Genomic signatures highlight stress-response evasion and translational tuning as key drivers of Escherichia coli adaptation to fluorinated tryptophans.

Fluorinated amino acids profoundly perturb cellular physiology because they enter the proteome while differing from their natural counterparts in subtle but functionally important ways. Here we investigated how Escherichia coli adapts to the biosynthesis and proteome-wide incorporation of fluorinated tryptophans derived from 4-, 5-, 6-, and 7-fluoroindoles using adaptive laboratory evolution (ALE). Whole-genome sequencing of independently evolved populations revealed convergent adaptive solutions. All 6- and 7-fluoroindole lineages acquired disruptive mutations in the stringent starvation regulator (sspA), effectively attenuating stress signaling and allowing continued expression of housekeeping functions despite proteotoxic pressure. In parallel, recurrent mutations in tryptophanyl-tRNA synthetase (trpS), which charges tRNATrp with tryptophan, pointed to translational tuning consistent with improved handling of fluorinated substrates, with Q27P emerging most prominently. In several 6-fluoroindole populations, additional defects in mutS, involved in DNA mismatch repair, allowed replication errors to accumulate, generating transient hypermutator states that accelerated evolutionary exploration but were not required for successful adaptation. Reconstruction experiments confirmed that loss of stringent response control and altered TrpRS function together increased fitness during fluorotryptophan incorporation. Together, these results suggest that adaptation does not appear to primarily rely on establishing a fundamentally new fluorine-based biochemistry, but rather on adjustment of stress-response regulation and translational control. More broadly, this work establishes a general framework for understanding, and ultimately engineering, microbial adaptation to non-natural metabolites through targeted modification of regulatory and translational control nodes rather than metabolic redesign.