Adaptive Laboratory Evolution of Product-Tolerant Hosts for Enhanced Production of Biobased Chemicals

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 loss-of-function mutations were determined by screening knockout libraries and generation of combinatorial knockouts, while causative coding mutations were determined in several strains by conjugation-based genome shuffling. Cross-compound screening of all evolved isolates has also revealed specific sets of mutations that confer tolerance toward multiple classes of compounds.  We have inferred numerous novel mechanisms of tolerance to different compound classes through the use of these strategies.

There is a risk that cells that have evolved tolerance to high concentrations of an exogenously added chemical can exhibit alterations in metabolic flux, cell wall properties, and other cellular processes that render them to be poorer endogenous producers of that chemical.  Possible solutions to this problem are screening introduced production pathways in many isolates from distinct evolutionary lineages, or reconstructing only minimal sets of a variety of causal mutations for tolerance in production strains. An example case will be presented where significantly improved endogenous production of isobutyrate was achieved using evolved strains as hosts.