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.

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The Embden–Meyerhof–Parnas (EMP) pathway is generally considered to be the biochemical standard for glucose catabolism. Alas, its native genomic organization and the control of gene expression in Escherichia coli are both very intricate, which limits the portability of the EMP pathway to other biotechnologically important bacterial hosts that lack the route. In this work, the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (GlucoBrick) that enables their grouping in the form of functional modules at the user’s will. After verifying their activity in several glycolytic mutants of E. coli, the versatility of these GlucoBricks was demonstrated in quantitative physiology tests and biochemical assays carried out in Pseudomonas putida KT2440 and P. aeruginosa PAO1 as the heterologous hosts. Specific configurations of GlucoBricks were also adopted to streamline the downward circulation of carbon from hexoses to pyruvate in E. coli recombinants, thereby resulting in a 3-fold increase of poly(3-hydroxybutyrate) synthesis from glucose. Refactoring whole metabolic blocks in the fashion described in this work thus eases the engineering of biochemical processes where the optimization of carbon traffic is facilitated by the operation of the EMP pathway—which yields more ATP than other glycolytic routes such as the Entner–Doudoroff pathway.

The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (i.e., GlucoBrick) that facilitates their grouping in the form of functional modules that can be combined at the user's will. This novel genetic tool allows for the à la carte implementation or boosting of EMP pathway activities into different Gram-negative bacteria. The potential of the GlucoBrick platform is further illustrated by engineering novel glycolytic activities in the most representative members of the Pseudomonas genus (Pseudomonas putida and Pseudomonas aeruginosa).