Tunable Control of an Escherichia coli Expression System for the

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Tunable control of an Escherichia coli expression system for the overproduction of membrane proteins by titrated expression of a mutant lac repressor Seong Keun Kim, Dae-Hee Lee, Oh Cheol Kim, Jihyun F Kim, and Sung Ho Yoon ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017

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ACS Synthetic Biology

Tunable control of an Escherichia coli expression system for the overproduction of membrane proteins by titrated expression of a mutant lac repressor Seong Keun Kim, Yoon*,∥

†,‡,#

Dae-Hee Lee,

†,‡,#

†

Oh Cheol Kim, Jihyun F. Kim,§ and Sung Ho

†

Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea ‡ Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea §Department

of Systems Biology, Yonsei University, Seoul 03722, Republic of Korea Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea ∥

ACS Paragon Plus Environment

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ABSTRACT Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operatorbased expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of L-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

KEYWORDS: lac repressor, mutant LacI, Escherichia coli, membrane protein, expression system

GRAPHICAL ABSTRACT


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■ INTRODUCTION Membrane proteins are involved in a variety of essential cellular processes, including transport of nutrients and metabolites across the cellular membrane, maintenance of cell morphology, and cell-to-cell communication such as signal transduction.1 Malfunctioning of integral membrane proteins has been often associated with numerous human diseases, and membrane proteins are targeted by about 70% of current drugs.2 Despite their physiological significance, structure-based functional studies of membrane proteins lag behind that of their soluble counterparts principally because of their low abundance in native host cells.3 Therefore, heterologous expression is the primary route to produce sufficient amounts of membrane proteins for biophysical and structural studies. Escherichia coli has been widely used as a production host for both globular and membrane proteins because of its fast growth,4 ease of genetic manipulation and cultivation 5, and high yields of many heterologous proteins.6,7 However, overexpression of membrane proteins in E. coli stimulates a cascade of deleterious events that ends in cell death and extremely low protein yields.8,9 Moreover, most of the protein expression systems for E. coli are leaky, even when the heterologous protein expression is not induced. Because leaky expression of toxic membrane proteins leads to transformation toxicity, it is difficult to obtain transformants.10,11 Leaky expression is problematic for E. coli BL21(DE3) cells harboring a series of T7 promoter-based plasmids which are widely implemented systems for the overexpression of heterologous proteins. This expression system was originally developed for the overproduction of soluble proteins.12-14 Protein overexpression in BL21(DE3) cells is governed by the bacteriophage T7 RNA polymerase (RNAP), which recognizes the T7 promoter that regulates the expression of the target protein in the plasmid and elongates transcripts much faster than E. coli RNAP.13,14 The principle behind the use of BL21(DE3) cells with T7 promoter-mediated plasmids is the more mRNA is synthesized, the more protein can be produced. However, this simple logic does not work for most membrane proteins. The overexpression of membrane proteins is usually toxic to BL21(DE3) cells, mainly because of the adverse effects of saturation of membrane protein biogenesis and protein secretion machinery.15 Developing a rational approach to membrane protein overproduction is challenging, as membrane protein biogenesis is an intricate multistep process that requires coordinated and balanced transcription, translation, folding, and insertion into membranes of host cells.1618 Miroux and Walker isolated spontaneous mutants of E. coli BL21(DE3) cells, named Walker strains C41(DE3) and C43(DE3), by expressing a particular membrane protein in BL21(DE3) cells in the presence of an inducer and selecting cells that survived and thus could cope with the toxic effects of membrane protein overexpression.7 The C41(DE3) and C43(DE3) strains are commonly employed to overproduce a wide array of heterologous membrane proteins. However, the mechanism underlying their improved capacity for membrane protein overproduction is not fully understood. Wagner et al. found that a mutation in the lacUV5 promoter, which reduced the activity of T7 RNAP in strains C41(DE3) and C43(DE3), was responsible for improved membrane protein overproduction. To mimic this effect, they developed Lemo21(DE3), a BL21(DE3) derivative, that controlled the activity of T7 RNAP by titratable expression of its natural inhibitor, T7 lysozyme.19 Lemo21(DE3) overcame the problem of leaky expression in T7 RNAP-based protein expression systems and showed improved target protein yields, especially for membrane proteins. However, use of Lemo21(DE3) cells is restricted to T7 RNAP expression systems and cannot be applied to numerous other expression systems that use endogenous E. coli RNAP. The same is true for a recently developed tunable recombinant expression system.20


