FUNGAL STRAINS

• US 20150252342A1
• Filed: 05/20/2015
• Published: 09/10/2015
• Est. Priority Date: 11/02/2010
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Baidyaroy, Dipnath, Dhawan, Ish K.
• Granted Patent: US 9,528,098 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• 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 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/12 Disaccharides
• 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 2203/00 Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source ethanol C12P7/10
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 101/99018 Cellobiose oxidase (1.1.99.18)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C13K 1/02 obtained by saccharification of cellulosic materials manufacture of fodder A23K10/32
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention provides improved fungal strains. In some embodiments, the improved fungal strains find use in hydrolyzing cellulosic material to glucose.

Claims

1. An enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is expressed by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is produced by the fungal cell.

3. The enzyme mixture of claim 1, comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is expressed by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell and further to increase the expression of at least one saccharide hydrolyzing enzyme.

4. The enzyme mixture of claim 1, wherein the enzyme mixture is a cell-free mixture.

5. The enzyme mixture of claim 1, wherein a substrate of the enzyme mixture comprises pretreated lignocellulose.

6. The enzyme mixture of claim 1, wherein the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of said cellulase enzyme.

7. The enzyme mixture of claim 6, wherein the enzyme mixture comprises at least one beta-glucosidase.

8. The enzyme mixture of claim 1, further comprising at least one cellobiose dehydrogenase.

9. The enzyme mixture of claim 8, wherein said cellobiose dehydrogenase is CDH1 and/or CDH2.

10. The enzyme mixture of claim 1, further comprising at least one cellulase enzyme and/or at least one additional enzyme.

11. The enzyme mixture of claim 1, wherein the enzyme mixture has been subjected to a purification process to selectively remove one or more glucose and/or cellobiose oxidizing enzymes from the enzyme mixture.

12. The enzyme mixture of claim 11, wherein the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzymes from other enzymes present in the enzyme mixture.

13. The enzyme mixture of claim 1, wherein the enzyme mixture comprises an inhibitor of one or more glucose and/or cellobiose oxidizing enzymes.

2. An enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is expressed by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell.

14. A method for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15%, or about 20% of the cellobiose and/or glucose is oxidized after 10 hours.

16. The method of claim 14, wherein the enzyme mixture is produced by a fungal cell has that been genetically modified to reduce the amount of one or more endogenous glucose and/or cellobiose oxidizing enzymes that is secreted by the fungal cell.

17. The method of claim 14, wherein the enzyme mixture has been subjected to a purification process to selectively remove at least one glucose and/or cellobiose oxidizing enzyme from the enzyme mixture.

18. The method of claim 14, wherein the enzyme mixture comprises an inhibitor of the glucose and/or cellobiose oxidizing enzyme.

15. A method for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell and wherein, of the cellulose hydrolyzed by the enzyme mixture, at least about 80%, about 85%, or about 90% is present in the form of cellobiose and/or glucose.