NOVEL BETA-GLUCOSIDASE ENZYMES

• US 20100304438A1
• Filed: 05/28/2010
• Published: 12/02/2010
• Est. Priority Date: 05/29/2009
• Status: Active Grant

• Current Assignee: Iogen Energy Corporation (Iogen Corporation)
• Original Assignee: Iogen Energy Corporation (Iogen Corporation)
• Inventors: Liu, Chengsong, Lavigne, James, Scott, Brian R., Tomashek, John J.
• Granted Patent: US 8,486,683 B2
• Time in Patent Office: Days
• US Class Current: 435/72
• CPC Class Codes: C12N 1/22 Processes using, or culture media containing, cellulose or hydrolysates thereof
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• 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: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Provided are modified beta-glucosidase enzymes, derived from the Trichoderma reesei Cel3A beta-glucosidase, that exhibit improvements in one or more kinetic parameters (K_G, K_G2, k_cat) comprising amino acid substitutions at one or more of positions 43, 101, 260 and 543. Also provided are genetic constructs comprising nucleotide sequences encoding for modified beta-glucosidase enzymes, methods for the production of modified beta-glucosidase enzymes from host strains and the use of the modified beta-glucosidase enzymes in the hydrolysis of cellulose.

Claims

1. A modified Trichoderma reesei TrCel3A beta-glucosidase comprising one or more amino acid substitutions selected from the group consisting of V43X, V101X, F260X and I543X, the modified TrCel3A beta-glucosidase comprising an amino acid sequence which is from about 80% to about 99.9% identical to SEQ ID NO:

1.

2. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 20% increase in K_G,

b. at least a 20% decrease in K_G2, or

c. at least a 10% increase in k_cat

relative to the K_G, K_G2, or k_cat of a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

3. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 30% increase in K_G,

b. at least a 30% decrease in K_G2, or

c. at least a 20% increase in k_cat

relative to the K_G, K_G2, or k_cat of a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

4. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, comprising an amino acid sequence which from about 90% to about 99.9% identical to SEQ ID NO:

1.

5. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the one or more of the amino acid substitutions is selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.

6. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2, wherein the one or more of the amino acid substitutions is selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.

7. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 5, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 20% increase in K_G,

b. at least a 20% decrease in K_G2, or

c. at least a 10% increase in k_cat

relative to a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

8. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 7, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 30% increase in K_G,

b. at least a 30% decrease in K_G2, or

c. at least a 20% increase in k_cat

relative to a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

9. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, further comprising one or more amino acid substitutions selected from the group consisting of V66X, S72X, F96X, T235X, N248X, N369X and A386X.

10. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2, further comprising one or more amino acid substitutions selected from the group consisting of V66X, S72X, F96X, T235X, N248X, N369X and A386X.

11. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 5, further comprising one or more amino acid substitutions selected from the group consisting of V66X, S72X, F96X, T235X, N248X, N369X and A386X.

22. A process for enzymatic hydrolysis of a cellulose substrate comprising contacting said substrate with one or more cellulase enzymes and the modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1.

23. A process for enzymatic hydrolysis of a cellulose substrate comprising contacting said substrate with one or more cellulase enzymes and the modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2.

24. The process of claim 22, wherein the cellulose substrate is a pretreated lignocellulosic feedstock and wherein the process produces fermentable sugars.

25. The process of claim 23, wherein the cellulose substrate is a pretreated lignocellulosic feedstock and wherein the process produces fermentable sugars.

26. The process of claim 24, wherein the pretreated lignocellulose feedstock is selected from the group consisting of corn stover, wheat straw, barley straw, rice straw, oat straw, canola straw, sugarcane straw, soybean stover, corn fiber, sugar beet pulp, pulp mill fines and rejects, sugar cane bagasse, hardwood, softwood, sawdust, switch grass, miscanthus, cord grass, and reed canary grass.

27. The process of claim 25, wherein the pretreated lignocellulose feedstock is selected from the group consisting of corn stover, wheat straw, barley straw, rice straw, oat straw, canola straw, sugarcane straw, soybean stover, corn fiber, sugar beet pulp, pulp mill fines and rejects, sugar cane bagasse, hardwood, softwood, sawdust, switch grass, miscanthus, cord grass, and reed canary grass.

12. An isolated genetic construct comprising a nucleic acid sequence encoding a modified Trichoderma reesei TrCel3A beta-glucosidase comprising one or more of the amino acid substitutions selected from the group consisting of V43X, V101X, F260X and I543X, wherein the amino acid sequence of the modified TrCel3A beta-glucosidase encoded by said construct comprises a sequence which is from about 80% to about 99.9% identical to SEQ ID NO:

1.

14. An isolated genetically modified microbe comprising the genetic construct of claim 12.

16. The isolated genetically modified microbe of claim 14, wherein said microbe is a species of yeast or filamentous fungus.

