The Entner–Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress

Abstract

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner-Doudoroff (ED) pathway due to the absence of 6-phosphofructokinase. In order to activate the Embden-Meyerhof-Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild-type strain as well as to an eda mutant, i.e. lacking 2-keto-3-deoxy-6-phosphogluconate aldolase. PfkA(E. coli) failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA(E. coli) was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP(+) ratio. Pseudomonas putida cells carrying PfkA(E. coli) became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA(E. coli) could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative-related insults.

Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression

“…In either case, control mock assays were made by spiking M9 minimal medium with different amounts of the C sources under examination. Biomass yields (CDW measurements), specific growth rates, and C consumption during exponential growth were calculated from growth parameters in each culture condition as described elsewhere [24,57,69,[84][85][86][87]. The acetylCoA content was determined by liquid chromatography coupled to mass spectrometry as previously detailed [88].…”

“…It is not casual that the species that host such plasmids (frequently encoding several oxidative biotransformations of complex organic compounds) do not belong to Enterobacteriaceae, but they are often members of the genus Pseudomonas [21][22][23]. One of the reasons for this occurrence is the vigorous Entner-Doudoroff and pentose phosphate pathways present in most Pseudomonas species, thus resulting in high rates of NADPH regeneration, which in turn helps counteracting both endogenous and exogenous oxidative stress [24,25]. This trait provides the right metabolic frame for running enzymatic pathways that other bacterial would be unable to cope with.…”

Large-Scale 13 C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

“…Many habitats in the marine environment impart oxidative stress; therefore, antioxidant mechanisms are important traits of marine microorganisms (58). To study the tolerance of marine bacteria to such oxidative conditions, we studied the tolerance to diamide (59). Diamide causes oxidative stress by oxidizing sulfhydryl bonds in the cytoplasm, which must be reduced at the expense of NADPH (60,61).…”

“…5A). P. putida has robust resistance to oxidative stress (59). Thus, the tolerance of P. putida was used as the reference (100%) to classify the other tested bacteria according to their stress tolerance.…”

“…Particularly, NADPH is an oxidative stress protectant that is required by many important antioxidant defense mechanisms (75) and is important for counteracting oxidative stress (61,76,77). A recent study revealed the importance of the ED pathway for oxidative stress protection in P. putida (59). As impressively shown, the introduction of a functional phosphofructokinase forced the organism to shift the flux from the natural ED pathway to the EMP pathway, which significantly decreased its tolerance to oxidative stress.…”