Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida

The core metabolism for glucose assimilation of the soil bacterium and platform strain Pseudomonas putida KT2440 has been reshaped from the native, cyclically-operating Entner-Doudoroff (ED) pathway to a linear Embden-Meyerhof-Parnas (EMP) glycolysis. The genetic strategy deployed to obtain a suitable host for the synthetic EMP route involved not only eliminating enzymatic activities of the ED pathway, but also erasing peripheral reactions for glucose oxidation that divert carbon skeletons into the formation of organic acids in the periplasm. Heterologous glycolytic enzymes, recruited from Escherichia coli, were genetically knocked-in in the mutant strain to fill the metabolic gaps for the complete metabolism of glucose to pyruvate through a synthetic EMP route. A suite of genetic, physiological, and biochemical tests in the thereby-refactored P. putida strain-which grew on glucose as the sole carbon and energy source-demonstrated the functional replacement of the native sugar metabolism by a synthetic catabolism. 13C-labelling experiments indicated that the bulk of pyruvate in the resulting strain was generated through the metabolic device grafted in P. putida. Strains carrying the synthetic glycolysis were further engineered for carotenoid synthesis from glucose, indicating that the implanted EMP route enabled higher carotenoid content on biomass and yield on sugar as compared with strains running the native hexose catabolism. Taken together, our results highlight how conserved metabolic features in a platform bacterium can be rationally reshaped for enhancing physiological traits of interest.