Expression of Hexose Kinase in Recombinant Host Cells

Abstract

The invention relates to a recombinant host cell having (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. Additionally, the invention relates to methods of making and using such recombinant host cells including, for example, methods of increasing glucose consumption, methods of improving redox balance, and/or methods of increasing the production of a product of a pyruvate-utilizing pathway.

1 . A recombinant yeast host cell comprising:
 (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity in the host cell wherein the activity of the polypeptide of (a) is reduced or substantially eliminated; and   (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity.   
     
     
         2 . The recombinant host cell of  claim 1  wherein the glucose consumption rate is increased as compared to that of the host cell comprising (a) but not (b). 
     
     
         3 . The recombinant host cell of  claim 1  wherein the modification of (a) is a deletion. 
     
     
         4 . The recombinant host cell of  claim 1  wherein the recombinant host cell further comprises (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. 
     
     
         5 . The recombinant host cell of  claim 4  wherein pyruvate decarboxylase activity is reduced or substantially eliminated. 
     
     
         6 . The recombinant host of  claim 1  wherein the polypeptide of (a) is HXK2, and wherein the recombinant host cell is  S. cerevisiae.    
     
     
         7 . The recombinant host cell of  claim 1 ,
 wherein the polypeptide of (a) is RAG5, and wherein the recombinant host cell is  K. lactis , or   wherein the polypeptide of (a) is HPGLK1, and wherein the recombinant host cell is  H. polymorpha , or   wherein the polypeptide of (a) is HXK2, and wherein the recombinant host cell is  S. pombe.      
     
     
         8 - 9 . (canceled) 
     
     
         10 . The recombinant host cell of  claim 1  wherein the heterologous polynucleotide of (b) comprises the polypeptide of (a) having a deletion of a protein interaction domain that prevents function as a transcriptional regulator. 
     
     
         11 . The recombinant host cell of  claim 1  wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 2, 115, 117, 119, 4, 6, 8, 121, or 123. 
     
     
         12 . The recombinant host cell of  claim 6  wherein the heterologous polynucleotide of (b) encodes a polypeptide of SEQ ID NO: 4, 6, 8, 121, or 123. 
     
     
         13 . The recombinant host cell of  claim 5  wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 4, 6, 8, 121, or 123. 
     
     
         14 . The recombinant host cell of  claim 5  wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 130. 
     
     
         15 . The recombinant host cell of  claim 1  wherein the polypeptide encoded by the heterologous polynucleotide of (b) is constitutively expressed. 
     
     
         16 . The recombinant host cell of  claim 1  wherein the heterologous polynucleotide of (b) comprises i) a promoter region derived from the  S. cerevisiae  ADH1 promoter region or ii) a promoter region having at least about 85% identity to SEQ ID NO: 131. 
     
     
         17 . The recombinant host cell of  claim 6  wherein the heterologous polynucleotide of (b) comprises a conditional promoter and encodes a polypeptide having at least 85% identity to SEQ ID NO: 4 or SEQ ID NO: 2. 
     
     
         18 . The recombinant host cell of  claim 17  wherein the conditional promoter comprises a sequence derived from the OLE1 promoter region. 
     
     
         19 . The recombinant host cell of  claim 1  further comprising a pyruvate utilizing biosynthetic pathway which forms a product selected from the group consisting of: 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malate, isoamyl alcohol, and isoprenoids. 
     
     
         20 . The recombinant host cell of  claim 19  wherein the product is isobutanol. 
     
     
         21 - 26 . (canceled) 
     
     
         27 . A method of increasing glucose consumption of a recombinant host cell comprising:
 (i) providing the recombinant host cell of  claim 1 ; and   (ii) growing the recombinant host cell under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form;   wherein the glucose consumption of the recombinant host cell is greater than the glucose consumption of a host cell comprising (a) but not (b).   
     
     
         28 . A method of increasing the formation of a product of a pyruvate-utilizing biosynthetic pathway comprising;
 (i) providing the recombinant host cell of  claim 19 , or combinations thereof; and   (ii) growing the recombinant host cell under conditions wherein the product of the pyruvate-utilizing pathway is formed;   wherein the amount of product formed by the recombinant host cell is greater than the amount of product formed by a host cell comprising (a) but not (b).   
     
     
         29 . The method of  claim 28  wherein the product is butanol. 
     
     
         30 . The butanol of  claim 29 , wherein said butanol is isobutanol. 
     
     
         31 . (canceled)