides; and   (b) culturing a fermenting microorganism in the presence of the monosaccharides produced in step (a) under fermentation conditions, thereby producing ethanol.   
     
     
         3 . The method of  claim 2 , further comprising, prior to step (a), pretreating the biomass. 
     
     
         4 . The method of  claim 2 , wherein said fermenting microorganism is a bacterium or a yeast. 
     
     
         5 . The method of  claim 4 , wherein said fermenting microorganism is a bacterium selected from  Zymomonas mobilis, Escherichia coli  and  Klebsiella oxytoca.    
     
     
         6 . The method of  claim 4 , wherein said fermenting microorganism is a yeast selected from  Saccharomyces cerevisiae, Saccharomyces uvarum, Kluyveromyces fragilis, Kluyveromyces lactis, Candida pseudotropicalis , and  Pachysolen tannophilus.    
     
     
         7 . The method of  claim 2 , wherein said biomass is corn stover, bagasses, sorghum, giant reed, elephant grass,  miscanthus , Japanese cedar, wheat straw, switchgrass, hardwood pulp, softwood pulp, crushed sugar cane, energy cane, or Napier grass. 
     
     
         8 . A method for generating a nucleic acid that encodes a product tolerant variant CBH I polypeptide, comprising modifying the nucleotide sequence of a CBH I-encoding nucleic acid so that the nucleic acid encodes a variant CBH I polypeptide, wherein said variant CBH I polypeptide comprises:
 (i) an R268 substitution;   (ii) an R411 substitution; or   (iii) both an R268 substitution and an R411 substitution,   thereby generating a nucleic acid that encodes a product tolerant variant CBH I polypeptide.   
     
     
         9 . The method of  claim 8 , wherein the modification is by site directed mutagenesis. 
     
     
         10 . The method of  claim 8 , wherein variant CBH I polypeptide comprises an R268 substitution. 
     
     
         11 . The method of  claim 10 , wherein the R268 substituent is a lysine. 
     
     
         12 . The method of  claim 10 , wherein the R268 substituent is an alanine. 
     
     
         13 . The method of  claim 8 , which comprises an R411 substitution. 
     
     
         14 . The method of  claim 13 , wherein the R411 substituent is a lysine. 
     
     
         15 . The method of  claim 13 , wherein the R411 substituent is an alanine. 
     
     
         16 . A method for producing ethanol, comprising:
 (a) treating biomass with a fermentation broth according to  claim 1 , thereby producing monosaccharides; and   (b) culturing a fermenting microorganism in the presence of the monosaccharides produced in step (a) under fermentation conditions, thereby producing ethanol.