Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose

• US 20070238155A1
• Filed: 07/13/2006
• Published: 10/11/2007
• Est. Priority Date: 10/10/1996
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Burlingame, Richard, Sinitsyn, Arkady, Baez, Marco, Salanovich, Tatyana, Okunev, Oleg, Ustinov, Boris, Gusakov, Alexander, Emalfarb, Mark, Antonov, Alexey
• Granted Patent: US 7,883,872 B2
• Time in Patent Office: Days
• US Class Current: 435/101
• 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: C12N 9/244 Endo-1,3(4)-beta-glucanase (3.2.1.6)
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• 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: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01006 Endo-1,3(4)-beta-glucanase (3.2.1.6)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

This invention provides novel enzyme compositions using newly identified and isolated C. lucknowense enzymes, including CBH Ib CBH IIb, EG II, EG VI, β-glucosidase, and xylanase II in conjunction with previously identified enzymes CBH Ia, CBH IIa (previously described as Endo 43), and EG V. These enzyme compositions demonstrate an extremely high ability to convert lignocellulosic biomass (e.g., Avicel, cotton, Douglas fir wood pretreated by organosolv) to glucose. CBH Ia and IIb, which both have a cellulose-binding module (CBM) displayed a pronounced synergism with three major endoglucanases (EG II, EG V, EG VI) from the same fungus in hydrolysis of cotton as well as a strong synergy with each other. The enzyme compositions are effective in hydrolysis of the lignocellulosic biomass.

Claims

1. An enzyme formulation, said formulation comprising at least one enzyme selected from the group consisting of CBH Ib (SEQ ID NO. 2), CBH IIb (SEQ ID NO. 4), EG II (SEQ ID NO. 10), EG VI (SEQ ID NO. 16), BGL (SEQ ID NO 12), and Xyl II (SEQ ID NO. 18).

2. A method of producing fermentable sugars from lignocellulosic material, said method comprising

(a) providing an enzyme formulation, wherein said enzyme formulation comprises at least one enzyme selected from the group consisting of CBH Ib (SEQ ID NO. 2), CBH IIb (SEQ ID NO. 4), EG II (SEQ ID NO. 10), EG VI (SEQ ID NO. 16), BGL (SEQ ID NO 12), and Xyl II (SEQ ID NO. 18);

(b) applying said enzyme formulation to lignocellulosic material to produce fermentable sugars.

3. The method according to claim 2, wherein the fermentable sugars comprise at least one sugar from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.

4. The method according to claim 2, wherein the lignocellulosic material is selected from the group consisting of orchard prunings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, prairie grass, gamagrass, foxtail;

sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, hard and soft woods, organic waste materials generated from agricultural processes, forestry wood waste, or combinations thereof.

5. A method of producing a fermentation product or a starting material for a fermentation product from a fermentable sugar, wherein said method comprises

(a) providing an enzyme formulation, wherein said enzyme formulation comprises at least one enzyme selected from the group consisting of CBH Ib (SEQ ID NO. 2), CBH IIb (SEQ ID NO. 4), EG II (SEQ ID NO. 10), EG VI (SEQ ID NO. 16), BGL (SEQ ID NO 12), and Xyl II (SEQ ID NO. 18);

(b) applying said enzyme formulation to lignocellulosic material to produce a fermentable sugar; and

(c) fermenting said fermentable sugar to produce a fermentation product.

6. The method according to claim 5, wherein the fermentable sugar is selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.

7. The method according to claim 5, wherein the lignocellulosic material is selected from the group consisting of orchard prunings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, prairie grass, gamagrass, foxtail;

sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, hard and soft woods, organic waste materials generated from agricultural processes, forestry wood waste, or combinations thereof.

8. The method according to claim 5, wherein said fermentation product is a biofuel.

9. The method according to claim 5, wherein said fermentation product is selected from the group consisting of lactic acid;

plastics;

specialty chemicals, organic acids, solvents, animal feed supplements, pharmaceuticals, vitamins;

amino acids, industrial enzymes, and chemical feedstocks.

10. A method of producing energy from a fermentable sugar, said method comprising

(a) providing an enzyme formulation, wherein said enzyme formulation comprises at least one enzyme selected from the group consisting of CBH Ib (SEQ ID NO. 2), CBH IIb (SEQ ID NO. 4), EG II (SEQ ID NO. 10), EG VI (SEQ ID NO. 16), BGL (SEQ ID NO 12), and Xyl II (SEQ ID NO. 18);

(b) applying said enzyme formulation to lignocellulosic material to produce a fermentable sugar;

(c) fermenting said fermentable sugar to produce a combustible fermentation product;

(d) combusting said combustible fermentation product to produce energy.

