- Open Access
Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production
© The Author(s) 2019
- Received: 20 March 2019
- Accepted: 26 April 2019
- Published: 7 May 2019
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.
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.
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.
- Fluorescent proteins
- Recombinant protein production
- Synthetic biology
- Metabolic engineering
- Microbial cell factory
Further advances in the field of recombinant protein production require continuous efforts concerning not only expression vector design but also rational genome engineering . Typical host-related aspects affecting recombinant protein production are related to the availability of energy and protein synthesis components comprising amino acids, free ribosomal subunits and other translational factors. Limitations related to such host parameters are commonly observed as reduced cell growth, plasmid loss and poor production yields . In many cases, though, it is difficult to predict and distinguish various host physiological effects, and there is a need for tools enabling high-throughput strain analysis in order to obtain more precise understanding of host-related responses related to overproduction of recombinant proteins [2, 3].
Depletion of amino acids is linked to the stringent stress response mediated by the guanosine tetraphosphate (ppGpp) alarmone, which among others down-regulates transcription of ribosomal protein (r-protein) and rRNA operons . Although its mode of regulation may differ among microbial species , in Gram-negative bacteria, ppGpp primarily interacts with RNA polymerase (RNAP), in synergy with the RNAP-binding transcriptional factor DksA, to directly affect transcription [6, 7]; and the ppGpp binding site regions are well conserved in representatives of the Gammaproteobacteria . In Escherichia coli, overproduction of recombinant proteins can lead to formation of inclusion bodies if there is an imbalance between the rates of protein synthesis and folding. Consequently, a σ32-related heat shock-like response is induced, which results in upregulation of the heat-shock proteins such as the chaperone DnaK [9–11]. Analysis of the rpoH genes encoding homologs of σ32 protein from the Alpha- and Gammaproteobacteria subgroups revealed sequence similarities that should reflect conserved function and regulation of σ32 in the heat-shock response . These data are consistent with further observations that expression of σ32 homologs from Serratia marcescens, Proteus mirabilis and Pseudomonas aeruginosa in E. coli mutant strain lacking its own σ32 leads to the transcriptional activation of the heat-shock genes from the start sites normally used in E. coli .
A reporter system for monitoring the heat-shock like response to recombinant protein production has been reported previously , as a σ32-dependent tandem promoter, Pibpfxs, generated by fusion of ibpAB and fxsA promoters. The ibpAB operon encodes two small heat shock proteins involved in resistance to heat and superoxide stresses  and FxsA is an inner membrane protein whose expression is reported to be strongly associated with accumulation of misfolded proteins in the cells . The Pibpfxs tandem promoter is characterised by a low basis level and strong transcriptional activation by the accumulation of aggregation prone proteins in inclusion bodies [11, 16].
Also, a fluorescence-based reporter system for the analysis of ribosome assembly was previously created and extensively characterized  and further used to measure the ribosome dynamics in E. coli .
We characterized applicability of the constructed reporter plasmid as a novel tool to analyse host responses relevant for recombinant protein production in E. coli. Moreover, the broad-host range properties of pAG032 were confirmed by performing additional analyses in two other biotechnologically relevant Gammaproteobacteria, Pseudomonas putida and Azotobacter vinelandii.
Design and construction of the broad-host-range reporter plasmid pAG032
As described above, the plasmid system was designed to analyze metabolic responses associated with availability of cellular resources utilized for recombinant protein production. We constructed the system (Fig. 1) by placing a gene encoding RFP under control of the Pm promoter positively regulated by XylS transcriptional factor. XylS/Pm has been widely used for regulated and high-level recombinant protein production in E. coli and other Gram-negative bacteria , and among all functional elements of the reporter plasmid RFP then serves as an indicator of the target protein production. In addition to inducible XylS/Pm, we used two stress-responsive promoters, PrpsJ and Pibpfxs. The expression of yfp is driven by the PrpsJ, which is significantly upregulated in response to optimal nutrient conditions and deficiencies of ribosomal components. Finally, expression of cfp relies upon the σ32-dependent Pibpfxs promoter  responding to heat shock and misfolded proteins. Transcriptional terminators were placed downstream of each reporter gene to avoid problems related to transcriptional read-through . The pAG032 plasmid is based on the broad-host-range RK2 minimal replicon thereby ensuring that the plasmid is applicable in many different Gram-negative bacterial species . The minimal RK2 replicon consists of the origin of vegetative replication (oriV) and its cognate trfA gene encoding TrfA replication initiator protein [19, 25]. The copy number of the plasmid is 5–7 per genome, and this number can easily be changed to different numbers up to above 100 copies per genome by replacing trfA with available mutant versions of trfA gene . The latter option could be valuable to monitor metabolic burden and other responses associated with increased recombinant protein production and increased plasmid copy numbers. The pAG032 further contains oriT enabling conjugative transfer and the hok-sok suicide elements previously demonstrated to ensure segregational stability of RK2-based plasmid also under high-cell density cultivations .
