Transformed cells that ferment pentose sugars and methods of their use

Abstract

The present invention relates to host cells transformed with a nucleic acid sequence encoding a eukaryotic xylose isomerase obtainable from an anaerobic fungus. When expressed, the sequence encoding the xylose isomerase confers to the host cell the ability to convert xylose to xylulose which may be further metabolized by the host cell. Thus, the host cell is capable of growth on xylose as carbon source. The host cell preferably is a eukaryotic microorganism such as a yeast or a filamentous fungus. The invention further relates to processes for the production of fermentation products such as ethanol, in which a host cell of the invention uses xylose for growth and for the production of the fermentation product. The invention further relates to nucleic acid sequences encoding eukaryotic xylose isomerases and xylulose kinases as obtainable from anaerobic fungi.

What is claimed: 
     
         1 . A process for producing ethanol, comprising:
 (a) fermenting, at a temperature less than 38° C., in a medium comprising
 a source of xylose and 
 a  Saccharomyces  host cell transformed with a nucleic acid construct comprising nucleotide sequence encoding a xylose isomerase, whereby the nucleic acid construct confers to the transformed host cell the ability to isomerize xylose to xylulose, 
 wherein the transformed host cell produces said xylose isomerase having a specific xylose isomerase activity of at least 100 nmol xylose/min/mg protein at 30° C., whereby the transformed host cell ferments xylose to ethanol, and 
 wherein the amino acid sequence of the encoded xylose isomerase is at least 45% identical to SEQ ID NO:1, comprises a first xylose isomerase signature pattern at positions corresponding to residues 185-194 of SEQ ID NO:1, a second xylose isomerase signature pattern at positions corresponding to residues 230-237 of SEQ ID NO:1, a catalytic triad including four residues corresponding to His 102, Asp 105, Lys 235 and Asp 340 of SEQ ID NO:1, and further comprises at least one Mg-binding site that corresponds to residue Glu 233 of SEQ ID NO:1, and 
   (b) optionally recovering the ethanol.   
     
     
         2 . The process according to  claim 1 , wherein the medium also contains a source of glucose. 
     
     
         3 . The process according to  claim 1 , wherein the production of ethanol occurs at a rate of at least 0.5 g ethanol per liter per hour. 
     
     
         4 . The process according to  claim 1 , wherein the ethanol yield is at least 50%. 
     
     
         5 . The process of  claim 1 , wherein the amino acid sequence of the encoded xylose isomerase is at least 50% identical to SEQ ID NO:1. 
     
     
         6 . The process of  claim 1 , wherein said xylose isomerase has a K m  for xylose that is less than 50 mM. 
     
     
         7 . The process of  claim 1 , wherein said  Saccharomyces  host cell has been further genetically modified to confer on the cell one or more of the following properties: (a) increased transport of xylose into the host cell; (b) increased xylulose kinase activity; (c) increased flux of the pentose phosphate pathway; (d) decreased sensitivity to catabolite repression; (e) increased tolerance to ethanol, osmolarity or organic acids; or (f) reduced production of by-products, in comparison to a  Saccharomyces  host cell that has not undergone said genetic modification. 
     
     
         8 . The process of  claim 7 , wherein the genetic modification that results in said properties (a)-(e) is overexpression of an endogenous gene or expression of a heterologous gene, said gene selected from the group consisting of a hexose transporter, a pentose transporter, a xylulose kinase, an enzyme from the pentose phosphate pathway, a glycolytic enzyme, or an ethanologenic enzyme. 
     
     
         9 . The process of  claim 8 , wherein the genetic modification is overexpression of a xylulose kinase. 
     
     
         10 . The process of  claim 7 , wherein the genetic modification that results in said properties (a)-(e) is one that causes inactivation of one of the following endogenous genes: (i) a gene encoding a hexose kinase (ii)  Saccharomyces  MIG1 gene; (iii)  Saccharomyces  MIG2 gene; (iv)  Saccharomyces  GRE3 gene; (v)  Saccharomyces  glycerol-phosphate dehydrogenase 1 gene; (vi)  Saccharomyces  glycerol-phosphate dehydrogenase 1 gene; or (vii) a gene homologous to one of (i)-(vii) and which hybridizes thereto. 
     
     
         11 . The process of  claim 10 , wherein the genetic modification is deletion of  Saccharomyces  GRE3 gene.