Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

• US 10,308,921 B2
• Filed: 10/31/2012
• Issued: 06/04/2019
• Est. Priority Date: 10/31/2011
• Status: Expired due to Fees

• Current Assignee: Novozymes
• Original Assignee: Novozymes
• Inventors: Tang, Lan, Liu, Ye, Zhang, Yu, Duan, Junxin, Shaghasi, Tarana
• Primary Examiner(s): Bui, Phuong T
• Application Number:
  US14/352,875
• Publication Number:
  US 20140256000A1
• Time in Patent Office: 2,407 Days
• Field of Search: None
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/37 from fungi
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/2405 Glucanases
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/244 Endo-1,3(4)-beta-glucanase (3.2.1.6)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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

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.

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

3. 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.

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

5. 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.

9. 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.

10. 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.

11. 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.

35. 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.

36. 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.

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.

12. 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.

13. 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.

14. 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.

15. 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.

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

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

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

19. 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.

37. 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.

38. 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.

39. 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.

40. 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.

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.

20. 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.

21. 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.

22. 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.

23. 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.

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

25. 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.

26. 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.

41. 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.

42. 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.

43. 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.

44. 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.

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.

27. 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.

28. 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.

29. 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.

30. 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.

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

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

33. 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.

34. 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.

45. 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.

46. 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.

47. 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.

48. 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.