Antibiotic resistance and its related process, antibiotic tolerance, are leading causes of treatment failures and continue to threaten healthcare systems worldwide. While many researchers are exploring the underlying mechanisms of resistance, little is known about tolerance. There is a demand to gain a deeper understanding of how tolerance is activated, and to find new targets for antibiotic development. Using the model organism Vibrio cholerae, which happens to be tolerant to β-lactam antibiotics, I interrogated how tolerance is activated with a transposon mutagenesis screen. I found that when a key enzyme in central carbon metabolism, pgi, was deleted, tolerance was induced. Combining microscopy, genetic manipulation, growth dynamics experiments, and viability assays, I found this deletion resulted in insignificant cell-wall associated defects and an increase in susceptibility to cell-wall acting antibiotics. Suppressor mutants hinted at sugar toxicity from glycolytic sugar accumulation, and notable defects could be restored with the addition of external GlcNAc. This initial study opened the doors to exploring how metabolic disruptions lead to altered antibiotic susceptibility. Seeking a greater mechanistic understanding of the observed defects seen in a Δpgi, I conducted targeted metabolomics in different growth medium. I found that there was a bottleneck around an enzyme that synthesizes early cell wall precursors, GlmU. This suggests that GlmU’s activity is repressed in a Δpgi background, presumably from the accumulation of glucose phosphates. In another study, I sought a more holistic view of the interconnectedness of metabolism and antibiotic tolerance. I collaborated on a large-scale multi-omics report on the effects of penicillin treatment on wild type V. cholerae. Combining RNAseq with metabolomic data, we find nucleotide synthesis products are decreased, yet transcripts of enzymes involved in nucleotide synthesis increase in response to tolerance. Collectively my work provides a new framework for the development of metabolic drug targets, as well as showcasing the global network of β- lactam tolerance.