GH61 glycoside hydrolase protein variants and cofactors that enhance GH61 activity

• US 8,877,474 B2
• Filed: 08/22/2012
• Issued: 11/04/2014
• Est. Priority Date: 08/20/2010
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Yang, Jie, Zhang, Xiyun, Yoon, Jungjoo, Rao, Kripa, Grate, John H., Elgart, David, Baidyaroy, Dipnath
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US13/592,024
• Publication Number:
  US 20130052713A1
• Time in Patent Office: 804 Days
• Field of Search: 435/188, 435/196, 435/198, 435/320.1, 435/254.11, 435/254.2, 536/23.2
• US Class Current: 435/188
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• 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 9/2445 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: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• 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

The present invention provides various GH61 protein variants comprising various amino acid substitutions. The GH61 protein variants have an improved ability to synergize with cellulase enzymes, thereby increasing the yield of fermentable sugars obtained by saccharification of biomass. In some embodiments, sugars obtained from saccharification are fermented to produce numerous end-products, including but not limited to alcohol.

Claims

1. A glycoside hydrolase 61 (GH61) variant protein comprising an amino acid sequence that is at least at least 90%, identical to SEQ ID NO:

2 or a fragment of SEQ ID NO;

2 having GH61 activity, wherein the amino acid sequence of the variant protein has one or more amino acid substitutions with respect to SEQ ID NO;

2 or said fragment.

2. The GH61 variant protein of claim 1, wherein said GH61 variant protein is at least 95% identical to SEQ ID NO:

2 or a fragment of SEQ ID NO;

2 having GH61 activity.

3. The GH61 variant protein of claim 1, wherein said GH61 variant protein has increased thermoactivity, thermostability, and/or activity as compared to the GH61 wild-type protein of SEQ ID NO:

2.

4. The GH61 variant protein of claim 1, wherein said GH61 variant protein comprises at least one substitution(s) at one or more of the following amino acid positions:

H20, N35, W42, Q44, P45, F68, T87, V97, P103, E104, S127, W131, F132, K133, I134, A137, Y139, A142, A143, I162, P163, S164, D165, L166, K167, A168, G169, N170, Y171, V172, L173, R174, H175, E176, I177, I178, A179, L180, H181, Q190, A191, Y192, Y192, S205, A212, S215, K218, S232, T236, G239, A244, A246, T258, G270, P273, N317, P322, T323, G328, S330, and/or C341, wherein the amino acid positions are numbered with reference to SEQ ID NO;

2.

5. The GH61 variant protein of claim 4, wherein said GH61 variant protein comprises at least one substitution(s) at one or more of the following amino acid positions:

H20, N35, W42, E104, I134, S164, K167, A168, V172, I177, A179, and/or A191, wherein the amino acid positions are numbered with reference to SEQ ID NO;

2.

6. The GH61 variant protein of claim 1, wherein said GH61 variant protein comprises the substitution set N35/E104/A168, wherein the amino acid positions are numbered with reference to SEQ ID NO:

2.

7. The GH61 variant protein of claim 1, wherein said amino acid substitution(s) comprise one or more of the following substitutions numbered with reference to SEQ ID NO:

2;

N35G, E104C/D/H/Q, and A168P/X, wherein the amino acid positions are numbered with reference to SEQ ID NO;

2.

8. The GH61 variant protein of claim 1, wherein said GH61 variant protein comprises one or more of the following substitutions:

N35G, E104H, and A168P, wherein the amino acid positions are numbered with reference to SEQ ID NO;

2.

9. The GH61 variant protein of claim 1, wherein said GH61 variant protein comprises the substitution set N35G/E104H/A168P, wherein the amino acid positions are numbered with reference to SEQ ID NO:

2.

10. The GH61 variant protein of claim 1, wherein said variant protein comprises the sequence set forth in any of SEQ ID NOS:

4, 6, and/or 8.

11. The GH61 variant protein of claim 1, wherein said GH61 variant protein is encoded by at least one polynucleotide sequence set forth in SEQ ID NOS:

3, 5, and/or 7.

