Large-Scale Determination of Causative Mutations for Enhanced Chemical Tolerance in Evolved Strains of Escherichia coli

Overcoming product toxicity remains one of the largest barriers toward achieving economically viable cell factory processes. Even relatively non-toxic products impose significant stresses at the economically-relevant titers ultimately required for commodity chemical production, which are often in excess of 100 g/L. We have utilized a robotic platform to rapidly evolve multiple parallel populations of E. coli K-12 MG1655 for enhanced growth in the presence of toxic concentrations of 11 chemicals representing diverse functional classes that have significant interest as polymer precursors, biofuels, and bulk chemical intermediates.  These include dicarboxylic acids, diamines, monocarboxylic acids, diols, and aromatic acids. Whole genome resequencing of over 200 strains isolated from nearly 90 independently evolved populations has enabled the determination of the genetic basis of tolerance to each chemical.  The mutations in evolved strains were biased toward loss-of-function mutations, which were reconstructed in combinations and shown to be major drivers of tolerance in many cases. Additional combinations of probable gain-of-function mutations were reconstructed using oligonucleotide-mediated recombineering and a conjugation-based genome shuffling approach. Based on successful reconstructions of tolerance phenotypes, novel mechanisms of tolerance have been inferred, and numerous new gene targets conferring increased tolerance have been discovered. Cross-compound screening of all evolved isolates has also revealed specific sets of mutations that confer tolerance toward multiple classes of compounds.  Ongoing work includes continuing to explore the mechanistic basis for tolerance and testing tolerant mutants for endogenous production capability.