Genetically modified strain of s. cerevisiae engineered to ferment xylose and arabinose

Patent metadata

Field	Value
Publication number	US-2017191088-A1
Application number	US-201715454275-A
Country	US
Kind code	A1
Filing date	Mar 9, 2017
Priority date	Nov 10, 2011
Publication date	Jul 6, 2017
Grant date	—

Abstract

The present invention provides a microorganism capable of fermenting arabinose to a desired product such as ethanol. In some embodiments, the organism is also capable of fermenting xylose. In some embodiments, the organism is capable of fermenting arabinose and xylose, and expresses one or more cellulases.

First claim

1 . A recombinant eukaryotic host cell comprising a heterologous polynucleotide encoding an arabinose transporter (AraT), a heterologous polynucleotide encoding an arabinose isomerase (AI), a heterologous polynucleotide encoding a ribulokinase (RK) and a heterologous polynucleotide encoding a ribulose 5-phosphate epimerase (R5PE).

2 . The recombinant eukaryotic host cell of claim 1 , wherein the AraT is derived from an AraT of an organism selected from the group consisting of Kluveromyces lactis, Kluveromyces thermotolerans, Zygosaccharomyces rouxii, Vanderwaltozyma polyspora, Debaryomyces hansenii, Aspergillus niger, Penicillium chrysogenum, Pichia guilermondii, Aspergillus flavus, Candida lusitnaea, Candida albicans (SC5314), Kluveromyces marxianus, Pichia stipites , and Candida arbinofermentans.

3 . The recombinant eukaryotic host cell of claim 1 , wherein the AraT comprises an amino acid sequence at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 9-22.

4 . The recombinant eukaryotic host cell of claim 1 , wherein one or more of the AI, RK and R5PE is derived from an AI, RK and R5PE of B. thetaiotamicron.

5 . A recombinant eukaryotic host cell comprising a heterologous polynucleotide encoding an arabinose isomerase (AI), a heterologous polynucleotide encoding a ribulokinase (RK) and a heterologous polynucleotide encoding a ribulose 5-phosphate epimerase (R5PE), wherein one or more of the AI, RK and R5PE is derived from an AI, RK and R5PE of B. thetaiotamicron.

6 . The recombinant eukaryotic host cell of claim 4 , wherein
a) the AI comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 6; b) the RK comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 7; and c) the R5PE comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

7 . The recombinant eukaryotic host cell of claim 1 , wherein expression of the heterologous polynucleotide confers an ability to ferment arabinose to the recombinant host cell.

8 . The recombinant eukaryotic host cell of claim 1 , further comprising a heterologous polynucleotide encoding a xylose isomerase (XI).

9 . The recombinant eukaryotic host cell of claim 8 , wherein the XI is derived from an XI of B. thetaiotamicron.

10 . The recombinant eukaryotic host cell of claim 8 , wherein the XI comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26.

11 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell is a yeast cell.

12 . The recombinant eukaryotic host cell of claim 11 , wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis.

13 . The recombinant eukaryotic host cell of claim 11 , wherein the yeast cell comprises a heterologous sequence encoding a xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phophate epimerase, transketolase and transaldolase, and wherein the yeast cell does not express an aldose reductase that is capable of catalyzing the conversion of xylose to xylitol.

14 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell is capable of fermenting xylose, arabinose, or a combination thereof.

15 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell is capable of fermenting arabinose from a cellulosic substrate.

16 . The recombinant eukaryotic host cell of claim 14 , wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, hydrogen, butyric acid, acetone, and butanol.

17 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell is an industrial strain exhibiting high ethanol tolerance.

18 . The recombinant eukaryotic host cell of claim 17 , wherein the host cell further exhibits high temperature tolerance.

19 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell produces an ethanol yield of at least about 10 g/l ethanol after 24 hours of fermentation from a medium containing 20 g/l xylose and 21 g/l arabinose.

20 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell produces an ethanol yield of at least about 13 g/l ethanol after 24 hours of fermentation from a medium containing 20 g/l glucose and 21 g/l arabinose.

21 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell produces an ethanol yield of at least about 15 g/l ethanol after 24 hours of fermentation from a medium containing 10 g/l glucose, 10 g/l xylose and 21 g/l arabinose.

22 . The recombinant eukaryotic host cell of claim 1 , wherein the host cell further comprises one or more heterologous polynucleotides encoding a cellulase.

23 . The recombinant eukaryotic host cell of claim 22 , wherein the one or more cellulases is selected from the group consisting of endoglucanases, exoglucanases, and β-glucosidases.

24 . The recombinant eukaryotic host cell of claim 23 , wherein the host cell comprises: (a) a first heterologous polynucleotide that encodes an endoglucanase; (b) a second heterologous polynucleotide that encodes a β-glucosidase; (c) a third heterologous polynucleotide that encodes a first cellobiohydrolase; and, (d) a fourth heterologous polynucleotide that encodes a second cellobiohydrolase.

25 . The recombinant eukaryotic host cell of claim 24 , wherein (a) the first heterologous polynucleotide that encodes an endoglucanase is derived from A. fumigatus ; (b) the second heterologous polynucleotide that encodes a β-glucosidase is derived from S. fibuligera ; (c) the third heterologous polynucleotide that encodes a first cellobiohydrolase is derived from T. emersonii ; and, (d) the fourth heterologous polynucleotide that encodes a second cellobiohydrolase is derived from C. lucknowense.

26 . A recombinant yeast cell comprising a heterologous polynucleotide encoding an arabinose transporter (AraT), wherein the yeast cell is capable of taking up at least about 5 g/l of arabinose in 24 hours.

27 . The recombinant yeast cell of claim 26 , wherein the AraT is derived from an AraT of an organism selected from the group consisting of Kluveromyces lactis, Kluveromyces thermotolerans , and Aspergillus niger.

28 . The recombinant yeast cell of claim 26 , wherein the AraT comprises an amino acid sequence at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 9-22.

29 . The recombinant host cell of claim 1 , wherein at least one of the heterologous polynucleotides is integrated into the genome of the host cell.

30 . A method of producing a fermentation product comprising:
a) combining the recombinant eukaryotic host cell of claim 1 with a substrate; b) allowing the host cell to ferment the substrate; and, c) recovering one or more products of the fermentation,
wherein the substrate is a cellulosic substrate and the yield of fermentation product is increased by virtue of the host cell's ability to ferment arabinose.

Assignees

• Lallemand Hungary Liquidity Man Llc

Inventors

• Argyros D Aaron
• Caiazza Nicky
• Barrett Trisha F
• Warner Anne K

Classifications

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title

• Y02E50/16

  Cross-Sectional Technologies · mapped topic

• C12P7/10 Primary

  substrate containing cellulosic material · CPC title