(234b) Elucidating Acetate Tolerance Mechanisms in E. Coli Using Genome-Scale Tools for Improved Cellulosic Biofuel Production

Biofuels from cellulosic biomass will play an integral role in the transition to the 2nd generation of renewable energy. Using cellulosic feedstock 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, sufficient tolerance to acetate and other inhibitors must be conferred upon the fermenting organism. The SCalar Analysis of Library Enrichments (SCALEs) method, created previously by the Gill laboratory, combines a traditional genomic library selection with DNA microarray technology providing genotypic data for selected clones. Our objective is to find mechanisms of acetate tolerance via the SCALEs method. We report here a 72 hour serial transfer selection using an E. coli K12 library with 1.75 g/L acetate in minimal media culture at neutral pH. Microarray studies were performed using the SCALEs method. Top selected clones, constructed based on SCALEs data, show an increase in growth rate between 60% to over 100%. Top clones contained both genes known in acid tolerance as well as genomic regions that could not have been predicted. Metabolic maps coupled with genome-wide fitness data were constructed. Growth rate studies of rational combinations of top clones were also done based on these maps.