Dissecting Metabolic Rewiring and Gene-Metabolite Interactions by Utilizing Untargeted Metabolomics and Single-Gene Knockouts in the Model Microorganism <i>E. coli</i>

Central carbon metabolism, comprising glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP), is essential for <i>Escherichia coli</i> survival and growth. While disruptions in these pathways are known to affect cellular physiology, the system-wide metabolite-level consequences of single-gene knockouts remain incompletely understood. Using untargeted LC-MS metabolomics, we systematically profiled <i>E. coli</i> knockouts of TCA core enzymes, isoforms, subunits, bypass routes, and TCA-associated pathways. Core TCA knockouts separated into two major metabolic clusters, with cluster 1 strains displaying strong divergence in amino acid metabolism and cluster 2 retaining partial similarity to the parent strain. Isoform-specific deletions revealed differential roles of aconitases (Δ<i>acnA</i> vs Δ<i>acnB</i>) and fumarases (Δ<i>fumA</i> vs Δ<i>fumC</i>), while subunit knockouts of 2-oxoglutarate dehydrogenase (Δ<i>sucA</i>, Δ<i>sucB</i>) and succinate dehydrogenase (Δ<i>sdhA-D</i>) produced localized but distinct metabolite shifts, particularly around glutamate- and 2-oxoglutarate-linked metabolism. Bypass enzyme deletions (Δ<i>aceA</i>, Δ<i>aceB</i>, Δ<i>glcB</i>, and Δ<i>maeB</i>) disrupted carbohydrate- and redox-related metabolites, underscoring their role as metabolic safety nets. Importantly, knockouts also triggered off-target effects in glycolysis, PPP, and the electron transport chain, highlighting the interconnectivity of central carbon metabolism. Our systematic approach demonstrated the possibility of utilizing comprehensive and untargeted metabolomics to map gene-metabolite associations and decipher potential metabolic interlinks.