12. The GH61 variant protein of claim 1, wherein said GH61 variant protein, comprises at least one substitution(s) at one or more of the following amino acid positions:

S24, V28, Y32, R34, N35, T40, Q44, P45, N46, T49, I51, T54, A55, A56, Q58, E64, N66, S67, G69, T70, P71, S78, T80, G82, G83, V88, K93, N95, E101, E104, A116, N118, S128, R130, G136, A137, K141, A142, G144, R145, A150, G155, Q161, S164, A168, Q184, N187, R199, G203, S205, A212, K218, A219, V230, S231, S232, P233, D234, T236, V237, G245, S253, A263, P266, G267, G268, G269, G270, A271, A280, T281, S282, R290, S295, A297, P303, G305, K310, N317, T320, V324, A326, P327, S329, S330, S332, V333, E336, W337, and/or S339, wherein the amino acid positions are numbered with reference to SEQ ID NO;

2.

13. The GH61 variant protein of claim 1, wherein said GH61 variant protein comprises a plurality of amino acid substitutions.

14. The GH61 variant protein of claim 1, wherein said GH61 variant protein comprises a polypeptide sequence that is at least 90% identical to any of SEQ ID NOS:

2, 3, 5, 6, 8, and/or 9, and/or a biologically active fragment of any of SEQ ID NOS;

2, 3, 5, 6, 8, and/or 9, wherein said fragment has GH61 activity.

16. An enzyme composition comprising at least one GH61 variant protein as set forth in claim 1.

17. The enzyme composition of claim 16, further comprising one or more enzymes selected from wild-type GH61 enzymes, endoglucanases (EG), β

-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), and/or Type 2 cellobiohydrolases (CBH2), cellulases, hemicellulases, xylanases, xylosidases, amylases, glucoamylases, proteases, esterases, and lipases.

18. The enzyme composition of claim 17, further comprising at least one adjunct material.

19. The enzyme composition of claim 18, wherein said adjunct material is selected from divalent metal cations, reductants, surfactants, buffers, culture media, and enzyme stabilizing systems.

20. The composition of claim 16, wherein the cellulosic substrate is selected from wheat grass, wheat straw, barley straw, sorghum, rice grass, sugarcane straw, bagasse, switchgrass, corn stover, corn fiber, grains, or any combination thereof.

21. A composition comprising at least one variant GH61 protein of claim 1, one or more cellulase enzymes, a cellulosic substrate, and Cu⁺⁺, wherein the variant GH61 protein is at least 90% identical to SEQ ID NO:

5, and/or a biologically fragment thereof with GH61 activity.

22. A composition comprising at least one GH61 protein of claim 1, one or more cellulase enzymes, a cellulosic substrate, and gallic acid, wherein the GH61 protein is at least 90% SEQ ID NO:

5, and/or a biologically fragment thereof with GH61 activity.

15. A GH61 variant protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

5, or a fragment of SEQ ID NO;

5, having GH61 activity, wherein the amino acid sequence of the variant protein has one or more amino acid substitutions with respect to SEQ ID NO;

2 or said fragment, and wherein the substitution(s) in the amino acid sequence result in the variant protein having increased GH61 activity in a reaction where crystalline cellulose undergoes saccharification by cellulase enzymes that are contained in culture broth from M. thermophile cells, compared with a reference protein comprising SEQ ID NO;

2, or said fragment, without any of said substitutions.

23. A GH61 variant protein encoded by a polynucleotide, wherein the protein comprises an amino acid sequence that is at least or about 85% identical to any of SEQ ID NO:

5, or a fragment of SEQ ID NO;

5, having GH61 activity, wherein the amino acid sequence of the variant protein has one or more amino acid substitutions with respect to SEQ ID NO;

2, or said fragment, and wherein the polynucleotide encoding the GH61 variant protein comprises at least one mutation set comprising t60c/c573g, wherein the nucleotide positions are numbered with reference to SEQ ID NO;

1.