Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichiacoli K12 and its implications on understanding the metabolism of Escherichia coliBL21 (DE3)

BMC Microbiology

Research Article

Helena Moens¹  Marjan De Mey¹  Wim Soetaert¹  Jo Maertens¹  Hendrik Waegeman¹  Joeri Beauprez¹  Joseph J Heijnen²  Daniel Charlier³  Maria R Foulquié-Moreno³ 

[1] Centre of Expertise-Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000, Ghent, Belgium;Kluyver Laboratory for Biotechnology, Department of Biochemical Engineering, Delft University of Technology Julianalaan 67, 2628, BC, Delft;Laboratory for Genetics and Microbiology, Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050, Brussels, Belgium;

关键词: Glyoxylate;    Metabolic Flux Analysis;    Isocitrate Lyase;    Glyoxylate Shunt;    Glucose Limitation;   

DOI  :  10.1186/1471-2180-11-70

 received in 2010-09-13, accepted in 2011-04-11,  发布年份 2011

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Waegeman et al; licensee BioMed Central Ltd. 2011