Table 4 Comparison of the codon usage in the arcA gene between E. coli K12 MG1655 and BL21 (DE3) based on Chen & Texada, [66]. AA OSI-906 strain Codon Frequency tRNA content L MG1655 CUG 54.1 1   BL21

CUA 2.97 Minor S MG1655 UCU 10.47 0.25   BL21 UCC 9.43 Minor P MG1655 CCA 8.12 Major   BL21 CCG 23.91 Major I MG1655 AUC 26.97 1   BL21 AUU 27.27 1 C MG1655 UGU 4.8 Minor   BL21 UGC 6.07 Minor Each codon is expressed as the frequency per 1000 codons. The content is the relative amount to that of tRNALeu1(CUG), which is normalized to 1 and approximately in the order of 104 molecules per cell for normally selleckchem growing E. coli cells Conclusions Under glucose abundant conditions the double knockout strain E. coli MG1655 ΔarcAΔiclR exhibits an increased biomass yield of 0.63 c-mole/c-mole glucose, which approximates the maximum theoretical yield of 0.65 c-mole/c-mole glucose. Also under glucose limitation a higher biomass yield was observed, but effects were less distinct due to a fixed growth rate and a higher maintenance. The higher biomass formation is accompanied by a decrease in acetate formation and PI3K inhibitor CO2 production. Only a small part of the higher yield was attributed to an increased glycogen content. Furthermore, enzyme activity measurements showed an increased transcription of glyoxylate enzymes, implying the activation of this

pathway in the ΔarcAΔiclR strain even under glucose abundant conditions, when Crp-activation is absent. This PAK5 was confirmed by 13 C metabolic flux analysis, showing that 30% of isocitrate molecules were channeled through the glyoxylate pathway when iclR was knocked out. Deletion of arcA results in loss of repression on transcription of TCA genes, which provokes a higher flux through the TCA cycle. This explains the lower acetate formation observed. Because many physiological and metabolic properties observed in the double knockout strains are also attributed to E. coli BL21, the metabolic fluxes of the two strains were compared

under glucose abundant conditions. Almost all fluxes in central metabolism seemed to be similar, which can be explained by mutations in the promoter region of iclR and a less efficient codon usage of arcA in BL21, resulting in lower activity of the corresponding enzymes. Methods Strains The strains used in this study are listed in Table 5. Escherichia coli MG1655 [λ-, F -, rph -1] and BL21 were obtained from the Coli Genetic Stock Center (CGSC). The single and double knockout strains were constructed using a one-step disruption protocol [68]. In order to confirm the mutations, polymerase chain reaction (PCR) was used to amplify fragments containing the modified sequences. Lengths of amplified fragments were tested by agarose gel electrophoresis and compared with those of the wild type strain (WT). PCR products were also sequenced to confirm knockouts and sequence substitutions.