Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)

Abstract

BackgroundGene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3).ResultsThe deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ΔarcAΔiclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ΔiclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ΔarcA ΔiclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a comparison of the genome sequences of the two strains showed that BL21 possesses two mutations in the promoter region of iclR and rare codons are present in arcA implying a lower tRNA acceptance. Both phenomena presumably result in a reduced ArcA and IclR synthesis in BL21, which contributes to the similar physiology as observed in E. coli K12 ΔarcAΔiclR.ConclusionsThe deletion of arcA results in a decrease of repression on transcription of TCA cycle genes under glucose abundant conditions, without significantly affecting the glyoxylate pathway activity. IclR clearly represses transcription of glyoxylate pathway genes under glucose abundance, a condition in which Crp activation is absent. Under glucose limitation, Crp is responsible for the high glyoxylate flux, but IclR still represses transcription. Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.

Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G

“…When IclR is inactivated, repression of the expression glyoxylate genes is released, and the glyoxylate pathway is activated. This strategy could be used to reduce the flux through the TCA cycle (15). PG05/H7 (△sucA△poxB::acs△aceA) and PG07/H7 (△sucA△poxB::acs△iclR) were constructed to investigate the effects of increasing glyoxylate pathway on DAOCS catalyzed reactions.…”

Metabolic Control of the TCA cycle by the YdcI Transcriptional Regulator in Escherichia coli

“…For example, in l-lysine fermentation, a reduced carbon flux was expected to increase the l-lysine-production yield [42], and higher carbon flux into the TCA cycle is expected to lead to higher l-glutamic acid production [31]. In E. coli, arcA gene deletion increases the carbon flux into the TCA cycle [13], and we confirmed that this deletion (ΔarcA) indeed improves l-glutamic acid production in the E. coli MG1655 ΔsucA strain (unpublished data).…”

“…To validate our method, we chose the ArcAB two-component regulatory system of Escherichia coli, as ArcAB regulates the bacterial transition from aerobic to anaerobic growth and controls more than 100 genes involved in the TCA cycle and energy metabolism [12,13]. DNA-array data for both arcA and arcB gene-deletion strains were used to classify genes with increased expression levels in ΔarcA and ΔarcB strains [14].…”

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

“…More specific details about the metabolic flux calculations can be found in (Waegeman et al, 2011a) www.intechopen.com…”

“…Furthermore, only a small part of the tremendously elevated biomass yield was attributed to increased glycogen content (Waegeman et al, 2011a) making this strain an attractive candidate for recombinant protein production.…”