Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.
The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats.
From the Systems and Synthetic Biology Program, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain.
Pablo I
PI
Nikel
the Escuela de Química, Universidad de Costa Rica, 2060 San José, Costa Rica, and.
Max
M
Chavarría
the Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland.
Tobias
T
Fuhrer
the Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland.
Uwe
U
Sauer
From the Systems and Synthetic Biology Program, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain, vdlorenzo@cnb.csic.es.
Víctor
V
de Lorenzo
D005947:Glucose / Q000378:metabolism*; D010427:Pentose Phosphate Pathway; D016958:Pseudomonas putida / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*
D005947:Glucose
Entner-Doudoroff; Pseudomonas; Pseudomonas putida; bacterial metabolism; glycolysis; metabolic engineering; metabolic flux analysis; redox regulation