Although metabolism plays an active role in antibiotic lethality, antibiotic resistance is generally associated with drug target modification, enzymatic inactivation, and/or transport rather than metabolic processes. Evolution experiments of
Escherichia coli
rely on growth-dependent selection, which may provide a limited view of the antibiotic resistance landscape. We sequenced and analyzed
E. coli
adapted to representative antibiotics at increasingly heightened metabolic states. This revealed various underappreciated noncanonical genes, such as those related to central carbon and energy metabolism, which are implicated in antibiotic resistance. These metabolic alterations lead to lower basal respiration, which prevents antibiotic-mediated induction of tricarboxylic acid cycle activity, thus avoiding metabolic toxicity and minimizing drug lethality. Several of the identified metabolism-specific mutations are overrepresented in the genomes of >3500 clinical
E. coli
pathogens, indicating clinical relevance.
