Strategy for directing combinatorial genome engineering in Escherichia coli

• DOI: 10.1073/pnas.1206299109
• Publication Date: 2012-06-12T05:53:56Z
• Authors: Nicholas R. Sandoval; Jaoon Y. H. Kim; Tirzah Y. Glebes; Philippa J. Reeder; Hanna R. Aucoin; Joseph R. Warner; Ryan T. Gill
• Keywords: Genome Engineering

We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856-862] with strategies rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894-898]. This should prove broadly applicable various efforts focused on improving production fuels, chemicals, and pharmaceuticals, among other products. used barcoded promoter map the effect increased or decreased expression nearly every gene in Escherichia coli onto growth several model environments (cellulosic hydrolysate, low pH, high acetate). Based these data, we created evaluated RBS mutant (containing greater than 100,000,000 mutations), targeting identified most affect growth. On laboratory timescales, successfully broad range mutations (>25 growth-enhancing confirmed), which improved rate 10-200% different conditions. Although successful, our identify superior combinations emphasized importance epistatic interactions (synergistic, antagonistic) taking full advantage this genome engineering.