β-glucosidase variants

• US 8,956,845 B2
• Filed: 05/28/2014
• Issued: 02/17/2015
• Est. Priority Date: 06/16/2009
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Zhang, Xiyun, Yang, Jie, Baidyaroy, Dipnath, Postlethwaite, Sally R., Andor, Attila, Clark, Louis A., Newman, Lisa Marie
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US14/289,021
• Publication Number:
  US 20140256018A1
• Time in Patent Office: 265 Days
• Field of Search: None
• US Class Current: 435/200
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12N 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention relates to recombinantly produced β-glucosidase variants with enhanced thermoactivity compared to naturally occurring proteins. The invention also provides methods for producing a variant β-glucosidase polypeptide with improved thermoactivity by identifying performance sensitive positions in a target β-glucosidase polypeptide and substituting the residue at that position with a thermoactivity enhancing residue.

Claims

1. A method of producing a variant β

-xylosidase polypeptide with improved thermoactivity, comprising;

a) identifying a first performance sensitive position (PSP) in a target β

-xylosidase polypeptide, comprising the steps of;

i) aligning the primary sequence of the target β

-xylosidase polypeptide with one or both of a GH3 consensus sequence SEQ ID NO;

53 or portion thereof and a GH3-C consensus sequence SEQ ID NO;

54 or portion thereof; and

ii) identifying a position in the target β

-xylosidase polypeptide that corresponds in the alignment to a PSP in the one or more a β

-glucosidase polypeptides, wherein the position so identified is a PSP; and

b) expressing a variant β

-xylosidase polypeptide in which the residue at the first performance sensitive position is replaced with a thermoactivity enhancing residue, wherein the variant β

-xylosidase polypeptide has greater thermoactivity than the target β

-xylosidase polypeptide.

2. The method of claim 1, wherein the target β

-xylosidase polypeptide has the sequence of a naturally occurring protein.

3. The method of claim 1, wherein the target β

-xylosidase polypeptide is a variant of a naturally occurring protein, wherein said variant has at least 80% sequence identity to a naturally occurring protein.

4. The method of claim 1, further comprising:

c) identifying a second performance sensitive position in the target β

-xylosidase polypeptide; and

d) expressing a polypeptide in which the residues at both the first and second performance sensitive positions are replaced with thermoactivity enhancing residues.

5. The method of claim 4, wherein steps (c) and (d) are carried out simultaneously with steps (a) and (b).

6. The method of claim 1, further comprising:

c) identifying a second performance sensitive position in the variant β

-xylosidase polypeptide; and

d) expressing a second variant polypeptide, in which the residues at both the first and second performance sensitive positions are replaced with thermoactivity enhancing residues.

7. The method of claim 1, wherein the step of identifying a performance sensitive position comprises:

a) aligning the primary sequence of the target β

-xylosidase polypeptide with one or more a β

-glucosidase polypeptides in which performance sensitive positions (“

PSP”

) have been defined; and

b) identifying a position in the target β

-xylosidase polypeptide that corresponds in the alignment to a PSP in the one or more β

-glucosidase polypeptides, wherein the position so identified is a PSP.

8. The method of claim 7, wherein the step of identifying a performance sensitive position comprises aligning the primary sequence of the target β

-xylosidase polypeptide with at least a portion of C1 BGL1 SEQ ID NO;

1, T. aurantiacus BGL SEQ ID NO;

2, or A. irakense CelA SEQ ID NO;

3.

9. The method of claim 7, comprising aligning the target β

-xylosidase polypeptide with a plurality of naturally occurring β

-glucosidase polypeptides in at least one of which performance sensitive positions have been defined.

10. The method of claim 1, wherein the target β

-xylosidase polypeptide has a sequence from 70% to 99% identical to any one of SEQ ID NOS;

58-82.

11. A non-naturally occurring β

-xylosidase polypeptide produced according to the method of claim 1.