Cellulolytic enzyme compositions and uses thereof

Patent metadata

Field	Value
Publication number	US-10081824-B2
Application number	US-201615265460-A
Country	US
Kind code	B2
Filing date	Sep 14, 2016
Priority date	Aug 24, 2011
Publication date	Sep 25, 2018
Grant date	Sep 25, 2018

Abstract

The present invention relates to recombinant filamentous fungal host cells producing cellulolytic enzyme compositions and methods of producing and using the compositions.

First claim

What is claimed is:

1. A process for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition comprising: (a) an Aspergillus fumigatus cellobiohydrolase I; (b) an Aspergillus fumigatus cellobiohydrolase II; (c) an Aspergillus fumigatus beta-glucosidase or a variant thereof; and (d) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof;
wherein the Aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of:
(i) a cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID NO: 2;
(ii) a cellobiohydrolase I comprising an amino acid sequence having at least 90% sequence identity to amino acids 27 to 532 of SEQ ID NO: 2;
(iii) a cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 79 to 1596 of SEQ ID NO: 1; and
(iv) a cellobiohydrolase I encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of to nucleotides 79 to 1596 of SEQ ID NO: 1, 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.;
wherein the Aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of:
(i) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID NO: 4;
(ii) a cellobiohydrolase II comprising an amino acid sequence having at least 90% sequence identity to amino acids 20 to 454 of SEQ ID NO: 4;
(iii) a cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 58 to 1700 of SEQ ID NO: 3; and
(iv) a cellobiohydrolase II encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 58 to 1700 of SEQ ID NO: 3, 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.;
wherein the Aspergillus fumigatus beta-glucosidase or homolog thereof is selected from the group consisting of:
(i) a beta-glucosidase comprising amino acids 20 to 863 of SEQ ID NO: 6;
(ii) a beta-glucosidase comprising an amino acid sequence having at least 90% sequence identity to amino acids 20 to 863 of SEQ ID NO: 6;
(iii) a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 58 to 2580 of SEQ ID NO: 5; and
(iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 58 to 2580 of SEQ ID NO: 5,
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.;
wherein the Aspergillus fumigatus beta-glucosidase variant comprises one or more substitutions selected from the group consisting of F100D, S283G, N456E, and F512Y of amino acids 20 to 863 of SEQ ID NO: 6; and
wherein the Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of:
(i) a GH61 polypeptide having cellulolytic enhancing activity comprising to amino acids 26 to 253 of SEQ ID NO: 8;
(ii) a GH61 polypeptide having cellulolytic enhancing activity comprising an amino acid sequence having at least 90% sequence identity to amino acids 26 to 253 of SEQ ID NO: 8;
(iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 76 to 832 of SEQ ID NO: 7; and
(iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of nucleotides 76 to 832 of SEQ ID NO: 7, 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.

2. The process of claim 1 , wherein the cellulosic material is pretreated.

3. The process of claim 1 , further comprising recovering the degraded cellulosic material.

4. The process of claim 1 , wherein the degraded cellulosic material is a sugar.

5. The process of claim 4 , wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.

6. The process of claim 1 , wherein the cellobiohydrolase I comprises an amino acid sequence having at least 95% sequence identity to amino acids 27 to 532 of SEQ ID NO: 2.

7. The process of claim 1 , wherein the cellobiohydrolase I comprises amino acids 27 to 532 of SEQ ID NO: 2.

8. The process of claim 1 , wherein the cellobiohydrolase II comprises an amino acid sequence having at least 95% sequence identity to amino acids 20 to 454 of SEQ ID NO: 4.

9. The process of claim 1 , wherein the cellobiohydrolase II comprises amino acids 20 to 454 of SEQ ID NO: 4.

10. The process of claim 1 , wherein the beta-glucosidase comprises an amino acid sequence having at least 95% sequence identity to amino acids 20 to 863 of SEQ ID NO: 6.

11. The process of claim 1 , wherein the beta-glucosidase comprises amino acids 20 to 863 of SEQ ID NO: 6.

12. The process of claim 1 , wherein the beta-glucosidase variant comprises the substitutions F100D, S283G, N456E, and F512Y of amino acids 20 to 863 of SEQ ID NO: 6.

13. The process of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 95% sequence identity to amino acids 26 to 253 of SEQ ID NO: 8.

14. The process of claim 1 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises amino acids 26 to 253 of SEQ ID NO: 8.

15. The process of claim 1 , wherein the enzyme composition further comprises an endoglucanase.

16. The process of claim 15 , wherein the endoglucanase is a Trichoderma endoglucanase I, a Trichoderma endoglucanase II, or a Trichoderma endoglucanase I and a Trichoderma endoglucanase II.

17. The process of claim 16 , wherein the Trichoderma endoglucanase I is a Trichoderma reesei endoglucanase I.

18. The process of claim 16 , wherein the Trichoderma endoglucanase II is a Trichoderma reesei endoglucanase II.

19. The process of claim 15 , wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of: (a) an Aspergillus fumigatus xylanase or homolog thereof, (b) an Aspergillus fumigatus beta-xylosidase or homolog thereof; or (iii) a combination of (a) and (b);
wherein the Aspergillus fumigatus xylanase or homolog thereof is selected from the group consisting of:
(i) an Aspergillus fumigatus xylanase comprising amino acids 18 to 364 of SEQ ID NO: 10, amino acids 20 to 323 of SEQ ID NO: 12, or amino acids 20 to 397 of SEQ ID NO: 14;
(ii) a xylanase comprising an amino acid sequence h

Assignees

• Novozymes Inc

Inventors

• Shasky Jeffrey
• Maiyuran Suchindra
• Fischer Amanda

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12Y302/01021

  Beta-glucosidase (3.2.1.21) · CPC title

• C12Y302/01004 Primary

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C12P19/14 Primary

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12N9/2445

  Beta-glucosidase (3.2.1.21) · CPC title