# Adaptive Evolution of GatC, a Component of the Galactitol Phosphotransferase System, for Glucose Transport in Escherichia coli

## TL;DR

This study shows how Escherichia coli can evolve to use alternative glucose transport systems when its main ones are disabled, revealing new insights into microbial metabolism.

## Contribution

The study identifies a gain-of-function mutation in gatC that enables glucose transport via the galactitol PTS in E. coli.

## Key findings

- Loss-of-function mutations in malI and nagC enhance alternative glucose transport pathways.
- A gatC missense mutation (F340C) expands the sugar specificity of the galactitol PTS for glucose.
- Succinate becomes the main fermentation product in strains using alternative glucose transport systems.

## Abstract

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 ptsG-deficient Escherichia coli, this study investigates strains lacking recognized glucose transporters (ptsG, manX, and exuT). Successive rounds of experimental evolution revealed key genetic adaptations, including loss-of-function mutations in malI and nagC, which encode repressors of the maltose and N-acetylglucosamine phosphotransferase systems (PTS), respectively. Additionally, a gain-of-function mutation in gatC, 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 gatC 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 E. coli and offers insights into regulatory networks, fermentative metabolism, and substrate specificity, which can be leveraged for evolutionary engineering in biotechnological applications.