Fermentative glycerol-free ethanol production

Abstract

The present invention relates to a yeast cell, in particular a recombinant yeast cell, the cell lacking enzymatic activity needed for the NADH-dependent glycerol synthesis or the cell having a reduced enzymatic activity with respect to the NADH-dependent glycerol synthesis compared to its corresponding wild-type yeast cell, the cell comprising one or more heterologous nucleic acid sequences encoding an NAD + -dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity. The invention further relates to the use of a cell according to the invention in the preparation of ethanol.

1 . Transgenic yeast cells comprising one or more recombinant heterologous, nucleic acid sequences encoding an NAD + -dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10),
 wherein said cells lack enzymatic activity needed for the NADH-dependent glycerol synthesis, or   said cells have a reduced enzymatic activity with respect to the NADH-dependent glycerol synthesis   compared to a corresponding wild-type yeast cell.   
     
     
         2 . The cells of  claim 1 , wherein at least one said NAD + -dependent acetylating acetaldehyde dehydrogenase is represented by SEQ ID NO: 2 or by SEQ ID: 29 or a functional homolog of SEQ ID NO: 2 or SEQ ID NO: 29, said homolog having a sequence identity of at least 60% with SEQ ID NO: 2 or SEQ ID NO: 29. 
     
     
         3 . The cells of  claim 2 , wherein at least one nucleic acid sequence encoding said dehydrogenase comprises a sequence according to SEQ ID NO: 1, SEQ ID NO: 28 or SEQ ID NO: 32 or a functional homolog of SEQ ID NO: 1, SEQ ID NO:28 or SEQ ID NO: 32, said homolog having a sequence identity of at least 60% with SEQ ID NO: 1. 
     
     
         4 . The cells of  claim 1 , wherein said cells are free of NAD-dependent glycerol 3-phosphate dehydrogenase activity or have reduced NAD-dependent glycerol 3-phosphate dehydrogenase activity compared to corresponding wild-type cells, and/or
 wherein the cells are either free of glycerol phosphate phosphatase activity or have reduced glycerol phosphate phosphatase activity compared to corresponding wild-type cells.   
     
     
         5 . The cells of  claim 4 , which comprise a genomic mutation in at least one gene selected from the group consisting of GPD1, GPD2, GPP1 and GPP2. 
     
     
         6 . The cells of  claim 4 , which are free of genes encoding NAD + -dependent glycerol 3-phosphate dehydrogenases (EC 1.1.1.8). 
     
     
         7 . The cells of  claim 1 , further comprising one or more nucleic acid sequences encoding an acetyl-Coenzyme A synthetase activity (EC 6.2.1.1) and one or more nucleic acid sequences encoding NAD + -dependent alcohol dehydrogenase activity (EC 1.1.1.1). 
     
     
         8 . The cells of  claim 1  which are Saccharomycetaceae,  Kluyveromyces, Pichia, Zygosaccharomyces , or  Brettanomyces.    
     
     
         9 . The cells of  claim 8  which are the  S. cerevisiae  strain deposited on 16 Jul. 2009 at the Centraal Bureau voor Schimmelcultures having deposit number CBS125049. 
     
     
         10 . A vector for the functional expression of a nucleotide sequence encoding a polypeptide having enzymatic activity for converting acetyl-Coenzyme A into acetaldehyde in the cytosol of said yeast cell comprising said sequence operably linked to a promoter functional in the yeast cell, wherein said polypeptide comprises a sequence according to SEQ ID NO: 2, SEQ ID NO: 29, or a functional homolog of said sequences. 
     
     
         11 . A method to prepare ethanol from acetate and a fermentable carbohydrate which comprises culturing the yeast cells of  claim 1  under anaerobic conditions. 
     
     
         12 . The method of  claim 11 , wherein said culturing is carried out in a fermentation medium comprising acetate and carbohydrate in a molar ratio is 0.7 or less. 
     
     
         13 . The method of  claim 12 , wherein at least part of the carbohydrate and at least part of the acetate has been obtained by hydrolysing a polysaccharide selected from the group of lignocelluloses, celluloses, hemicelluloses, and pectins. 
     
     
         14 . The method of  claim 13 , wherein lignocellulosic biomass has been hydrolyzed thereby obtaining the fermentable carbohydrate and acetate. 
     
     
         15 . The cells of  claim 5 , wherein at least one said mutation is a complete deletion of said gene in comparison to the corresponding wild-type yeast gene.