Quantifying the Intracellular Metabolic Network that Establishes the Simultaneous Utilization of Sugars and Aromatic Substrates in Pseudomonas putida KT2440

Kukurugya, Matthew A.

Quantifying the Intracellular Metabolic Network that Establishes the Simultaneous Utilization of Sugars and Aromatic Substrates in Pseudomonas putida KT2440

Abstract

The ability of Pseudomonas to use both aromatic compounds and sugars is being explored and exploited for emerging biotechnological applications, such as bioremediation as well as biofuel and chemical synthesis. The present study investigates the intracellular metabolism in the biotechnologically-important soil bacterium Pseudomonas putida KT2440 during feeding on a mixture containing the cellulosic hexose sugar (glucose) and an aromatic carboxylic acid (benzoate). Through a combination of 13C tracer experiments with metabolic flux analysis (MFA), I elucidated and quantified the discriminate metabolic routing of each substrate throughout central carbon metabolism. The results determined that glucose-derived carbon was primarily routed to the Entner-Doudoroff (ED) pathway, reverse Embden–Meyerhof–Parnas (EMP) pathway, and Pentose Phosphate (PP) pathway, while benzoate-derived carbon was routed almost exclusively to the tricarboxylic acid (TCA) cycle. I found that benzoate catabolism influenced the routing of glucose in the ED pathway, reverse EMP pathway, and PP pathway, despite the absence of benzoate-derived carbon in these pathways. In the TCA cycle, the simultaneous catabolism of benzoate and glucose led to the activation of the glyoxylate shunt. Accordingly, kinetic isotopic flux revealed a decreased flux through isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. In addition, feeding on the substrate mixture induced carbon overflow from the TCA cycle primarily through a significant involvement of malate dehydrogenase activity over the ED pathway in the biosynthesis of pyruvate. In sum, the data revealed that a preferential flux of substrate-derived carbons through different metabolic pathways was necessary to optimize biomass growth. These findings will contribute to an emerging framework for understanding soil carbon metabolism and advancing novel biotechnological applications in soil bacteria.

Date Issued

2017-01-30

Keywords

aromatic; benzoate; glucose; intracellular; Pseudomonas putida; Cellular biology; Biochemistry; Metabolism

Committee Chair

Aristilde, Ludmilla

Committee Member

Thies, Janice E.
Hay, Anthony G.

Degree Discipline

Biological and Environmental Engineering

Degree Name

M.S., Biological and Environmental Engineering

Degree Level

Master of Science

Types

dissertation or thesis