Cellobiohydrolase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10144920-B2
Application number	US-201715446676-A
Country	US
Kind code	B2
Filing date	Mar 1, 2017
Priority date	Oct 23, 2009
Publication date	Dec 4, 2018
Grant date	Dec 4, 2018

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.

First claim

What is claimed is:

1. An isolated variant of a parent cellobiohydrolase II, comprising a substitution at a position corresponding to position 347 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 347 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.

3. The variant of claim 2 , wherein the substitution is Ile.

4. The variant of claim 1 , wherein the variant has at least 96% sequence identity to residues 18-481 of SEQ ID NO: 2.

5. The variant of claim 1 , wherein the variant has at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2.

6. The variant of claim 1 , wherein the variant has at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2.

7. The variant of claim 1 , wherein the variant has at least 99% sequence identity to residues 18-481 of SEQ ID NO: 2.

8. The variant of claim 1 , 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.

9. The variant of claim 1 , wherein the parent cellobiohydrolase II has at least 96% sequence identity to residues 18-481 of SEQ ID NO: 2.

10. The variant of claim 1 , wherein the parent cellobiohydrolase II has at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2.

11. The variant of claim 1 , wherein the parent cellobiohydrolase II has at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2.

12. The variant of claim 1 , wherein the parent cellobiohydrolase II has at least 99% sequence identity to residues 18-481 of SEQ ID NO: 2.

13. The variant of claim 1 , wherein the parent cellobiohydrolase II comprises residues 18-481 of SEQ ID NO: 2, or a fragment thereof having cellobiohydrolase activity.

14. The variant of claim 1 , wherein the parent cellobiohydrolase II comprises residues 18-481 of SEQ ID NO: 2.

15. The variant of claim 1 , wherein the variant has increased thermostability relative to the parent.

16. An isolated polynucleotide encoding the variant of claim 1 .

17. A recombinant host cell comprising the isolated polynucleotide of claim 16 .

18. 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 16 under conditions suitable for the expression of the variant; and
(b) recovering the variant.

19. A transgenic plant, plant part or plant cell transformed with the isolated polynucleotide of claim 16 .

20. A method of producing the variant of claim 1 , 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.

21. A method for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition comprising the variant of claim 1 .

22. The method of claim 21 , further comprising recovering the degraded cellulosic material.

23. A method for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition comprising 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.

24. 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 1 .

25. The method of claim 24 , wherein the fermenting of the cellulosic material produces a fermentation product.

26. The method of claim 25 , further comprising recovering the fermentation product from the fermentation.

Assignees

• Novozymes Inc

Inventors

• Wogulis Mark

Classifications

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• C12Y302/01091

  Cellulose 1,4-beta-cellobiosidase (3.2.1.91) · CPC title