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

摘要

The genetic characterization of Pseudomonas protegens Pf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed ~(13)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 P. protegens 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 P. protegens Pf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways.IMPORTANCE Species of the Pseudomonas 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 Pseudomonas protegens 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 P. protegens is important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untapped Pseudomonas species. KEYWORDS: Pseudomonas , carbohydrate co-utilization, hexose sugar, metabolic flux analysis, metabolomicsINTRODUCTIONSpecies of the genus Pseudomonas , which are ubiquitous in the environment, are metabolically diverse and often touted for industrial bioproduction (1). The elucidation of the cellular network of carbon fluxes through metabolic pathways of these bacterial species is critical both to understand their role in carbon processing in environmental matrices and to engineer these species to optimize their use in agriculture, industry, and medicine. Gaining importance in bioremediation, Pseudomonas protegens Pf-5 was identified to produce enzymes that degrade polyurethane, a plastic polymer (2). Furthermore, P. protegens Pf-5 is also known to synthesize and release several antimicrobials and exoenzymes that are toxic to plant pathogens (3,–6). Recently, P. protegens Pf-5 was characterized and annotated at the genomic level (7). However, the metabolic network of P. protegens Pf-5 has only been inferred from the genome annotation and has not yet been investigated experimentally.Given the importance and ubiquity of carbohydrate-containing feedstocks, we sought to unravel the metabolic network structure for carbohydrate metabolism in P. protegens Pf-5 by combining ~(13)C-assisted cellular carbon mapping with ~(13)C metabolic flux analysis (MFA). Previous studies on other Pseudomonas species (i.e., P. putida and P. fluorescens ) focused on elucidating the metabolic fluxes during growth on glucose, a prototypical carbohydrate substrate (8,–11). In a similar fashion, we also studied the innate carbohydrate metabolism in P. protegens Pf-5 during feeding on glucose alone. However, carbon feedstocks are typically composed of other carbohydrates in addition to glucose. Therefore, we also investigated carbon assimilation and fluxes when the P. protegens Pf-5 cells were fed on mixtures of glucose with other hexoses (mannose, fructose, and galactose) or pentoses (xylose and arabinose).Previous reports showed that P. protegens strains were able to grow on glucose, mannose, or fructose as a single carbon source but not on galactose, xylose, or arabinose (6). In the genome of P. protegens Pf-5, genes for the following transporters were found: a phosphoenolpyruvate (PEP)/