Metabolic Flux Analysis Reveals Entner-Doudoroff Pathway Dominance in Heterotrophic Deep-Sea Bacterial Isolates

Deep-sea microorganisms adapt to extreme conditions by diverse metabolic strategies, yet their intracellular carbon fluxes remain largely unexplored. Here, we investigated the central carbon metabolic fluxes of four bacterial strains isolated from Pacific Ocean sediments and deep waters, including Stutzerimonas marianensis PS1, Shewanella piezotolerans WP3, Georhizobium profundi WS11 and Parasedimentitalea marina W43. Using ¹³C metabolic flux analysis with multiple ¹³C-labelled glucose isotopologues, all strains preferentially degraded glucose via the Entner-Doudoroff pathway (EDP) with fluxes accounting for 66.7%-94.0% of total glycolytic flux, indicating that EDP is a conserved and dominant glycolytic route among deep-sea heterotrophs. S. piezotolerans WP3 uniquely exhibited substantial flux through both oxidative and non-oxidative branches of the pentose phosphate pathway, suggesting heightened precursor and redox demands. The strains also displayed diverse anaplerotic strategies, engaging either phosphoenolpyruvate or pyruvate carboxylation to replenish tricarboxylic acid cycle intermediates and enhance carbon utilisation efficiency. Oxidative stress assays further revealed a link between intracellular energy status and tolerance to hydrogen peroxide. Collectively, these findings provide comparative fluxomic evidence for central carbon metabolism in deep-sea bacteria and highlight metabolic traits that support survival in carbon-limited, high-pressure marine environments.