Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress

Abstract

As a frequent inhabitant of sites polluted with toxic chemicals, the soil bacterium and plant-root colonizer Pseudomonas putida can tolerate high levels of endogenous and exogenous oxidative stress. Yet, the ultimate reason of such phenotypic property remains largely unknown. To shed light on this question, metabolic network-wide routes for NADPH generation—the metabolic currency that fuels redox-stress quenching mechanisms—were inspected when P. putida KT2440 was challenged with a sub-lethal H2O2 dose as a proxy of oxidative conditions. 13C-tracer experiments, metabolomics, and flux analysis, together with the assessment of physiological parameters and measurement of enzymatic activities, revealed a substantial flux reconfiguration in oxidative environments. In particular, periplasmic glucose processing was rerouted to cytoplasmic oxidation, and the cyclic operation of the pentose phosphate pathway led to significant NADPH-forming fluxes, exceeding biosynthetic demands by ~50%. The resulting NADPH surplus, in turn, fueled the glutathione system for H2O2 reduction. These properties not only account for the tolerance of P. putida to environmental insults—some of which end up in the formation of reactive oxygen species—but they also highlight the value of this bacterial host as a platform for environmental bioremediation and metabolic engineering.

Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes inPseudomonas putidareveals a general principle underlying glycolytic strategies in bacteria

“…The specific activity was almost lost in mutants deficient in G6PDH-A when grown on glucose, indicating that this isozyme supplies by far the most activity under glycolytic conditions. The specific G6PDH activities in the wild-type strain were similar to previous reports (21,22), and the observed activity of the zwfABC triple mutant in cell-free extracts was very low, indicating that only low promiscutive G6PDH of other proteins is present (Fig. 5A).…”

“…Significant differences between the G6PDH isoforms were observed when the relative NADH-to-NADPH output was simulated as a function of the redox ratios at two G6P concentrations spanning one order of magnitude [i.e. 100 M and 1 mM, compatible with values reported for glucose-grown strain KT2440 (21,22)]. While the activity of G6PDH-A yields essentially the same amounts of NADH and NADPH regardless of the G6P concentration (NADH produced per NADPH close to 1) ( Fig.…”

“…1), employing the ED and PP pathways in combination with gluconeogenesis through the EMP route to oxidize hexoses (21). This structure, termed EDEMP cycle, was described in P. putida KT2440 as a biochemical mechanism that adjusts NADPH production levels through cycling the flux through the upper glycolysis (21,22). Besides this main set of reactions in the cytoplasm, P. putida harbors periplasmic enzymes for the direct oxidation of glucose to gluconate.…”

Micro‐aerobic production of isobutanol with engineered Pseudomonas putida

“…The total NADPH supply was estimated using the ratio of 1.7 mol NADPH mol glucose ‐1 , and a NADPH surplus of 14 to 21% was assumed for growth on glucose [21, 39, 42]. The additional NADPH demand in the isobutanol production pathway was calculated based on the extracellular uptake and formation rates of KIV, l ‐valine and isobutanol.…”

Friss oder stirb! Erweiterung des Substratspektrums von P. putida

“…ó Aufgrund seiner Robustheit, der Resistenz gegenüber einer Vielzahl von Stressfaktoren, seiner genetischen Zugänglichkeit und seines breiten Substratspektums, erfreut sich Pseudomonas putida zunehmenden Interes ses für biotechnologische Anwendungen [1][2][3][4]. Die intrinsische Resistenz von P. putida liegt u. a. in der einzigartigen metabolischen Architektur begründet, die es dem Organismus erlaubt die Menge der zur Verfügung stehenden Reduktionsäquivalente anzupassen [5,6] sowie in der verhältnismäßig dichten äußeren Mem bran.…”