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Team:SCAU-China/ppsA-promoter
From 2014.igem.org
Utilization of sugar responsive promoter of ppsA
Microcalorimetric and respiratory measurements show that the contitutive production of TCA cycle enzymes in the ΔarcA mutant results in an elevated respiration rate and total metabolic activity during carbon starvation. The ΔarcA mutant is severely impaired in surviving prolonged periods of exogenous carbon starvation. In addition, the mutant strain fails to perform normal reductive division (Nystrom et al. 1996) .
When the ΔarcA strain is applied for industrial usage it can significantly reduce the frequency of exchanging new strain due to low survival rate. Wastewater nutrient is not always sufficient for a high metabolic consumption. We want our ΔarcA strain to alter its metabolic rate by its own regulatory system.
Catabolic Receptor Activator (CRA) and cAMP Receptor Protein (CRP) are global regulation factor for carbon source utilization in E. coli (Saier and Ramseier 1996, Shimada et al. 2011). They control the synthesis of enzymes for glycolysis and gluconeogenesis, thus directs the flow of carbon source. ppsA encodes PEP (phosphoenolpyruvate) synthetase, which is regulated directly by FruR, a conserved sequence in CRA, and indirectly by RNAP related factors (Nègre et al. 1998). The ppsA promoter is responsive to external sugar level. CRA senses the nutrient status by catabolite effectors (fructose-1-phosphate and fructose-1, 6-bisphosphate) resembling the function of FruR (Fig. 1). CRP responses to cellular cAMP level and interacts with ppsA promoter though RNA Polymerase (Fig. 2).
In our project, the arcA was driven by the ppsA promoter region in order to restore the ΔarcA strain under low nutrient condition. When the transcription factor ArcA exists, the rate of catabolism is turned down under anaerobic condition, Therefore, the ΔarcA strain can tolerance starvation (Fig.3). Once nutrient becomes ample again, the expression of arcA will be repressed by CRA and CRP to achieve a fine-tuned balance.
[2] Saier, M. H., Jr. and T. M. Ramseier. The catabolite repressor/activator (Cra) protein of enteric bacteria. J. Bacteriol. 1996, 178(12): 3411-3417.
[3] Shimada, T., N. Fujita, K. Yamamoto and A. Ishihama. Novel Roles of cAMP Receptor Protein (CRP) in Regulation of Transport and Metabolism of Carbon Sources. PLoS ONE. 2011, 6(6): e20081.
[4] Nègre, D., C. Oudot, J.-F. Prost, K. Murakami, A. Ishihama, A. J. Cozzone and J.-C. Cortay. FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli. Journal of Molecular Biology. 1998, 276(2): 355-365.
Fig.1 The regulatory scheme of CRA.
Catabolic Receptor Activator (CRA) and cAMP Receptor Protein (CRP) are global regulation factor for carbon source utilization in E. coli (Saier and Ramseier 1996, Shimada et al. 2011). They control the synthesis of enzymes for glycolysis and gluconeogenesis, thus directs the flow of carbon source. ppsA encodes PEP (phosphoenolpyruvate) synthetase, which is regulated directly by FruR, a conserved sequence in CRA, and indirectly by RNAP related factors (Nègre et al. 1998). The ppsA promoter is responsive to external sugar level. CRA senses the nutrient status by catabolite effectors (fructose-1-phosphate and fructose-1, 6-bisphosphate) resembling the function of FruR (Fig. 1). CRP responses to cellular cAMP level and interacts with ppsA promoter though RNA Polymerase (Fig. 2).
Fig.2 A fine-tune circuit for sugar starvation
Fig.3 A fine-tune circuit for sugar starvation motabolic regulation.
References
[1] Nystrom, T., C. Larsson and L. Gustafsson. Bacterial defense against aging: role of the Escherichia coli ArcA regulator in gene expression, readjusted energy flux and survival during stasis. Embo J. 1996, 15(13): 3219-3228.[2] Saier, M. H., Jr. and T. M. Ramseier. The catabolite repressor/activator (Cra) protein of enteric bacteria. J. Bacteriol. 1996, 178(12): 3411-3417.
[3] Shimada, T., N. Fujita, K. Yamamoto and A. Ishihama. Novel Roles of cAMP Receptor Protein (CRP) in Regulation of Transport and Metabolism of Carbon Sources. PLoS ONE. 2011, 6(6): e20081.
[4] Nègre, D., C. Oudot, J.-F. Prost, K. Murakami, A. Ishihama, A. J. Cozzone and J.-C. Cortay. FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli. Journal of Molecular Biology. 1998, 276(2): 355-365.