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Recently, we sequenced the C41(DE3) and C43(DE3) genomes and found genetic mutations responsible for a reduction in cellular toxicity caused by membrane protein overexpression that led to an enhanced capacity for membrane protein overproduction.21 Among the mutations detected, one occurring in the lac repressor (lacI) gene in the λDE3 chromosomal region was critical to membrane protein overproduction. Although the LacI variant (LacIV192F, referred to as mLacI hereafter) can bind to the lac operator (lacO) site, mLacI cannot bind to inducer molecules such as isopropyl β-D-1-thiogalactopyranoside (IPTG) and cannot transmit allosteric signals to the DNA binding domain.21,22 Therefore, upon addition of an inducer, the accumulation of mLacI considerably reduced T7 RNAP expression, leading to reduced toxicity from membrane protein overexpression.21 In this study, we developed a tunable bacterial expression system for overproduction of membrane proteins through controlled expression of mLacI upon addition of L-rhamnose as an inducer. Performance of the tunable mLacI expression system was evaluated in conjunction with commonly used lac operator-based expression vectors that employ the T7 or tac promoter. The novel expression system was successfully applied to overproduce three membrane proteins, E. coli cytosine transporter protein (CodB), bovine 2-oxoglutarate carrier protein (OGCP), and E. coli F-ATPase subunit b (Ecb), which have been reported to be difficult to produce in E. coli strains.7,23

■ RESULTS AND DISCUSSION Construction of the tunable mLacI expression system Our basic premise here is that T7 RNAP activity for the expression of a target gene can be controlled by balancing the speed of transcription and translation. A transcriptional burst in response to high activity of T7 RNAP leads to desynchronization of transcriptiontranslation.7,24 In the case of overexpression of membrane proteins, the high transcription rate of T7 RNAP leads to the accumulation of membrane proteins in cytoplasm that cannot be translocated into the host membrane, resulting in saturation of the Sec translocon and inefficient ATP synthesis.19 To address this, we designed a dual control expression system in which the transcriptional rate of a target gene is modulated by RNAP activity that can be controlled by the amount of L-rhamnose added to the culture medium. We chose an Lrhamnose inducible promoter (PrhaBAD) for the tunable expression of mLacI, as it offers tight control of gene expression with negligible background expression.25 In contrast to the all-ornone response to IPTG-inducible promoters, protein expression from PrhaBAD is highly correlated with the concentration of L-rhamnose and is homogenous throughout the cell population.25 In addition, L-rhamnose, a naturally occurring deoxy-hexose, is harmless to bacterial hosts, unlike IPTG or anhydrotetracycline, which are toxic to cells.26 Although the E. coli PrhaBAD promoter has been used for production of some membrane proteins,25 Hjelm et al. recently reported that tunability of protein production using PrhaBAD promoter was attributed to L-rhamnose consumption rates rather than production rates.27 To overexpress membrane proteins, they had to construct an L-rhamnose transport and catabolism deficient double mutant to regulate protein production rates in an L-rhamnose concentration-dependent manner. In this study, we aimed to provide an auxiliary plasmid that can simply be added to existing expression systems. We constructed pAR-mLacI plasmid containing the mLacI gene (mlacI) that was placed under the control of the PrhaBAD promoter on the low-copy plasmid pACYCDuet-1 carrying the p15A replicon (Figure 1A). Thus, the pAR-mLacI is fully compatible with a variety of T7 promoter-based pET plasmids containing a ColE1 or pMB1 origin. For comparison, we also constructed the pAR-wtLacI plasmid expressing the wildtype (wt) lacI gene under the control of PrhaBAD on pACYCDuet-1.


