Reduced DNA synthesis costs, advances in next-generation sequencing, and the advent of oligo-mediated allelic replacement have pioneered the way for rapid library preparation, selection, and elucidation of beneficial genotypes.  Here, we have applied two genome-wide library techniques, SCalar Analysis of Library Enrichments (Lynch et al. 2007, Nat. Methods) and TRackable Multiplex Recombineering (Warner et al. 2010, Nat. Biotechnology), to engineer tolerance to furfural, an inhibitory compound of hydrolysate.  SCALEs utilizes plasmid-based genomic libraries of defined size and enriched alleles are tracked via microarray.  TRMR is comprised of two chromosomally-based mutant libraries harboring increased or decreased expression of virtually every gene within E. coli, for ~8,000 mutants.  In addition, we have developed genome-editing approach by targeting modular alleles enriched within these selections, as well as acetate (Sandoval et al. 2012, PNAS) and hydrolysate TRMR selections.  We will present our findings and compare and contrast the enrichment patterns between these various selections.

Genome-editing approaches for improving tolerance to hydrolysate inhibitors in <i>E. coli</i>

245th National Meeting (2013)

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Genome-editing approaches for improving tolerance to hydrolysate inhibitors in E. coli