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 (σ⁵⁴)-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 (σ⁷⁰) TFs. Here, we show that these unique characteristics of σ⁵⁴-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 σ⁵⁴ 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 σ⁵⁴-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.

Figure 1

Schematic depiction of the GESS and involved transcriptional factors in the host. Under stress conditions, (p)ppGpp can be synthesized by RelA/SpoT, which controls the expression levels of several transcription factors. Along with the target enzyme, the GESS consists of sigma 70 (σ⁷⁰, rpoD)-dependent DmpR/enzyme expression systems and a σ⁵⁴-dependent reporter expression system, including RNA polymerase (subunit α, β, β′ and ω corresponds to rpoA, rpoB, rpoC and rpoZ, respectively), SpoT (bifunctional (p)ppGpp synthase/hydrolase), RelA ((p)ppGpp synthase), σ⁵⁴ (sigma 54 factor, rpoN) and IHF (heterodimer subunits, ihfA and ihfB).

Figure 2

Fluorescence intensity of the GESS using various E. coli hosts. (a) Microscopic image of colonies harbouring the plasmid pGESS grown on LB agar plates containing 100 μM phenol; Con: control strain (DH5α) harbouring the pUC19 plasmid. Image processing and analysis were performed using Nikon’s NIS-Elements AR 4.2 software. (b) Fluorescence intensity of cells harbouring the pGESS grown on LB broth plates containing 100 μM phenol. (c) Microscopic image of colonies harbouring pGESS in BL21 and DH5α on LB agar plates.

Figure 3

Time-lapse cell growth and fluorescence intensity profiles of GESS with various culture media. (a) Cell growth and corresponding fluorescence intensity profiles of DH5α harbouring the pGESS grown in LB and M9 4 g/L glucose broth; “phenol” indicates that the broth contained 100 μM phenol. (b) Cell growth and fluorescence profiles of DH5α harbouring pGESS grown in various carbon sources in M9 broth containing 100 μM phenol.

Figure 4

Fluorescence intensity of colonies harbouring pGESS or pHCEIIB-egfp on LB and M9 4 g/L glucose agar plates. (a) Microscopic image of single colonies harbouring the pHCEIIB-EGFP on LB and M9 agar plates. Image processing and analysis were performed using Nikon’s NIS-Elements AR 4.2 software. (b) Fluorescence intensity and colony density profile. (c) Microscopic image of single colonies harbouring pGESS on LB and M9 agar plates containing 100 μM phenol. (d) Fluorescence intensity and colony density profile.

Figure 5

Cell growth and fluorescence intensity of GESS expressing TPL determined with a two-step reaction. (a) Fluorescence intensity of GESS in response to TPL activity at different cell states in LB broth. (b) Time-lapse cell growth and fluorescence intensity by the two-step reaction. The cells were cultured in LB broth until the late exponential growth phase as the first step, and the detection reaction was performed in M9 acetate medium with tyrosine as the second step. Circles represent the OD₆₀₀ value, and triangles represent the specific fluorescence intensity (GFP/OD₆₀₀).

Figure 6

Application of GESS to screen mutant TTPL using M9 minimal medium plates and the two-step reaction. (a) Microscopic image of colonies harbouring pGESS and mutant TTPL. Image processing and analysis were performed using Nikon’s NIS-Elements AR 4.2 software. (b) Normalized fluorescence intensity of mutant TTPLs by the GESS two-step reaction for screening high activity. Final candidates are represented as yellow circles. (c) Normalized enzyme activity of mutant TTPLs by heat inactivation and 4-AAP measurement for screening high stability.