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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).

Waegeman H, Beauprez J, Moens H, Maertens J, De Mey M, Foulquié-Moreno MR, Heijnen JJ, Charlier D, Soetaert W - BMC Microbiol. (2011)

Bottom Line: 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.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.Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre of Expertise-Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Hendrik.Waegeman@ugent.be

ABSTRACT

Background: Gene 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).

Results: The 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.

Conclusions: The 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.

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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 O2 is actually consumed during the experiments. The values represented in the graph are the average of at least two separate experiments and the errors are standard deviations calculated on the yields.
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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 O2 is actually consumed during the experiments. The values represented in the graph are the average of at least two separate experiments and the errors are standard deviations calculated on the yields.

Mentions: 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 maximum growth rate. The arcA-iclR double knockout strain exhibits a reduction of as much as 38% in μmax. 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.


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).

Waegeman H, Beauprez J, Moens H, Maertens J, De Mey M, Foulquié-Moreno MR, Heijnen JJ, Charlier D, Soetaert W - BMC Microbiol. (2011)

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 O2 is actually consumed during the experiments. The values represented in the graph are the average of at least two separate experiments and the errors are standard deviations calculated on the yields.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094197&req=5

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 O2 is actually consumed during the experiments. The values represented in the graph are the average of at least two separate experiments and the errors are standard deviations calculated on the yields.
Mentions: 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 maximum growth rate. The arcA-iclR double knockout strain exhibits a reduction of as much as 38% in μmax. 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.

Bottom Line: 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.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.Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre of Expertise-Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Hendrik.Waegeman@ugent.be

ABSTRACT

Background: Gene 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).

Results: The 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus