Endoglucanase 1B (EG1B) variants

• US 9,611,462 B2
• Filed: 12/18/2012
• Issued: 04/04/2017
• Est. Priority Date: 12/20/2011
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Ang, Ee Lui, Eberhard, Ellen D., Zhang, Xiyun, Zhang, Wei, Tian, Jing, Smith, Derek J., Mitchell, Vesna
• Primary Examiner(s): Nashed, Nashaat
• Assistant Examiner(s): Moore, William W
• Application Number:
  US14/366,761
• Publication Number:
  US 20140370552A1
• Time in Patent Office: 1,568 Days
• Field of Search: None
• 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 19/14 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

Abstract

The present invention provides endoglucanase 1b (EG1b) variants suitable for use in saccharification reactions. The present application further provides genetically modified fungal organisms that produce EG1b variants, as well as enzyme mixtures exhibiting enhanced hydrolysis of cellulosic material to fermentable sugars, enzyme mixtures produced by the genetically modified fungal organisms, and methods for producing fermentable sugars from cellulose using such enzyme mixtures.

Claims

1. A variant endoglucanase or enzymatically active fragment thereof comprising

(a) an amino acid sequence at least 80% identical to the wild-type endoglucanase amino acid sequence of SEQ ID NO:

3; and

(b) an amino acid substitution introducing an amino acid other than phenylalanine at position 63,

wherein the residue positions are numbered with reference to SEQ ID NO;

2.

2. The variant endoglucanase of claim 1, wherein the variant comprises one or more amino acid substitutions selected from P34, T38, W39, T43, Q49, E66, S75, D85, N126, G137, V143, Q201, N218, K228, S241, S269, M274, D282, S328, L342, L342, G350, D397, T398, G399, D400, G401, N403, N403, N404, G405, G407, P408, P408, N409, S411, S412, T413, T414, T415, T417, T417, A418, T419, T420, P425, P428, Y433, G434, Q435, Q435, G438, K439, G440, G443, P444, R446, N457, and L464, wherein the residues are numbered with reference to SEQ ID NO:

2.

3. The variant endoglucanase of claim 1, wherein the variant comprises one or more amino acid substitutions selected from 34R, 38A, 39R, 43C, 49R, 66R, 75P, 85V, 126K, 137T, 143M, 201L, 201M, 218S, 228F, 241K, 269L, 274V, 282G, 328Q, 342F, 342A, 350S, 397Y, 398M, 399C, 400R, 401D, 403D, 403M, 404L, 405C, 405Y, 405W, 407R, 408E, 408G, 409W, 411A, 412Q, 413E, 413P, 414L, 415G, 417G, 417H, 418C, 418S, 419G, 420G, 420I, 420L, 420K, 425S, 425L, 428C, 428V, 433F, 433V, 434D, 434P, 435G, 435K, 435R, 438A, 439P, 440D, 440R, 440T, 443E, 443S, 444Q, 446G, 457H, and 464Q, wherein the residues are numbered with reference to SEQ ID NO:

2.

4. The variant endoglucanase of claim 1, wherein the variant comprises at least one substitution selected from N403D/M, T419G, P425L, N404L, P428V, L340M, D400R, A418S, G440D, N346D, G401D, Q435G, R446G, and T417G, wherein the residues are numbered with reference to SEQ ID NO:

2.

6. The variant endoglucanase of claim 1, wherein the variant is a variant of the Myceliophthora thermophila EG 1b amino acid sequence of SEQ ID NO:

2.

7. The variant endoglucanase of claim 1, wherein the variant has increased thermoactivity at pH of about 4-5 and a temperature of about 65°

C. in comparison to the wild-type endoglucanase 1b set forth in SEQ ID NO;

3.

8. The variant endoglucanase of claim 1, wherein the variant has increased activity on cellulose in comparison to the wild-type endoglucanase 1b set forth in SEQ ID NO:

3.

9. An enzyme composition comprising the variant endoglucanase of claim 1.

10. The enzyme composition of claim 9, further comprising at least one additional enzyme.

11. The enzyme composition of claim 9, further comprising one or more enzyme(s) selected from other endoglucanases (EG), β

-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), and/or Type 2 cellobiohydrolases (CBH2).

12. A method for producing at least one fermentable sugar from a biomass substrate, comprising contacting the biomass substrate with the enzyme composition according to claim 9, under culture conditions whereby fermentable sugars are produced.

13. The method of claim 12, wherein the enzyme composition comprises at least one enzyme selected from other endoglucanases (EG), β

-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), and Type 2 cellobiohydrolases (CBH2).

5. A variant endoglucanase comprising an enzymatically active endoglucanase amino acid sequence wherein an amino acid substitution introduces an amino acid other than phenylalanine at position 63 and having at least 80% identity to an amino acid sequence set forth in any of SEQ IDs NOs:

5, 7 or 9.