Metabolic plasticity enables deep-sea microorganisms to survive under simultaneous fluctuations in carbon availability, hydrostatic pressure, and redox conditions, yet the regulatory mechanisms underlying these adaptations remain poorly understood. Here, we combined ¹³C-metabolic flux analysis, transcriptomics, metabolomics, and electrochemical analyses to investigate how the deep-sea bacterium Shewanella piezotolerans WP3 reorganizes central carbon metabolism across multiple environmental gradients.

¹³C-metabolic flux analysis revealed that WP3 preferentially utilizes the Entner–Doudoroff pathway for glucose catabolism while maintaining substantial flux through both oxidative and non-oxidative branches of the pentose phosphate pathway. Compared with other deep-sea isolates, phosphoenolpyruvate carboxylation serves as the major anaplerotic route, supporting precursor replenishment and redox balance. Integrative transcriptomic and metabolomic analyses further demonstrated substrate-specific metabolic reprogramming. Glucose preferentially activated the oxidative pentose phosphate pathway to maximize NADPH production, pyruvate promoted gluconeogenesis and biomass synthesis, acetate induced the glyoxylate shunt for carbon conservation, whereas N-acetyl-D-glucosamine stimulated amino acid biosynthesis and nitrogen metabolism. These findings reveal a non-classical resource allocation strategy that optimizes energy production, biosynthesis, and carbon conservation according to substrate availability, also further confirming the core node of PEP carboxylation between upstream glycolytic process and downstream energy-producing way.

Under elevated hydrostatic pressure, WP3 exhibited a piezotolerant phenotype, with optimal growth at 20 MPa followed by growth inhibition at higher pressures. Pressure adaptation involved coordinated transcriptional activation of amino acid metabolism, membrane remodeling, and fatty acid degradation, accompanied by repression of energetically costly processes including flagellar assembly and chemotaxis. Under variable redox conditions, WP3 maintained efficient extracellular electron transfer through the OmcA–MtrCAB pathway and riboflavin secretion, supported by enhanced glycolytic and TCA cycle activity.

Together, these results demonstrate that environmental adaptation in WP3 is driven by dynamic reprogramming of central carbon metabolism rather than isolated stress responses. This metabolic flexibility provides an integrated framework for understanding how deep-sea bacteria coordinate carbon utilization, energy metabolism, and respiratory adaptation in heterogeneous deep-ocean environments.