Sridhar Ranganathan, Ting Wei Teeb, Anupam Chowdhuryc, Ali R. Zomorrodic, Jong Moon Yoon, Yanfen Fu, Jacqueline V. Shanksb, Costas D. Maranas
Affiliations
Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA, USA and Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA
Keywords
Metabolic flux analysis, E. coli, fatty acids overproduction, MG1655
Sample preparation for 2-dimensional NMR analysis - Cells were prepared as described previously [1]. Cells are centrifuged, washed twice with saline water containing 0.9% NaCl, then hydrolyzed with 6 N hydrochloric acid at 110 °C for 18–24 h. Acids were evaporated, the resid
... ue reconstituted in nanopure water and filtered, then lyophilized. Finally, the sample was dissolved in deuterium oxide for NMR analysis.
NMR measurement - 2D [13C, 1H] Heteronuclear Single Quantum Correlation (HSQC) spectra were acquired on a Bruker Avance DRX 500 MHz spectrometer at 298 K and processed as described previously [1,2]. Nonoverlapping multiplets on the spectrum were quantified using NMRView [3]. Overlapping multiplets (α amino acids) were analyzed using a peak deconvolution software [1]. The amino acids isotopomer abundances measured by 2D HSQC NMR are related to the precursor metabolites by using amino acids biosynthesis pathways as described by Szyperski [4].
Flux evaluation methodology - Fluxes were quantified using NMR2Flux software developed by Sriram et al. [2]. NMR2 Flux employs isotopomer balancing and aglobal optimization routine to find stoichiometrically feasible fluxes set consistent with experimental measurements.
----------------References---------------------
[1] Choudhary, M.K., Yoon,J.M., Gonzalez,R., Shanks,J.V., 2011. Re-examination of metabolic fluxes in Escherichia coli during anaerobic fermentation of glucose using (13)C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. Biotechnol. Bioprocess Eng.16,419–437. http://doi.org/bpdmkg [2] Sriram, G., Fulton,D.B., Iyer,V.V., Peterson,J.M., Zhou,R.L., Westgate, M.E., Spalding, M.H.,Shanks,J.V.,2004.Quantification of compartmented metabolic fluxes in developing soybean embryos by employing Biosynthetically directed fractional C-13 labeling, C-13, H-1 two-dimensional nuclear magnetic resonance, and comprehensive isotopomer balancing. Plant Physiol.136, 3043–3057. http://dx.doi.org/10.1104/pp.104
[3] Johnson, B.A., Blevins,R.A.,1994. NMR View — A computer-program for the visualization and analysis of NMR data. J. Biomolecular NMR 4, 603–614. http://doi.org/btdckd [4] Szyperski, T.,1995. Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. Eur. J. Biochem. 232, 433–448. http://doi.org/bdmpc2
An integrated computational and experimental study for overproducing fatty acids in Escherichia coli.
Pubmed ID
23036703
Journal
Metabolic Engineering
Year
2012
Authors
Sridhar Ranganathana, Ting Wei Teeb, Anupam Chowdhuryc, Ali R. Zomorrodic, Jong Moon Yoon, Yanfen Fu, Jacqueline V. Shanksb, Costas D. Maranas
Affiliations
Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA, USA and Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA
Keywords
Metabolic flux analysis, E. coli, fatty acids overproduction, MG1655
Sample preparation for 2-dimensional NMR analysis - Cells were prepared as described previously [1]. Cells are centrifuged, washed twice with saline water containing 0.9% NaCl, then hydrolyzed with 6 N hydrochloric acid at 110 °C for 18–24 h. Acids were evaporated, the residue reconstituted in nanopure water and filtered, then lyophilized. Finally, the sample was dissolved in deuterium oxide for NMR analysis.
NMR measurement - 2D [13C, 1H] Heteronuclear Single Quantum Correlation (HSQC) spectra were acquired on a Bruker Avance DRX 500 MHz spectrometer at 298 K and processed as described previously [1,2]. Nonoverlapping multiplets on the spectrum were quantified using NMRView [3]. Overlapping multiplets (α amino acids) were analyzed using a peak deconvolution software [1]. The amino acids isotopomer abundances measured by 2D HSQC NMR are related to the precursor metabolites by using amino acids biosynthesis pathways as described by Szyperski [4].
Flux evaluation methodology - Fluxes were quantified using NMR2Flux software developed by Sriram et al. [2]. NMR2 Flux employs isotopomer balancing and aglobal optimization routine to find stoichiometrically feasible fluxes set consistent with experimental measurements.
----------------References---------------------
[1] Choudhary, M.K., Yoon,J.M., Gonzalez,R., Shanks,J.V., 2011. Re-examination of metabolic fluxes in Escherichia coli during anaerobic fermentation of glucose using (13)C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. Biotechnol. Bioprocess Eng.16,419–437.
[2] Sriram, G., Fulton,D.B., Iyer,V.V., Peterson,J.M., Zhou,R.L., Westgate, M.E., Spalding, M.H.,Shanks,J.V.,2004.Quantification of compartmented metabolic fluxes in developing soybean embryos by employing Biosynthetically directed fractional C-13 labeling, C-13, H-1 two-dimensional nuclear magnetic resonance, and comprehensive isotopomer balancing. Plant Physiol.136, 3043–3057.
[3] Johnson, B.A., Blevins,R.A.,1994. NMR View — A computer-program for the visualization and analysis of nmr data. J. Biomolecular NMR 4, 603–614.
[4] Szyperski, T.,1995. Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. Eur. J. Biochem. 232, 433–448.
KiMoSys (https://kimosys.org). Data EntryID 67 (Escherichia coli). [online], [Accessed 21 November 2024]. Available from: https://doi.org/10.34619/7bxr-2h93
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