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

Patent metadata

Field	Value
Publication number	US-10047380-B2
Application number	US-201715454275-A
Country	US
Kind code	B2
Filing date	Mar 9, 2017
Priority date	Nov 10, 2011
Publication date	Aug 14, 2018
Grant date	Aug 14, 2018

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

What is claimed is:

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), wherein the AraT is an AraT of an organism selected from the group consisting of Kluveromyces lactis, Kluyveromyces thermotolerans, Zygosaccharomyces rouxii, Vanderwaltozyma polyspora, Debaryomyces hansenii, Aspergillus niger, Penicillium chrysogenum, Pichia guillermondii, Aspergillus flavus, Candida lusitnaea, Candida albicans (SC5314), Kluveromyces marxianus , and Candida arabinofermentans.

2. 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 22.

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

4. The recombinant eukaryotic host cell of claim 1 , 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.

5. 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.

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

7. The recombinant eukaryotic host cell of claim 6 , wherein the XI is an XI of B. thetaiotamicron.

8. The recombinant eukaryotic host cell of claim 6 , wherein the XI comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25.

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

10. The recombinant eukaryotic host cell of claim 9 , wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia slipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidenialis.

11. The recombinant eukaryotic host cell of claim 9 , 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.

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

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

14. The recombinant eukaryotic host cell of claim 12 , wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, hydrogen, butyric acid, acetone, and butanol.

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

16. The recombinant eukaryotic host cell of claim 15 , wherein the host cell further exhibits high temperature tolerance.

17. 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/I xylose and 21 g/l arabinose.

18. 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.

19. 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.

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

21. The recombinant eukaryotic host cell of claim 20 , wherein the one or more cellulases is selected from the group consisting of endoglucanases, exoglucanases, and β-glucosidases.

22. The recombinant eukaryotic host cell of claim 21 , 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.

23. The recombinant eukaryotic host cell of claim 22 , wherein (a) the first heterologous polynucleotide that encodes an endoglucanase is A. fumigatus ; (b) the second heterologous polynucleotide that encodes a β-glucosidase is S. fibuligera ; (c) the third heterologous polynucleotide that encodes a first cellobiohydrolase is T. emersonii ; and, (d) the fourth heterologous polynucleotide that encodes a second cellobiohydrolase is C. lucknowense.

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

25. A composition comprising a carbon source and the recombinant eukaryotic host cell of claim 1 , wherein the carbon source is a cellulosic substrate that contains at least about 1% 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

• C12P2203/00

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

• C12N9/90

  Isomerases (5.) · CPC title

• C07K14/38

  from Aspergillus · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title