Adaptive Evolution of GatC, a Component of the Galactitol Phosphotransferase System, for Glucose Transport in <i>Escherichia coli</i>

Microbial adaptive laboratory evolution is a powerful approach for uncovering novel gene functions within metabolic pathways. Building on our previous discovery of ExuT as a glucose transporter in <i>ptsG</i>-deficient <i>Escherichia coli</i>, this study investigates strains lacking recognized glucose transporters (<i>ptsG</i>, <i>manX</i>, and <i>exuT</i>). Successive rounds of experimental evolution revealed key genetic adaptations, including loss-of-function mutations in <i>malI</i> and <i>nagC</i>, which encode repressors of the maltose and N-acetylglucosamine phosphotransferase systems (PTS), respectively. Additionally, a gain-of-function mutation in <i>gatC</i>, a component of the galactitol PTS EIIC, was identified. The functional significance of these mutations was validated through transcript analysis, genetic knockouts, and CRISPR-Cas9-mediated site-specific genome mutagenesis, with a particular focus on the <i>gatC</i> missense mutation (F340C). The resulting modifications were examined for their effects on sugar specificity and metabolic flux. Furthermore, our findings identified succinate as the predominant fermentation product in engineered strains utilizing alternative glucose transport pathways, including the maltose, N-acetylglucosamine, and galactitol PTS. This study advances our understanding of sugar transport mechanisms in <i>E. coli</i> and offers insights into regulatory networks, fermentative metabolism, and substrate specificity, which can be leveraged for evolutionary engineering in biotechnological applications.