Ethanol production

Abstract

The present invention relates to the production of ethanol as a product of bacterial fermentation. In particular this invention relates to a novel method of gene inactivation and gene expression based upon homologous recombination.

1 - 12 . (canceled)  
     
     
         13 . A method of inactivating a native gene encoding lactate dehydrogenase and inserting one or more expressible genes comprising homologous recombination.  
     
     
         14 . The method according to  claim 13  wherein the one or more expressible genes are a gene encoding pyruvate decarboxylase and a gene encoding alcohol dehydrogenase.  
     
     
         15 . The method according to  claim 14  wherein the gene encoding pyruvate decarboxylase and the gene encoding alcohol dehydrogenase form part of a pyruvate decarboxylase (PDC) operon operatively linked to an insertion element (IE) sequence comprising nucleotides 651-3800 of SEQ ID NO:1.  
     
     
         16 . The method according to  claim 13  in which the gene encoding pyruvate decarboxylase is from a  Zymomonas  sp.  
     
     
         17 . The method according to  claim 16  in which the gene encoding pyruvate decarboxylase is from  Zymomonas mobilis.    
     
     
         18 . The method according to  claim 15  wherein the gene encoding alcohol dehydrogenase is from  Bacillus  strain LN.  
     
     
         19 . The method according to any one of  claims 13  to  18  wherein an insertion sequence and a PDC operon, or portions thereof, are stably integrated into a chromosome of a recombinant bacterium.  
     
     
         20 . The method according to any one of  claims 13  to  14  comprising using a shuttle vector which is able to replicate in  E. coli  and  Bacillus  strains.  
     
     
         21 - 46 . (canceled)  
     
     
         47 . The method of  claim 20  wherein the shuttle vector is pUBUC-IE.