Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels

Abstract

Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate starvation. The machinery involves a cascade with a two-component system (TCS) that relays the external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative understanding of this system has implications in synthetic biology and biotechnology, for example, in applications for wastewater treatment. To this aim, we present a computational model and experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together with the mechanisms of gene expression. The model is parameterised within the feasible range, and fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this network that is used in alkaline phosphatase production. Deterministic and stochastic simulations with our model predict the regulation dynamics in higher external phosphate concentrations while reproducing the experimental observations. In a cycle of simulations and experimental verification, our model predicts and explores phenotypes with various synthetic promoter designs that can optimise the inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes have a significant influence over the phosphate response. Together with experimental findings, our model should thus provide insights for the investigations on engineering new sensors and regulators for living technologies.

Mechanisms of Switching Response to External Phosphate Levels in Escheria coli

“…However, when the external P i is in excess, the E. coli begins to enhance P i accumulation and switches the TCS off. To explain this system, we have previously designed and analysed a two component system model that quantifies the dynamic mechanisms of auto-regulation in E. coli, and explored and verified emerging phenotypes with synthetic promoters (Uluşeker et al, 2018(Uluşeker et al, , 2017. Moreover, we have analysed the response of the model to variations in the external P i levels.…”

“…The signal is thus propagated to PhoR, resulting in its inhibition state P hoR r . has been quantified in our previous work (Uluşeker et al, 2018(Uluşeker et al, , 2017. In the current work, this framework is used as a basis to describe mechanistically the effect of external P i concentration change and TCS inhibition.…”

“…The models have been tested with 0, 50, and 100 µM external P i . The concentrations of proteins PhoR and ABC are set to 0.22 µM based on literature and verified by our previous work (Van Dien and Keasling, 1997;Uluşeker et al, 2018Uluşeker et al, , 2017. The parameters for the rates are listed in Table 1.…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…Transcriptomic analysis revealed a substantial proportion of upregulated genes in the nisIPRKFEG cluster and other genes involved in amino acids biosynthesis. 68 Constructing TF-containing genetic circuits for protein sciences The discovery and in-depth elucidation of the lac operon opened up an era for scientists to utilize the genetic circuits that naturally exist in the system to sense different compounds 69 or environmental factors, 70,71 to detect gene deletion 72 and bacterial cell division. 73 Engineered TFs are generally indispensable components in designed genetic circuits.…”

“…The discovery and in‐depth elucidation of the lac operon opened up an era for scientists to utilize the genetic circuits that naturally exist in the system to sense different compounds 69 or environmental factors, 70,71 to detect gene deletion 72 and bacterial cell division 73 . Engineered TFs are generally indispensable components in designed genetic circuits 74,75 …”