Genome-Wide Approach to Engineering Inhibitor Tolerance In E. Coli for Biofuel Applications

The microbial conversion of lignocellulosic biomass to biofuels is a promising field for renewable energy.  Large-scale implementation of this technology is partially thwarted by biocatalyst sensitivity to hydrolysate components like furfural and acetate.  Escherichia coli is a widely used model organism for the conceptual understanding of complex phenotypes and is used here to explore inhibitor toxicity of hydrolysate components.  We report the use of genome-wide tools developed by our lab, namely SCalar Analysis of Library Enrichments (SCALEs) and TRackable Multiplex Recombineering (TRMR), to determine genetic targets for the engineering of tolerant clones.  One such target that has been identified is the overexpression of D-sedoheptulose 7-phosphate isomerase (lpcA), which improves growth by 430% in 1 g/l furfural.  This overexpression also increases growth in acetate (124% at 15 g/l) and ethanol (180% at 25 g/l), potentially lending itself to robust applications.  We use the insight gained from multiple selections to elucidate synergistic tolerant functionalities while also revealing novel mechanisms of toxicity.  The advantage of this approach grants trackable knowledge that might otherwise be missed by directed engineering or untraced mutational studies.