Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

Patent metadata

Field	Value
Publication number	US-9605037-B2
Application number	US-201615182170-A
Country	US
Kind code	B2
Filing date	Jun 14, 2016
Priority date	Mar 10, 2011
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

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. An isolated recombinant host cell transformed with 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 polynucleotide encoding 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 having at least 90% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6;
(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the full-length complement of nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (ii) the cDNA of (i), 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 (i) a polynucleotide having at least 90% sequence identity to nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (ii) the cDNA of (i); and
(d) a fragment of the GH61 polypeptide of (a), (b), or (c), wherein the fragment has cellulolytic enhancing activity.

2. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 90% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

3. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 91% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

4. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 92% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

5. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 93% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

6. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 94% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

7. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 95% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

8. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 96% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

9. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 97% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

10. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 98% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

11. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 99% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6.

12. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises (i) the polypeptide of SEQ ID NO: 6; (ii) amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6; or (iii) a fragment of (i) or (ii) having cellulolytic enhancing activity.

13. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the full-length complement of nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (ii) the cDNA of (i), 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.

14. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) the full-length complement of nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (ii) the cDNA of (i), 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.

15. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by (i) a polynucleotide comprising SEQ ID NO: 5, (ii) a polynucleotide comprising nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (iii) the cDNA of (i) or (ii).

16. The recombinant host cell of claim 1 , wherein one or more of the control sequences is heterologous to the polynucleotide encoding the GH61 polypeptide having cellulolytic enhancing activity.

17. A method of producing a GH61 polypeptide having cellulolytic enhancing activity, comprising cultivating the recombinant host cell of claim 1 under conditions conducive for production of the polypeptide.

18. 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 having at least 90% sequence identity to amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6;
(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the full-length complement of nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (ii) the cDNA of (i), 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 (i) a polynucleotide having at least 90% sequence identity to nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5, or (ii) the cDNA of (i); and
(d) a fragment of the GH61 polypeptide of (a), (b), or (c), wherein the fragment has cellulolytic enhancing activity.

19. The nucleic acid construct of claim 18 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises (i) the polypeptide of SEQ ID NO: 6; (ii) amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6; or (iii) a fragment of (i) or (ii) having cellulolytic enhancing activity.

20. A method of producing a GH61 polypeptide having cellulolytic enhancing activity, comprising cultivating a recombinant host cell transformed with the nucleic acid construct of claim 18 under conditions conducive for production of the polypeptide.

21.

Assignees

• Novozymes As
• Novozymes Inc

Inventors

• Schnorr Kirk
• Kramer Randall

Classifications

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · 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

• C07K14/37 Primary

  from fungi · CPC title

• C12N9/2437

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

• C12P7/14

  Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms · CPC title