Use of glycoside hydrolase 61 family proteins in processing of cellulose

• US 8,298,795 B2
• Filed: 08/22/2011
• Issued: 10/30/2012
• Est. Priority Date: 08/20/2010
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Yang, Jie, Clark, Louis, Campopiano, Onorato, Rao, Kripa, Szabo, Lorand, Baidyaroy, Dipnath, Torok, Janos
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US13/215,193
• Publication Number:
  US 20120083019A1
• Time in Patent Office: 435 Days
• Field of Search: 530/350, 530/356, 536/23.1, 435/320.1, 435/209, 435/254.1, 435/99, 435/41, 435/162, 435/163, 623/13.17, 623/14.12, 604/214
• US Class Current: 435/99
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• 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: C12P 7/14 Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention provides recombinant GH61 proteins obtained from Myceliophtora thermophila, and nucleic acids that encode such proteins. The invention also provides protein fractions isolated from M. thermophila supernatant that have GH61 protein activity. These preparations can be used to increase yield of products from reactions in which a cellulose-containing substrate undergoes saccharification by one or more cellulase enzymes, such as endoglucanase, β-glucosidase, or cellobiohydrolase. Combinations of GH61 protein and cellulases can be used to break down cellulosic biomass into fermentable sugars in the production of ethanol.

Claims

1. A method for increasing yield of fermentable sugars in a reaction in which a cellulose-containing substrate undergoes saccharification by cellulase enzymes comprising an endoglucanase (EG), a β

-glucosidase (BGL), and a cellobiohydrolase (CBH), the method comprising

conducting the reaction in the presence of a recombinant GH61 protein that comprises an amino acid sequence that is at least 90% identical to residues 11-323 of SEQ ID NO;

2 or to a biologically active fragment thereof, whereby the reaction results in a glucose yield that is at least 20% higher than a glucose yield obtained from a saccharification reaction under the same conditions in the absence of said GH61 protein.

2. The method of claim 1, wherein the GH61 protein comprises an amino acid sequence that is at least 95% identical to residues 11-323 of SEQ ID NO:

2, or to a biologically active fragment thereof.

3. The method of claim 1 wherein said cellulase enzymes comprise a combination of an endoglucanase (EG), a β

-glucosidase (BGL), a Type 1 cellobiohydrolase (CBH

     1), and a Type 2 cellobiohydrolase (CBH2).

4. The method of claim 1 wherein the GH61 protein comprises an amino acid sequence that is at least 98% identical to residues 11-323 of SEQ ID NO:

2.

5. The method of claim 1, wherein the recombinant GH61 protein is a variant GH61 protein wherein one or more of residues 11-323 of SEQ ID NO:

2 is changed to increase cellulase enhancing activity, as compared with the cellulase enhancing activity of GH61a (residues 11-323 of SEQ ID NO;

2) without any of said residues changed.

13. The method of claim 1 or claim 6, wherein the cellulose containing substrate is obtained from wheat, wheat straw, sorghum, rice, barley, sugar cane straw, sugar cane bagasse, grasses, switchgrass, corn grain, corn cobs, corn fiber, corn stover, or a combination thereof.

14. The method of claim 1 or claim 6, wherein the substrate is obtained from wheat straw.

6. A method of producing a biofuel comprising ethanol, the method comprising:

a) contacting a cellulose containing substrate with;

i) a plurality of cellulase enzymes comprising an endoglucanase (EG), a β

-glucosidase, and a cellobiohydrolase (CBH); and

ii) a recombinant GH61 protein comprising an amino acid sequence that is at least 90% identical to residues 11-323 of SEQ ID NO;

2 or to a biologically active fragment thereof;

under conditions whereby simple sugars are produced from the substrate;

b) combining simple sugars produced in step (a) with fungal cells under conditions whereby fermentation occurs and ethanol is produced;

wherein the recombinant GH61 protein is a variant GH61 protein wherein one or more of residues 11-323 of SEQ ID NO;

2 is changed to increase cellulase enhancing activity, as compared with the cellulase enhancing activity of GH61a (residues 11-323 of SEQ ID NO;

2) without any of said residues changed.

7. The method of claim 6, wherein the GH61 protein comprises an amino acid sequence that is at least 95% identical to residues 11-323 of SEQ ID NO:

2 or to a biologically active fragment thereof.

8. The method of claim 6, wherein the GH61 protein comprises an amino acid sequence that is at least 98% identical to residues 11-323 of SEQ ID NO:

2 or to a biologically active fragment thereof.

9. The method of claim 6, wherein the GH61 protein comprises an amino acid sequence that is 100% identical to residues 11-323 of SEQ ID NO:

2 or to a biologically active fragment thereof.

10. The method of claim 6, wherein the GH61 protein comprises an amino acid sequence that is at least 95% identical to residues 11-323 of SEQ ID NO:

2.

11. The method of claim 6, wherein said cellulase enzymes are from M. thermophila.

12. The method of claim 6, wherein said fungal cells are yeast cells.

15. A method of producing a biofuel comprising ethanol, the method comprising:

a) contacting a cellulose containing substrate with;

i) a plurality of cellulase enzymes comprising an endoglucanase (EG), a β

-glucosidase, a cellobiohydrolase (CBH); and

ii) a recombinant GH61 protein comprising an amino acid sequence that is at least 90% identical to residues 11-323 of SEQ ID NO;

2 or to a biologically active fragment thereof;

under conditions wherein said GH61 protein enhances cellulase activity of said cellulase enzymes, thereby producing simple sugars from the substrate;

b) combining simple sugars produced in step (a) with fungal cells under conditions whereby fermentation occurs and ethanol is produced;

wherein step (a) comprises contacting the cellulose containing substrate with one or more cellulase enzymes selected from SEQ ID NOs;

61 to 68.

16. The method of claim 15, wherein said fungal cells are yeast cells.

17. A method of producing fermentable sugars from a cellulose containing substrate, comprising combining the substrate with:

a) an enzyme composition comprising one or more β

-glucosidases and one or more cellobiohydrolases; and

b) a recombinant GH61 protein that comprises an amino acid sequence that is at least 90% identical to residues 11-323 of SEQ ID NO;

2 or to a biologically active fragment thereof;

wherein the enzyme composition is substantially free of recombinant endoglucanase.

18. A method for increasing yield of fermentable sugars in a reaction in which a cellulose-containing substrate undergoes saccharification by cellulase enzymes comprising an endoglucanase (EG), a β

-glucosidase (BGL), and a cellobiohydrolase (CBH), the method comprising

conducting the reaction in the presence of a recombinant GH61 protein that comprises an amino acid sequence that is at least 90% identical to residues 11-323 of SEQ ID NO;

2 or to a biologically active fragment thereof,

wherein the recombinant GH61 protein is a variant GH61 protein wherein one or more of residues 11-323 of SEQ ID NO;

2 is changed to increase cellulase enhancing activity, as compared with the cellulase enhancing activity of GH61a (residues 11-323 of SEQ ID NO;

2) without any of said residues changed.

19. The method of claim 18, wherein the GH61 protein comprises an amino acid sequence that is at least 95% identical to residues 11-323 of SEQ ID NO:

2, or to a biologically active fragment thereof.

20. The method of claim 18 wherein said cellulase enzymes comprise a combination of an endoglucanase (EG), a β

-glucosidase (BGL), a Type 1 cellobiohydrolase (CBH

     1), and a Type 2 cellobiohydrolase (CBH2).

21. The method of claim 18 wherein the GH61 protein comprises an amino acid sequence that is at least 98% identical to residues 11-323 of SEQ ID NO:

2.