Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

• US 20100306879A1
• Filed: 05/27/2010
• Published: 12/02/2010
• Est. Priority Date: 06/02/2009
• Status: Active Grant

• Current Assignee: Novozymes, Novozymes A/S (Novozymes)
• Original Assignee: Novozymes, Novozymes A/S (Novozymes)
• Inventors: Wu, Wenping, Liu, Ye, Harris, Paul, Tang, Lan
• Granted Patent: US 8,278,507 B2
• Time in Patent Office: Days
• US Class Current: 800/295
• CPC Class Codes: C12N 1/22 Processes using, or culture media containing, cellulose or hydrolysates thereof
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• 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: 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: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• CPC Class Codes: C12Y 302/01099 Arabinan endo-1,5-alpha-L-arabinosidase (3.2.1.99)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to isolated polypeptides having cellobiohydrolase 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

1. An isolated polypeptide having cellobiohydrolase activity, selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO;

2 or SEQ ID NO;

4;

(b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO;

1 or SEQ ID NO;

3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO;

1 or SEQ ID NO;

3, or (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO;

1 or SEQ ID NO;

3;

(d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO;

2 or SEQ ID NO;

4; and

(e) a fragment of a polypeptide of (a), (b), (c), or (d) that has cellobiohydrolase activity.

28. The polypeptide of claim 1, which is encoded by the polynucleotide contained in plasmid pGEM-T-CBHII45097 which is contained in E. coli DSM 22598 or plasmid pGEM-T-CBHII45178 which is contained in E. coli DSM 22599.

29. A composition comprising the polypeptide of claim 1.

30. An isolated polynucleotide encoding the polypeptide of claim 1.

32. A recombinant host cell comprising the polynucleotide of claim 30 operably linked to one or more control sequences that direct the production of the polypeptide.

33. A method of producing the polypeptide of claim 1, comprising:

(a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

34. A method of producing a polypeptide having cellobiohydrolase activity, comprising:

(a) cultivating the host cell of claim 32 under conditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

35. A transgenic plant, plant part or plant cell transformed with a polynucleotide encoding the polypeptide of claim 1.

36. A method of producing a polypeptide having cellobiohydrolase activity, comprising:

(a) cultivating the transgenic plant or plant cell of claim 35 under conditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

37. A method of producing a mutant of a parent cell, comprising inactivating a polynucleotide encoding the polypeptide of claim 1, which results in the mutant producing less of the polypeptide than the parent cell.

41. A double-stranded inhibitory RNA (dsRNA) molecule comprising a subsequence of the polynucleotide of claim 30, wherein optionally the dsRNA is an siRNA or an miRNA molecule.

43. A method of inhibiting the expression of a polypeptide having cellobiohydrolase activity in a cell, comprising administering to the cell or expressing in the cell the double-stranded inhibitory RNA (dsRNA) molecule of claim 41.

52. A method for degrading or converting a cellulosic material, comprising:

treating the cellulosic material with an enzyme composition in the presence of the polypeptide having cellobiohydrolase activity of claim 1.

57. The method of claim 52, further comprising recovering the degraded cellulosic material.

60. A method for producing a fermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition in the presence of the polypeptide having cellobiohydrolase activity of claim 1;

(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.

67. A method 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 in the presence of a polypeptide having cellobiohydrolase activity of claim 1.

68. The method of claim 67, wherein the fermenting of the cellulosic material produces a fermentation product.

69. The method of claim 68, further comprising recovering the fermentation product from the fermentation.

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48. An isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 17 of SEQ ID NO:

2 or amino acids 1 to 17 of SEQ ID NO;

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51. A method of producing a protein, comprising:

(a) cultivating a recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of claim 48, wherein the gene is foreign to the polynucleotide encoding the signal peptide, under conditions conducive for production of the protein; and

(b) recovering the protein.

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