Recombinant Yeast Strains For Pentose Fermentation

Abstract

Described herein are recombinant yeast cells expressing a xylulose kinase (XK) which are suitable for fermentation of pentoses. Also described are recombinant yeast cells with higher tolerance to formic and/or acetic acid and suitable for fermentation of pentoses. Also described are recombinant yeast cells expressing an enolase, a phosphofructokinase beta subunit, a 6-phosphofructo-2-kinase, a glucose-6-phosphate isomerase, a phosphoglycerate mutase and/or a triose-phosphate isomerase, and suitable for fermentation of pentoses. Also described are recombinant yeast cells expressing a a phosphoglucomutase and/or phosphoribomutase which are suitable for fermentation of pentoses. Further described are are methods of using or producing such recombinant yeast cells and related materials.

1 - 27 . (canceled) 
     
     
         28 . A recombinant  Saccharomyces  cell which is capable of fermenting xylose and which comprises a heterologous gene encoding a xylulokinase (XK), wherein the XK:
 provides an enzymatic activity for converting D-xylulose to xylulose 5-phosphate at least twice that provided by  S. cerevisiae  XK (SEQ ID NO: 32), and   provides for an anaerobic growth rate of the recombinant cell on xylose which is higher than that provided by a  S. cerevisiae  XK.   
     
     
         29 . The recombinant cell of  claim 28 , wherein the XK further provides for an aerobic growth rate of the cell on xylose which is higher than that provided by  S. cerevisiae  XK. 
     
     
         30 . The recombinant cell of  claim 28 , wherein the XK comprises the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 22 or a catalytically active variant or fragment of any thereof. 
     
     
         31 . The recombinant cell of  claim 28 , wherein the XK has a sequence identity of atleast 80% with the amino acid sequence of SEQ ID NO: 6. 
     
     
         32 . A method for producing a fermentation product, comprising
 (a) contacting the recombinant cell of  claim 28  with a medium comprising a carbon source comprising xylose or arabinose under anaerobic conditions, and   (b) isolating the fermentation product from the medium.   
     
     
         33 . A recombinant yeast cell which is capable of fermenting xylose and which comprises a heterologous gene encoding an Yme2p polypeptide comprising the amino acid sequence of SEQ ID NO: 50 or a catalytically active variant, fragment or yeast ortholog thereof, wherein the Yme2p polypeptide provides for an increased tolerance of the recombinant cell to formic acid, acetic acid, or both. 
     
     
         34 . The recombinant yeast cell of  claim 33 , wherein the Yme2p polypeptide provides for
 (a) an increased anaerobic growth on xylose,   (b) an increased xylose consumption rate,   (c) an increased ethanol production rate, or   (d) a combination of two or all of (a) to (c),   of the recombinant cell in the presence of formic acid.   
     
     
         35 . The recombinant yeast cell of  claim 33 , wherein the Yme2p polypeptide has a sequence identity of at least 70% with the amino acid sequence of SEQ ID NO: 50. 
     
     
         36 . The recombinant yeast cell of  claim 33 , wherein the Yme2p polypeptide comprises the amino acid sequence of SEQ ID NO: 50. 
     
     
         37 . A method for producing a fermentation product, comprising
 (a) contacting the recombinant cell of  claim 33  with a medium comprising a carbon source comprising xylose or arabinose under anaerobic conditions, and   (b) isolating the fermentation product from the medium.   
     
     
         38 . A method for increasing the tolerance of a  Saccharomyces  cell to formic acid, comprising transforming the cell with a gene encoding a Yme2p polypeptide comprising the amino acid sequence of SEQ ID NO: 50 or a catalytically active variant, fragment or yeast ortholog thereof, and expressing the gene. 
     
     
         39 . A recombinant yeast cell which is capable of fermenting xylose and which comprises a heterologous gene encoding
 (a) an enolase comprising the amino acid sequence of SEQ ID NO: 132,   (b) a phosphofructokinase beta subunit polypeptide comprising the amino acid sequence of SEQ ID NO: 134,   (c) a 6-phosphofructo-2-kinase comprising the amino acid sequence of SEQ ID NO: 136,   (d) a glucose-6-phosphate isomerase comprising the amino acid sequence of SEQ ID NO: 138,   (e) a phosphoglycerate mutase comprising the amino acid sequence of SEQ ID NO: 140,   (f) a triose-phosphate isomerase comprising the amino acid sequence of SEQ ID NO: 142,   (g) a catalytically active variant, fragment or yeast ortholog of any one of (a) to (f), or   (h) a combination of any two or more of (a) to (g).   
     
     
         40 . The recombinant yeast cell of  claim 39 , wherein the heterologous gene or combination provides for an increased anaerobic growth rate on xylose, an increased aerobic growth rate on xylose, an increased ethanol production from xylose, or a combination of any two or all thereof. 
     
     
         41 . A method for producing a fermentation product, comprising
 (a) contacting the recombinant cell of  claim 39  with a medium comprising a carbon source comprising xylose or arabinose under anaerobic conditions, and   (b) isolating the fermentation product from the medium.   
     
     
         42 . A recombinant yeast cell which is capable of fermenting xylose and which comprises a heterologous gene encoding a phosphoglucomutase and/or phosphoribomutase comprising the amino acid sequence of SEQ ID NO: 150 or a catalytically active variant or fragment thereof. 
     
     
         43 . The recombinant yeast cell of  claim 42 , wherein the heterologous gene provides for an increased anaerobic growth rate on xylose, an increased xylose consumption, an increased ethanol production from xylose, or a combination of any two or all thereof. 
     
     
         44 . The recombinant yeast cell of  claim 42 , wherein the catalytically active variant has a sequence identity of at least 80% with the amino acid sequence of SEQ ID NO: 150. 
     
     
         45 . The recombinant yeast cell of  claim 42 , wherein the phosphoglucomutase and/or phosphoribomutase comprises the amino acid sequence of SEQ ID NO: 150. 
     
     
         46 . A method for producing a fermentation product, comprising
 (a) contacting the recombinant cell of  claim 42  with a medium comprising a carbon source comprising xylose or arabinose under anaerobic conditions, and   (b) isolating the fermentation product from the medium.