Cellobiohydrolase variants

Patent metadata

Field	Value
Publication number	US-8945903-B2
Application number	US-201213549300-A
Country	US
Kind code	B2
Filing date	Jul 13, 2012
Priority date	Aug 23, 2011
Publication date	Feb 3, 2015
Grant date	Feb 3, 2015

Abstract

The present invention relates to cellobiohydrolase variants having improved thermostability in comparison to wild-type CBH2a.

First claim

What is claimed is:

1. A recombinant cellobiohydrolase type 2a (CBH2a) variant comprising at least about 90% sequence identity to SEQ ID NO:1 and comprising an amino acid substitution, relative to SEQ ID NO:1, at one or more positions selected from 15, 204, and 340, wherein the position is numbered with reference to SEQ ID NO:1.

2. The variant of claim 1 , wherein the amino acid residue at position 15 is phenylalanine, isoleucine, or serine (X15F/I/S); the amino acid residue at position 204 is tryptophan (X204W); and/or the amino acid residue at position 340 is serine (X340S).

3. The variant of claim 1 , further comprising an amino acid substitution, relative to SEQ ID NO:1, at one or more positions selected from 39, 113, and 250.

4. The variant of claim 3 , wherein the amino acid residue at position 39 is alanine or cysteine (X39A/C); the amino acid residue at position 113 is leucine or arginine (X113L/R); and/or the amino acid residue at position 250 is proline or valine (X250P/V).

5. The variant of claim 1 , wherein the variant is a Myceliophthora thermophila cellobiohydrolase.

6. The variant of claim 1 comprising at least 95% sequence identity to SEQ ID NO:1.

7. The variant of claim 1 that has increased thermostability after incubation at pH 5.0 and 55° C. for 1 hour in comparison to wild-type Myceliophthora thermophila CBH2a (SEQ ID NO:1).

8. A recombinant cellobiohydrolase type 2 (CBH2) variant comprising at least 90% sequence identity to SEQ ID NO:1 and comprising an amino acid substitution selected from:
a glutamine, arginine, or tryptophan residue at position 204 (X204Q/R/W);

wherein the position is numbered with reference to SEQ ID NO:1, and wherein the cellobiohydrolase variant has increased thermostability in comparison to the wild-type cellobiohydrolase from which the variant is derived.

9. An enzyme composition comprising the variant cellobiohydrolase polypeptide of claim 1 .

10. A method of producing a fermentable sugar from a cellulosic substrate, comprising contacting the cellulosic substrate with a β-glucosidase (Bgl), a type 2 endoglucanase (EG2), a type 1a cellobiohydrolase (CBH1a), a glycoside hydrolase 61 protein (GH61), and the CBH2a variant of claim 1 under conditions in which the fermentable sugar is produced.

11. The method of claim 10 , comprising:
contacting the fermentable sugar with a microorganism in a fermentation to produce an end-product.

12. The method of claim 11 , wherein the end-product is an alcohol, an amino acid, an organic acid, a diol, or glycerol.

13. The variant of claim 8 , wherein the variant further comprises one or more amino acid substitutions selected from:
an alanine, cysteine, lysine, proline, arginine, or valine residue at position 39 (X39A/C/K/P/R/V); and
a cysteine, histidine, lysine, asparagine, proline, threonine, or valine at position 250 (X250C/H/K/N/P/T/V).

14. The variant of claim 8 , wherein the variant further comprises an amino acid substitution at one or more positions selected from 15, 39, 113, 250, and 340, wherein the position is numbered with reference to SEQ ID NO:1.

15. The variant of claim 14 , wherein:
the amino acid substitution at position 15 is phenylalanine, isoleucine, or serine (X15F/I/S);
the amino acid substitution at position 39 is alanine or cysteine (X39A/C/K/P/R/V);
the amino acid substitution at position 113 is leucine or arginine (X113L/R/T);
the amino acid substitution at position 250 is proline or valine (X250P/V); and/or
the amino acid residue at position 340 is serine (X340S).

Assignees

• Baidyaroy Dipnath
• Clark Louis
• Elgart David
• Voladri Rama
• Zhang Xiyun
• Codexis Inc

Inventors

• Baidyaroy Dipnath
• Clark Louis
• Elgart David
• Voladri Rama
• Zhang Xiyun

Classifications

• C12P7/58

  Aldonic, ketoaldonic or saccharic acids (uronic acids C12P19/00) · CPC title

• Y02E50/16

  Cross-Sectional Technologies · mapped topic

• C12P7/16

  Butanols · CPC title

• C12Y302/01091 Primary

  Cellulose 1,4-beta-cellobiosidase (3.2.1.91) · CPC title

• C12P7/20

  Glycerol · CPC title