Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system

Abstract

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ 54 )-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ 70 ) TFs. Here, we show that these unique characteristics of σ 54 -dependent tfs can be used to control the host cell state to be more appropriate for high-throughput screening. the acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ 54 tf-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ 54 -dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. this combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.

Antagonistic Control of Genetic Circuit Performance for Rapid Analysis of Targeted Enzyme Activity in Living Cells

“…In this experiment, benzene, 3,5-dimethylphenol, 2,4-dichlorophenol, and p NP were administered along with phenol to test the inhibitory effect of DmpR. As DmpR is a σ54-dependent transcriptional regulator, it responds to metabolism of alternative carbon sources such as acetate (Kwon et al, 2020 ). To activate DmpR in the genetic circuit, cells harboring pDmpR-GESS were cultured in LB at and the culture media were changed to fresh M9 media containing 4 g/L acetate, 1 mM aromatic compounds, and various concentrations of phenol.…”

“…TPL was expressed in LB, after which the cells were transferred to M9 minimal media for two-step induction to maximize fluorescence intensity (Kwon et al, 2020 ). Figure 4B shows the fluorescence intensity induced by different concentrations of alanine, as measured by flow cytometry.…”

“…Cells harboring pDmpR-GESS or pmDmpR-GESS were cultivated in lysogeny broth (LB) medium (10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter) and M9 minimal medium (12.8 g Na 2 HPO 4 ·7H 2 O, 3 g KH 2 PO 4 , 0.5 g NaCl, 1 g NH 4 Cl, 2 mM MgSO 4 , 0.1 mM CaCl 2 , and 0.01% (w/v) thiamine per later) supplemented with 4 g/L acetate as a carbon source and 50 μg/mL ampicillin. For the two-step phenol reaction, the cells were grown in LB at 37°C until an OD 600 of 2.0 was reached, and then the culture media was changed to fresh M9 with 1 mM aromatic compounds and various concentrations of phenol by mild centrifugation (1,000 × g, 5 min) (Kwon et al, 2020 ). After 15 h of incubation at 37°C, the fluorescence intensities of the cells were measured using a FACSAriaIII (BD Biosciences, Franklin Lakes, NJ, USA) with a blue laser source (488 nm) and an FL1 (530/30 nm) photomultiplier tube.…”

“…We previously reported a genetic enzyme screening system (GESS) consisting of DmpR as a phenol-dependent transcriptional regulator and green fluorescent protein (GFP) as a reporter protein (Choi et al, 2014 ; Kwon et al, 2020 ). The quantitative range of a genetic circuit relying on an enzyme and regulator could be adjusted by using an enzyme-inhibitor pair and regulator-antagonist pair.…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…81 These examples demonstrated the HTS of enzyme mutants enabled by the utilization of a TF-based genetic circuit. 82…”