Microorganisms Having Enhanced Tolerance To Inhibitors and Stress
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.