# US 9,751,916 B2 · Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

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- Number: US 9,751,916 B2
- Document ID: US9751916B2
- Status: Granted
- Kind: B2
- Application number: 15/188,731
- Filed: 2016-06-21
- Published: 2016-10-06
- Granted: 2017-09-05
- Earliest claimed: 2004-01-30
- Assignees: Novozymes, Inc.
- Applicants: Novozymes, Inc.
- Inventors: Kimberly Brown; Paul Harris; Elizabeth Zaretsky; Edward Re; Elena Vlasenko; Keith McFarland; Alfredo Lopez de Leon
- Web page: https://patentgrep.com/patent/US9751916B2

## CPC

- C07K 14/37: from fungi
- C11D 3/38645: {containing cellulase}
- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 1/16: Yeasts; Culture media therefor
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12P 7/14: Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
- 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 302/01021: Beta-glucosidase (3.2.1.21)
- C12P 2201/00: Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- 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
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

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

## Claims

### Claim 1

An isolated recombinant host cell transformed with a nucleic acid construct comprising a polynucleotide encoding a GH61 polypeptide having cellulolytic enhancing activity, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide, wherein the GH61 polypeptide is heterologous to the recombinant host cell, and wherein the GH61 polypeptide having cellulolytic enhancing activity is selected from the group consisting of:
(a) a GH61 polypeptide comprising an amino acid sequence having at least 90% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8;
(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7, wherein 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 65° C.; and
(c) a GH61 polypeptide encoded by a polynucleotide having at least 90% sequence identity to nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7.

### Claim 2 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide comprises an amino acid sequence having at least 90% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 3 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide comprises an amino acid sequence having at least 95% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 4 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide comprises an amino acid sequence having at least 97% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 5 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide comprises an amino acid sequence having at least 98% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 6 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide comprises an amino acid sequence having at least 99% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 7 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide comprises SEQ ID NO: 8 or amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 8 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide is encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7, wherein 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 65° C.

### Claim 9 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide is encoded by a polynucleotide that hybridizes under very high stringency conditions with the full-length complement of nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7, 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.

### Claim 10 (depends on 1)

The recombinant host cell of claim 1, wherein the GH61 polypeptide is encoded by the polynucleotide contained in plasmid pTter61E which is contained in E. coli NRRL B-30814.

### Claim 11 (depends on 1)

A method for producing a GH61 polypeptide having cellulolytic enhancing activity, comprising (a) cultivating the recombinant host cell of claim 1 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

### Claim 12

A nucleic acid construct comprising a polynucleotide encoding a GH61 polypeptide having cellulolytic enhancing activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide, and wherein the GH61 polypeptide having cellulolytic enhancing activity is selected from the group consisting of:
(a) a GH61 polypeptide comprising an amino acid sequence having at least 90% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8;
(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7, wherein 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 65° C.; and
(c) a GH61 polypeptide encoded by a polynucleotide having at least 90% sequence identity to nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7.

### Claim 13 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide comprises SEQ ID NO: 8 or amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 14 (depends on 12)

A recombinant host cell transformed with the nucleic acid construct of claim 12.

### Claim 15 (depends on 12)

A method for producing a GH61 polypeptide having cellulolytic enhancing activity, comprising (a) cultivating a recombinant host cell comprising the nucleic acid construct of claim 12 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

### Claim 16 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide comprises an amino acid sequence having at least 90% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 17 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide comprises an amino acid sequence having at least 95% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 18 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide comprises an amino acid sequence having at least 97% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 19 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide comprises an amino acid sequence having at least 98% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 20 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide comprises an amino acid sequence having at least 99% sequence identity to amino acids 19 to 226 of SEQ ID NO: 8.

### Claim 21 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide is encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7, wherein 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 65° C.

### Claim 22 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide is encoded by a polynucleotide that hybridizes under very high stringency conditions with the full-length complement of nucleotides 55 to 678 of the polynucleotide of SEQ ID NO: 7, 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.

### Claim 23 (depends on 12)

The nucleic acid construct of claim 12, wherein the GH61 polypeptide is encoded by the polynucleotide contained in plasmid pTter61E which is contained in E. coli NRRL B-30814.