Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose

Patent metadata

Field	Value
Publication number	US-10294484-B2
Application number	US-201514936840-A
Country	US
Kind code	B2
Filing date	Nov 10, 2015
Priority date	Jun 3, 2010
Publication date	May 21, 2019
Grant date	May 21, 2019

Abstract

The present invention is directed to a yeast strain, or strains, secreting a full suite, or any subset of that full suite, of enzymes to hydrolyze corn starch, corn fiber, lignocellulose, (including enzymes that hydrolyze linkages in cellulose, hemicellulose, and between lignin and carbohydrates) and to utilize pentose sugars (xylose and arabinose). The invention is also directed to the set of proteins that are well expressed in yeast for each category of enzymatic activity. The resulting strain, or strains can be used to hydrolyze starch and cellulose simultaneously. The resulting strain, or strains can be also metabolically engineered to produce less glycerol and uptake acetate. The resulting strain, or strains can also be used to produce ethanol from granular starch without liquefaction. The resulting strain, or strains, can be further used to reduce the amount of external enzyme needed to hydrolyze a biomass feedstock during an Simultaneous Saccharification and Fermentation (SSF) process, or to increase the yield of ethanol during SSF at current saccharolytic enzyme loadings. In addition, multiple enzymes of the present invention can be co-expressed in cells of the invention to provide synergistic digestive action on biomass feedstock. In some aspects, host cells expressing different heterologous saccharolytic enzymes can also be co-cultured together and used to produce ethanol from biomass feedstock.

First claim

We claim:

1. A recombinant yeast host cell comprising
a heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 445; and
at least one heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of: SEQ ID NO: 443, SEQ ID NO: 496, SEQ ID NO: 498 and SEQ ID NO: 504
wherein the recombinant yeast host cell expresses the polypeptides.

2. The recombinant yeast host cell of claim 1 , wherein the heterologous polynucleotide encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 445 and
at least one heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of: SEQ ID NO: 443, SEQ ID NO: 496, SEQ ID NO: 498 and SEQ ID NO: 504.

3. The recombinant yeast host cell of claim 2 , wherein the heterologous polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 445 and
at least one heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 443, SEQ ID NO: 496, SEQ ID NO: 498 and SEQ ID NO: 504.

4. The recombinant yeast host cell of claim 1 , wherein the yeast strain is a Saccharomyces cerevisiae strain.

5. The recombinant yeast host cell of claim 1 , further comprising one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 287, SEQ ID NO: 450, SEQ ID NO: 451, and SEQ ID NO: 452.

6. The recombinant yeast host cell of claim 5 , wherein the one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 287, SEQ ID NO: 450, SEQ ID NO: 451, and SEQ ID NO: 452.

7. The recombinant yeast host cell of claim 5 , wherein the one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 287, SEQ ID NO: 450, SEQ ID NO: 451, and SEQ ID NO: 452.

8. The recombinant yeast host cell of claim 5 , wherein the yeast strain is a Saccharomyces cerevisiae strain.

9. The recombinant yeast host cell of claim 1 , wherein the host cell further expresses a xylose isomerase.

10. The recombinant yeast host cell of claim 9 , wherein the host cell is capable of fermenting a pentose sugar.

11. The recombinant yeast host cell of claim 10 , wherein the pentose sugar is xylose or arabinose.

12. The recombinant yeast host cell of claim 9 , wherein the xylose isomerase is isolated from an organism selected from the group consisting of: Piromyces, Bacterioides thetaiotaomicron, Chitinophaga pinensis, Parabacteroides distasonis , and combinations thereof.

13. The recombinant yeast host cell of claim 1 , further comprising one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, and SEQ ID NO: 465.

14. The recombinant yeast host cell of claim 13 , further comprising one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, and SEQ ID NO: 465.

15. The recombinant yeast host cell of claim 14 , further comprising one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, and SEQ ID NO: 465.

16. The recombinant yeast host cell of claim 13 , wherein the yeast strain is a Saccharomyces cerevisiae strain.

17. The recombinant yeast host cell of claim 1 , further comprising one or more additional heterologous polynucleotides encoding one or more additional polypeptides comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide selected from the group consisting of: SEQ ID NO: 447, SEQ ID NO: 448, and SEQ ID NO: 449.

18. The recombinant yeast host cell of claim 1 , wherein the quantity of ethanol that is produced at 35.degree. C. on a substrate of corn mash, corn flour or combinations thereof is at least about 125 grams per liter in 72 hours.

19. The recombinant yeast host cell of claim 1 , wherein the quantity of ethanol that is produced at 35.degree. C. on a substrate of corn flour is at least about 1.9 grams per liter per hour.

20. The recombinant yeast host cell of claim 1 , wherein the quantity of ethanol that is produced at 35.degree. C. on a substrate of corn mash is at least about 2.2 grams per liter per hour.

21. The recombinant yeast host cell of claim 1 , where the ethanol is produced at a temperature of at least about 35.degree. C.

22. The recombinant yeast host cell of claim 17 , comprising one or more copies of each of the heterologous polynucleotides encoding the polypeptides comprising amino acid sequences at least 90% identical to the amino acid sequences of the polypeptides encoded by: SEQ ID NO: 447, SEQ ID NO: 448, and SEQ ID NO: 449.

23. The recombinant host cell of claim 17 , comprising four or more copies of the heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 445 and one or more copies of each of the heterologous polynucleotides encoding the polypeptides comprising amino acid sequences at least 90% identical to the amino acid sequences of the polypeptides encoded by: SEQ ID NO: 447, SEQ ID NO: 448, and SEQ ID NO: 449.

24. The recombinant yeast host cell of claim 17 , further comprising a down-regulation of at least one gene encoding an endogenous glycerol-producing enzyme selected from the group consisting of: glycerol 3-phosphate dehydrogenase 1 and glycerol-3 phosphate dehydrogenase 2.

25. The recombinant host cell of claim 1 , comprising two or more copies of the heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 445.

26. The recombinant host cell of claim 1 , comprising four or more copies of the heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 445.

Assignees

• Lallemand Hungary Liquidity Man Llc
• Univ Stellenbosch

Inventors

• Brevnova Elena
• Mcbride John E
• Wiswall Erin
• Wenger Kevin S
• Caiazza Nicky
• Lau Heidi
• Argyros Aaron
• Agbogbo Frank
• Rice Charles F
• Barrett Trisha
• Bardsley John S
• Foster Abigail
• Warner Anne K
• Mellon Mark
• Skinner Ryan
• Shikhare Indraneel
• Den Haan Riaan
• Gandhi Chhayal V
• Belcher Alan
• Rajgarhia Vineet B
• Froehlich Allan C
• Deleault Kristen M
• Stonehouse Emily
• Tripathi Shital A
• Gosselin Jennifer
• Chiu Yin-Ying
• Xu Haowen

Classifications

• C12Y302/01091

  Cellulose 1,4-beta-cellobiosidase (3.2.1.91) · CPC title

• C12N9/2482

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

• C12N9/2405

  Glucanases · CPC title

• C12P7/06

  Ethanol, i.e. non-beverage · CPC title

• C12Y302/01008

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