Fungal beta-xylosidase variants

• US 8,980,578 B2
• Filed: 06/10/2013
• Issued: 03/17/2015
• Est. Priority Date: 06/11/2012
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Fong, Ryan, Zhang, Xiyun, Agard, Nicholas, Gohel, Anupam, Smith, Derek, Noriega, Chris
• Primary Examiner(s): Prouty, Rebecca
• Assistant Examiner(s): EPSTEIN, TODD MATTHEW
• Application Number:
  US13/914,482
• Publication Number:
  US 20140051119A1
• Time in Patent Office: 645 Days
• Field of Search: None
• US Class Current: 435/41
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12N 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• 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: C12P 7/14 Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01037 Xylan 1,4-beta-xylosidase (3.2.1.37)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention provides fungal xylanase and/or beta-xylosidase enzymes suitable for use in saccharification reactions. The present application further provides genetically modified fungal organisms that produce xylanase and/or beta-xylosidases, as well as enzyme mixtures exhibiting enhanced hydrolysis of cellulosic material to fermentable sugars, enzyme mixtures produced by the genetically modified fungal organisms, and methods for producing fermentable sugars from cellulose using such enzyme mixtures.

Claims

1. A recombinant beta-xylosidase variant and/or biologically active recombinant beta-xylosidase variant fragment comprising at least one amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:

2 and comprising at least one mutation at position 31, 108, 115, 174, 177, 203, 209, 211, 219, 235, 264, 280, 309, 320, 322, 345, 347, 375, 379, 389, 394, 398, 431, 438, 449, 475, 482, 484, 499, 525, 539, 560, 565, 571, 572, 589, 662, 727, 761, 763, 798, and/or 842, wherein the positions are numbered with reference to SEQ ID NO;

2, and wherein said recombinant beta-xylosidase variant comprises SEQ ID NO;

27.

2. An enzyme composition comprising the beta-xylosidase variant of claim 1.

3. The enzyme composition of claim 2, further comprising at least one additional enzyme.

4. The enzyme composition of claim 3, further comprising one or more glycosyl hydrolase family 61 (GH61) enzymes.

5. A method for producing at least one fermentable sugar from a feedstock, comprising contacting the feedstock with at least one enzyme composition of claim 2, under culture conditions whereby fermentable sugars are produced.

6. The method of claim 5, wherein the enzyme composition further comprises at least one enzyme selected from endoglucanases (EG), β

-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), Type 2 cellobiohydrolases (CBH2), glycosyl hydrolase family 61 (GH61) enzymes, and xylanases.

7. The method of claim 5, wherein the fermentable sugar comprises glucose and/or xylose.

8. The method of claim 5, further comprising recovering at least one fermentable sugar.

9. The method of claim 5, further comprising contacting the at least one fermentable sugar with a microorganism under conditions such that said microorganism produces at least one fermentation end product.

10. The method of claim 9, wherein said fermentation end product is selected from alcohols, fatty acids, lactic acid, acetic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric acid, succinic acid, amino acids, 1,3-propanediol, ethylene, glycerol, and β

-lactams.

11. A method of producing an end product from a feedstock, comprising:

a) contacting the feedstock with at least one enzyme composition of claim 2, under conditions whereby at least one fermentable sugar is produced from the substrate; and

b) contacting the fermentable sugar with a microorganism under conditions such that the microorganism uses the fermentable sugar to produce an end-product.

12. The method of claim 11, wherein the method comprises simultaneous performance of steps a) and b) of claim 11 or separate performance of steps a) and b) of claim 11.

13. A method of producing a fermentation end product from a feedstock, comprising:

a) obtaining at least one fermentable sugar produced according to the method of claim 5; and

b) contacting the fermentable sugar with a microorganism in a fermentation to produce at least one fermentation end product.

14. The method of claim 13, wherein said fermentation end product is selected from alcohols, fatty acids, lactic acid, acetic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric acid, succinic acid, amino acids, 1,3-propanediol, ethylene, glycerol, and β

-lactams.

15. The method of claim 13, further comprising recovering the fermentation end product.

16. A method for producing at least one fermentable sugar from a feedstock, comprising contacting the feedstock with the composition provided in claim 2.

17. The method of claim 16, wherein the enzyme composition further comprises at least one enzyme selected from endoglucanases (EG), β

-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), Type 2 cellobiohydrolases (CBH2), glycosyl hydrolase family 61 (GH61) enzymes, and xylanases.

18. The method of claim 16, further comprising recovering at least one fermentable sugar.

19. The method of claim 16, further comprising contacting the at least one fermentable sugar with a microorganism under conditions such that said microorganism produces at least one fermentation end product.

20. The method of claim 19, wherein said fermentation end product is selected from alcohols, fatty acids, lactic acid, acetic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric acid, succinic acid, amino acids, 1,3-propanediol, ethylene, glycerol, and β

-lactams.