Filamentous fungal host cells and methods of recombinantly producing proteins

Patent metadata

Field	Value
Publication number	US-9969993-B2
Application number	US-13083808-A
Country	US
Kind code	B2
Filing date	May 30, 2008
Priority date	May 31, 2007
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

The present invention relates to cellulolytic compositions for degrading or converting cellulose-containing material and methods of producing and using the compositions.

First claim

What is claimed is:

1. An isolated recombinant filamentous fungal host cell for producing a cellulolytic protein composition, comprising: (a) a first polynucleotide encoding a heterologous GH61 polypeptide having cellulolytic enhancing activity; (b) a second polynucleotide encoding a heterologous beta-glucosidase; and (c) polynucleotides encoding native or heterologous cellulolytic enzymes selected from the group consisting of a cellobiohydrolase I, a cellobiohydrolase II, and an endoglucanase I;
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 mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14;
(b) a GH61 polypeptide encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2X SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;
(c) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; and
(d) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14;
wherein the beta-glucosidase is selected from the group consisting of:
(a) a beta-glucosidase comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105;
(b) a beta-glucosidase encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 104 or SEQ ID NO: 106, or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2X SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;
(c) a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 104 or SEQ ID NO: 106; and
(d) a beta-glucosidase comprising the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105;
wherein the cellobiohydrolase I is selected from the group consisting of:
(a) a cellobiohydrolase I comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 52;
(b) a cellobiohydrolase I encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 51 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2X SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;
(c) a cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 51; and
(d) a cellobiohydrolase I comprising the mature polypeptide of SEQ ID NO: 52;
wherein the cellobiohydrolase II is selected from the group consisting of:
(a) a cellobiohydrolase II comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 54;
(b) a cellobiohydrolase II encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 53 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2X SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;
(c) a cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 53; and
(d) a cellobiohydrolase II comprising the mature polypeptide of SEQ ID NO: 54; and
wherein the endoglucanase I is selected from the group consisting of:
(a) an endoglucanase I comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 56;
(b) an endoglucanase I encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 55 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2X SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;
(c) an endoglucanase I encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 55; and
(d) an endoglucanase I comprising the mature polypeptide of SEQ ID NO: 56;
wherein the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , or Trichoderma cell.

2. The filamentous fungal host cell of claim 1 , further comprising polynucleotides encoding cellulolytic enzymes selected from the group consisting of an endoglucanase II, an endoglucanase III, and an endoglucanase V;
wherein the endoglucanase II is selected from the group consisting of:
(a) an endoglucanase II comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 58;
(b) an endoglucanase II encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 57 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sp

Assignees

• Merino Sandra
• Mcfarland Keith
• Cherry Joel
• Teter Sarah
• Novozymes Inc

Inventors

• Merino Sandra
• Mcfarland Keith
• Cherry Joel
• Teter Sarah

Classifications

• C12N9/2445

  Beta-glucosidase (3.2.1.21) · CPC title

• Y02E50/17

  Cross-Sectional Technologies · mapped topic

• C12P7/10

  substrate containing cellulosic material · CPC title

• C12N9/2437 Primary

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

• C12P19/16

  produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin (non-biological hydrolysis of starch C08B30/00) · CPC title