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Performance of the mLacI titratable system in conjunction with commonly used expression vectors To evaluate the performance of the mLacI titratable expression system with commonly used E. coli expression vectors, a kinetic analysis of target expression and cell growth was performed using different concentrations of L-rhamnose. To this end, a green fluorescent protein (GFP) gene28 was inserted into two different plasmids, a high-copy plasmid pMW7 under the control of the T7 promoter and T7 RNAP (pMW-GFP) and a medium-copy plasmid pMAL-c2X using the tac promoter and E. coli RNAP (pMH-GFP) (Figure 1B). Plasmid pMW-GFP does not have lacO downstream of the promoter, whereas pMH-GFP does. Each of the constructed plasmids was transformed into E. coli BL21(DE3). To verify control of the mLacI titratable expression system over the target gene on the T7 promoter-based and lacO-free plasmids, pAR-mLacI or pAR-wtLacI was transformed into E. coli BL21(DE3) cells harboring pMW-GFP (Figure 2A). E. coli cells containing the dual plasmids were induced by varying the L-rhamnose concentration (ranging from 0 to 4 mM) and maintaining a fixed IPTG concentration of 0.7 mM to remove repression by the endogenous wild-type LacI protein, and then were monitored for growth and fluorescence for 5 h at 37°C. When L-rhamnose was added to the culture medium at varitions, GFP production from pMW-GFP varied accordingly in cells containing pAR-mLacI (Figure 2B) and was not changed in those containing pAR-wtLacI (Figure 2C). As expected, temporal expression of GFP showed a negative correlation with L-rhamnose concentration, and its maximum was 2.7-fold higher than the minimum in the 5 h after induction. Similarly, GFP overexpression from the high-copy plasmid pMW-GFP in the absence of L-rhamnose was also associated with reduced growth and therefore fluorescence after 2 h (Figure 2B). The reduced growth seems to be GFP overexpression which is toxic to E. coli host.29 However, this growth defect disappeared when the high-level GFP expression was modulated by mLacI. The mLacI regulation system was tested for its potential to be broadly applied to any lacO-based expression system such as those employing T7, T5, trc, tac, lacUV5, and lac promoters. As for the mLacI expression system in conjunction with expression plasmids based on lacO and tac promoters, pAR-mLacI or pAR-wtLacI was transformed into E. coli BL21(DE3) cells harboring pMH-GFP (Figure 3A). The same experiment described above was performed. Temporal GFP expression level varied by concentration of L-rhamnose not only in cells containing mLacI (Figure 3B) but also in those containing wtLacI (Figure 3C). However, GFP expression levels through time were distinct with mLacI expression at different concentrations of L-rhamnose. Cell growth was almost the same in all cases. The effect of mLacI on GFP expression was more pronounced in lacO/tac-based pMH-GFP than in T7 promoter-based pMW-GFP, in terms of the discernable temporal GFP expression level and its dynamic range. Remarkably, GFP expression from pMH-GFP was almost completely repressed when mLacI was induced with 4 mM L-rhamnose, indicating tight basal control of target expression by the mLacI titratable expression system. The exceptional dose-dependent response of the mLacI inducible expression system in conjunction with lacO-based pMHGFP can be explained by the fact that pMH-GFP has a lacO site for the target gene and the tac promoter is not dependent on T7 RNAP. BL21(DE3) cells express wtLacI from the endogenous lacI in DE3 and lac operon regions and the pMH-GFP plasmid contains additional lacI with an enhanced promoter (lacIq) (Figure 1B).30 Considering wtLacI is abundant in the host cells, it is remarkable that relatively small amount of mLacI can finely tune for GFP expression. Cell-to-cell homogenous gene expression mediated by the mLacI titratable expression


