Fig. 6: Modular deconstruction of the NADPH metabolism in P. putida.
A Simplified scheme of central metabolism in P. putida. Four core reactions deliver NADPH, i.e., glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (MaeB), and isocitrate dehydrogenase (ICTDH). The PntAB and SthA transhydrogenases catalyze electron transfer between nicotinamide nucleotides to buffer cofactor imbalances, while the reversible glutamate dehydrogenase (GDH) can supply NADPH when glutamate is fed. B Three redox editing modules were defined as to constrain NADPH supply either through transhydrogenation (TH), upper metabolism (UM; G6PDH and 6PGDH), or lower metabolism (LM; Icd, MaeB, and GhrB). NADPH formation within the UM when cells are grown on gluconeogenic substrates was prevented by interrupting the two phosphoglucoisomerase (Pgi) genes. In some experiments, a NADPH-dependent formate dehydrogenase (FDHe) gene was constitutively expressed in a NADPH-depleted strains to restore redox balance. C P. putida KT2440 and genome-edited strains with a combination of the UM, LM, and TH modules were grown in de Bont minimal medium with 30 mM pyruvate. D The TH + UG strain was further engineered by knocking out icd, ghrB, and maeB, or their combination (i.e., the NADPH-depleted P. putida strain). These strains were grown in de Bont minimal medium with 30 mM pyruvate and 50 mM α-ketoglutarate. E Growth profile of the NADPH-depleted P. putida strain in de Bont minimal medium with 30 mM pyruvate and varying glutamate concentrations as indicated in the graphic. D Growth profile of the NADPH-depleted P. putida strain, carrying either a NADP+-dependent formate dehydrogenase (solid line) or an empty plasmid (dashed line), in de Bont minimal medium with 30 mM pyruvate and varying formate concentrations (concn.). Data represent mean values of three independent biological experiments, and doubling times ± standard deviations were derived from the optical density at 600 nm (OD600) measurements. Source data underlying panels C–F are provided as a Source Data file.
