Cellobiohydrolase variants and polynucleotides encoding same

Abstract

The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

1 - 27 . (canceled) 
     
     
         28 . An isolated variant of a parent cellobiohydrolase II, comprising a substitution at a position corresponding to position 325 of SEQ ID NO: 2, wherein the variant has cellobiohydrolase II activity and is selected from the group consisting of:
 (a) a variant having at least 95% sequence identity to residues 18-481 of SEQ ID NO: 2;   (b) a variant encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) nucleotides 52-1443 of SEQ ID NO: 1, or (ii) the full-length complement of (i), wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.; and   (c) a variant encoded by a polynucleotide having at least 95% sequence identity to nucleotides 52-1443 of SEQ ID NO: 1 or the genomic DNA sequence thereof.   
     
     
         29 . The variant of  claim 28 , which comprises a substitution at a position corresponding to position 325 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. 
     
     
         30 . The variant of  claim 29 , wherein the substitution is Asp. 
     
     
         31 . The variant of  claim 28 , wherein the variant has at least 96% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         32 . The variant of  claim 28 , wherein the variant has at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         33 . The variant of  claim 28 , wherein the variant has at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         34 . The variant of  claim 28 , wherein the variant has at least 99% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         35 . The variant of  claim 28 , wherein the parent cellobiohydrolase II is selected from the group consisting of:
 (a) a polypeptide having at least 95% sequence identity to residues 18-481 of SEQ ID NO: 2;   (b) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) nucleotides 52-1443 of SEQ ID NO: 1, or (ii) the full-length complement of (i), wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.; and   (c) a polypeptide encoded by a polynucleotide having at least 95% sequence identity to nucleotides 52-1443 of SEQ ID NO: 1.   
     
     
         36 . The variant of  claim 28 , wherein the parent cellobiohydrolase II has at least 96% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         37 . The variant of  claim 28 , wherein the parent cellobiohydrolase II has at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         38 . The variant of  claim 28 , wherein the parent cellobiohydrolase II has at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         39 . The variant of  claim 28 , wherein the parent cellobiohydrolase II has at least 99% sequence identity to residues 18-481 of SEQ ID NO: 2. 
     
     
         40 . The variant of  claim 28 , wherein the parent cellobiohydrolase II comprises residues 18-481 of SEQ ID NO: 2, or a fragment thereof having cellobiohydrolase activity. 
     
     
         41 . The variant of  claim 28 , wherein the parent cellobiohydrolase II comprises residues 18-481 of SEQ ID NO: 2. 
     
     
         42 . The variant of  claim 28 , wherein the variant has increased thermostability relative to the parent. 
     
     
         43 . An isolated polynucleotide encoding the variant of  claim 28 . 
     
     
         44 . A recombinant host cell comprising the isolated polynucleotide of  claim 43 . 
     
     
         45 . A method of producing a variant of a parent cellobiohydrolase II, the method comprising:
 (a) cultivating an isolated host cell comprising the isolated polynucleotide of  claim 43  under conditions suitable for the expression of the variant; and   (b) recovering the variant.   
     
     
         46 . A transgenic plant, plant part or plant cell transformed with the isolated polynucleotide of  claim 43 . 
     
     
         47 . A method of producing the variant of  claim 28 , the method comprising:
 (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and   (b) recovering the variant.   
     
     
         48 . A method for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition comprising the variant of  claim 28 . 
     
     
         49 . The method of  claim 48 , further comprising recovering the degraded cellulosic material. 
     
     
         50 . A method for producing a fermentation product, comprising:
 (a) saccharifying a cellulosic material with an enzyme composition comprising the variant of  claim 28 ;   (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and   (c) recovering the fermentation product from the fermentation.   
     
     
         51 . A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition comprising the variant of  claim 28 . 
     
     
         52 . The method of  claim 51 , wherein the fermenting of the cellulosic material produces a fermentation product. 
     
     
         53 . The method of  claim 52 , further comprising recovering the fermentation product from the fermentation.