18. The isolated genetically modified microbe of claim 16, wherein said microbe is Saccharomyces cerevisiae or Trichoderma reesei.

20. A process for producing a modified Trichoderma reesei TrCel3A beta-glucosidase, comprising the steps of (i) providing a genetically modified microbe comprising the genetic construct of claim 12 (ii) culturing the genetically modified microbe in submerged liquid fermentations under conditions that induce the expression of the modified Trichoderma reesei TrCel3A beta-glucosidase, and (iii) recovering the modified Trichoderma reesei TrCel3A beta-glucosidase.

13. An isolated genetic construct comprising DNA encoding a modified Trichoderma reesei TrCel3A beta-glucosidase comprising one or more of the amino acid substitutions selected from the group consisting of V43X, V101X, F260X and I543X, wherein the amino acid sequence of the modified TrCel3A beta-glucosidase encoded by said construct is from about 80% to about 99.9% identical to SEQ ID NO:

1 and the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 20% increase in K_G,

b. at least a 20% decrease in K_G2, or

c. at least a 10% increase in k_cat

relative to a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

15. An isolated genetically modified microbe comprising the genetic construct of claim 13.

17. The isolated genetically modified microbe of claim 15, wherein said microbe is a species of yeast or filamentous fungus.

19. The isolated genetically modified microbe of claim 17, wherein said microbe is Saccharomyces cerevisiae or Trichoderma reesei.

21. A process for producing a modified Trichoderma reesei TrCel3A beta-glucosidase, comprising the steps of (i) providing a genetically modified microbe comprising the genetic construct of claim 13 (ii) culturing the genetically modified microbe in submerged liquid fermentations under conditions that induce the expression of the modified Trichoderma reesei TrCel3A beta-glucosidase, and (iii) recovering the modified Trichoderma reesei TrCel3A beta-glucosidase.

28. A modified Trichoderma reesei TrCel3A beta-glucosidase comprising the amino acid sequence of one of:

SEQ ID NO;

2 (TrCel3A-V43I);

SEQ ID NO;

3 (TrCel3A0V43C);

SEQ ID NO;

4 (TrCel3A-V101A);

SEQ ID NO;

5 (TrCel3A-V101G);

SEQ ID NO;

6 (TrCel3A-F260I);

SEQ ID NO;

7 (TrCel3A-F260V);

SEQ ID NO;

8 (TrCel3A-F260Q);

SEQ ID NO;

9 (TrCel3A-F260D);

SEQ ID NO;

10 (TrCel3A-I543N);

SEQ ID NO;

11 (TrCel3A-I543W);

SEQ ID NO;

12 (TrCel3A-I543A);

SEQ ID NO;

13 (TrCel3A-I543S);

SEQ ID NO;

14 (TrCel3A-I543G);

SEQ ID NO;

15 (TrCel3A-I543L);

SEQ ID NO;

16 (TrCel3A-572N-V101M-F260I);

SEQ ID NO;

17 (TrCel3A-V43I-572N-V101M);

SEQ ID NO;

18 (TrCel3A-572N-V101M-I543N);

SEQ ID NO;

19 (TrCel3A-572N-V101M-I543D);

SEQ ID NO;

20 (TrCel3A-572N-V101M-I543L);

SEQ ID NO;

21 (TrCel3A-V43′

-572N-V101M-F260I);

SEQ ID NO;

22 (TrCel3A-V43′

-572N-V101M-F260I-I543N);

SEQ ID NO;

23 (TrCel3A-V43I-572E-V101M-I543N);

SEQ ID NO;

69 (TrCel3A-572N-V101M-F260I-I543N);

or

SEQ ID NO;

70 (TrCel3A-572N-V101M-F260I-I543L).

29. A modified Family 3 beta-glycosidase comprising one or more of the amino acid substitutions selected from the group consisting of selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L, said position determined from alignment of a parental Family 3 beta-glycosidase with the TrCel3A amino acid sequence as defined in SEQ ID NO:

1, wherein the amino acid sequence of the modified Family 3 beta-glycosidase comprises a sequence which is from about 80% to about 99.9% identical to an amino acid sequence of a parental Family 3 beta-glycosidase from which the modified Family 3 beta-glycosidase is derived.

30. The modified Family 3 beta-glycosidase of claim 29, wherein the amino acid sequence of the modified Family 3 beta-glycosidase is from about 80% to about 99.9% identical to an amino acid sequence of a parental Family 3 beta-glycosidase from which the modified Family 3 beta-glycosidase is derived, the modified Family 3 beta-glycosidase exhibiting

a. an increase in K_P,

b. a decrease in K_S, or

c. an increase in k_cat

relative to the K_P, K_S, or k_cat of a parental Family 3 beta-glycosidase from which the modified Family 3 beta-glycosidase is derived.