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Inducible bacterial expression systems often show all-or-none induction of gene expression. At sub-saturating levels of an inducer, expression levels vary greatly within the population of genetically identical cells, resulting in two subpopulations: one fraction that is fully induced and the other fraction that is not induced at all.31,32 To investigate the effects of mLacI expression on cellular homogeneity and gene expression stability, we performed a flow cytometric analysis to measure wtLacI- or mLacI-mediated GFP expression in individual cells (Figure 4). The analysis revealed that cell-to-cell GFP expression from pARmLacI/pMH-GFP and pAR-mLacI/pMW-GFP was homogenous at all test concentrations of L-rhamnose, which ranged from 0 to 4 mM. Remarkably, in overall, GFP expression from pMH-GFP showed symmetrically distributed fluorescent intensities, and the distribution curve tended to have a greater width and lower height at increased L-rhamnose concentrations (Figure 4A). Similar GFP expression patterns were observed for cells containing pMW-GFP, although the distribution curves were less distinct between the different concentrations of Lrhamnose (Figure 4B). These results indicate that the homogeneous expression of a target gene over a wide expression range can be controlled by titratable expression of mLacI. In our GFP overexpression protocols, mLacI expression was tuned by various Lrhamnose concentrations from an initial inoculation, then GFP was induced with IPTG when the cell density reached 0.4–0.7 in OD600. In these conditions, the GFP production rates were dependent on L-rhamnose concentrations (Figures 2B and 3B). It should be mentioned that the genetic background of host cells relating L-rhamnose metabolism is carefully considered to ensure homogenous and tunable protein expression.27 Comparison with another expression system based on the modulation of T7 RNAP activity Performance of the mLacI titratable system was compared to that of Lemo21(DE3) system, which was developed specifically for membrane protein production.19 In Lemo21(DE3), T7 RNAP activity can be modulated by the titrated expression of its natural inhibitor, T7 lysozyme (T7LysY), from the pLemo plasmid, which is induced by L-rhamnose. Both the mLacI and T7LysY inducible systems modulate T7 RNAP activity; however, regulation occurs at the transcriptional and posttranslational level, respectively. It should be noted that use of the Lemo21(DE3) is limited to T7 RNAP-based expression systems and cannot be applied to other lacO-based expression systems that use endogenous E. coli RNAP. For comparison, Lemo21(DE3) was transformed with T7 promoter-based pMW-GFP. A kinetic analysis of Lemo21(DE3) containing pMW-GFP was performed with varying concentrations of L-rhamnose (0 to 4 mM) and a fixed IPTG concentration of 0.7 mM (Figure 5). Compared to BL21(DE3) cells containing the plasmids pAR-mLacI and pMW-GFP (Figure 2), the dynamic expression range (max/min) of Lemo21(DE3) was similar. However, the GFP expression levels at different concentrations of L-rhamnose were inconsistent at the various sampling time points. The negative correlation between L-rhamnose concentration and fluorescence intensity was less, and the extent was different at various times after induction (Figure 5). The tighter control of GFP expression in the mLacI inducible system can be attributed to repression of T7 RNAP at the transcription level, rather than through timeelapsed posttranslation. The mLacI titratable expression and Lemo21(DE3) systems employ different strategies to tune the transcription of target genes. Enhanced production of membrane proteins We next explored the performance of our system in expressing three membrane proteins, E. coli cytosine transporter protein (CodB), bovine 2-oxoglutarate carrier protein (OGCP), and E. coli F-ATPase subunit b (Ecb). For each of these membrane proteins, we carefully chose E. coli host strains based on previous attempts to express these proteins. For example, CodB


