# US 9,518,253 B2 · Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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- Number: US 9,518,253 B2
- Document ID: US9518253B2
- Status: Granted
- Kind: B2
- Application number: 14/747,460
- Filed: 2015-06-23
- Published: 2015-12-24
- Granted: 2016-12-13
- Earliest claimed: 2011-01-26
- Assignees: Novozymes, Inc.
- Applicants: Novozymes Inc.
- Inventors: Nikolaj Spodsberg
- Web page: https://patentgrep.com/patent/US9518253B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- 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)
- 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

## Abstract

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

## Claims

### Claim 1

A process for degrading a cellulosic material, comprising: treating the cellulosic material with a polypeptide having cellobiohydrolase activity selected from the group consisting of:
(a) apolypeptide having at least 90% sequence identity to amino acids 19 to 464 of SEQ ID NO: 2;
(b) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) nucleotides 55 to 1895 of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii), wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/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.;
(c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence thereof; and
(d) a fragment of the polypeptide of (a), (b), or (c) that has cellobiohydrolase activity.

### Claim 2

A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide
selected from the group consisting of:
(1) a polypeptide having at least 90% sequence identity to amino acids 19 to 464 of SEQ ID NO: 2;
(2) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) nucleotides 55 to 1895 of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii), wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide and washing three times each for 15minutes using 2×SSC. 0.2% SDS at 70° C.;
(3) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1; or the cDNA sequence thereof; and
(4) a fragment of the polypeptide of (1), (2), or (3) that has cellobiohydrolase activity; and
(b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product.

### Claim 3

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 in the presence of the polypeptide having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.

### Claim 4

A isolated man-made polynucleotide encoding the polypeptide having at least 95% sequence identity to amino acids 19 to 464 of SEQ ID NO: 2.

### Claim 5 (depends on 4)

A nucleic acid construct or expression vector comprising the polynucleotide of claim 4 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

### Claim 6 (depends on 4)

A man-made recombinant host cell comprising the polynucleotide of claim 4 operably linked to one or more control sequences that direct the production of the polypeptide.

### Claim 7 (depends on 6)

A method of producing a polypeptide having cellobiohydrolase activity, comprising:
cultivating the host cell of claim 6 under conditions conducive for production of the polypeptide.

### Claim 8

A man-made polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 18 of SEQ ID NO: 2.

### Claim 9 (depends on 8)

A recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of claim 8, wherein the gene is foreign to the polynucleotide encoding the signal peptide.

### Claim 10 (depends on 8)

A method of producing a protein, comprising:
cultivating a recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of claim 8, wherein the gene is foreign to the polynucleotide encoding the signal peptide, under conditions conducive for production of the protein.