Use of glycohydrolase 61 protein variants with improved thermostability for processing cellulose

• US 9,493,802 B2
• Filed: 08/22/2011
• Issued: 11/15/2016
• 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, Torok, Janos, Baidyaroy, Dipnath
• Primary Examiner(s): Holland, Paul
• Application Number:
  US13/640,471
• Publication Number:
  US 20140186896A1
• Time in Patent Office: 1,912 Days
• US Class Current: 1/1
• 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, said method comprising:

contacting a cellulose containing substrate with;

(i) a plurality of cellulase enzymes comprising one or more β

-glucosidases (BGL) and one or more cellobiohydrolases (CBH), and

(ii) a purified variant glycosidase hydrolase 61 (GH61) protein comprising an amino acid sequence that is at least 90% identical but not 100% identical to the amino acid residues corresponding to amino acid residues 11-323 of the amino acid sequence of SEQ ID NO;

2 and having glycosidase hydrolase (GH61) activity,

thereby increasing said yield of fermentable sugars.

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

2.

7. The method of claim 1, wherein said cellulase enzymes are from Myceliophthora thermophila.

8. The method of claim 1, wherein said cellulase enzymes are each selected from the amino acid sequence of SEQ ID NOs:

61 to 68.

9. The method of claim 1, wherein said cellulose containing substrate is selected from the group consisting of wheat, wheat straw, sorghum, corn, rice, barley, sugar cane bagasse, grasses, switchgrass, corn grain, corn cobs, corn fiber, and a combination thereof.

10. The method of claim 1, wherein said cellulose containing substrate is wheat straw.

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

2.

16. The method of claim 1, wherein said plurality of cellulase enzymes further comprises an endoglucanase (EG).

17. The method of claim 1, wherein said plurality of cellulase enzymes is free of added endoglucanase (EG).

2. A method of producing a biofuel containing ethanol, said method comprising:

(a) contacting a cellulose containing substrate with;

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

-glucosidase (BGL), a cellobiohydrolase (CBH); and

(ii) a purified variant glycosidase hydrolase 61 (GH61) protein comprising an amino acid sequence that is at least 90% identical but not 100% identical to the amino acid residues corresponding to amino acid residues 11-323 of the amino acid sequence of SEQ ID NO;

2 and having glycosidase hydrolase (GH61) activity,

under conditions where simple sugars are produced from said cellulose containing substrate;

(b) combining said simple sugars produced in step (a) with fungal cells under fermentation conditions to produce ethanol.

3. The method of claim 2, wherein said fungal cells are yeast cells.

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

2.

5. The method of claim 2, wherein said purified variant GH61 protein comprises an amino acid sequence that is at least 98% identical to the amino acid residues corresponding to amino acid residues 11-323 of the amino acid sequence of SEQ ID NO:

2.

12. The method of claim 2, wherein said cellulase enzymes are from Myceliophthora thermophila.

13. The method of claim 2, wherein said cellulase enzymes are each selected from the amino acid sequence of SEQ ID NOs:

61 to 68.

14. The method of claim 2, wherein said cellulose containing substrate is selected from the group consisting of wheat, wheat straw, sorghum, corn, rice, barley, sugar cane bagasse, grasses, switchgrass, corn grain, corn cobs, corn fiber, and a combination thereof.

15. The method of claim 2, wherein said cellulose containing substrate is wheat straw.

18. A method for increasing yield of fermentable sugars, said method comprising:

contacting a cellulose containing substrate with;

(i) a plurality of cellulase enzymes comprising one or more β

-glucosidases (BGL) and one or more cellobiohydrolases (CBH), and

(ii) a purified variant glycosidase hydrolase 61 (GH61) protein, wherein said GH61 protein consists of an amino acid sequence that is at least 90% identical to a fragment corresponding to amino acid residues 11-323 of the amino acid sequence of SEQ ID NO;

2 and having glycosidase 61 (GH61) activity,

thereby increasing said yield of fermentable sugars.

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

2.

20. The method of claim 18, wherein said purified variant GH61 protein consists of an amino acid sequence that is at least 98% identical to a fragment corresponding to amino acid residues 11-323 of the amino acid sequence of SEQ ID NO:

2.

21. The method of claim 18, wherein said cellulase enzymes are from Myceliophthora thermophila.

22. The method of claim 18, wherein said cellulase enzymes are each selected from the amino acid sequence of SEQ ID NOs:

61 to 68.

23. The method of claim 18, wherein said cellulose containing substrate is selected from the group consisting of wheat, wheat straw, sorghum, corn, rice, barley, sugar cane bagasse, grasses, switchgrass, corn grain, corn cobs, corn fiber, and a combination thereof.

24. The method of claim 18, wherein said cellulose containing substrate is wheat straw.