Methods for improving the efficiency of simultaneous saccharification and fermentation reactions

• US 10,138,499 B2
• Filed: 12/17/2010
• Issued: 11/27/2018
• Est. Priority Date: 12/23/2009
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Gutierrez, Christina, Mitchinson, Colin, Huang, Tom T., Diner, Bruce A., Fagan, Paul Joseph, Hitz, William D.
• Primary Examiner(s): Mondesi, Robert B
• Assistant Examiner(s): Epstein, Todd Matthew
• Application Number:
  US13/516,610
• Publication Number:
  US 20130143277A1
• Time in Patent Office: 2,902 Days
• Field of Search: None
• CPC Class Codes: C12N 1/22 Processes using, or culture media containing, cellulose or hydrolysates thereof
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2477 Hemicellulases not provided in a preceding group
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/44 Preparation of O-glycosides, e.g. glucosides polysaccharides and not substituted disaccharides C12P19/04, C12P19/12
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/18 polyhydric
• 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 disclosure is directed, in a first aspect, to the use of inverting beta-xylosidase enzymes to reduce byproduct formation and increase the yield of fermentation products, as well as, in a second aspect, to the use of retaining beta-xylosidase enzymes to improve production of alkyl-beta-xylopyranoside compounds, in a simultaneous saccharification and fermentation reactions.

Claims

1. A method for simultaneous saccharification and fermentation (SSF) comprising culturing a complete fermentation medium, said complete fermentation medium comprising at least one fermenting microorganism, at least one xylan-containing biomass, at least one cellulase, at least one hemicellulase, at least one retaining β

-xylosidase, and at least one inverting β

-xylosidase, for a period and under conditions suitable for producing a fermentation product, which is an alcohol, wherein the at least one inverting β

-xylosidase comprises an Fv43D polypeptide having at least 90% sequence identity to SEQ ID NO;

2, or to residues 21 to 350 of SEQ ID NO;

2, and the complete fermentation medium comprises a greater amount of the at least one inverting β

-xylosidase than that of the at least one retaining β

-xylosidase on a mole basis.

2. The method of claim 1, wherein the complete fermentation medium comprises an effective amount of the at least one inverting β

-xylosidase such that the complete fermentation medium produces less short chain alkyl-β

-xylopyranoside (“

AXP”

) than does a control fermentation medium cultured for the period and under the conditions of the complete fermentation medium, wherein the control fermentation medium comprises the at least one fermenting microorganism, the at least one xylan-containing biomass, the at least one cellulase, the at least one hemicellulase, and the at least one retaining β

-xylosidase, and lacks the at least one inverting β

-xylosidase.

3. The method of claim 2, wherein the complete fermentation medium comprises an effective amount of the at least one inverting β

-xylosidase such that the complete fermentation medium produces at least 50% less AXP than does the control fermentation medium.

4. The method of claim 3, wherein the complete fermentation medium comprises an effective amount of the at least one inverting β

-xylosidase such that the complete fermentation medium produces at least 60% less AXP than does the control fermentation medium.

5. The method of claim 4, wherein the complete fermentation medium comprises an effective amount of the at least one inverting β

-xylosidase such that the complete fermentation medium produces at least 70% less AXP than does the control fermentation medium.

6. The method of claim 2, wherein the AXP is a methyl-β

-xylopyranoside (MXP), an ethyl-β

-xylopyranoside (EXP), a propyl-β

-xylopyranoside (PXP), or a butyl-β

-xylopyranoside (BXP).

7. The method of claim 2, wherein the complete fermentation medium comprises an effective amount of the at least one inverting β

-xylosidase such that the complete fermentation medium produces at least 40% less AXP than does the control fermentation medium.

8. The method of claim 1 wherein the at least one inverting β

-xylosidase comprises residues 21 to 350 of SEQ ID NO;

2.

9. The method of claim 1, wherein the complete fermentation medium comprises an effective amount of the at least one inverting β

-xylosidase to increase the yield of the fermentation product, as compared to the yield of the fermentation product from a control fermentation medium cultured for the period and the under conditions of the complete fermentation medium, wherein the control fermentation medium comprises the at least one fermenting microorganism, the at least one xylan-containing biomass, the at least one cellulase, the at least one hemicellulase, and the at least one retaining β

-xylosidase, and lacks the at least one inverting β

-xylosidase, wherein the yield of the fermentation product is increased by at least 1%.

