Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA

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Abstract

© 2015 Lien et al. Background: The bacteriumPseudomonas fluorescensswitches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, inmucAdeletion mutants compared to the wild-type. In this study, theP. fluorescensSBW25 wild-type and an alginate non-producingmucA-ΔalgCdouble-knockout mutant are investigated through model-based < sup > 13 < /sup > C-metabolic flux analysis ( < sup > 13 < /sup > C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for < sup > 13 < /sup > C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry.Results: From mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in themucA-ΔalgCmutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in themucA- ΔalgCmutant and up-regulation of the glyoxylate shunt.Conclusions: Absolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using amucA-mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.

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Lien, S. K., Niedenführ, S., Sletta, H., Nöh, K., & Bruheim, P. (2015). Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA. BMC Systems Biology, 9(1). https://doi.org/10.1186/s12918-015-0148-0

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