Expression of enzymes in yeast for lignocellulose derived oligomer CBP

Patent metadata

Field	Value
Publication number	US-9745560-B2
Application number	US-201314424560-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Aug 29, 2012
Publication date	Aug 29, 2017
Grant date	Aug 29, 2017

Abstract

The present invention provides a multi-component enzyme system that hydrolyzes hemicellulose oligomers from hardwood which can be expressed, for example, in yeast such as Saccharomyces cerevisiae . In some embodiments, this invention provides for the engineering of a series of biocatalysts combining the expression and secretion of components of this enzymatic system with robust, rapid xylose utilization, and ethanol fermentation under industrially relevant process conditions for consolidated bioprocessing. In some embodiments, the invention utilizes co-cultures of strains that can achieve significantly improved performance due to the incorporation of additional enzymes in the fermentation system.

First claim

We claim:

1. A recombinant yeast host cell, comprising:
(a) a heterologous polynucleotide comprising a nucleic acid which encodes an acetylxylanesterase;
(b) a heterologous polynucleotide comprising a nucleic acid which encodes a xylanase; and
(c) a heterologous polynucleotide comprising a nucleic acid which encodes a xylosidase.

2. The recombinant yeast host cell of claim 1 , wherein the heterologous polynucleotide of (a) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:6-10.

3. The recombinant yeast host cell of claim 1 , wherein the heterologous polynucleotide of (b) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:37-62.

4. The recombinant yeast host cell of claim 1 , wherein the heterologous polynucleotide of (c) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:78-92.

5. The recombinant yeast host cell of claim 1 , wherein at least one of the acetylxylanesterase, the xylanase, or the xylosidase comprises a histidine tag.

6. The recombinant yeast host cell of claim 1 , further comprising:
(d) a heterologous polynucleotide comprising a nucleic acid which encodes a galactosidase; and either
(e) a heterologous polynucleotide comprising a nucleic acid which encodes a mannosidase, or
(f) a heterologous polynucleotide comprising a nucleic acid which encodes an alpha-glucuronidase.

7. The recombinant yeast host cell of claim 6 , wherein the heterologous polynucleotide of (d) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:108-122.

8. The recombinant yeast host cell of claim 6 , wherein the heterologous polynucleotide of (e) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:146-168.

9. The recombinant yeast host cell of claim 6 , wherein the heterologous polynucleotide of (f) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:184-198.

10. The recombinant yeast host cell of claim 1 , further comprising:
(d) a heterologous polynucleotide comprising a nucleic acid which encodes an acetyl esterase; and
(e) a heterologous polynucleotide comprising at least one of a nucleic acid which encodes a beta-glucosidase, an endoglucanase, and an alpha-glucoronidase.

11. The recombinant yeast host cell of claim 10 , wherein the heterologous polynucleotide of (d) comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:223-225.

12. The recombinant yeast host cell of claim 10 , wherein the heterologous polynucleotide that encodes the alpha-glucoronidase comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:185-198.

13. The recombinant yeast host cell of claim 10 , wherein the heterologous polynucleotide that encodes the beta-glucosidase comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:92, 164-168, 226 and 227.

14. The recombinant yeast host cell of claim 10 , wherein the heterologous polynucleotide that encodes the endoglucanase comprises a nucleic acid which encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:289-345.

15. The recombinant yeast host cell of claim 1 , wherein at least one of the heterologous polynucleotides is expressed.

16. The recombinant yeast host cell of claim 1 , wherein at least one of the heterologous polynucleotides express a polypeptide that is secreted by the recombinant yeast host cell.

17. The recombinant yeast host cell of claim 1 , wherein the recombinant yeast host cell ferments a lignocellulosic material to produce a fermentation product comprising at least one of ethanol, lactic acid, hydrogen, butyric acid, acetone, and butanol.

18. The recombinant yeast host cell of claim 17 , wherein the lignocellulosic material is insoluble cellulose, crystalline cellulose, pretreated hardwood, paper sludge, pretreated corn stover, pretreated sugar cane bagasse, pretreated corn cobs, pretreated switchgrass, pretreated municipal solid waste, pretreated distiller's dried grains, pretreated wheat straw, corn fiber, or agave.

19. The recombinant yeast host cell of claim 17 , wherein the recombinant yeast host cell ferments at least about 20% of xylo-oligomers in the lignocellulosic material.

20. The recombinant yeast host cell of claim 17 , wherein about 20% to about 80% of xylo-oligomers in the lignocellulosic material are hydrolyzed to monomers during fermentation of the recombinant yeast host cell.

21. The recombinant yeast host cell of claim 17 , wherein the yeast strain has a specific growth rate (h −1 ) of at least about 0.05 in a culture medium containing xylose as the primary sugar source.

22. The recombinant yeast host cell of claim 21 , wherein the xylose in the culture medium is fermented in about 40 hours or less and is at an initial concentration of at least 30 g/L.

23. The recombinant yeast host cell of claim 1 , wherein fermentation of the recombinant yeast host cell produces an ethanol yield of at least about 15% more ethanol than is produced by a nonrecombinant yeast.

24. The recombinant yeast host cell of claim 1 , wherein the recombinant yeast host cell further comprises a deletion or alteration of one or more glycerol producing enzymes.

25. The recombinant yeast host cell of claim 1 , wherein the recombinant yeast host cell further comprises a deletion or alteration of GPD1.

26. A composition comprising a lignocellulosic material and a recombinant yeast host cell of claim 1 .

27. A method of producing a fermentation product, comprising:
(i) combining a recombinant yeast host cell of claim 1 with a lignocellulosic material;
(ii) allowing the recombinant yeast host cell to ferment the lignocellulosic material; and
(iii) recovering a fermentation product produced by the recombinant yeast host cell,
wherein the lignocellulosic material is insoluble cellulose, crystalline cellulose, pretreated hardwood, paper sludge, pretreated corn stover, pretreated sugar cane bagasse, pretreated corn cobs, pretreated switchgrass, pretreated municipal solid waste, pretreated distiller's dried grains, pretreated wheat straw, corn fiber, or agave, and
wherein the fermentation product is ethanol, lactic acid, hydrogen, butyric acid, acetone, or butanol.

28. A recombinant host cell, comprising an acetylxylanesterase, xylanase, and xylosidase.

29. The recombinant host cell of claim 28 , wherein the acetylxylanesterase comprises an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:6-10,
wherein the xylanase comprises an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:37-62, and
wherein the xylosidase comprises an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:78-92.

30. The recombinant host cell

Assignees

• Lallemand Hungary Liquidity Man Llc

Inventors

• Mcbride John E
• Wiswall Erin
• Shikhare Indraneel
• Xu Haowen
• Thorngren Naomi
• Hau Heidi H
• Stonehouse Emily

Classifications

• C12N9/2465

  acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22) · CPC title

• C12N9/2482

  Endo-1,4-beta-xylanase (3.2.1.8) · CPC title

• C12Y302/01131

  Xylan alpha-1,2-glucuronosidase (3.2.1.131) · CPC title

• C12P3/00

  Preparation of elements or inorganic compounds except carbon dioxide {(recovery of carbon dioxides as by-products C12F3/02)} · CPC title

• C12Y302/01072

  Xylan 1,3-beta-xylosidase (3.2.1.72) · CPC title