Plasmids used in this study
cfp under control of PLtetO-1, yfp under control of PLlacO-1, rfp under control of Plac/ara-1, Kanr
ibpfxs::lucA reporter unit, Ampr
RK2 replicon, trfA gene, XylS/Pm regulator/promoter system, Ampr
RK2 replicon, trfA gene with cop-271 mutation, XylS/Pm regulator/promoter system, hok-sok suicide system, Ampr
RK2 replicon, trfA gene, hok-sok suicide system, Ampr
pAG001 plasmid with rfp under control of XylS/Pm, Ampr
pAG001 plasmid with cfp under control of ibpfxs, Ampr
pAG001 plasmid with yfp under control of rpsJ, Ampr
pAG001 plasmid with rfp under control of XylS/Pm and yfp under control of rpsJ, Ampr
pAG001 plasmid with rfp under control of XylS/Pm and cfp under control of ibpfxs, Ampr
pAG001 plasmid with cfp under control of ibpfxs and yfp under control of rpsJ, Ampr
pAG001 plasmid with rfp under control of XylS/Pm, cfp under control of ibpfxs and yfp under control of rpsJ, Ampr
Changes of yfp expression controlled by PrpsJ promoter in E. coli cells exposed to sub-lethal concentrations of translation-inhibiting antibiotic chloramphenicol
In order to confirm that chloramphenicol treatment affects transcriptional upregulation of ribosomal protein genes, we measured transcription levels of the chromosomal rplC gene, which is part of the rpsJ (S10) operon in E. coli and encodes the 50S ribosomal subunit protein L3. The rplC transcript level was measured by quantitative PCR in RNA samples extracted from untreated E. coli cells and cells cultivated for 15 min after addition of increasing doses (2, 4 and 8 μM) of chloramphenicol . The results showed that rplC transcript levels increased with increasing concentrations of chloramphenicol (Fig. 2b), in agreement with the YFP activity results (Fig. 2a).
Elevated expression of cfp from Pibpfxs in cells exposed to azetidine is likely caused by protein misfolding and aggregation
Simultaneous fluorescence measurement of reporter proteins YFP and CFP in cells cultivated in different growth media indicates host effects related to ribosome synthesis rates and σ32-mediated responses
Inducible expression of rfp from XylS/Pm varies under different growth conditions
Plasmid pAG032 functions in other Gammaproteobacteria, Pseudomonas putida and Azotobacter vinelandii, indicating its potential as a broad-host-range tool
The broad-host range properties of the XylS/Pm system have been well documented, and importantly the inducer, m-toluate, enters cells via passive diffusion and most bacteria cannot metabolize it . The PrpsJ and Pibpfxs promoters of pAG032 as well as the reporter genes used are not well studied in species other than E. coli. Therefore pAG032 was transformed to Pseudomonas putida KT2440 (TOL plasmid cured strain) and to Azotobacter vinelandii OP (UW) to explore its broad-host range potential. These two organisms were selected as relevant model hosts due their biotechnological applications for production of recombinant proteins and biopolymers [37, 38].
