(507a) Combinatorial Design of Hydrolysate-Tolerant E. Coli Mutants

Lignocellulosic biomass is a cheap, renewable, and sustainable feedstock for biofuel production.  For routes utilizing biological conversion of the lignocellulosic hydrolysate, microbial strains need to be engineered for the efficient production of the desired product, as well as designed for tolerance to the variety of inhibitors in hydrolysate (e.g., acetate and furfural).  Here, we provide data from genome-wide searches based on the TRackable Multiplex Recombineering (Warner et al. 2010) approach.  TRMR utilizes two barcoded-libraries, with ~ 4,000 members each, coding for either an increased or decreased expression of virtually every gene within E. coli.  Selections were performed using TRMR libraries in the presence of acetate, furfural, and hydrolysate.  Mutations that were enriched during the course of any selection were tracked via microarray.  Targets that were conserved for enrichment among these selections were identified and individually confirmed for improved tolerance to these inhibitors.  In addition, select targets were combinatorially designed into mutant strains to further improve tolerance to hydrolysate.  Based on these conserved enrichments, hypotheses about toxicity mechanisms of hydrolysate were formulated and tested.  We will provide a summary of these individual mutations and the combinatorial construction in this work, highlighting the aspects of designing targeted pools to enhance epistatic effects of combinatorial mutants.