PMC

Comparison of TALE evolved IL tolerant clones MG1655#4.7 and MG1655#3.10 to previously engineering tolerant strains JBEI-13314 and JBEI-10101 in LB (a, b) and M9 (c, d) media containing either 300 mM of [C₂C₁Im][OAc] [5.1% (w/v)] or [C₂C₁Im][Cl] [4.4% (w/v)]. TALE evolved clones exhibited improved growth compared to rationally-designed strains, particularly in M9, where JBEI-13314 and JBEI-10101 were severely inhibited

Plots of population growth rate versus IL concentration over the course of two TALE experiments for increasing IL tolerance. a E. coli K-12 MG1655 population ALE #1 evolved on glucose minimal medium with ([C₄C₁Im]Cl) and, b E. coli DH1 population ALE #11 evolved on glucose minimal medium with ([C₂C₁Im][OAc]). Depicted are fitness trajectories and IL concentration versus cumulative cell divisions (CCD) experienced by the cultures for two out of the total sixteen individual experiments. IL concentration was increased step-wise when the growth rate for the population increased

Performance of evolved clones in batch culture under M9 minimal media conditions with various [C₂C₁Im][OAc] IL loadings. Shown is the final optical density (OD_600nm) of the selected best performing clones (Table ) under different concentrations of [C₂C₁Im][OAc]: a first, a screen with 250 mM and all best performing clones, b second, a follow up screen with 300, 500 and 700 mM utilizing the highest final density clones from the 250 mM screen. Surprisingly, a number of clones that were originally evolved for [C₄C₁Im]Cl tolerance showed high cross-tolerance to elevated [C₂C₁Im][OAc] concentrations. At the highest concentration (700 mM), only the MG1655 derived strains showed measurable growth

A diagram of the cell showing processes associated with key mutations. A cartoon diagram of key mutations in potential causal genetic regions identified in the evolved strains. A total of eight mutations are represented amongst different genetic regions. A Δ120 bp deletion was found in the non-coding region of mdtJ, near the promoter, and its neighboring gene tqsA. Two other structural changes were found in the tqsA gene—one was an intragenic in-frame Δ12 bp deletion, the other was a Δ3035 bp deletion which included a major section of tqsA and the pntB and pntA genes located next to tqsA on the chromosome. Finally, five structural changes were identified in yhdP gene. These were two out-of-frame short deletions (Δ2 bp and Δ4 bp), two intergenic IS mobile element insertions, and a short 7 bp insertion/duplication. These mutations indicate a probable loss-of-function of yhdP