(175c) Towards a Semi-Synthetic Stress Response System to Engineer Solvent Tolerance

Many desirable cellular phenotypes are affected by multiple genes and systems functioning together. These so-called complex phenotypes require new approaches which utilize multiple genes simultaneously to engineer an optimal result. To that end we have utilized a method employing multiple coexisting plasmids to generate a semi-synthetic stress response system to engineer an Escherichia coli (E. coli) strain capable of tolerating high levels of toxic solvents. We found that not only can overexpression of multiple genes produce a beneficial phenotype for the complex trait of alcohol tolerance but the expression level of these genes, regulated through induction level of inducible promoters and plasmid copy number, can have a significant effect on the overall success of a tolerance system. Through plasmid based expression of autologous heat shock proteins in E. coli we have produced a tunable stress response system to improve tolerance to a variety of toxic chemicals.

Overexpression of the autologous GroESL proteins in E. coli enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-hour cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 hours of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. Simultaneous overexpression of GrpE and GroESL produced a 2-fold increase in viable cells from a double plasmid control in 5% (v/v) ethanol, overexpression of GroESL and ClpB on multiple plasmids produced a 11.3-fold increase in viable cells after 24 hours of exposure to 5% (v/v) ethanol, and overexpression of GrpE, GroESL, and ClpB on a single plasmid produced a 2.5-fold increase in viable cells after 24 hours of exposure to 7% (v/v) ethanol. Through the production of engineered strains designed to increase expression of the heat shock proteins GroES, GroEL, ClpB, and GrpE we have produced a semi-synthetic stress response system capable of greatly increasing the tolerance of E. coli to a selection of useful alcohols, and potentially providing a platform to generate tolerance to a wide variety of toxic chemicals.