Clostridia are of industrial importance due to their broad and flexible systems for substrate utilization such as C. thermocellum which metabolizes cellulose and C. ljungdahlii that metabolizes syngas to produce biofuels. But these organisms remain poorly characterized with significant uncertainty in their metabolic repertoire. In this study, we develop dynamic metabolic models for the two organisms using the Ensemble Modeling (EM) paradigm. For C. thermocellum, we constructed a core kinetic model k-ctherm118, parameterized by simultaneously imposing fermentation yield data in lactate, malate, acetate and hydrogen production pathways for 19 measured metabolites spanning a library of 19 distinct single and multiple gene knockout mutants along with 18 intracellular metabolite concentration data for a Δgldh mutant and ten experimentally measured Michaelis-Menten kinetic parameters. k-ctherm118 captures significant metabolic changes caused by (i) nitrogen limitation leading to increased yields for lactate, pyruvate and amino acids, and (ii) ethanol stress causing an increase in intracellular ammonia and sugar phosphate concentrations due to downregulation of fermentation pathways. Robustness analysis of k-ctherm118 alludes to the presence of a secondary activity of ketol-acid reductoisomerase and possible regulation by valine and/or leucine pool levels. A similar effort is also underway for C. ljungdahlii. A core metabolic model composed of 77 reactions and 63 metabolites was constructed with 41 substrate level regulatory interactions from BRENDA based on other Clostridia species. The network spans glycolysis, the Wood-Ljungdahl pathway, and TCA cycle. Experimental flux data for a wild-type strain grown under two different substrates fructose and CO2:H2 was used to generate an ensemble of core model parameters and identify the enzyme level perturbation strategies to which the ensemble is more sensitive. The predicted strategies will guide experiments and accelerate kinetic model parameterization. The constructed kinetic models will be ultimately used to explore metabolic drivers that underpin the over-production of iso-butanol in C.thermocellum and acetate in C. ljungdahlii.

Kinetic modeling of metabolism in clostridia

253rd National Meeting (2017)

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