So far, in vivo only the effects of arcA-fnr [12], arcA-cra [24], and crp-fur [25] knockout combinations have been studied. Recently, two studies focused on the effect of the deletion of genes coding for a global regulator and a local regulator, i.e. cra-iclR and crp-iclR [26, 27], on gene expression and activities of key metabolic enzymes. However, the effect of the knockouts
on the metabolic fluxes were not investigated. This study investigates such a knockout combination and shows that the combined INCB018424 molecular weight deletion of arcA and iclR has a profound effect on metabolism and redirects carbon fluxes in such a way that the biomass content increases remarkably both under glucose abundant and glucose limiting conditions as opposed to its parent strain E. coli K12 MG1655. Many of the observed characteristics
in the double knockout strain are also ascribed to E. coli BL21 (DE3), which is why fluxes between these learn more two strains were investigated as well. Results and Discussion Physiological effects of arcA and iclR deletions Wild type MG1655, single and double knockout strains were first cultivated in a 2L bioreactor under glucose abundant (batch) and limiting (chemostat, D = ±0.1 h -1) conditions in order to precisely determine extracellular fluxes and growth rates. The growth rates are shown in Table 1. The arcA and iclR single knockout strains have a slightly lower mTOR inhibitor maximum growth rate. The arcA-iclR double knockout strain exhibits a reduction of as much as 38% in μ max. Figure 1 shows the effects of these mutations on various product yields under batch and chemostat conditions for the different strains. The corresponding average redox and carbon balances close very well (data shown in Additional file 1). The phenotypic effects will be discussed below. Table 1 Average maximum growth rates (batch) and dilution rates Fossariinae (chemostat) of the different strains. Batch Chemostat Strain μ max ( h -1 ) D influent ( h -1) D effluent ( h -1 ) Wild type 0.66 ± 0.02 0.099 ± 0.001 0.100 ±
0.001 ΔarcA 0.60 ± 0.01 0.118 ± 0.001 0.120 ± 0.001 ΔiclR 0.61 ± 0.02 0.085 ± 0.001 0.090 ± 0.001 ΔarcAΔiclR 0.44 ± 0.03 0.090 ± 0.001 0.093 ± 0.001 Under chemostat conditions, the apparent growth rate equals the dilution rate of the influent. Differences between D influent and D effluent are due to addition of base and acid for pH correction and sampling. Figure 1 Product yields of the wild type and knockout strains. Product yields in c-mole/c-mole glucose of the wild type MG1655, the derived single knockout strains ΔarcA and ΔiclR, and the double knockout strain ΔarcAΔiclR under glucose abundant, batch (A) and glucose limiting, chemostat (B) conditions. Oxygen yield is shown as a positive number for a clear representation, but O 2 is actually consumed during the experiments.
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