Visualizing evolution in real time to determine the molecular mechanisms of n-butanol tolerance in Escherichia coli.

Toxicity of products or feedstock components poses a challenge in the biocatalyst-based production of fuels and chemicals. The genetic determinants that are involved in increased resistance to an inhibitor form the adaptive landscape for the phenotype; so in order to engineer more robust biocatalysts, a better understanding of the adaptive landscape is required. Here, we used an adaptive laboratory evolution method called visualizing evolution in real time (VERT) to help map out part of the adaptive landscape of Escherichia coli tolerance to the biofuel n-butanol. VERT enables identification of adaptive events (population expansions triggered by adaptive mutants) via visualization of the relative proportions of different fluorescently-labeled cells. Knowledge of the occurrence of adaptive events allows for a more systematic isolation of adaptive mutants while simultaneously reducing the number of missed adaptive mutants (and the underlying adaptive mechanisms) that result from clonal interference during the course of in vitro evolution. Based on the evolutionary dynamics observed, clonal interference was found to play a significant role in shaping the population structure of E. coli during exposure to n-butanol, and VERT helped to facilitate the isolation of adaptive mutants from the population. We further combined adaptive laboratory evolution with genome shuffling to significantly enhance the desired n-butanol tolerance phenotype. Subsequent transcriptome analysis of the isolated adaptive mutants revealed different mechanisms of n-butanol resistance in different lineages. In one fluorescently-marked subpopulation, members of the Fur regulon were upregulated; which was not observed in the other subpopulation. In addition, genome sequencing of several adaptive mutants revealed the genetic basis for some of the observed transcriptome profiles. We further elucidated the potential role of the iron-related gene in n-butanol tolerance via overexpression and deletion studies and hypothesized that the upregulation of the iron-related genes indirectly led to modifications in the outer membrane, which contributed to enhanced n-butanol tolerance.