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was hardly produced in E. coli K-12,23 and historically, when OGCP was expressed in BL21(DE3), most of the host cells died.7 From the BL21(DE3) survivors expressing OGCP, C41(DE3) cells were screened to successfully produce toxic membrane proteins, and C43(DE3) cells were screened from C41(DE3) cells expressing Ecb.7 To measure temporal expression levels by whole-cell fluorescence, each target gene was inserted into the pMHGFP or pMW-GFP plasmid to generate target proteins fused with GFP at their C-terminal. Then, the resulting plasmids was transformed into the host strain harboring pAR-mLacI. Fluorescence from the strains harboring the dual plasmids was monitored at 37°C for 6 h post induction, with 0.7 mM IPTG and various concentrations of L-rhamnose. The GFP fusion strategy has long been successful in functional and structural studies of various membrane proteins as it allows direct and convenient monitoring and visualization of membrane proteins of interest.27,33-38 In this study, we used the GFP fusion to analyze real-time kinetics of membrane proteins, which can further determine the optimal expression condition for each of the tested membrane proteins. For CodB production, pMH-CodB-GFP was transformed into E. coli K-12 MG1655 harboring pAR-mLacI. As expected, without the addition of L-rhamnose, the engineered strain showed a nearly 50% reduction in cell growth at 6 h post induction with 0.7 mM IPTG. Cell growth was restored by expression of mLacI induced with 1 or 4 mM L-rhamnose (Figure 6A). The highest level of CodB production was obtained with the addition of 4 mM L-rhamnose, which was 4.5-fold the level observed in a culture without L-rhamnose (Figure 6B). Because severe growth inhibition was observed with CodB overexpression, we performed a flow cytometric analysis after 24 h of cultivation. As expected, almost all of the cells (89%) lost florescence in the absence of L-rhamnose, whereas higher L-rhamnose concentrations (0.25, 1, and 4 mM) resulted in homogenous cultures with all cells expressing the CodB-GFP fusion protein (Figure 6C). In contrast, suboptimal concentrations of Lrhamnose led to two subpopulations: fluorescent cells that expressed the CodB-GFP fusion protein and nonfluorescent cells that did not express any fusion protein. Interestingly, the flow cytometry analysis indicated that cell size (forward scatter, FSC) greatly increased with 4 mM L-rhamnose (Figure 6D). Further, microscopy of two cell populations induced with 4 mM L-rhamnose confirmed that filamentous cells were associated with cell division failure (Figure 6E). Membrane localization of CodB-GFP was observed in these filamentous cells, as well as in normal cells (Figures 6E and 6F). This filamentous growth phenotype was also observed with the overexpression of other membrane proteins.15 For OGCP expression, pMH-OGCP-GFP was transformed into E. coli BL21(DE3) cells harboring pAR-mLacI or pAR-wtLacI. In cells harboring pAR-mLacI, GFP production decreased and cell growth increased with increasing L-rhamnose concentrations (Supplementary Figure S1A). In contrast, in cells harboring pAR-wtLacI, GFP production was not discernable, and a cell growth defect was evident at all tested L-rhamnose concentrations (Supplementary Figure S1B). As for Ecb expression, C41(DE3) cells were transformed with pAR-mLacI and pMHEcb-GFP (Supplementary Figure S2). The Ecb-GFP fusion protein was highly expressed in C41(DE3) pAR-mLacI/pMH-Ecb-GFP without any L-rhamnose. These examples strongly suggest that titrated expression of mLacI decreases the toxicity of the membrane protein overproduction and is versatile in terms of the host strain and expression vector used. As the productivity of target proteins is proportional to the final cell density, it would be helpful to separate the growth and production phases by controlling L-rhamnose in culture media. For example, high cell density can be obtained by keeping Lrhamnose concentration high to block promoter leakiness, which is then transferred to production media containing optimum concentration of L-rhamnose to derepress T7 RNAP. Overcoming transformation toxicity by the mLacI expression system


