Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-9499806-B2
Application number	US-201615139007-A
Country	US
Kind code	B2
Filing date	Apr 26, 2016
Priority date	Feb 23, 2011
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

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 of producing and using the polypeptides.

First claim

What is claimed is:

1. A nucleic acid construct comprising an isolated polynucleotide encoding a GH61 polypeptide having cellulolytic enhancing activity, wherein the polynucleotide is operably linked to control sequences that direct expression of the polynucleotide in a suitable host cell and wherein one or more of the control sequences are foreign to the polynucleotide, wherein the GH61 polypeptide having cellulolytic enhancing activity is selected from the group consisting of:
(a) a GH61 polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6;
(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-length complementary strand of (i) or (ii), 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.;
(c) a GH61 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, the mature polypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequence thereof, or the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; and
(d) a fragment of the GH61 polypeptide of (i), (ii), or (iii), that has cellulolytic enhancing activity.

2. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

3. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 96% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

4. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

5. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 98% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

6. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

7. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or a fragment thereof having cellulolytic enhancing activity.

8. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity is a fragment of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6; wherein the fragment has cellulolytic enhancing activity.

9. The nucleic acid construct of claim 1 , wherein the GH61 polypeptide is encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-length complementary strand 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 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.

10. An isolated recombinant host cell transformed with the nucleic acid construct of claim 1 .

11. A method of producing a GH61 polypeptide having cellulolytic enhancing activity, comprising:
(a) cultivating the recombinant host cell of claim 10 under conditions conducive for production of the GH61 polypeptide; and optionally
(b) recovering the GH61 polypeptide.

12. An isolated recombinant host cell transformed with a nucleic acid construct comprising an isolated polynucleotide encoding a GH61 polypeptide having cellulolytic enhancing activity, wherein the polynucleotide is operably linked to control sequences that direct expression of the polynucleotide in the host cell, wherein the GH61 polypeptide having cellulolytic enhancing activity is selected from the group consisting of:
(a) a GH61 polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6;
(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-length complementary strand of (i) or (ii), 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.;
(c) a GH61 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, the mature polypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequence thereof, or the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; and
(d) a fragment of the GH61 polypeptide of (i), (ii), or (iii), that has cellulolytic enhancing activity.

13. The recombinant host cell of claim 12 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

14. The recombinant host cell of claim 12 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 96% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

15. The recombinant host cell of claim 12 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.

16. The recombinant host cell of claim 12 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 98% sequence identity to the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of

Assignees

• Novozymes Inc

Inventors

• Zhang Yu
• Duan Junxin
• Tang Lan
• Wu Wenping

Classifications

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• 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

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C12P2203/00

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