|Lin (2005) - Chemostat culture characterization of Escherichia coli mutant strains metabolically engineered for aerobic succinate production: a study of the modified metabolic network based on metabolite profile, enzyme activity, and gene expression profile||sdhAB, inactivation of succinate dehydrogenase (SDH), (ackA-pta), inactivation of acetate kinase-phosphotransacety-lase, poxB, inactivation of pyruvate oxidase, iclR, inactivation of aceBAK operon repressor, and ptsG, inactivation of glucose phosphotransferase||0.91 mol/mol ||pKK313||Luria-Bertani broth (LB), 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl (Sam-brook et al., 1989), added with 2 g/L NaHCO3 and 20 g/L of glucose. The medium used for inoculum preparation was also the same medium, except glucose was not added. NaHCO3was added to the culture medium because it promoted cell growth and succinate production due to its pH-buffering capacity and its ability to supply CO2||The working volume was maintained at 600 ml in a 1.0-L New Brunswick Scientific Bioflo 110 fermenter.
Dilution rate- 0.1/h
The pH was controlled at 7.0 using 1.5 N HNO3 and 2 N Na2CO3.
Temperature- 37 C
Agitation speed- 500 rpm
Inlet air flow- 0.6 L/min
Oxygen maintained at 50% above saturation level.||1. OD measurements
2. Intracellular and extracellular metabolite concentrations- Glucose, Succinate, Acetate, Pyruvate, Glyoxylate, Acetyl-CoA
3. Enzyme assays-Citrate synthase (CS), Malate dehydrogenase (MDH), Phosphoenolpyruvate carboxylase, Isocitrate dehydrogenase, Malate Synthase, Isocitrate Lysase
4. Gene expression analysis||Methods need detailed review. Mainly individual assays|
|Kang (2010) - A novel strategy for succinate and polyhydroxybutyrate co-production in Escherichia coli||Knockouts - sdhA, ptsG, poxB, pta and iclR Heterologous expression - phbCAB genes from Ralstonia eutropha||Succinate - 0.83 (mol/mol) 9.25 g/L/day PHB - 0.23 (mol/mol) 1.86 g/L/day Residual biomass - 7.05 g/L ||Stress-induced plasmid pQKZ103|