Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production

Abstract

Background: Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield.

Results: Here, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ³²-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts.

Conclusions: The plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.

Keywords: Fluorescent proteins; Metabolic engineering; Microbial cell factory; Recombinant protein production; Synthetic biology.

Fig. 1

Genetic map of the pAG032 reporter plasmid (11,238 nt large) including representation of the control mechanisms of the three promoter/reporter pairs. The XylS/Pm regulator/promoter is inducible by benzoic acid derivatives such as m-toluic acid, while the PrpsJ and Pibpfxs promoters are responsive to intracellular concentrations of ppGpp and σ³², respectively. oriT, origin for conjugative transfer; bla, gene conferring ampicillin resistance; oriV, origin of vegetative replication; trfA; gene encoding an essential initiator protein TrfA; T, transcription terminators; hok/sok RNA, hok-sok suicide elements; Ps, σ⁷⁰-dependent constitutive promoter; xylS, gene encoding the positive transcriptional regulator XylS

Fig. 2

Transcriptional upregulation of PrpsJ in response to cell growth in various sub-lethal doses of chloramphenicol, measured as a correlation of PrpsJ-dependent expression of YFP activity and growth rate (a) or rplC transcript accumulation (b). The growth rates were calculated based on change in OD_600nm values measured every 1 h over a period of 3 h after adding chloramphenicol. OD_600nm normalized fluorescence values were calculated from measurements taken 3 h after adding chloramphenicol. RNA samples collection was performed after 15-min incubation of the recombinant cells in the presence of chloramphenicol. Relative gene expression (ΔΔCt) is presented as the level of rplC transcript in samples treated with chloramphenicol (2, 4, 8 μM) relative to its abundance in reference samples (0 μM). The data presented are from three independent biological replica (average ± SD)

Fig. 3

Time-course-activity measurements of YFP expressed from Pibpfxs in E. coli BW25113 (pAG032) cultivated in the presence of different concentrations (0, 4, 8, 16, 32 and 64 μg/ml) of azetidine. Incorporation of azetidine during protein synthesis promotes formation of abnormal proteins that tend to misfold and aggregate. The OD_600nm normalized fluorescence values were calculated based on the measurements taken every 1 h up to 4 h after addition of azetidine. The data presented are from three independent biological replicas (average ± SD)

Fig. 4

Recombinant cells were cultivated in minimal medium supplemented with glycerol (M63/glycerol), glucose (M63/glucose) and glucose + casamino acids (M63/glucose + CAA), to ensure different growth rates (Additional file 1: Table S1). The OD_600nm normalized fluorescence values were calculated based on measurements taken 3 h after a time point when all the cultures displayed approximate OD_600nm (~ 0.35). The data presented are from three independent biological replicas (average ± SD)

Fig. 5

Responsiveness of the XylS/Pm reporter unit during cultivation of E. coli BW25113 in M63 medium supplemented with either glycerol (M63/glycerol), glucose (M63/glucose) or glucose and casamino acids (M63/glucose + CAA). The OD_600nm normalized fluorescence values were calculated from measurements taken at the time point corresponding to 3 h after the induction. The OD_600nm of the samples at the point of induction was similar for all cultures despite differences in the growth rate. The data presented are from three independent biological replica (average ± SD)

Fig. 6

Responsiveness of the PrpsJ and XylS/Pm reporter units during cultivation of P. putida KT2440 under induced conditions (a) and the course of the Pibpfxs activity during cultivation at 42 °C (b). Data represent relative fluorescence levels shown as a ratio of OD_600nm normalized fluorescence values at different time points under conditions of induction or heat shock to the OD_600nm normalized fluorescence intensity of the reference cultures (R) growing in the absence of the inducer at 30 °C. The OD_600nm normalized fluorescence values were calculated based on measurements taken every 1 h up to 4 h after the induction and every 0.5 h up to 2 h after the temperature shift. The data presented are from three independent biological replica (average ± SD)

Fig. 7

Responsiveness of the Pibpfxs and XylS/Pm reporter units during cultivation of A. vinelandii OP (UW) under induced conditions (a) and the activity of PrpsJ ribosomal promoter after the addition of ammonium acetate (b). Data represent relative fluorescence levels expressed as a ratio of OD_600nm normalized fluorescence values at different time points under conditions of induction or ammonium acetate supplementation to the OD_600nm normalized fluorescence intensity of the reference cultures (R) growing in the absence of the inducer or ammonium acetate. The OD_600nm normalized fluorescence values were calculated based on measurements taken up to 24 h after the induction or ammonium acetate supplementation. The data presented are from technical triplicates (average ± SD)