The advent of efficient and precise genome engineering tools such as CRISPR/Cas9 significantly accelerated the traditional endonuclease- and recombineering-based strain development processes useful for metabolic engineering [40, 41]. According to Mahalik et al.  engineering of bacterial hosts for improved capability for recombinant protein production requires removing translational bottlenecks and redirecting the metabolic flux away from biomass formation towards target protein synthesis. Moreover, the engineered cells should be characterized for their ability to efficiently fold and export proteins in order to prevent aggregation of the newly synthesized proteins inside the cytoplasm. In this work, we created the reporter system pAG032 that allows monitoring of translational capacity of bacterial cells and stresses related to protein misfolding. Our results confirmed that the activity of the PrpsJ ribosomal promoter of pAG032 corresponded to transcriptional upregulation of the chromosomal ribosome synthesis gene rplC in E. coli. Furthermore, by combining the PrpsJ ribosomal promoter reporter with the well-characterized σ32-dependent Pibpfxs on pAG032, we enabled monitoring of heat shock-like stress responses that may result from genome engineering aiming solely for improved translational capacity. The responsiveness of the Pibpfxs promoter controlling expression of cfp reporter gene was confirmed in our study by analysis of CFP fluorescence in cell cultures cultivated in a presence of the proline analogue azetidine. The activity of these two stress-responsive reporter units was further quantified by using different growth conditions of the recombinant E. coli cells. As PrpsJ-dependent YFP fluorescence intensity was monitored during the exponential growth phase with nutrient concentrations sufficient for steady growth rate [10, 18], the activity of this ribosomal reporter unit increased in correspondence to the increased growth rate. These results are in agreement with previously reported data indicating a positive correlation of ribosomal protein synthesis with growth rate in E. coli . Our results using A. vinelandii as an alternative host also showed the expected growth-related expression of YFP from PrpsJ. Moreover, it has previously been proposed that high bacterial growth rate promotes increased rates of cellular protein synthesis, which may become a burden to the protein folding machinery [34, 35]. This may explain why the expression of cfp from σ32-dependent Pibpfxs promoter also increased in response to elevated growth rate of the E. coli cells.
The third reporter unit of pAG032 is the inducible XylS/Pm system controlling expression of RFP, and we chose its non-mutated version previously shown to display very low background expression to ensure tightly regulated and high-level induced expression of, in principle, any target protein using pAG032 . Contrary to YFP and CFP, expression level of RFP from the XylS/Pm promoter decreased with the increasing growth rate. According to a model proposed by Klumpp et al. , the concentrations of constitutively expressed regulators is reduced with increased cell growth rate, mainly because of the increase of cell volume. It was plausible to assume that such effects can explain our results with RFP.
We constructed the reporter system to monitor product formation under different growth conditions by using the XylS/Pm promoter system, and we demonstrated that this expression cassette can be used in combination with the two other stress-responsive reporter units. As we see it, expression of the individual reporter genes used in this study is likely not independent. For example, a dependence is indicated by the fact the σ32-mediated heat shock response may induce production of ppGpp [45, 46] leading to the PrpsJ inhibition or by influence of inducer on upregulation of stress-responsive promoters (Additional file 1: Figure S1). Still, even under the assumption that recombinant expression of YFP and CFP may impose some stress to the host cells, in addition to the stress caused by inducible expression from XylS/Pm, the reporter system can discriminate between the effects dependent on different strain background during comparative studies. Such analysis of variation in expression of three interdependent reporter units in a subset of different genetic variants of E. coli has been previously demonstrated by Cardinale et al. .
One important aim was to design and construct pAG032 with promoters and a replicon suitable for broad-host range applications, and we were able to demonstrate its usefulness and detect activity and functionality of all promoters and concomitant reporter proteins in P. putida and A. vinelandii both representing the Gammaproteobacteria subgroup. The detected expression levels of the reporter proteins in A. vinelandii were low compared to those obtained in E. coli and P. putida, possibly due to non-optimal codons of reporter genes in this host. Moreover, plasmid derivative pAG028, containing the two promoter/reporter cassettes PrpsJ/yfp and Pibpfxs/cfp, can also possibly prove to be useful for analysing host effects in combination with overproduction of any target protein of interest expressed from a separate compatible expression vector and in any Gram-negative species.
In this study, we created a novel reporter system suitable for monitoring of native stress responses and translational capacity in E. coli as well as in other Gram-negative bacteria. The system is based on individual expression of three different fluorescent proteins from three different promoters, which makes it useful for high-throughput screening accompanying effective strain development studies and for on-line monitoring of host parameters during cultivation. Characterization of cells in terms of the stringent and heat shock-like stress responses should allow for selection of strains displaying improved translational capacity and ability to synthesise correctly folded proteins.
