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 is: 
     
         1 . A eukaryotic host cell transformed with a nucleic acid construct comprising a nucleotide sequence encoding a xylose isomerase (XI) enzyme, wherein, expression of the nucleic acid construct in the host cell confers on the host cell the ability to isomerize xylose to xylulose. 
     
     
         2 . The transformed host cell according to  claim 1 , wherein the nucleotide sequence is selected from the group consisting of:
 (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO:1;   (b) a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence of SEQ ID NO:2;   (c) a nucleotide sequence the complementary strand of which hybridizes to the nucleic acid sequence of (a) or (b); and   (d) a nucleotide sequence which differs from the sequence of (c) due to degeneracy of the genetic code.   
     
     
         3 . The transformed host cell according to  claim 2 , wherein the host cell is a yeast cell. 
     
     
         4 . A transformed host cell according to  claim 3 , wherein the yeast cell belongs to one of the following genera:  Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces,  or  Yarrowia.    
     
     
         5 . The transformed host cell according to  claim 4 , wherein the yeast cell is a member of one of the following species:  Saccharomyces cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces uvarum, Saccharomyces diastaticus, Kluyveromyces lactis, Kluyveromyces marxianus,  and  Kluyveromyces fragilis.    
     
     
         6 . The transformed host cell according to  claim 2 , that is a filamentous fungus cell. 
     
     
         7 . A transformed host cell according to  claim 6 , wherein the filamentous fungus cell belongs to one of the following genera:  Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium,  or  Penicillium.    
     
     
         8 . The transformed host cell of  claim 1 , wherein the nucleotide sequence encoding the XI enzyme is operably linked to a promoter that drives sufficient expression of the XI enzyme in the host cell, that confers-on the host cell the ability to isomerize xylose to xylulose. 
     
     
         9 . The transformed host cell according to  claim 8 , wherein the promoter is insensitive to catabolite repression in the host cell. 
     
     
         10 . The transformed host cell according to  claim 1  that further comprises a genetic modification that results in:
 (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) decreased production of by-products, 
 
       which increase or decrease is in comparison to a similar cell that does not comprise said genetic modification. 
     
     
         11 . The transformed host cell according to  claim 10 , wherein the genetic modification results in (i) overexpression of an endogenous gene, (ii) expression of a heterologous gene, or (iii) a combination of (i) and (ii), and
 wherein the gene being expressed or overexpressed is selected from the group consisting of a gene encoding:   (a) a hexose transporter;   (b) a pentose transporter;   (c) a xylulose kinase;   (d) an enzyme from the pentose phosphate pathway,   (e) a glycolytic enzyme, and   (f) an ethanologenic enzyme.   
     
     
         12 . The transformed host cell according to  claim 10 , wherein the genetic modification results in inactivation of an endogenous gene which is selected from the group consisting of:
 (a) a gene encoding a hexose kinase   (b) the  Saccharomyces  MIG1 gene;   (c) the  Saccharomyces  MIG2 gene; and   (d) a gene homologous to (a), (b) or (c) and which hybridizes thereto.   
     
     
         13 . The transformed host cell according to  claim 1  that further expresses one or more enzymes that confers on the cell the ability to produce lactic acid, acetic acid, succinic acid, amino acids, 1,3-propanediol, ethylene, glycerol, a 13-lactam antibiotic or a cephalosporin. 
     
     
         14 . The transformed host cell according to  claim 13  that further comprises a genetic modification that results in decreased alcohol dehydrogenase activity. 
     
     
         15 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding an XI enzyme, which nucleic acid molecule is selected from the group consisting of:
 (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence that has at least 53% sequence identity with the amino acid sequence of SEQ ID NO:1;   (b) a nucleic acid molecule comprising a nucleotide sequence that has at least 57% sequence identity with the nucleotide sequence of SEQ ID NO:2;   (c) a nucleic acid molecule that comprises the complementary strand of, and that hybridizes to, the nucleic acid molecule of (a) or (b); and,   (d) a nucleic acid molecule the sequence of which differs from the sequence of the nucleic acid molecule of (c) due to degeneracy of the genetic code.   
     
     
         16 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding an XI enzyme, which nucleic acid molecule is selected from the group consisting of:
 (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence that has at least 47% sequence identity with the amino acid sequence of SEQ ID NO:3;   (b) a nucleic acid molecule comprising a nucleotide sequence that has at least 37% sequence identity with the nucleotide sequence of SEQ ID NO:4;   (c) a nucleic acid molecule that comprises the complementary strand of, and that hybridizes to, the nucleic acid molecule of (a) or (b); and,   (d) a nucleic acid molecule the sequence of which differs from the sequence of the nucleic acid molecule of (c) due to degeneracy of the genetic code.   
     
     
         17 . A process for producing ethanol, comprising the steps of:
 (a) fermenting a medium containing a source of xylose with the transformed host cell of  claim 1 , which host cell ferments xylose to ethanol, and, optionally,   (b) recovering the ethanol.   
     
     
         18 . The process according to  claim 17 , wherein the medium also contains a source of glucose. 
     
     
         19 . The process according to  claim 17 , wherein the production of ethanol occurs at a rate of at least 0.5 g ethanol per liter per hour. 
     
     
         20 . The process according to  claim 17 , wherein the ethanol yield is at least 50%. 
     
     
         21 . A process for producing, as a fermentation product, lactic acid, acetic acid, succinic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic or a cephalosporin, which process comprises the steps of:
 (a) fermenting a medium containing a source of xylose with the transformed host cell of  claim 13 , which host cell ferments xylose to yield the fermentation product, and, optionally, 
 (b) recovering the fermentation product. 
 
     
     
         22 . The process according to  claim 21 , wherein the medium also contains a source of glucose.