title: Comparative fluxome and metabolome analysis for overproduction of succinate in Escherichia coli.
abstract: An aerobic succinate-producing Escherichia coli mutant was compared to its wild-type by quantitatively analyzing both the metabolome and fluxome, during glucose-limited steady-state and succinate excess dynamic conditions, in order to identify targets for further strain engineering towards more efficient succinate production. The mutant had four functional mutations under the conditions investigated: increased expression of a succinate exporter (DcuC), deletion of a succinate importer (Dct), deletion of succinate dehydrogenase (SUCDH) and expression of a PEP carboxylase (PPC) with increased capacity due to a point mutation. The steady-state and dynamic patterns of the intracellular metabolite levels and fluxes in response to changes were used to locate the quantitative differences in the physiology/metabolism of the mutant strain. Unexpectedly the mutant had a higher energy efficiency, indicated by a much lower rate of oxygen consumption, under glucose-limited conditions, caused by the deletion of the transcription factors IclR and ArcA. Furthermore the mutant had a much lower uptake capacity for succinate (26-fold) and oxygen (17-fold under succinate excess) compared to the wild-type strain. The mutant strain produced 7.9 mmol.CmolX(-1).h(-1) succinate during chemostat cultivation, showing that the choice of the applied genetic modifications was a successful strategy. Furthermore, the applied genetic modifications resulted in multiple large changes in metabolite levels (FBP, pyruvate, 6PG, NAD(+) /NADH ratio, α-ketogluarate) corresponding to large changes in fluxes. Compared to the wild-type a considerable flux shift occurred from the tricarboxylic acid (TCA) cycle to the oxidative part of the pentose phosphate pathway, including an inversion of the pyruvate kinase flux. The mutant responded very differently to excess of succinate, with a remarkable possible reversal of the TCA cycle. The mutant and the wild-type both showed homeostatic behaviour with respect to the energy charge. In contrast, large changes in redox ratios (NAD(+) /NADH) occurred in the wild-type, while the mutant showed even larger changes. This large redox change can be associated to the reversal of flux directions. The observed large flexibility in the central metabolism following genetic (deletions) and environmental (substrate excess) perturbations of the mutant, indicates that introducing a more efficient succinate exporter could result in an even higher succinate production rate.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands. hilal.taymaz@gmail.com.|Present address: Department of Chemical Engineering, Bogazici University, 34342 Bebek, Istanbul, Turkey. hilal.taymaz@gmail.com.
authors.forename: Hilal
authors.initials: H
authors.lastname: Taymaz-Nikerel
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Ghent, Belgium.
authors.forename: Marjan
authors.initials: M
authors.lastname: De Mey
authors.affiliation: Department of Applied Mathematics, Biometrics and Process Control, Ghent University, Ghent, Belgium.|Present address: Leuven Institute for Beer Research, CMPG Lab for Genetics and Genomics, University of Leuven, Leuven, Belgium.
authors.forename: Gino J E
authors.initials: GJ
authors.lastname: Baart
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Ghent, Belgium.|Department of Applied Mathematics, Biometrics and Process Control, Ghent University, Ghent, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Maertens
authors.affiliation: Department of Applied Biological Sciences, Research Group of Microbiology, Vrije Universiteit Brussel, Brussels, Belgium.|Present address: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Flanders, Belgium.|Present address: Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, Belgium.
authors.forename: Maria Remedios
authors.initials: MR
authors.lastname: Foulquié-Moreno
authors.affiliation: Department of Applied Biological Sciences, Research Group of Microbiology, Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Daniel
authors.initials: D
authors.lastname: Charlier
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Heijnen
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Walter M
authors.initials: WM
authors.lastname: van Gulik
mesh_terms: D000332:Aerobiosis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D064688:Metabolic Flux Analysis; D055442:Metabolome; D009154:Mutation; D019802:Succinic Acid / Q000378:metabolism*
chemical_list: D019802:Succinic Acid
keywords: Escherichia coli; chemostat culture; dynamic changes; metabolic flux analysis; metabolomics; succinate