IP Library › Granted Patent US 9,725,489
Granted Patent B2
US 9,725,489 · App. 15/439,569 · Granted Aug 8, 2017

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Inventors: Kirk Schnorr (Holte, DK); Randall Kramer (Lincoln, CA)
Assignees: Novozymes A/S; Novozymes, Inc.
C07K14/37C12P19/02C12P19/14
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Quick Facts
Patent No.
US 9,725,489
App. No.
15/439,569
Granted
Aug 8, 2017
Kind
B2
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.

Claims (44)

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 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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, 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 nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9; 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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

12. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises (i) the polypeptide of SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 10; (ii) amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10; 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 comprises amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

14. 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 the full-length complement of nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, 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.

15. 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 the full-length complement of nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, 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.

16. The recombinant host cell of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide comprising nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9.

17. 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.

18. 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, and optionally recovering the GH61 polypeptide.

19. 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 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, 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 nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9; and

(d) a fragment of the GH61 polypeptide of (a), (b), or (c), wherein the fragment has cellulolytic enhancing activity.

20. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 90% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

21. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 91% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

22. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 92% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

23. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 93% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

24. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 94% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

25. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 95% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

26. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 96% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

27. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 97% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

28. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 98% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

29. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity has at least 99% sequence identity to amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

30. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises (i) the polypeptide of SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 10; (ii) amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10; or (iii) a fragment of (i) or (ii) having cellulolytic enhancing activity.

31. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10.

32. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, 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.

33. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide that hybridizes under very high stringency conditions with the full-length complement of nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, 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.

34. The nucleic acid construct of claim 19 , wherein the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide comprising nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9.

35. An isolated recombinant host cell transformed with the nucleic acid construct of claim 19 .

36. A method of producing a GH61 polypeptide having cellulolytic enhancing activity, comprising cultivating the recombinant host cell of claim 35 under conditions conducive for production of the polypeptide and optionally recovering the GH61 polypeptide.

Assignments (1)
CONFIRMATORY LICENSE Recorded Jul 22, 2019
From: NOVOZYMES, INC.
To: UNITED STATES DEPARTMENT OF ENERGY
Reel/Frame 049825/0619 →
Continuity (4)
Division 15182170 · Jun 14, 2016
Division 14004141
Provisional Application 61451413 · Mar 10, 2011
Related Publication 20170166613A1 · Jun 15, 2017