(449b) Engineering Acetate Tolerance in E. Coli Using Multiple Genome-Wide Tools for Improved Cellulosic Biofuel Production

Biofuels from cellulosic biomass are an integral part in the transition to the 2nd generation of renewable energy. Using cellulosic feedstocks has more obstacles when compared to first generation biofuels. These include inhibitory compounds formed during pretreatment, the most prevalent of which is acetate. In order to make second generation biofuels an economically viable option, the fermenting organism must have sufficient tolerance to acetate and other inhibitors. Here we utilize two tools created in our group to identify mechanisms of acetate tolerance and engineer an improved strain. The SCalar Analysis of Library Enrichments (SCALES) method combines a traditional genomic library selection with DNA microarray technology providing genotypic data for selected clones. The TRackable Multiplex Recombineering (TReMR) method uses directed chromosomal insertions in front of nearly every gene in E. coli. These insertions are of two types: a strong promoter with a ribosomal binding site that increases gene expression or an inert sequence replacing the ribosomal binding site that decreases gene expression. The two libraries of mutants (?up' and ?down') each represent >98% of the E. coli genome. Each insertion is contains a unique DNA barcode for use with microarrays in order to identify the population genotype. Presented here are SCALEs and TReMR selections with acetate titrated to neutral pH. Serial transfers were used to prevent cultures reaching stationary phase. Mutants were identified via microarray analysis and traditional colony picking. Mutants identified after selection showed a significant increase in growth rate compared to control strain. Genome-wide data was analyzed; we identified various genetic modifications and supplementation strategies that increase tolerance.