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Leaky expression of heterologous proteins is a major problem in most protein expression systems using E. coli. Leaky expression of toxic membrane proteins results in transformation toxicity and difficulty in obtaining transformants.10,11 To examine whether our system could reduce the transformation toxicity, we transformed the pMW-GFP plasmid into BL21(DE3) strain harboring the pAR-mLacI and plated on LB solid medium supplemented with 0 or 4 mM L-rhamnose for mLacI induction. In the absence of L-rhamnose, most colonies grown on the LB agar medium colored green under visible light, which indicates the severe leaky expression of GFP (Supplementary Figure S3A). In contrast, mLacI induction with 4 mM Lrhamnose completely repressed the leaky expression by visual inspection. To test transformation toxicity of a membrane protein, we chose E. coli F-ATPase subunit b (Ecb) encoded by atpF, as this membrane protein that made it difficult to obtain BL21(DE3) transformants.21 BL21(DE3) cells harboring the pAR-mLacI were transformed with the pMW-Ecb-GFP plasmid and were plated on LB agar medium supplemented with 0 or 4 mM L-rhamnose (Supplementary Figure S3B). While most colonies were small in the absence of L-rhamnose, uniform and large colonies were formed at 4 mM L-rhamnose. These examples demonstrate that the titrated mLacI expression can overcome the transformation toxicity caused by leaky expression of the toxic proteins.

■ CONCLUSIONS Mass production of membrane proteins is hard to achieve because of the poor solubility, low expression levels, incorrect folding, and cellular toxicity of these proteins. In this study, we developed a new method to construct a robust production host tolerant of toxic membrane protein overexpression. We showed that the titrated expression of mutant LacI could be mediated by the addition of the small molecule inducer L-rhamnose and provide dynamic control of the expression levels of a target gene and homogenous gene expression in a cell population. Importantly, the mLacI inducible expression plasmid can be applied to commonly used expression systems based on both T7 RNAP and endogenous E. coli RNAP. The ease of use and versatility of this expression system make it useful for metabolic engineering and synthetic biology purposes.

■ METHODS Strains, plasmids, and culture conditions E. coli strains and plasmids used in this study are summarized in Table 1. Cells were grown aerobically in low-salt LB medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl). The medium was supplemented with ampicillin (100 µg/ml) if the cells contained pMW7- or pMH7-derived plasmids and with chloramphenicol (30 µg/ml) if the cells harbored pACYCderived plasmids or pLemo. Cell growth was measured at OD600 with an Optizen POP spectrophotometer (Mecasys, Daejeon, Republic of Korea). In all culture conditions, target genes were induced by the addition of 0.7 mM IPTG at a cell density of 0.4–0.7 in OD600. Various concentrations of L-rhamnose were added at the beginning of each culture. Construction of tunable mLacI expression plasmids Primer sequences are listed in Supplementary Table S1. To construct plasmids expressing wild-type lacI or lacIV192F under the control of an L-rhamnose-inducible promoter, a portion of the rhamnose promoter region was amplified from K-12 MG1655 genomic DNA using RhamL-F/RhamL-R primers and then cloned into the NdeI and XhoI sites of pACYCDuet-1 (Merck Millipore). This low-copy plasmid was designated pAR. Wild-type lacI or lacIV192F


