DataEntryID 103
    General information
      Manuscript title: Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition.
      PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/15781419
      Journal: Metabolic Engineering
      Year: 2005
      Authors: Jiangfeng Zhu, Kazuyuki Shimizu
      Affiliations: Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan
      Keywords: Escherichia coli, Enzyme activity, Metabolite concentration, Metabolic flux analysis, Lactate production, Pyruvate metabolism
      Full text article: https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBc1lFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--b80939e3ed45ef558a60173cd02f7091fc780622/Zhu_2005.pdf
      Project name: not specified

    Experiment description
      Organism: Escherichia coli
      Strain: BW25113, pflA, pta, ppc, adhE and pykF mutants
      Data type: metabolites at steady-state
      Data units: mM/g DCW
      Execution date: not specified

    Experimental details
      Temperature (°C): 37
      pH: 7.0
      Carbon source: glucose
      Culture mode: batch
      Process condition: aerobic
      Dilution rate (h⁻¹): —
      Working volume: 1.0 L
      Biomass concentration (g/L): Cell dry weight was determined by measuring the optical density at 600 nm.
      Medium composition: Reactor medium was a minimal medium containing (per liter) 2 g of Na2SO4, 2.5 g of (NH4)2SO4, 0.5 g of NH4Cl, 7.3 g of K2HPO4, 3.6 g of NaH2PO4. The following components were sterilized by passing through a 0.2 μm pore-size filter (per liter of final medium): 3 ml of 1 M MgSO4, and 3 ml trace element solution containing (per liter) 0.5 g of CaCl2·H2O, 0.18 g of ZnSO4·7H2O, 0.1 g of MnSO4·H2O, 21.1 g of Na2EDTA, 16.7 g of FeCl3·6H2O, 0.16 g of CuSO4·5H2O and 0.18 g of CoCl2·6H2O. Glucose was used as a carbon source and the initial concentration was about 10 g/L.
      General protocol information: Sampling Method: About 10 ml of the culture broth was quickly taken as the sample, and immediately injected into 40 ml of pre-cooled 60% methanol. The mixture was kept at −20 °C. ; Quenching: The quenched samples were centrifuged at 10,000×g at a temperature of −15 °C for 10 min. The pellets were kept at −20 °C for 30 min and then lyophilized. The dried biomass was kept in a −20 °C freezer for further processing.; Extraction list: perchloric acid, boiling ethanol; Analysis list: enzymatic;
      Methods description: Intracellular metabolites except NADH and NADPH were extracted by perchloric acid in ethanol. The extract was placed on ice-bath for 10 min and then neutralized with K2CO3. Then, the cell debris and KClO4 were removed by centrifugation [1]. For NADH measurement, alkaline extraction was utilized, and acid extraction method was used for other metabolite measurements. Nucleotides concentrations were measured according to [1]. The concentrations of glucose 6-phosphate (G6P), fructose 1,6-bisphosphate (FDP), phosphoenolpyruvate (PEP) and pyruvate (PYR) were measured according to [2]. The concentration of acetyl coenzyme A (AcCoA) was measured following to [3], and ATP, ADP, AMP as well as NAD+ were measured based on [4,5].                                                                                                                                                                                                                 --------------------------------------------------References----------------------------------------
[1]  J.R. Williamson, B.E. Corkey, J.M. Lowenstein (Ed.), Citric Acid Cycle, Methods in Enzymology, XIII, Academic Press, New York (1969), pp. 434-513 Chapter 65. http://doi.org/d2cnhf
[2] U. Schaefer, W. Boos, R. Takors, D. Weuster-Botz, Anal. Biochem., 270 (1999), pp. 88-96. http://doi.org/dt38q9                                                               
[3] C. Riondet, R. Cachon, Y. Waché, G. Alcaraz, C. Diviès.  J. Bacteriol., 182 (2000), pp. 620-626. http://doi.org/cf5kb7
[4] Bergmeyer, H.U., 1984. Methods of enzymatic analysis. third ed, vol. 6. VCH, Weinheim, Germany.                                                                                                                                                  [5] Bergmeyer, H.U., 1985. Methods of enzymatic analysis. third ed, vol. 7. VCH, Weinheim, Germany.                                                                                                                                                                                                                                                 
      Data file: http://kimosys.org/repository/103/download?parameter=1221; 
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    Submission and curation
      Entered by: Administrator KiMoSys
      Created: 2018-07-20 14:51:02 UTC
      Updated: 2020-04-24 16:10:36 UTC
      Version: 1
      Status: (reviewed) 2018-07-20 14:53:30 UTC
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