# US 8,541,651 B2 · Cellobiohydrolase variants and polynucleotides encoding same

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- Number: US 8,541,651 B2
- Document ID: US8541651B2
- Status: Granted
- Kind: B2
- Application number: 12/908,339
- Filed: 2010-10-20
- Published: 2011-04-28
- Granted: 2013-09-24
- Earliest claimed: 2009-10-23
- Assignees: Novozymes, Inc.
- Applicants: Mark Wogulis
- Inventors: Mark Wogulis
- Web page: https://patentgrep.com/patent/US8541651B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12Y 302/01091: Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- Y02P 20/52: using catalysts, e.g. selective catalysts
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## 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.

## Claims

### Claim 1

An isolated variant of a parent cellobiohydrolase II, comprising a substitution at a position corresponding to position 357 of residues 18-481 of SEQ ID NO: 2, wherein the variant has cellobiohydrolase II activity and 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, (ii) the genomic DNA sequence of nucleotides 52-1443 of SEQ ID NO: 1, or (iii) the full-length complementary strand of (i) or (ii); and
(c) a polypeptide encoded by a polynucleotide having at least 99% identity to nucleotides 52-1443 of SEQ ID NO: 1 or the genomic DNA sequence thereof.

### Claim 2 (depends on 1)

The variant of claim 1, which comprises a substitution at a position corresponding to position 357 of the mature polypeptide 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 or comprises a substitution at position 357 of the mature polypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr.

### Claim 3 (depends on 1)

The variant of claim 1, which further comprises a substitution at a position corresponding to position 435 of the mature polypeptide 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 or further comprises a substitution at position 435 of the mature polypeptide of SEQ ID NO: 2 with Ala, Am, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr.

### Claim 4 (depends on 1)

An isolated polynucleotide encoding the variant of claim 1.

### Claim 5 (depends on 4)

A method of producing a variant of a parent cellobiohydrolase II, said method comprising:
(a) cultivating an isolated host cell comprising the polynucleotide of claim 4 under conditions suitable for the expression of the variant; and
(b) recovering the variant.

### Claim 6 (depends on 4)

A transgenic plant, plant part or plant cell transformed with the isolated polynucleotide of claim 4.

### Claim 7 (depends on 1)

A method for degrading or converting a cellulosic material, said method comprising treating the cellulosic material with an enzyme composition in the presence of the variant of claim 1.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 2)

The variant of claim 2, wherein the substitution is Asn.

### Claim 11 (depends on 1)

The variant of claim 1, wherein the parent cellobiohydrolase II has at least 95% sequence identity to residues 18-481 of SEQ ID NO:2.

### Claim 12 (depends on 1)

The variant of claim 1, wherein the parent cellobiohydrolase II is encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) nucleotides 52-1443 of SEQ ID NO:1, (ii) the genomic DNA sequence of nucleotides 52-1443 of SEQ ID NO:1, or (iii) the full-length complementary strand of (i) or (ii).

### Claim 13 (depends on 1)

The variant of claim 1, wherein the parent cellobiohydrolase II is encoded by a polynucleotide at least 99% sequence identity to nucleotides 52-1443 of SEQ ID NO:1.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 7)

The method of claim 7, said method further comprising recovering the degraded cellulosic material.

### Claim 17 (depends on 9)

The method of claim 9, wherein the fermenting of the cellulosic material produces a fermentation product.

### Claim 18 (depends on 1)

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