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was PCR-amplified from BL21(DE3) genomic DNA using lacI-F/lacI-R primers and C43(DE3) genomic DNA using lacI-F/lacUV5-R primers, and then these products were inserted into the NdeI and NarI sites of the pAR plasmid, generating pAR-wtLacI and pARmLacI, respectively. Cloning of membrane proteins Three proteins, GFP, cytosine permease, and E. coli F-ATPase subunit b were used to investigate our tunable lacIV192F regulation system. For production of mildly toxic GFP, gfp was PCR-amplified from pMW(Ecb-GFP) using frGFP-F/frGFP-R primers.21 The forward primer added an NdeI site, and the reverse primer added a HindIII site. The purified PCR product was digested and subsequently cloned into pMW(Ecb-GFP) and pMAL-c2X that were digested with NdeI and HindIII. The resulting plasmids were designated pMW-GFP and pMH-GFP, respectively. E. coli F-ATPase subunit b and cytosine permease were produced as GFP fusions from a pMW7-derived plasmid and pMAL-c2X. E. coli codB was amplified from K-12 MG1655 genomic DNA using codB-F/codB-R primers and subsequently ligated into pMW(Ecb-GFP) digested with NdeI and BamHI, which replaced atpF with codB, producing pMW-CodB-GFP. For pMH-CodB-GFP plasmid construction, the pMW-CodBGFP plasmid was digested with NdeI and BamHI, and the CodB-GFP fragment was subsequently cloned into pMAL-c2X digested with NdeI and HindIII. For E. coli F-ATPase subunit b overexpression, pMW(Ecb-GFP) was used as previously described.21 GFP fluorescence detection and flow cytometry analysis Whole-cell GFP fluorescence was determined using a fluorescence plate reader as previously described.21 Briefly, 500 µl of cells was centrifuged at 13,000 rpm for 5 min and then resuspended in 500 µl of phosphate-buffered saline (PBS). One hundred microliters of the resuspensions were transferred to 96-well Black Optiplate 96F plates (Perkin Elmer, Waltham, MA, USA), and whole cell fluorescence was detected using a Fusion AlphaFluorescence Microplate Analyzer (Perkin Elmer) with a 485-nm excitation wavelength and 535-nm emission wavelength. To prevent fluorescence saturation, the voltage of the photo multiplier tube was adjusted to 600 for pMW7-derived plasmids (high-copy) and 900 for pMAL-c2X-derived plasmids (medium-copy). For time-course monitoring of cell growth and fluorescence, E. coli colonies were inoculated into LB medium containing the appropriate antibiotics and 4 mM L-rhamnose, and cultured at 37°C and 200 rpm overnight. Then, the cultures were diluted (1:99) in fresh LB medium containing appropriate antibiotics and various concentrations of L-rhamnose in black-walled 96-well polystyrene plates. Cell growth and fluorescence were measured using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland). Cell images were obtained by laser scanning confocal microscopy (LSM 5 Live, Carl Zeiss, Jena, Germany) using a 63 × 1.4 NA oil immersion objective and a laser 489 nm with 500-525 nm bandpass filter. The flow cytometric analysis was performed using a FACSCalibur instrument (Becton Dickinson & Co., Mountain View, CA, USA) as previously described.21 Briefly, cultured cells were diluted into PBS, and the gate was set according to side scatter channel (SSC) and forward scatter channel (FSC) parameters, then, approximately 10,000 events were recorded per sample. GFP fluorescence was detected at 488 nm excitation wavelength and the intensity was measured and analyzed by FL1 (530/30 nm) photomultiplier tubes (PMTs) and FlowJo software (Ashland, OR, USA), respectively.

■ ASSOCIATED CONTENT Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at XXX. Supplemental figures and tables references (PDF)

■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: + 82 2 450 3761. Fax: +82 2 450 0686. ORCID Sung Ho Yoon: 0000-0003-0171-944X Author Contributions S.H.Y. conceived and supervised the project; S.K.K., D.H.L., and O.C.K. performed the experiments; J.F.K. participated in analyzing the results. S.K.K., D.H.L., and S.H.Y. wrote the manuscript. #S.K.K. and D.H.L. contributed equally to this work. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS This work was supported by the National Research Foundation (NRF) of the Republic of Korea through the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (grant no. 2012M1A2A2026559) and the Ministry of Agriculture, Food, and Rural Affairs through the Strategic Initiative for Microbiomes in Agriculture and Food (grant no. 916006-2). The work of D.H.L. was supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) (grant no. 20163030091540) and the KRIBB Research Initiative Program.

