Redox stress reshapes carbon fluxes of Pseudomonas putida for cytosolic glucose oxidation and NADPH generation

Abstract

The soil bacterium and metabolic engineering platform Pseudomonas putida tolerates high levels of endogenous and exogenous oxidative stress, yet the ultimate reason of such property remains unknown. To shed light on this question, NADPH generation routes—the metabolic currency that fuels redox stress responses—were assessed when P. putida KT2440 was challenged with H2O2 as proxy of oxidative conditions. 13C-tracer experiments, metabolomics and flux analysis, together with inspection of physiological parameters and measurement of enzymatic activities, revealed a substantial flux reconfiguration under oxidative stress. In particular, periplasmic glucose processing was rerouted to cytoplasmic oxidation, and cyclic operation of the pentose phosphate pathway led to significant NADPH fluxes, exceeding biosynthetic demands by ~50%. This NADPH surplus, in turn, fuelled the glutathione system for H2O2 reduction. These properties not only contribute to the tolerance of P. putida to environmental stresses, but they also highlight the value of this host for harsh biotransformations.