FAMILY 6 CELLULASE WITH DECREASED INACTIVATION BY LIGNIN

• US 20100041100A1
• Filed: 07/30/2009
• Published: 02/18/2010
• Est. Priority Date: 08/01/2008
• Status: Active Grant

• Current Assignee: Iogen Energy Corporation (Iogen Corporation)
• Original Assignee: Iogen Energy Corporation (Iogen Corporation)
• Inventors: Lavigne, James A., Scott, Brian R., Tomashek, John J., Whissel, Martine
• Granted Patent: US 8,110,389 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)

Abstract

A modified Trichoderma reesei Family 6 (TrCel6A) cellulase enzyme comprising amino acid substitutions at one or more positions selected from the group consisting of 129, 322, 363 and 410 of SEQ ID NO: 1 is provided. Genetic constructs and genetically modified microbes comprising nucleic sequences encoding the modified TrCel6a cellulase are also provided. The modified TrCel6A cellulase of the invention display at least a 15% decrease in inactivation by lignin relative to a parental TrCel6A cellulase from which the modified TrCel6A is derived. Such cellulases find use in a variety of applications in industry requiring enzymatic hydrolysis of cellulose in the presence of lignin, e.g., the hydrolysis of pretreated lignocellulosic feedstocks for the production of fermentable sugars, sugar alcohols and fuel alcohols.

Claims

1. A modified Trichoderma reesei Family 6 (TrCel6A) cellulase comprising one or more amino acid substitutions selected from the group consisting of:

substitution of a basic amino acid at one or more of positions 129 and 410 by a charge-neutral or an acidic amino acid;

substitution of a charge neutral amino acid at one or more of positions 322 and 363 by an acidic amino acid; and

substitution of an amino acid at position 186 by a threonine;

wherein amino acids 83-447 of the modified TrCel6A are from about 47% to about 99.9% identical to amino acids 83-447 of SEQ ID NO;

1.

2. The modified TrCel6A cellulase of claim 1, wherein amino acids 83-447 of the modified TrCel6A cellulase are from about 70% to about 99.9% identical to amino acids 83-447 of SEQ ID NO:

1 and wherein the modified TrCel6A cellulase exhibits at least a 15% reduction in the extent of deactivation by lignin relative to that of a parental TrCel6A cellulase.

3. The modified TrCel6A cellulase of claim 2, wherein amino acids 83-447 of the modified TrCel6A cellulase are from about 90% to about 99.9% identical to amino acids 83-447 of SEQ ID NO:

1.

4. The modified TrCel6A cellulase of any one of claims 1 through 3, wherein the amino acid substitutions are selected from the group consisting of K129E, S186T, A322D, Q363E, R410G, and R410Q.

5. The modified TrCel6A cellulase of any one of claims 1 through 4 further comprising one or more amino acid substitutions selected from the group consisting of Y103H, Y103K, Y103R, Y103A, Y103V, Y103L, Y103P, L136V, L136I, and S413P.

6. An isolated genetic construct comprising a nucleic acid sequence encoding the modified TrCel6A cellulase of any one of claims 1 through 5.

7. An isolated genetically modified microbe comprising the genetic construct of claim 6.

8. The isolated genetically modified microbe of claim 7, wherein the microbe is a species of yeast or filamentous fungus.

9. The isolated genetically modified microbe of claim 8, wherein the microbe is Saccharomyces cerevisiae or Trichoderma reesei.

10. A process for producing a modified TrCel6A cellulase comprising the steps of growing the genetically modified microbe of claim 7 in a culture medium under conditions that induce expression and secretion of the modified TrCel6A cellulase and recovering the modified TrCel6A cellulase from the culture medium.

11. A process for the hydrolyzing a cellulose substrate comprising contacting the substrate with the modified TrCel6A cellulase of any one of claims 1 through 5 in the presence of lignin.

12. The process of claim 11, wherein the cellulose substrate is a pretreated lignocellulosic feedstock and wherein the enzymatic hydrolysis produces fermentable sugars.

13. The process of claim 12, wherein the pretreated lignocellulose feedstock is selected from the group consisting of corn stover, wheat straw, barley straw, rice straw, oat straw, canola 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.

14. A process for producing a modified TrCel6A cellulase comprising the steps of (i) transforming fungal host cells with a genetic construct as defined in claim 6 to produce recombinant fungal strains;

(ii) selecting the recombinant fungal strains expressing the modified TrCel6A cellulase; and

(iii) culturing the recombinant fungal strains selected in step (ii) in submerged liquid fermentations under conditions that induce expression of the modified TrCel6A cellulase.

15. A modified Family 6 glycosidase selected from the group consisting of:

TrCel6A-K129E-S413P (SEQ ID NO;

     37);

TrCel6A-S186T-S413P (SEQ ID NO;

     38);

TrCel6A-A322D-S413P (SEQ ID NO;

     39);

TrCel6A-Q363E-S413P (SEQ ID NO;

     40);

TrCel6A-R410G-S413P (SEQ ID NO;

     41);

TrCel6A-R410Q-S413P (SEQ ID NO;

     42);

TrCel6A-K129E-S186T-A322D-Q363E-S413P (SEQ ID NO;

     43); and

TrCel6A-K129E-S186T-A322D-Q363E-R410Q-S413P (SEQ ID NO;

     44).