Membrane transporter identification and modulation via adaptive laboratory evolution. Escherichia coli

In this project, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids and identify specific mutations which modulate the activities of these transporters. Escherichia coli was adaptively evolved under increasing concentrations of L-histidine, L-phenylalanine, L-threonine, and L-methionine separately with multiple individual replicate experiments. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations, four to six-fold increases, of the targeted amino acids. Further, strains evolved in the presence of elevated concentrations of L-methionine and L-phenylalanine showed a growth rate higher than the unstressed ancestral strain. Whole-genome sequencing of the evolved strains revealed a diverse number of key mutations including SNPs, small deletions, and copy number variants targeting the inner membrane transporters leuE for L-histidine, yddG for L-phenylalanine, yedA for L-methionine, and brnQ and rhtC for the transport of L-threonine. The functional roles of yedA and brnQ in the transport of L-methionine and L-histidine, respectively, are novel assignments. Reverse engineering the mutations in the ancestral strain established causality of the specific mutations for the tolerant phenotypes.