11. The method according to claim 10, wherein the fermentable sugar is selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.

12. The method according to claim 10, wherein the lignocellulosic material is selected from the group consisting of orchard prunings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, prairie grass, gamagrass, foxtail;

sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, hard and soft woods, organic waste materials generated from agricultural processes, forestry wood waste, or combinations thereof.

13. The method according to claim 10, wherein said combustible fermentation product is an alcohol.

14. The method according to claims 2, 5, or 10, wherein the lignocellulosic material is subjected to a pretreatment prior to being exposed to enzymes.

15. The method according to claim 14, wherein the pretreatment comprises exposing the lignocellulosic biomass to an acid, base, solvent, heat, peroxide, ozone, mechanical shredding, grinding, milling, rapid depressurization, or a combination thereof.

16. The method according to claim 15, wherein said solvent is an acetone/ethanol mixture or organosolv.

17. A mutant Chrysosporium lucknowense strain capable of expressing at least one cellobiohydrolase and at least one endo-1,4-β

-glucanase at higher levels than the corresponding non-mutant strain under the same conditions;

wherein said at least one cellobiohydrolase is selected from the group consisting of CBH Ia, CBH Ib, CBH IIa and CBH IIb; and

wherein said at least one endo-1,4-β

-glucanase is selected from the group consisting of EG II, EG V, and EG VI.

18. The mutant Chrysosporium lucknowense strain according to claim 17, wherein said strain is capable of expressing a β

-glucosidase and/or a xylanase at higher levels than the corresponding non-mutant strain under the same conditions.

19. The mutant Chrysosporium lucknowense strain according to claim 18, wherein said xylanase is Xyl II.

20. The mutant Chrysosporium lucknowense strain according to claim 17, wherein said mutant is or is derived from a Chrysosporium lucknowense mutant strain selected from the group consisting of C. lucknowense strain C1 (VKM F-3500 D), UV13-6 (VKM F-3632 D), NG7C-19 (VKM F-3633 D), and UV18-25 (VKM F-3631 D).

21. A protein exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the CBH Ib amino acid sequence of SEQ ID NO. 2 or a part thereof having at least 20 contiguous amino acids.

22. The protein according to claim 21, wherein the protein exhibits at least 70% amino acid identity as determined by the BLAST algorithm with the CBH Ib amino acid sequence of SEQ ID NO. 2 or a part thereof having at least 20 contiguous amino acids.

23. The protein according to claim 21, wherein said protein exhibits at least 75% amino acid identity as determined by the BLAST algorithm with the CBH Ib amino acid sequence of SEQ ID NO. 2 or a part thereof having at least 20 contiguous amino acids.

24. A nucleic acid sequence encoding a protein according to claim 21.

25. A nucleic acid sequence encoding a protein according to claim 22.

26. A nucleic acid sequence encoding a protein according to claim 23.

27. A protein exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the CBH IIb amino acid sequence of SEQ ID NO. 4 or a part thereof having at least 20 contiguous amino acids.

28. The protein according to claim 27, wherein the protein exhibits at least 70% amino acid identity as determined by the BLAST algorithm with the CBH IIb amino acid sequence of SEQ ID NO. 4 or a part thereof having at least 20 contiguous amino acids.

29. The protein according to claim 28, wherein said protein exhibits at least 75% amino acid identity as determined by the BLAST algorithm with the CBH IIb amino acid sequence of SEQ ID NO. 4 or a part thereof having at least 20 contiguous amino acids.

30. A nucleic acid sequence encoding a protein according to claim 27.

31. A nucleic acid sequence encoding a protein according to claim 28.

32. A nucleic acid sequence encoding a protein according to claim 29.

33. A protein exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the EG VI amino acid sequence of SEQ ID NO. 16 or a part thereof having at least 20 contiguous amino acids.

34. The protein according to claim 33, wherein the protein exhibits at least 70% amino acid identity as determined by the BLAST algorithm with the EG VI amino acid sequence of SEQ ID NO. 16 or a part thereof having at least 20 contiguous amino acids.

35. The protein according to claim 34, wherein said protein exhibits at least 75% amino acid identity as determined by the BLAST algorithm with the EG VI amino acid sequence of SEQ ID NO. 16 or a part thereof having at least 20 contiguous amino acids.

