Polynucleotides encoding recombinant C1 beta-glucosidase for production of sugars from cellulosic biomass

• US 8,900,847 B2
• Filed: 09/30/2010
• Issued: 12/02/2014
• Est. Priority Date: 09/30/2009
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Clark, Louis, Baidyaroy, Dipnath, Szabo, Lorand
• Primary Examiner(s): RAMIREZ, DELIA M
• Application Number:
  US13/496,368
• Publication Number:
  US 20120190076A1
• Time in Patent Office: 1,524 Days
• Field of Search: 435/320.1, 435/69.1, 435/252.3, 435/325, 435/200
• US Class Current: 435/200
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• 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 302/01021 Beta-glucosidase (3.2.1.21)

Abstract

The invention relates to expression of a recombinant C1 β-glucosidase. The invention also provides methods for producing a fermentable sugar from cellobiose by contacting celiobiose with a recombinant host cell comprising a polynucleotide sequence encoding C1 β-glucosidase, operably linked to heterologous promoter, under conditions in which β-glucosidase is expressed and secreted by the cell and the cellobiose is enzymatically converted by said β-glucosidase to glucose. Methods of the invention may be used for conversion of a biomass substrate to a fermentable sugar, and ultimately to ethanol or other biofuel.

Claims

1. An isolated polynucleotide encoding a recombinant β

-glucosidase polypeptide, wherein said isolated polynucleotide comprises SEQ ID NO;

9.

2. An expression vector comprising the polynucleotide of claim 1, operably linked to a heterologous promoter.

3. The expression vector of claim 2, wherein the heterologous promoter is the M. thermophile C1 cellobiohydrolase 1a (CBH1a) promoter.

4. A method of producing a β

-glucosidase polypeptide, comprising culturing a cell comprising the polynucleotide according to claim 1, operably linked to a heterologous promoter, under conditions in which β

-glucosidase is produced.

5. The method of claim 4, wherein the cell is Myceliophthora thermophila cell.

6. The method of claim 4, wherein the heterologous promoter is the M. thermophila C1 cellobiohydrolase 1a (CBH1a) promoter.

7. The method of claim 4, further comprising recovering the β

-glucosidase produced by the cell.

8. A method of producing a β

-glucosidase polypeptide, comprising culturing a cell comprising the expression vector according to claim 2, under conditions in which β

-glucosidase is produced.

9. The method of claim 8, wherein the heterologous promoter is the M. thermophila C1 cellobiohydrolase 1a (CBH1a) promoter.

10. The method of claim 8, wherein the cell is Myceliophthora thermophila cell.

11. The method of claim 8, further comprising recovering the β

-glucosidase produced by the cell.

12. A recombinant host cell comprising the polynucleotide according to claim 1, encoding a recombinant β

-glucosidase polypeptide, wherein the encoding sequence does not comprise introns, and wherein optionally the polynucleotide is operably linked to a heterologous promoter.

13. The recombinant host cell of claim 12, wherein the cell expresses at least one other recombinant cellulase enzyme.

14. The recombinant host cell of claim 12, wherein the cell is Myceliophthora thermophila cell.

15. The recombinant host cell of claim 12, wherein the heterologous promoter is the M. thermophila C1 cellobiohydrolase 1a (CBH1a) promoter.