title: Deciphering flux adjustments of engineered E. coli cells during fermentation with changing growth conditions.
abstract: Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this study,13C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37°C in stage 1, and then the temperature was transitioned to 20°C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (late production phase) in the high producing strain, reaching a final concentration of 1.5mmol/L. On the contrary, ~0.02mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed13C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).
authors.affiliation: Department of Energy, Environmental, and Chemical Engineering, Washington University, St. Louis, MO, USA.
authors.forename: Lian
authors.initials: L
authors.lastname: He
authors.affiliation: Department of Chemical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA; College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xiu
authors.affiliation: Department of Chemical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Chemistry, Hamilton College, Clinton, NY, USA.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Jones
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: Edward E K
authors.initials: EEK
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, CA, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA; California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé, DK2970-Hørsholm, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Department of Energy, Environmental, and Chemical Engineering, Washington University, St. Louis, MO, USA. Electronic address: yinjie.tang@wustl.edu.
authors.forename: Yinjie J
authors.initials: YJ
authors.lastname: Tang
authors.affiliation: Department of Chemical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA. Electronic address: koffam@rpi.edu.
authors.forename: Mattheos A G
authors.initials: MAG
authors.lastname: Koffas
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D053898:Biosynthetic Pathways / Q000502:physiology; D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods; D049109:Cell Proliferation / Q000502:physiology*; D004926:Escherichia coli / Q000166:cytology / Q000502:physiology*; D005285:Fermentation / Q000502:physiology*; D007211:Indoles / Q000302:isolation & purification / Q000378:metabolism*; D064688:Metabolic Flux Analysis / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D008954:Models, Biological
chemical_list: D007211:Indoles; C063155:violacein
keywords: (13)C-MFA; Channeling; Free metabolites; Promoter; Reflux; Tryptophan; Violacein