36. A nucleic acid sequence encoding a protein according to claim 33.

37. A nucleic acid sequence encoding a protein according to claim 34.

38. A nucleic acid sequence encoding a protein according to claim 35.

39. A protein exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the BGL amino acid sequence of SEQ ID NO. 12 or a part thereof having at least 20 contiguous amino acids.

40. The protein according to claim 39, wherein the protein exhibits at least 70% amino acid identity as determined by the BLAST algorithm with the BGL amino acid sequence of SEQ ID NO. 12 or a part thereof having at least 20 contiguous amino acids.

41. The protein according to claim 40, wherein said protein exhibits at least 75% amino acid identity as determined by the BLAST algorithm with the BGL amino acid sequence of SEQ ID NO. 12 or a part thereof having at least 20 contiguous amino acids.

42. A nucleic acid sequence encoding a protein according to claim 39.

43. A nucleic acid sequence encoding a protein according to claim 40.

44. A nucleic acid sequence encoding a protein according to claim 41.

45. A protein exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the Xyl II amino acid sequence of SEQ ID NO. 18 or a part thereof having at least 20 contiguous amino acids.

46. The protein according to claim 45, wherein the protein exhibits at least 70% amino acid identity as determined by the BLAST algorithm with the Xyl II amino acid sequence of SEQ ID NO. 18 or a part thereof having at least 20 contiguous amino acids.

47. The protein according to claim 46, wherein said protein exhibits at least 75% amino acid identity as determined by the BLAST algorithm with the Xyl II amino acid sequence of SEQ ID NO. 18 or a part thereof having at least 20 contiguous amino acids.

48. A nucleic acid sequence encoding a protein according to claim 45.

49. A nucleic acid sequence encoding a protein according to claim 46.

50. A nucleic acid sequence encoding a protein according to claim 47.

51. A nucleic acid sequence having at least 80% homology with the nucleic acid sequence encoding CBH Ib as shown in SEQ ID NO. 1.

52. A nucleic acid sequence having at least 80% homology with the nucleic acid sequence encoding CBH IIb as shown in SEQ ID NO. 3.

53. A nucleic acid sequence having at least 80% homology with the nucleic acid sequence encoding EG VI as shown in SEQ ID NO. 15.

54. A nucleic acid sequence having at least 80% homology with the nucleic acid sequence encoding BGL as shown in SEQ ID NO. 11.

55. A nucleic acid sequence having at least 80% homology with the nucleic acid encoding Xyl II as shown in SEQ ID NO. 17.

56. A method for degrading a lignocellulosic material to fermentable sugars, said method comprising

contacting the lignocellulosic material with an effective amount of a multi-enzyme product derived from a microorganism, to produce at least one fermentable sugar

wherein at least one of enzyme in the multi-enzyme product is selected from the group consisting of CBH Ia (SEQ ID NO. 6), CBH Ib (SEQ ID NO. 2), CBH IIa (SEQ ID NO 8), CBH IIb (SEQ ID NO. 4), EG II (SEQ ID NO. 10), EG V (SEQ ID NO. 14), EG VI (SEQ ID NO. 16), BGL (SEQ ID NO. 12), and Xyl II (SEQ ID NO. 18).

57. A microorganism or plant capable of expressing one or more of an enzyme selected from the group consisting of CBH Ia (SEQ ID NO.6), CBH Ib (SEQ ID NO. 2), CBH IIa (SEQ ID NO 8), CBH IIb (SEQ ID NO. 4), EG II (SEQ ID NO. 10), EG V (SEQ ID NO. 14), EG VI (SEQ ID NO. 16), BGL (SEQ ID NO. 12), and Xyl II (SEQ ID NO. 18).

58. The microorganism according to claim 57, wherein said microorganism is a fungus.

59. The microorganism according to claim 58, wherein said fungus is selected from the group consisting of Talaromyces, Aspergillus, Trichoderma, Neurospora, Penicillium, Fusarium, Humicola, Myceliophthora, Corynascus, Chaetomium, Tolypocladium, Thielavia, Acremonium, Sporotrichum, Thermoascus, and Chrysosporium.

60. The microorganism according to claim 59, wherein said fungus is Chrysosporium lucknowense.

61. The microorganism according to claim 60, wherein said Chrysosporium is a strain selected from the group consisting of the wild-type strain, Acc. No. VKM F-3500 D;

C1 UV13-6, Acc. No. VKM F-3632 D;

strain C1 NG7C-19, Acc. No. VKM F-3633 D; and

C1 UV18-25, Acc. No. VKM F-3631 D.

62. A nucleic acid sequence having at least 80% homology with the nucleic acid sequence encoding EG II as shown in SEQ ID NO. 9.