Method of Modifying a Yeast Cell for the Production of Ethanol

Abstract

The invention relates to a method of modifying a yeast cell for the production of ethanol. According to some embodiments of the invention, the activity of the Gpd1 protein and/or the Gpd2 protein is reduced.

1 . A method of modifying a yeast cell for the production of ethanol, characterized by reducing the activity of the Gpd1 protein and/or the Gpd2 protein. 
     
     
         2 . The method according to  claim 1 , characterized by reducing the activity of the Gpd1 and eliminating the activity of the Gpd2 protein. 
     
     
         3 . The method according to  claim 1 , characterized by eliminating the activity of the Gpd1 and reducing the activity of the Gpd2 protein. 
     
     
         4 . The method according to  claim 1 , characterized by reducing the activity of the Gpd1 and reducing the activity of the Gpd2 protein. 
     
     
         5 . The method according to  claim 1 ,
 characterized in that the reduction of the activity of the Gpd1 protein and/or the Gpd2 protein is achieved by   reducing the expression of the GPD1 gene and/or the GPD2 gene,   providing an antisense molecule to the GPD1 and/or the GPD2 mRNA,   providing an antagonist to the Gpd1 and/or the Gpd2 protein,   providing a mutated form of the Gpd1 and/or the Gpd2 protein, or   providing a small inhibitory molecule for inhibiting the Gpd1 and/or the Gpd2 protein.   
     
     
         6 . The method according to  claim 1 , characterized in that the GPD1 gene and/or the GPD2 gene is expressed by a promoter that is operably linked to the GPD1 gene or the GPD2 gene, wherein the promoter causes less than or equal to 20% transcription of the TEF1 promoter operably linked to the GPD1 gene or the GPD2 gene. 
     
     
         7 . The method according to  claim 6 , characterized in that the expression of the GPD1 gene and/or the GPD2 gene is reduced by at least 50% compared to the expression in a wild type yeast cell. 
     
     
         8 . The method according to  claim 6 , characterized in that the promoter is a promoter according to SEQ ID NO 5 or SEQ ID NO 6. 
     
     
         9 . The method according to  claim 5 , characterized in that the antisense molecule has a sequence that hybridizes with the mRNA according to SEQ ID NO 1 or SEQ ID NO 2. 
     
     
         10 . The method according to  claim 9 , characterized in that the antisense molecule hybridizes with any 10 to 30 bases of the mRNA according to SEQ ID NO 1 or SEQ ID NO 2. 
     
     
         11 . The method according to  claim 5 , characterized in that the mutated form of the Gpd1 protein and/or the Gpd2 protein bears a mutation in a functional domain of the protein. 
     
     
         12 . The method according to  claim 1 , characterized by additionally reducing the activity of the Gpp1 protein and/or the Gpp2 protein. 
     
     
         13 . The method according to  claim 12 , characterized by reducing the activity of the Gpp1 and eliminating the activity of the Gpp2 protein. 
     
     
         14 . The method according to  claim 12 , characterized by eliminating the activity of the Gpp1 and reducing the activity of the Gpp2 protein. 
     
     
         15 . The method according to  claim 12 , characterized by reducing the activity of the Gpp1 and reducing the activity of the Gpp2 protein. 
     
     
         16 . The method according to  claim 12 , characterized in that the reduction of the activity of the Gpp1 protein and/or the Gpp2 protein is achieved by
 reducing the expression of the Gpp1 and/or the Gpp2 protein,   providing an antisense molecule to the GPP1 and/or the GPP2 mRNA,   providing an antagonist to the Gpp1Δnd/or the Gpp2 protein,   providing a mutated form of the Gpp1 protein and/or the Gpp2 protein, or providing a small inhibitory molecule for inhibiting the Gpp1 protein and/or the Gpp2 protein.   
     
     
         17 . The method according to  claim 16 , characterized in that the GPP1 gene and/or the GPP2 gene is expressed by a promoter that is operably linked to the GPP1 gene or the GPP2 gene, wherein the promoter causes less than or equal to 20% transcription of the TEF1 promoter operably linked to the GPP1 gene or the GPP2 gene. 
     
     
         18 . The method according to  claim 17 , characterized in that the expression of the GPP1 gene and/or the GPP2 gene is reduced by at least 50% compared to the expression in a wild type yeast cell. 
     
     
         19 . The method according to  claim 17 , characterized in that the promoter is a promoter according to SEQ ID NO 5 or SEQ ID NO 6. 
     
     
         20 . The method according to  claim 16 , characterized in that the antisense molecule has a sequence that hybridizes with the mRNA according to SEQ ID NO 3 or SEQ ID NO 4. 
     
     
         21 . The method according to  claim 20 , characterized in that the antisense molecule hybridizes with any 10 to 30 bases, preferably with any 18 to 23 bases of the mRNA according to SEQ ID NO 3 or SEQ ID NO 4. 
     
     
         22 . The method according to  claim 16 , characterized in that the mutated form of the Gpp1 protein and/or the Gpp2 protein bears a mutation in a functional domain of the protein. 
     
     
         23 . A modified yeast cell, in which the activity of the Gpd1 and/or Gpd2 protein is reduced compared to a wild-type yeast cell. 
     
     
         24 . The modified yeast cell according to  claim 23 , characterized in that the reduced activity is achieved by
 reduced expression of the GPD1 gene and/or GPD2 gene,   a presence of an antisense molecule to the GPD1 and/or GPD2 mRNA,   a presence of an antagonist to the Gpd1 protein and/or Gpd2 protein, a presence of a mutated form of a Gpd1 protein and/or Gpd2 protein, or   a presence of a small inhibitory molecule for inhibiting the Gpd1 and/or the Gpd2 protein.   
     
     
         25 . The modified yeast cell according to  claim 24 , characterized in that the GPD1 and/or the GPD2 gene is expressed by a promoter that is operably linked to the GPD1 gene or the GPD2 gene, wherein the promoter is weak compared to the promoter in the wild-type yeast cell. 
     
     
         26 . The modified yeast cell according to  claim 30 , characterized in that the expression of the GPD1 gene and/or the GPD2 gene is reduced by at least 50% compared to the expression of the wild type gene. 
     
     
         27 . The modified yeast cell according to  claim 25 , characterized in that the promoter is a promoter according to SEQ ID NO 5 or SEQ ID NO 6. 
     
     
         28 . The modified yeast cell according to  claim 24 , characterized in that the antisense molecule to the GPD1 gene and/or the GPD2 mRNA has a sequence that hybridizes with the mRNA according to SEQ ID NO 1 or SEQ ID NO 2. 
     
     
         29 . The modified yeast cell according to  claim 28 , characterized in that the antisense molecule hybridizes with any 10 to 30 bases of the mRNA according to SEQ ID NO 1 or SEQ ID NO 2. 
     
     
         30 . The modified yeast cell according to  claim 24 , characterized in that the mutated form of a Gpd1 and/or Gpd2 protein bears a mutation in a functional domain of the protein. 
     
     
         31 . Use of a genetically modified yeast cell according to  claim 23  for producing ethanol. 
     
     
         32 . A method for the production of ethanol, comprising the following steps:
 providing a yeast cell according to  claim 23 ,   providing biomass,   growing the yeast cell in the presence of the biomass under conditions that allow for the production of ethanol.