Strains, media and growth conditions
Escherichia coli K-12 BW25113  served as an expression host for studying the responsiveness of the reporter system. E. coli DH5α was used for cloning purposes. P. putida KT2440 (TOL plasmid cured derivative ) and A. vinelandii OP (UW)  were utilized as alternative host organisms. E. coli and P. putida were routinely grown at 37 °C or 30 °C, respectively, with 225 rpm shaking in liquid LB medium or on solid LB plates, unless stated otherwise. The M63 medium used in this study was composed as described by others  and supplemented with 0.2% (w/v) glycerol or 0.2% (w/v) glucose and/or 0.1% (w/v) Casamino acids. A. vinelandii was routinely grown in liquid Burk’s medium (pH 7.2) at 30 °C, 225 rpm shaking . For plasmid selection in E. coli, P. putida and A. vinelandii, ampicillin was used at concentrations 100 μg/ml, 500 μg/ml and 25 μg/ml, respectively.
Oligonucleotide primers used in this study
Sequence (5′ → 3′)
a) PCR primers:
b) qPCR primers:
Expression studies and activity measurement of the reporters
Recombinant E. coli, P. putida and A. vinelandii strains were grown over-night in 2-10 ml of adequate liquid medium with ampicillin. Afterwards, 20 ml of the fresh medium with the antibiotic were inoculated with the overnight culture to an initial OD of 0.05 measured at 600 nm for E. coli and P. putida and at 660 nm for A. vinelandii. Following incubation in shake flasks, the XylS/Pm-mediated protein expression was induced in exponential phase (OD = 0.4–0.6) by adding m-toluic acid to a final concentration of 1 mM for E. coli and P. putida and 0.5 mM for A. vinelandii. Heat shock was applied by transferring the cultures of P. putida from 30 °C to 42 °C, 225 rpm shaking, at OD600 = 0.3–0.4. In order to reduce intracellular levels of ppGpp, 10 mM ammonium acetate was added to the cultures of A. vinelandii at OD660 = 0.3–0.4.
Fluorescence measurements were performed with the Tecan Infinite M200 (Tecan, Männedorf, Switzerland) plate reader. Fluorescence intensity was determined directly from the cultures (M63, Burk’s medium) or after resuspension in PBS (LB medium) in order to reduce background signal. The following fluorescent filter setup was used for the detection:  CFP excitation: 433 nm; emission: 475 nm;  YFP excitation: 505 nm; emission: 538 nm;  RFP excitation: 580 nm; emission: 615 nm. The gain was set to 100 for RFP and 80 for CFP and YFP.
Transcript analysis by qPCR
Following adequate incubation in shake flasks, 3 ml of cell cultures were stabilized with RNAlater stabilization solution (Qiagen). The subsequent total RNA isolation was achieve using RNAqueous® Total RNA Isolation Kit (Ambion). RNA samples were treated with TURBO™ DNase (Ambion) and used for cDNA synthesis with First-Strand cDNA Synthesis Kit (GE Healthcare Life Sciences). Two-step qPCR with the power PowerSYBR® Green PCR Master Mix (Applied Biosystems) in QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) was used for quantification of cysG and rplC transcripts. All steps were performed as described by the manufacturers. PCR cycles were 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s; 62 °C for 1 min) and one additional stage of amplification to generate a melt curve. Results were analyzed with QuantStudio™ Design & Analysis Software (Applied Biosystems) and data were normalized as described previously . Primer pairs used during amplification are listed in Table 2. Efficiency was verified for each pair of the primers (Table S2). Transcript generated from the cysG gene was used for normalization .
Most experimental work was done by AG, with input from KB and SH. HE, MI and PN has been involved in planning and discussions throughout, and the work has been leaded by TB together with AG. All authors contributed actively to writing of manuscript. All authors read and approved the final manuscript.
We are grateful to Rahmi Lale for helpful discussions and advice.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its additional files.
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This work was supported by the ERASysAPP project LeanProt financed by Research Council of Norway (257147), German Federal Ministry of Education and Research and Estonian Research Council.
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