DataEntryID 67
    General information
      Manuscript title: An integrated computational and experimental study for overproducing fatty acids in Escherichia coli.
      PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/23036703
      Journal: Metabolic Engineering
      Year: 2012
      Authors: 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
      Full text article: https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBb2NFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--7acb97ec7787d6b4a180b5251a7f1994f8a1ed55/Ranganathan_2012.pdf
      Project name: not specified

    Experiment description
      Organism: Escherichia coli
      Strain: MG1655
      Data type: flux measurements
      Data units: % substrate uptake
      Execution date: not specified

    Experimental details
      Temperature (°C): 37.0
      pH: 7.0
      Carbon source: glucose
      Culture mode: batch
      Process condition: aerobic
      Dilution rate (h⁻¹): —
      Working volume: 0.4 L
      Biomass concentration (g/L): 0.36 (gDW/L) = 1 OD550 
      Medium composition: MOPS medium: (8.37 g/L MOPS powder (Sigma-Alrich, Saint Louis, MO), 0.72 g Tricine (Sigma-Alrich, Saint Louis, MO), 2.92 g/L NaCl, 0.51 g/L NH4Cl, 1.6 g/L KOH, 0.215 mg/L Na2SeO3, 0.303 mg/L Na2MoO4, 0.17 mg/L ZnCl2, 50.3 mg/L MgCl2, 48.1 mg/L K2SO4, 0.348 g/L K2HPO4, and micronutrients containing 2.5 mg/L FeCl2·4H2O, 92 μg/L CaCl2·2H2O, 31 μg/L H3BO3, 20 μg/L MnCl2·4H2O, 9 μg/L CoCl2·6H2O, 2 μg/L CuCl2·2H2O, and 48.4 μM HCl) supplemented with 1% glucose.
      General protocol information: Type analysis list: 13C constrained MFA; Platform list: NMR;
      Methods description: 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. 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 
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      Created: 2013-06-20 16:59:55 UTC
      Updated: 2020-04-24 16:10:33 UTC
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