# US 10,308,921 B2 · Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

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- Number: US 10,308,921 B2
- Document ID: US10308921B2
- Status: Granted
- Kind: B2
- Application number: 14/352,875
- Filed: 2012-10-31
- Published: 2014-09-11
- Granted: 2019-06-04
- Earliest claimed: 2011-10-31
- Assignees: Novozymes, Inc.
- Applicants: Novozymes, Inc.; Novozymes A/S
- Inventors: Lan Tang; Ye Liu; Yu Zhang; Junxin Duan; Tarana Shaghasi
- Web page: https://patentgrep.com/patent/US10308921B2

## CPC

- C12N 9/2405: {Glucanases}
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07K 14/37: from fungi
- C12N 9/14: Hydrolases (3)
- C12N 9/2434: {acting on beta-1,4-glucosidic bonds}
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/244: {Endo-1,3(4)-beta-glucanase (3.2.1.6)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- 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}
- C07K 2319/02: containing a signal sequence
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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)
- 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)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention provides isolated polypeptides having cellulolytic enhancing activity and polynucleotides encoding the polypeptides, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

## Claims

### Claim 1

A nucleic acid construct comprising a polynucleotide encoding a GH61 polypeptide having cellulolytic enhancing activity operably linked to one or more heterologous control sequences that direct the production of the GH61 polypeptide, 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 95% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10;
(b) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 1008 of SEQ ID NO: 9;
(c) a GH61 polypeptide comprising an amino acid sequence having the amino acid sequence of amino acids 20 to 251 of SEQ ID NO: 10; and
(d) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 753 of SEQ ID NO: 9.

### Claim 2 (depends on 1)

A recombinant host cell comprising the nucleic acid construct of claim 1.

### Claim 3 (depends on 2)

A method of producing a GH61 polypeptide having cellulolytic enhancing activity, comprising:
(a) cultivating the recombinant host cell of claim 2 under conditions conducive for production of the polypeptide; and optionally
(b) recovering the GH61 polypeptide.

### Claim 4 (depends on 1)

A transgenic plant or plant cell transformed with the nucleic acid construct of claim 1.

### Claim 5 (depends on 4)

A method of producing a GH61 polypeptide having cellulolytic enhancing activity, comprising:
(a) cultivating the transgenic plant or plant cell of claim 4 under conditions conducive for production of the polypeptide; and optionally
(b) recovering the GH61 polypeptide.

### Claim 6

A process for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition comprising a GH61 polypeptide having cellulolytic enhancing activity, 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 95% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10;
(b) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 1008 of SEQ ID NO: 9;
(c) a GH61 polypeptide comprising an amino acid sequence having the amino acid sequence of amino acids 20 to 251 of SEQ ID NO: 10; and
(d) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to] the nucleotide sequence of nucleotides 58 to 753 of SEQ ID NO: 9.

### Claim 7

A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition comprising a polypeptide having cellulolytic enhancing activity, wherein the polypeptide having cellulolytic enhancing activity is selected from the group consisting of:
i) a GH61 polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10;
(ii) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 1008 of SEQ ID NO: 9;
(iii) a GH61 polypeptide comprising an amino acid sequence having the amino acid sequence of amino acids 20 to 251 of SEQ ID NO: 10; and
(iv) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 753 of SEQ ID NO: 9;
(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 8

A process 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 a GH61 polypeptide having cellulolytic enhancing activity, 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 95% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10;
(b) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 1008 of SEQ ID NO: 9;
(c) a GH61 polypeptide comprising an amino acid sequence having the amino acid sequence of amino acids 20 to 251 of SEQ ID NO: 10; and
(d) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of nucleotides 58 to 753 of SEQ ID NO: 9.

