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General Information info

Manuscript title Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition.
PubMed ID 15781419
Journal Metabolic Engineering
Year 2005
Authors Jiangfeng Zhu, Kazuyuki Shimizu
Affiliations Department of Biochemical Engineering & 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 Downloadarticle Zhu_2005.pdf
Project name not specified

Experiment Description info

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 info

Temperature (0C) 37
pH 7.0
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 1.0
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 procedure: 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 technique: perchloric acid, boiling ethanol

Sample analyzing method: enzymatic

Methods description - Notes

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.

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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2018-07-20 14:51:02 UTC

Updated 2018-07-20 14:53:12 UTC

Version 1

Status (reviewed) 2018-07-20 14:53:30 UTC




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