Metabolic adaptation enabling D-xylose assimilation via the Entner-Doudoroff pathway in glycolysis-impaired Escherichia coli strains.
In the model bacterium Escherichia coli, the Embden-Meyerhof-Parnas (EMP) glycolysis pathway is the primary route for carbohydrate metabolism. However, alternative metabolic routes can be activated depending on genetic configurations and available carbon sources. In this study, E. coli strains lacking key enzymes of the lower EMP pathway-phosphoglycerate kinase or enolase-were subjected to long-term adaptive evolution in continuous culture under a medium swap regime, for growth on D-xylose as the sole carbon and energy source. Through metabolic 13C-labeling experiments on evolved isolates, we found that carbon flux was predominantly rerouted via the Entner-Doudoroff pathway, known to remain silent when wild type cells are fed with D-xylose. To investigate the molecular basis of this growth adaptation, we identified the mutations fixed in the genomes of evolved prototrophic isolates and conducted comprehensive transcriptomic and proteomic analyses. Our study revealed that mutations in key enzymes and transcriptional regulators at various metabolic branching points were essential for effective growth on D-xylose, enabling optimized partitioning of carbon through central metabolism despite the loss of the canonical EMP glycolytic route. The multi-layered regulatory and metabolic adaptations identified in the evolved strains demonstrate the complex nature of evolutionary trajectories and underscore the potential of adaptive evolution to optimize metabolic network function and enzyme utilization in ways that extend rational engineering approaches.