Microorganisms Having Enhanced Resistance To Acetate And Related Compositions And Methods of Use

Abstract

The present invention provides isolated or genetically modified strains of microorganisms that display enhanced resistance to acetate as a result of increased expression of a sodium proton antiporter. The present invention also provides methods for producing such microbial strains, as well as related promoter sequences and expression vectors. Further, the present invention provides methods of producing alcohol from biomass materials by using microorganisms with enhanced resistance to acetate.

1 . A genetically modified microorganism, wherein the genetic modification results in increased expression of a sodium-proton antiporter in said microorganism and elevated resistance to acetate as compared to microorganism without the genetic modification. 
     
     
         2 . The microorganism of  claim 1 , wherein said microorganism is selected from bacteria or fungi. 
     
     
         3 . The microorganism of  claim 2 , wherein said microorganism is a bacterial species selected from  Acetobacterium, Bacillus, Streptococcus, Clostridium, Zymomonas  sp., and  Gluconobacter  sp. 
     
     
         4 . The microorganism of  claim 2 , wherein said microorganism is a fungal species selected from  Saccharomyces  sp.,  Kluyveromyces  sp.,  Pichia  sp.,  Candida  sp., and  Schizosaccharomycetes  sp. 
     
     
         5 . The microorganism of  claim 1 , wherein said sodium-proton antiporter is selected from the group consisting of a plasma membrane sodium-proton antiporter, an endosomal sodium-proton antiporter, and a vacuolar sodium-proton antiporter. 
     
     
         6 . The microorganism of  claim 5 , wherein said sodium-proton antiporter is a plasma membrane sodium-proton antiporter native to said microorganism. 
     
     
         7 . The microorganism of  claim 1 , wherein said microorganism is  Z. mobilis.    
     
     
         8 . The microorganism of  claim 7 , wherein said sodium-proton antiporter comprises the protein sequence as set forth in SEQ ID NO: 2. 
     
     
         9 . The microorganism of  claim 8 , wherein the genetic modification comprises a deletion in the 5′ region upstream of the genomic coding sequence of said antiporter, wherein said deletion is less than 1000 bp and comprises SEQ ID NO: 4, and wherein the modified 5′ region comprises SEQ ID NO: 5. 
     
     
         10 . The microorganism of  claim 8 , wherein said sodium-proton antiporter is expressed from an expression vector introduced into said microorganism. 
     
     
         11 . The microorganism of  claim 1 , wherein said microorganism is  S. cerevisiae.    
     
     
         12 . The microorganism of  claim 11 , wherein said sodium-proton antiporter is the plasma membrane sodium-proton antiporter comprising the amino acid sequence of SEQ ID NO: 15. 
     
     
         13 . The microorganism of  claim 12 , wherein said plasma membrane sodium-proton antiporter is expressed from an expression vector introduced into said  S. cerevisiae.    
     
     
         14 . A method of enhancing resistance of a microorganism to acetate, comprising genetically modifying said microorganism to increase expression of a sodium-proton antiporter in said microorganism. 
     
     
         15 . The method of  claim 14 , wherein the genetic modification comprises modification of the 5′ region upstream of the genomic coding sequence of said sodium-proton antiporter. 
     
     
         16 . The method of  claim 14 , wherein the genetic modification comprises introducing an expression vector into said microorganism, wherein said expression vector directs expression of said sodium-proton antiporter in said microorganism. 
     
     
         17 . The method of  claim 14 , wherein said sodium-proton antiporter is a plama membrane sodium-proton antiporter. 
     
     
         18 . The method of  claim 14 , said microorganism is selected from bacteria or fungi. 
     
     
         19 . An isolated nucleic acid molecule, comprising the nucleotide sequence of SEQ ID NO: 4. 
     
     
         20 . An isolated nucleic acid molecule, comprising the nucleotide sequence of SEQ ID NO: 5. 
     
     
         21 . A method of producing alcohol from a cellulosic biomass material, comprising adding a genetically modified microorganism according to any one of  claims 1 - 13  to a fermentation mixture comprising a cellulosic biomass material and/or fermentation substrates derived from said cellulosic biomass material, allowing said microorganism to ferment and produce alcohol, and recover alcohol produced.