The strong biocatalytic potential of species in the Pseudomonas genus is due to their diverse range of metabolic capabilities. Pseudomonas protegens Pf-5, a producer of several specialized metabolites that are antagonists to plant pathogens, was genetically characterized recently. However, the metabolic network structure inferred from the genomic analysis has not been experimentally evaluated. Due to the ubiquity of carbohydrates in carbon feedstocks for engineered bioconversion, the intracellular network for carbohydrate metabolism in P. protegens Pf-5 was determined using 13C-tracer experiments and quantitative metabolic flux analysis. Similar to other Pseudomonas species, P. protegens Pf-5 was found to utilize the Entner-Doudoroff (ED) pathway to connect initial glucose catabolism to downstream metabolic pathways. Instead of the direct phosphorylation of glucose by glucose kinase, flux quantitation revealed phosphorylation of oxidized products of glucose (gluconate and 2-ketogluconate) accounted for 80% of glucose uptake towards the ED pathway. When glucose was fed simultaneously with fructose or mannose, glucose was the preferred substrate over fructose (5:1) and over mannose (15:1). Both fructose and mannose entered metabolism via fructose-1,6-bisphosphate before being cycled back through the reverse Embden-Meyerhof-Parnas pathway to the ED pathway. Collectively, our findings shed light on the constitutive mixed-carbohydrate utilization in P. protegens and thus lay the foundation for engineering these untapped Pseudomonas species.

Metabolic network and mixed-carbohydrate utilization in Pseudomonas protegens Pf-5

256th National Meeting (2018)

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