Cellobiohydrolase variants and polynucleotides encoding same
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. An isolated variant of a parent cellobiohydrolase II, comprising a substitution at a position corresponding to position 363 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.
2. The variant of claim 1 , which comprises a substitution at a position corresponding to position 363 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val.
3. The variant of claim 2 , wherein said substitution is Lys.
4. The variant of claim 1 , wherein said variant has at least 99% sequence identity to residues 18-481 of SEQ ID NO: 2.
5. The variant of claim 1 , wherein said variant is encoded by a polynucleotide having at least 99% sequence identity to nucleotides 52-1443 of SEQ ID NO: 1.
6. The variant of claim 1 , wherein said 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.
7. The variant of claim 1 , wherein said parent cellobiohydrolase II comprises residues 18-481 of SEQ ID NO: 2, or a fragment thereof having cellobiohydrolase activity.
8. The variant of claim 1 , which further comprises a substitution at a position corresponding to position 435 of SEQ ID NO: 2.
9. The variant of claim 1 , wherein said variant has increased thermostability relative to the parent.
10. An isolated polynucleotide encoding said variant of claim 1 .
11. A recombinant host cell comprising said isolated polynucleotide of claim 10 .
12. A method of producing a variant of a parent cellobiohydrolase II, said method comprising:
(a) cultivating an isolated host cell comprising the isolated polynucleotide of claim 10 under conditions suitable for the expression of the variant; and
(b) recovering the variant.
13. A transgenic plant, plant part or plant cell transformed with the isolated polynucleotide of claim 10 .
14. A method of producing the variant of claim 1 , said 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.
15. A method for degrading a cellulosic material, said method comprising treating the cellulosic material with an enzyme composition in the presence of the variant of claim 1 .
16. The method of claim 15 , said method further comprising recovering the degraded cellulosic material.
17. A method for producing a fermentation product, said method comprising:
(a) saccharifying a cellulosic material with an enzyme composition in the presence of the variant of claim 1 ;
(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.
18. A method of fermenting a cellulosic material, said method comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of the variant of claim 1 .
19. The method of claim 18 , wherein the fermenting of the cellulosic material produces a fermentation product.
20. The variant of claim 1 , wherein said variant has at least 96% sequence identity to residues 18-481 of SEQ ID NO: 2.
21. The variant of claim 1 , wherein said variant has at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2.
22. The variant of claim 1 , wherein said variant has at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2.
23. The variant of claim 1 , wherein said parent cellobiohydrolase II has at least 96% sequence identity to residues 18-481 of SEQ ID NO: 2.
24. The variant of claim 1 , wherein said parent cellobiohydrolase II has at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2.
25. The variant of claim 1 , wherein said parent cellobiohydrolase II has at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2.
26. The variant of claim 1 , wherein said parent cellobiohydrolase II has at least 99% sequence identity to residues 18-481 of SEQ ID NO: 2.