Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis |
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| Institution: | 1. Department of Biotechnology, Delft University of Technology, Julianalaan 67 – 2628 BC Delft, The Netherlands;2. Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The Netherlands;3. Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands;1. Institute of Systems Biotechnology, Saarland University, Germany;2. Université de Toulouse, INSA, UPS, INP, Toulouse, France;3. INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France;4. CNRS, UMR5504, Toulouse, France;5. BASF SE, Fine Chemicals and Biotechnology, Ludwigshafen, Germany;1. Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA;2. Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA;3. Department of Chemistry, Hamilton College, 198 College Hill Rd., Clinton, NY 13323, USA;4. Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA;5. The Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711, USA;1. Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France;2. INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France;3. CNRS, UMR 5504, F-31400 Toulouse, France;4. Universität des Saarlande, Systembiotechnologie Campus, D-66123 Saarbrücken, Germany;1. Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055. Lomas 4a Sec, San Luis Potosí, 78216, México;2. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional Unidad Guadalajara de Ingeniería Avanzada, Av. del Bosque 1145, Colonia el Bajío, Zapopan 45019, Jalisco, México;3. Central Mining Institute, Pl. Gwarków 1, 40-166 Katowice, Poland |
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| Abstract: | Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13C metabolic flux analysis (13C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13C-MFA using the optimal tracers 1,2-13C]glucose, 1,6-13C]glucose, 1,2-13C]xylose and 5-13C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying U-13C]glucose and U-13C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH2, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology. |
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| Keywords: | Parallel labeling experiments Metabolism Model validation Flux estimation Co-factor balances |
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