Adaptive laboratory evolution of KT2440 improves -coumaric and ferulic acid catabolism and tolerance.

KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids -coumaric acid (CA) and ferulic acid (FA) individually and in combination (CA ​+ ​FA). The TALE experiments led to evolved strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L CA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. and were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of CA, but not an improved growth rate in high concentrations of FA. Deletion of part of the TtgABC efflux pump, severely inhibited growth in CA ​+ ​FA TALE-derived strains but did not affect growth in CA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving for microbial lignin valorization.