### Claim 9 (depends on 1)

The nucleic acid construct of claim 1, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 10 (depends on 1)

The nucleic acid construct of claim 1, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 11 (depends on 1)

The nucleic acid construct of claim 1, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 12 (depends on 6)

The process of claim 6, wherein the cellulosic material is pretreated by steam pretreatment with explosion, steam pretreatment without explosion, dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation pretreatment, wet explosion pretreatment, ammonia fiber explosion pretreatment, organosolv pretreatment, biological pretreatment, ammonia percolation pretreatment, ultrasound pretreatment, electroporation pretreatment, microwave pretreatment, supercritical CO2 pretreatment, supercritical H2O pretreatment, ozone pretreatment, ionic liquid pretreatment, or gamma irradiation pretreatment.

### Claim 13 (depends on 6)

The process of claim 6, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 14 (depends on 13)

The process of claim 13, wherein the enzyme is a cellulase, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

### Claim 15 (depends on 13)

The process of claim 13, wherein the enzyme is a hemicellulase, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

### Claim 16 (depends on 6)

The process of claim 6, further comprising recovering the degraded cellulosic material.

### Claim 17 (depends on 16)

The process of claim 16, wherein the degraded cellulosic material is a sugar.

### Claim 18 (depends on 17)

The process of claim 17, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.

### Claim 19 (depends on 6)

The process of claim 6, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 20 (depends on 7)

The process of claim 7, wherein the cellulosic material is pretreated by steam pretreatment with explosion, steam pretreatment without explosion, dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation pretreatment, wet explosion pretreatment, ammonia fiber explosion pretreatment, organosolv pretreatment, biological pretreatment, ammonia percolation pretreatment, ultrasound pretreatment, electroporation pretreatment, microwave pretreatment, supercritical CO2 pretreatment, supercritical H2O pretreatment, ozone pretreatment, ionic liquid pretreatment, or gamma irradiation pretreatment.

### Claim 21 (depends on 7)

The process of claim 7, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 22 (depends on 21)

The process of claim 21, wherein the enzyme is a cellulase, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

### Claim 23 (depends on 21)

The process of claim 21, wherein the enzyme is a hemicellulase, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

### Claim 24 (depends on 7)

The process of claim 7, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

### Claim 25 (depends on 7)

The process of claim 7, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.

### Claim 26 (depends on 7)

The process of claim 7, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 27 (depends on 8)

The process of claim 8, wherein the cellulosic material is pretreated by steam pretreatment with explosion, steam pretreatment without explosion, dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation pretreatment, wet explosion pretreatment, ammonia fiber explosion pretreatment, organosolv pretreatment, biological pretreatment, ammonia percolation pretreatment, ultrasound pretreatment, electroporation pretreatment, microwave pretreatment, supercritical CO2 pretreatment, supercritical H2O pretreatment, ozone pretreatment, ionic liquid pretreatment, or gamma irradiation pretreatment.

### Claim 28 (depends on 8)

The process of claim 8, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 29 (depends on 28)

The process of claim 28, wherein the enzyme is a cellulase, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

### Claim 30 (depends on 28)

The process of claim 28, wherein the enzyme is a hemicellulase, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

### Claim 31 (depends on 8)

The process of claim 8, wherein the fermenting of the cellulosic material produces a fermentation product.

### Claim 32 (depends on 31)

The process of claim 31, further comprising recovering the fermentation product from the fermentation.

### Claim 33 (depends on 32)

The process of claim 32, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.

### Claim 34 (depends on 8)

The process of claim 8, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 35 (depends on 1)

The nucleic acid construct of claim 1, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 36 (depends on 1)

The nucleic acid construct of claim 1, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 37 (depends on 6)

The process of claim 6, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 38 (depends on 6)

The process of claim 6, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having as at least 97% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 39 (depends on 6)

The process of claim 6, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 40 (depends on 6)

The process of claim 6, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 41 (depends on 7)

The process of claim 7, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having as at least 96% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 42 (depends on 7)

The process of claim 7, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 43 (depends on 7)

The process of claim 7, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 44 (depends on 7)

The process of claim 7, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 45 (depends on 8)

The process of claim 8, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having as at least 96% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 46 (depends on 8)

The process of claim 8, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 47 (depends on 8)

The process of claim 8, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.

### Claim 48 (depends on 8)

The process of claim 8, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having as at least 99% sequence identity to the amino acid sequence of amino acids 20 to 336 of SEQ ID NO: 10.