Adaptive laboratory evolution of product-tolerant hosts for biobased chemical production

Product toxicity is frequently encountered as a major barrier to achieving economically viable cell factory processes. Even relatively non-toxic products can impose significant cellular stresses at economically-relevant titers, often in excess of 100 g/L, that are required for bulk biobased chemicals such as polymer precursors and fuels. To address this issue, we have utilized a robotic platform to rapidly evolve parallel populations of E. coli K-12 MG1655 for enhanced growth in the presence of toxic concentrations of 11 chemicals representing diverse functional classes (including dicarboxylic acids, diamines, monocarboxylic acids, diols, and aromatic acids) that have significant interest as polymer precursors, chemical intermediates, and biofuel precursors.

Whole genome resequencing was performed for over 220 isolates from 85 independently evolved populations. Based on these results, causative mutations were determined by a number of techniques. Cross-compound screening of all evolved isolates has revealed specific sets of mutations that confer tolerance toward multiple classes of compounds, while RNA-sequencing and proteomics analysis of selected isolates has provided further insights into mechanisms of toxicity and tolerance.

As the ultimate goal is to develop strains with an improved capability to endogenously produce chemicals beyond inherently toxic levels, evolved isolates have also been investigated as production hosts. Due to the risk that mutations in evolved isolates could render cells poorer endogenous producers, production pathways were either introduced into distinct evolutionary lineages or minimal sets of causal tolerance mutations were reconstructed in production hosts. Example cases will be presented where significantly improved titers were achieved.