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 method of producing alcohol from a cellulosic biomass material, comprising:
a. adding a genetically modified fungus to a fermentation mixture comprising a cellulosic biomass material or fermentation substrates derived from said cellulosic biomass material, wherein said genetic modification to said fungus comprises introduction of an expression vector comprising the coding sequence of a yeast protein of the Sm-like superfamily, and wherein said genetic modification results in elevated tolerance to at least one inhibitory chemical compound formed during biomass pretreatment as compared to without the genetic modification;
b. allowing said microorganism to ferment and produce alcohol; and
c. recovering alcohol produced by fermentation with said fungus.
2. The method of claim 1 , wherein said fungus is a fungal species selected from Saccharomyces sp., Kluyveromyces sp., Pichia sp., Candida sp., and Schizosaccharomycetes sp.
3. The method of claim 2 , wherein said fungal species is yeast selected from S. cerevisiae or P. pastoris.
4. The method of claim 3 , wherein said yeast protein is a yeast Sm or Lsm protein.
5. The method of claim 4 , 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 protein sequence that shares at least 95% sequence identity therewith.
6. The method of claim 1 , wherein said expression vector is a replicative vector or an integrative vector.
7. The method of claim 1 , wherein said at least one inhibitory chemical compound formed during biomass pretreatment is selected from the group consisting of an acetate salt, vanillin, furfural, hydroxymethylfurfural (HMF) and H 2 O 2 .
8. The method of claim 7 , wherein said acetate salt is selected from the group consisting of sodium acetate, ammonium acetate and potassium acetate.
9. The method 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.