Compositions and methods for production of fermentable sugars

• US 8,309,328 B1
• Filed: 07/02/2012
• Issued: 11/13/2012
• Est. Priority Date: 11/02/2010
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Dhawan, Ish Kumar, Baidyaroy, Dipnath, Shaw, Andrew, Tanchak, Oleh, Hill, Christopher, Liu, Chengsong, Chokshi, Amala, Scott, Brian R.
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US13/539,799
• Publication Number:
  US 20120270278A1
• Time in Patent Office: 134 Days
• Field of Search: None
• US Class Current: 435/72
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• 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 19/12 Disaccharides
• 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 2203/00 Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source ethanol C12P7/10
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 101/99018 Cellobiose oxidase (1.1.99.18)
• CPC Class Codes: C13K 1/02 obtained by saccharification of cellulosic materials manufacture of fodder A23K10/32
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present application provides genetically modified fungal organisms that produce enzyme mixtures exhibiting enhanced hydrolysis of cellulosic material to glucose, enzyme mixtures produced by the genetically modified fungal organisms, and processes for producing glucose from cellulose using such enzyme mixtures.

Claims

1. A method for generating at least one fermentable sugar comprising contacting a substrate comprising cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a Myceliophthora fungal cell that has been genetically modified to reduce the amount of endogenous cellobiose oxidizing enzyme activity that is produced by the fungal cell, wherein the fungal cell has been genetically modified by deleting endogenous cellobiose dehydrogenase enzymes having at least about 90% or at least about 95% sequence identity to the sequences of SEQ ID NOS:

6 and 8.

2. The method of claim 1, wherein the Myceliophthora fungal cell is a Myceliophthora thermophile fungal cell.

3. The method of claim 1, wherein the cellulose comprises pretreated lignocellulose.

4. The method of claim 1, wherein the substrate is selected from wood, wood pulp, wood chips, sawdust, hardwood, softwood, newsprint, cardboard, paper pulp, corn fiber, corn grain, corn cobs, corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, oat straw, oat hulls, hay, rice, rice straw, switchgrass, cord grass, rye grass, miscanthus, reed carary grass, waste paper, paper, fruit pulp, vegetable pulp, distillers grain, rice hulls, rice straw, cotton, hemp, flax, sisal, sugar cane bagasse, sugar cane straw, beet pulp, sorghum, soy, soybean stover, canola straw, flowers, and any suitable mixtures thereof.

5. The method of claim 1, wherein the enzyme mixture comprises a cell-free mixture.

6. The method of claim 1, wherein said at least one fermentable sugar comprises glucose.

7. The method of claim 1, wherein said at least one fermentable sugar comprises cellobiose.

8. The method of claim 1, wherein the enzyme mixture comprises at least one inhibitor of said two or more cellulose hydrolyzing enzymes.

9. The method of claim 1, wherein the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of said cellulase enzyme.

10. The method of claim 9, wherein at least one cellulase enzyme is an enzyme that is endogenous to the Myceliophthora fungal cell.

11. The method of claim 10, wherein the enzyme mixture comprises at least one beta-glucosidase.

12. The method of claim 1, wherein the enzyme mixture comprises at least one cellobiose dehydrogenase.

13. The method of claim 12, wherein the cellobiose dehydrogenase comprises CDH1 and/or CDH2.

14. The method of claim 1, further comprising at least one cellulase enzyme and/or at least one additional enzyme.

15. The method of claim 1, wherein the enzyme mixture is subjected to a purification process to selectively remove one or more glucose and/or cellobiose oxidizing enzymes from the enzyme mixture.

16. The method of claim 15, wherein the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzymes from other enzymes present in the enzyme mixture.

17. The method of claim 1, wherein the enzyme mixture comprises at least one inhibitor of the cellobiose oxidizing enzyme.

18. The method of claim 1, wherein at least about 80%, about 85%, or about 90% of the cellulose hydrolyzed by the enzyme mixture is present in the form of cellobiose and/or glucose.

19. The method of claim 1, wherein the method is conducted in a reaction volume of at least 10,000 liters.

20. The method of claim 1, further comprising fermentation of the at least one fermentable sugar to at least one end product.

21. The method of claim 20, wherein said method for producing at least one fermentable sugar and said fermentation of said at least one fermentable sugar are conducted in a simultaneous saccharification and fermentation process.

22. The method of claim 20, wherein the enzyme mixture comprises a cell-free mixture.

23. The method of claim 20, wherein the cellulose comprises pretreated lignocellulose.

24. The method of claim 20, wherein the substrate is selected from wood, wood pulp, wood chips, sawdust, hardwood, softwood, newsprint, cardboard, paper pulp, corn fiber, corn grain, corn cobs, corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, oat straw, oat hulls, hay, rice, rice straw, switchgrass, cord grass, rye grass, miscanthus, reed carary grass, waste paper, paper, fruit pulp, vegetable pulp, distillers grain, rice hulls, rice straw, cotton, hemp, flax, sisal, sugar cane bagasse, sugar cane straw, beet pulp, sorghum, soy, soybean stover, canola straw, flowers, and any suitable mixtures thereof.

25. The method of claim 20, wherein the enzyme mixture comprises at least one inhibitor of the cellobiose oxidizing enzyme.

26. The method of claim 20, wherein said at least one fermentable sugar comprises glucose.

27. The method of claim 20, wherein said at least one fermentable sugar comprises cellobiose.

28. The method of claim 20, wherein the enzyme mixture comprises at least one inhibitor of said two or more cellulose hydrolyzing enzymes.

29. The method of claim 20, wherein the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of said cellulase enzyme.

30. The method of claim 29, wherein at least one cellulase enzyme is an enzyme that is endogenous to the Myceliophthora fungal cell.

31. The method of claim 30, wherein the enzyme mixture comprises at least one beta-glucosidase.

32. The method of claim 20, wherein the enzyme mixture comprises at least one cellobiose dehydrogenase.

33. The method of claim 32, wherein the cellobiose dehydrogenase comprises CDH1 and/or CDH2.

34. The method of claim 20, further comprising at least one cellulase enzyme and/or at least one additional enzyme.

35. The method of claim 20, wherein the enzyme mixture is subjected to a purification process to selectively remove one or more glucose and/or cellobiose oxidizing enzymes from the enzyme mixture.

36. The enzyme mixture of claim 35, wherein the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzymes from other enzymes present in the enzyme mixture.

37. The method of claim 20, wherein the enzyme mixture comprises at least one inhibitor of the cellobiose oxidizing enzyme.

38. The method of claim 20, wherein at least about 80%, about 85%, or about 90% of the cellulose hydrolyzed by the enzyme mixture is present in the form of cellobiose and/or glucose.

39. The method of claim 20, wherein the method is conducted in a reaction volume of at least 10,000 liters.

40. The method of claim 20, wherein the end product is a fuel alcohol or a precursor industrial chemical.

41. The method of claim 40, wherein the fuel alcohol is ethanol or butanol.