Metabolic engineering of xylose-fermenting eukaryotic cells

Abstract

The present invention relates to genetic modifications in eukaryotic host cells that have been transformed to express a xylose isomerase that confers on the host cell the ability to isomerize xylose to xylulose. These genetic modifications are aimed at improving the efficiency of xylose metabolism and include, e.g., reduction of nonspecific aldose reductase activity, increased xylulose kinase activity and increased flux of the pentose phosphate pathway. The modified host cells of the invention are suitable for the production of a wide variety of fermentation products, including ethanol, in fermentation processes in which a source of xylose or a source of xylose and glucose are used as carbon source.

What is claimed is: 
     
         1 . A eukaryotic host cell with the ability to directly isomerise xylose into xylulose, which host cell is genetically modified to increase the flux of the pentose phosphate pathway, so that specific xylose consumption rate in the cells is at least 346 mg xylose/gram biomass/hour. 
     
     
         2 . A host cell according to  claim 1 , in which the at least one gene of the non-oxidative branch of the pentose phosphate pathway is overexpressed. 
     
     
         3 . A host cell according to  claim 2 , in which the at least one gene is selected from the group consisting of a gene encoding ribulose-5-phosphate isomerase, a gene encoding ribulose-5-phosphate epimerase, a gene encoding transketolase and a gene encoding transaldolase. 
     
     
         4 . A host cell according to  claim 2 , in which at least the genes encoding transketolase and transaldolase are overexpressed. 
     
     
         5 . A host cell according to  claim 2  that further comprises a second genetic modification that increases specific xylulose kinase activity. 
     
     
         6 . A host cell according to  claim 5 , in which the second genetic modification results in overexpression of a gene encoding a xylulose kinase 
     
     
         7 . A host cell according to  claim 2  in which the overexpressed gene is endogenous to the host cell. 
     
     
         8 . A host cell according to  claim 1  comprising an additional genetic modification that reduces non-specific aldose reductase activity. 
     
     
         9 . A host cell according to  claim 8 , in which the additional genetic modification inactivates or results in reduced expression of a non-specific aldose reductase. 
     
     
         10 . A host cell according to  claim 9 , in which the aldose reductase gene is inactivated by deletion of at least part of, or by disruption of, the gene. 
     
     
         11 . A host cell according to  claim 8 , in which the expression of all genes encoding non-specific aldose reductase is reduced or the genes are inactivated. 
     
     
         12 . A host cell according to  claim 1  that is a yeast cell. 
     
     
         13 . A host cell according to  claim 12  wherein the yeast is a member of one of the following genera:  Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces , or  Yarrowia.    
     
     
         14 . A host cell according to  claim 13 , wherein the yeast is a member of the species  S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K lactis, K. marxianus , or  K. fragilis.    
     
     
         15 . A host cell according to  claim 1  that is a filamentous fungus. 
     
     
         16 . A host cell according to  claim 15  wherein the fungus is a member of one of the following genera:  Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium , or  Penicillium.    
     
     
         17 . A host cell according to  claim 1  that expresses one or more enzymes that confer on the host cell the ability to produce a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and a cephalosporin. 
     
     
         18 . A process for producing ethanol, comprising the steps of:
 (a) incubating a host cell according to  claim 1  in a medium containing a source of xylose so that the host cell ferments xylose to ethanol, and optionally,   (b) recovering the ethanol.   
     
     
         19 . A process according to  claim 18 , wherein the medium also contains a source of glucose. 
     
     
         20 . A process according to  claim 18  wherein ethanol is produced at a rate of at least 0.5 grams/liter/hour. 
     
     
         21 . A process according to  claim 18  wherein the ethanol yield is at least 50%. 
     
     
         22 . A process for producing a fermentation product comprising:
 (a) incubating host cells according to  claim 17  in a medium containing a source of xylose with, so that the host cells ferment xylose to the fermentation product, and, optionally,   (b) recovering the fermentation product   
       wherein the fermentation product is selected from the group consisting of lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and a cephalosporin. 
     
     
         23 . A process according to  claim 22 , wherein the medium also contains a source of glucose.