Table 3 Glycogen content of the wild type and the double knockout strain under glucose abundant (batch) and glucose limiting (chemostat) conditions. Strain Batch Chemostat MG1655 0.25 ± 0.26 0.50 ± 0.24 MG1655 ΔarcAΔiclR 1.47 ± 0.19 1.29 ± 0.16 Values are expressed as carbon relative to the total amount of biomass carbon.

The results shown are the averages of two cultures, measured 4 times. The wild type chemostat culture had a dilution rate of 0.17 ± 0.01 h – 1; the ΔarcAΔiclR strain had buy Emricasan a dilution rate of 0.33 ± 0.02 h – 1. The carbon balance and redox balance for these experiments are similar to the data shown in Additional file 1 Considering the product yield and storage compound results, it can be concluded that the increase in biomass yield in the double knockout strain is primarily the result of the lower acetate and CO2 production under glucose abundant conditions and of the lower CO2 production ALK inhibitor under

glucose limitation. Only a small and similar amount of the extra carbon is converted to storage molecules like glycogen under both growth conditions. Effect of arcA and iclR knockouts on metabolic fluxes The arcA and iclR gene deletions have a profound effect on the phenotype of the resulting strains and on the Doramapimod in vivo activity of some key central metabolic enzymes under the different growth conditions as shown in the previous sections. In order to understand the metabolic implications of these deletions and consequently to grasp the role of IclR and ArcA in central metabolism, metabolic flux ratios and the corresponding net fluxes were determined. Figure 4 shows the origin of different intermediate metabolites of the different strains Rebamipide grown in batch and continuous mode. Figure 4 Origin of metabolic

intermediates in E. coli MG1655 single knockout strains Δ arcA and Δ iclR , and the double knockout strain Δ arcA Δ iclR cultivated in glucose abundant (batch) and glucose limiting (continuous) condtions. Standard deviations are calculated on different samples originating from different cultivations. The serine through EMP and the pyruvate through ED results were obtained from experiments using 50% 1-13C glucose and 50% naturally labeled glucose. To determine the remaining values a mixture of 20% U-13C glucose and 80% naturally labeled glucose was used. To determine the fractions resulting in the formation of OAA a Monte-Carlo approach was applied. For chemostat experiments, a dilution rate of 0.1 h -1 was set. Under glucose abundant conditions, deleting arcA results in a decrease of the OAA from PEP fraction, indicating that a higher fraction of OAA originates from the TCA cycle (OAA from TCA = 1 – OAA from PEP – OAA from glyoxylate). This phenomenon is also observed in the double knockout strain. Deletion of iclR results in an increase of the OAA from glyoxylate fraction from 0 to 23%.