A Cyclic Metabolic Network in Pseudomonas protegens Pf-5 Prioritizes the Entner-Doudoroff Pathway and Exhibits Substrate Hierarchy during Carbohydrate Co-Utilization.

Authors:
Caroll M Mendonca
Caroll M Mendonca
Cornell University
Graduate Student Researcher
Ithaca, NY | United States
Dr. Ludmilla Aristilde, PhD
Dr. Ludmilla Aristilde, PhD
Cornell University
Associate Professor
Environmental Chemistry; Environmental Biochemistry; Environmental Engineering.
Ithaca, NY | United States

Appl Environ Microbiol 2019 Jan 13;85(1). Epub 2018 Dec 13.

Department of Biological and Environmental Engineering, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, USA

The genetic characterization of Pf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes in Pf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates in Pf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways. Species of the genus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism in Pf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network in is important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untapped species.

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http://dx.doi.org/10.1128/AEM.02084-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293094PMC
January 2019
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References

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Fructose metabolism in four Pseudomonas species
Van Dijken et al.
Arch Microbiol 1977

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