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In this study, we show that glucose catabolism in Pseudomonas putida occurs through the simultaneous operation
of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda
Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB
porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the
cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and
is then phosphorylated by glucokinase (encoded by the glk gene) and converted by glucose-6-phosphate dehydrogenase
(encoded by the zwf genes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the
cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate,
which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate.
In the wild-type strain, glucose was consumed at a rate of around 6 mmol g 1 h 1, which allowed a growth
rate of 0.58 h 1 and a biomass yield of 0.44 g/g carbon used. Flux analysis of 13C-labeled glucose revealed that, in
the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct
oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes,
regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in
the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and
found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop
were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose
catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters,
genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster
coevolved. The glk gene encoding glucokinase was located in an operon with the edd gene, whereas the zwf-1 gene,
encoding glucose-6-phosphate dehydrogenase, formed an operon with the eda gene. Therefore, the enzymes of the
glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously.
It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with
glucose. |
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