10. The method of claim 9, wherein the yield of the fermentation product is increased by at least 2%.

11. The method of claim 10, wherein the yield of the fermentation product is increased by at least 5%.

12. The method of claim 11, wherein the yield of the fermentation product is increased by at least 10%.

13. The method of claim 9, wherein the alcohol is methanol, ethanol, propanol, propane-1,3-diol, or butanol.

14. The method of claim 1, further comprising a Pf43A polypeptide wherein the Pf43A polypeptide has at least 90% sequence identity to SEQ ID NO:

8, or to residues 21 to 445 of SEQ ID NO;

8.

15. The method of claim 1, further comprising a Fv43E polypeptide wherein the Fv43E polypeptide has at least 90% sequence identity to SEQ ID NO:

10, or to residues 19 to 530 of SEQ ID NO;

10.

16. The method of claim 1, further comprising a Fv43B polypeptide wherein the Fv43B polypeptide has at least 90% sequence identity to to SEQ ID NO:

12, or to residues 17 to 574 of SEQ ID NO;

12.

17. The method of claim 1, further comprising a Af43A polypeptide wherein the Af43A polypeptide has at least 90% sequence identity to SEQ ID NO:

14, or to residues 15-558 of SEQ ID NO;

14.

18. The method of claim 1, further comprising a Fo43A polypeptide wherein the Fo43A polypeptide has at least 90% sequence identity to SEQ ID NO:

24, or to residues 21-348 of SEQ ID NO;

24.

19. The method of claim 1, further comprising a Gz43A polypeptide wherein the Gz43A polypeptide has at least 90% sequence identity to SEQ ID NO:

22, or to residues 19-340 of SEQ ID NO;

22.

20. The method of claim 1, further comprising a XynB3 polypeptide wherein the XynB3 polypeptide has at least 90% sequence identity to SEQ ID NO:

25.

21. The method of claim 1, wherein the at least one inverting β

-xylosidase is present in the complete fermentation medium at a concentration of 0.3 mg to 10 mg per gram of xylan in the xylan-containing biomass.

22. The method of claim 21, wherein the at least one inverting β

-xylosidase is present at a concentration of 0.4 mg to 10 mg per gram of xylan in the xylan-containing biomass.

23. The method of claim 21, wherein the at least one inverting β

-xylosidase is present at a concentration of 0.3 mg to 3 mg per gram of xylan in the xylan-containing biomass.

24. The method of claim 1, wherein the SSF is performed as a continuous, a batch, or a fed-batch process.

25. The method of claim 1, further comprising a step of forming the complete fermentation medium.

26. The method of claim 25, wherein the step of forming the complete fermentation medium comprises combining (a) the fermenting microorganism, (b) the xylan-containing biomass, (c) the cellulase, (d) the hemicellulase, (e) the at least one inverting β

-xylosidase, and (f) a medium lacking one or more or all of (a)-(e).

27. The method of claim 26, wherein the cellulase is present in the form of a whole cellulase preparation.

28. The method of claim 27, wherein the whole cellulase preparation comprises a hemicellulase.

29. The method of claim 27, wherein the whole cellulase preparation is a culture broth obtained from culturing a filamentous fungus.

30. The method of claim 29, wherein the filamentous fungus is a T. reesei.

31. The method of claim 1, wherein the fermenting microorganism is a fungus.

32. The method of claim 31, wherein the fungus is a Saccharomyces cerevisiae yeast.

33. The method of claim 1, wherein the fermenting microorganism is a bacterium.

34. The method of claim 33, wherein the bacterium is a Zymomonas mobilis.

35. The method of claim 1, wherein the xylan-containing biomass is corn stover, bagasses, sorghum, giant reed, elephant grass, miscanthus, Japanese cedar, wheat straw, switchgrass, hardwood pulp, or softwood pulp.

36. The method of claim 35, wherein the xylan-containing biomass is in a slurry.

37. The method of claim 35, wherein the xylan-containing biomass has been pretreated.

38. The method of claim 1, further comprising the step of recovering the fermentation product.

39. The method of claim 1, wherein the ratio of the at least one inverting β

-xylosidase to the at least one retaining β

-xylosidase in the complete fermentation medium is at least 2;

1, on a mole basis.