Microorganisms Having Enhanced Tolerance To Inhibitors and Stress

Abstract

The present invention provides genetically modified strains of microorganisms that display enhanced tolerance to stress and/or inhibitors such as sodium acetate and vanillin. The enhanced tolerance can be achieved by increasing the expression of a protein of the Sm-like superfamily such as a bacterial Hfq protein and a fungal Sm or Lsm protein. Further, the present invention provides methods of producing alcohol from biomass materials by using the genetically modified microorganisms of the present invention.

1 . A genetically modified microorganism, wherein said genetic modification comprises introduction of an expression vector comprising the coding sequence of a protein of the Sm-like superfamily, and wherein said genetic modification results in elevated tolerance to stress or at least one inhibitor as compared to 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 bacterium selected from the group consisting of  Acetobacterium, Bacillus, Streptococcus, Clostridium, Zymomonas, Anaerocellum, Caldicellulosiruptor, Thermoanaerobacter, Gluconobacter,  and  E. coli.    
     
     
         4 . The microorganism of  claim 3 , wherein said bacterium is selected from the group consisting of  C. thermocellum, Z. mobilis, Anaerocellum thermophilum, Caldicellulosiruptor saccharolyticus ),  Thermoanaerobacter  sp. X514, and  E. coli.    
     
     
         5 . The microorganism of  claim 2 , wherein said microorganism is a bacterium and wherein said protein of the Sm-like superfamily is a bacterial Hfq protein. 
     
     
         6 . The microorganism of  claim 5 , wherein said Hfq protein comprises an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8 or 10, or a functional derivative or homolog thereof that shares at least 95% sequence identity therewith. 
     
     
         7 . The microorganism of  claim 5 , wherein said microorganism is  Z. mobilis.    
     
     
         8 . The microorganism of  claim 7 , wherein said Hfq protein comprises the sequence as set forth in SEQ ID NO: 2. 
     
     
         9 . 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. 
     
     
         10 . The microorganism of  claim 9 , wherein said fungal species is yeast selected from  S. cerevisiae  or  P. pastoris.    
     
     
         11 . The microorganism of  claim 10 , wherein said protein of the Sm-like superfamily is a yeast Sm or Lsm protein. 
     
     
         12 . The microorganism of  claim 11 , wherein said protein comprises an amino acid sequence selected from any one of SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, or a functional derivative or homolog thereof that shares at least 95% sequence identity therewith. 
     
     
         13 . The microorganism of  claim 1 , wherein said expression vector is a replicative vector or an integrative vector. 
     
     
         14 . The microorganism of  claim 1 , wherein said stress is environmental stress selected from the group of high temperatures, low temperatures, low pH, oxidation, osmotic, and drought. 
     
     
         15 . The microorganism of  claim 1 , wherein said at least one inhibitor is selected from the group consisting of an acetate salt, vanillin, furfural, hydroxymethylfurfural (HMF) and H 2 O 2 . 
     
     
         16 . The microorganism of  claim 15 , wherein said acetate salt is selected from the group consisting of sodium acetate, ammonium acetate and potassium acetate. 
     
     
         17 . The microorganism of  claim 1 , wherein said enhanced tolerance is characterized by ability to grow in a media containing sodium acetate at a concentration of 195 mM. 
     
     
         18 . A method of producing alcohol from a cellulosic biomass material, comprising adding a genetically modified microorganism according to any one of  claims 1 - 17  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.