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Table 1. Strains and plasmids used in this study. Strains or plasmids Strains E. coli DH5α

Description

F- Φ80lacZ∆M15 endA1 recA1 hsdR17 (rK– mK+) thi-1 gyrA96 relA1 ∆(lacZYA-argF) U169 E. coli K-12 MG1655 F- ilvG- rfb-50 rph-1 λE. coli BL21(DE3) F- ompT hsdSB (rB- mB-) gal dcm (DE3) E. coli C41(DE3) C41(DE3) was derived from BL21(DE3) as described in Miroux and Walker, 1996 Plasmids pACYCDuet-1 p15A ori, CamR (Novagen) pAR pACYCDuet-1 derivative with NdeI/XhoI fragment of L-rhamnose inducible promoter amplified with primer pair RhamL-F and RhamLR from E. coli K-12 MG1655 genomic DNA (gDNA) pAR-wtLacI pAR derivative with NdeI/NarI fragment of wild-type lacI amplified with primer pair lacI-F and lacI-R from E. coli BL21(DE3) genomic DNA pAR-mLacI pAR derivative with NdeI/NarI fragment of lacIV192F amplified with primer pair lacI-F and lacUV5-R from E. coli C43(DE3) gDNA pMW(Ecb-GFP) T7 promoter, E. coli F-ATPase subunit b fused to folding reporter GFP (frGFP) at the C-terminus pMW-GFP pMW7 derivative with NdeI/HindIII fragment of frGFP amplified with primer pair frGFP-F and frGFP-R from pMW(Ecb-GFP) pMW-CodB-GFP pMW(Ecb-GFP) derivative with NdeI/BamHI fragment of cytosine transporter (codB) amplified with primer pair codB-F and codB-R from E. coli K-12 MG1655 gDNA pMAL-c2X pBR322 ori, tac promoter, AmpR pMH-GFP pMAL-c2X derivative with NdeI/HindIII fragment of frGFP amplified with primer pair frGFP-F and frGFP-R from pMW(EcbGFP) pMH-CodB-GFP pMAL-c2X derivative with NdeI/HindIII fragment of codB-frGFP from pMW-CodB-GFP


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FIGURE LEGENDS Figure 1. Construction of a tunable mLacI inducible expression system. (A) Plasmid map of pAR-mLacI and a compatible pET plasmid of E. coli. In the pAR-mLacI plasmid, mlacI was placed under the control of PrhaBAD on the low-copy plasmid pACYCDuet-1 carrying the p15A replicon, which is fully compatible with a variety of T7 promoter-based pET plasmids containing a ColE1 or pMB1 origin. (B) Fluorescence reporter plasmids. gfp was inserted into two different plasmids, a high-copy plasmid (pMW7) under control of the T7 promoter and T7 RNAP (pMW-GFP) and a medium-copy plasmid (pMAL-c2X) using the tac promoter and E. coli RNAP (pMH-GFP). Note that pMW-GFP does not have lacO downstream of the promoter, whereas pMH-GFP does. Figure 2. Tunable expression of GFP from the pMW-GFP plasmid by the mLacI inducible system. (A) Schematic representation of the mLacI inducible system for expression of a target gene from a T7 promoter-based and lacO-free plasmid. pAR-mLacI or pAR-wtLacI were transformed into E. coli BL21(DE3) harboring pMW-GFP. Kinetic analysis of Lrhamnose dose-dependent GFP expression and growth of cells containing pAR-wtLacI (B) or pAR-mLacI (C). E. coli BL21(DE3) was used as control. The error bar denotes the standard deviation of the mean from the three replicates. Figure 3. Tunable expression of GFP from the pMH-GFP plasmid by the mLacI inducible system. (A) Schematic representation of the mLacI inducible system for expression of a target gene from a tac promoter and lacO-based plasmid. pAR-mLacI or pAR-wtLacI were transformed into E. coli BL21(DE3) harboring pMH-GFP. Kinetic analysis of L-rhamnose dose-dependent GFP expression and growth of cells containing pAR-wtLacI (B) or pARmLacI (C). E. coli BL21(DE3) was used as control. The error bar denotes the standard deviation of the mean from the three replicates. Figure 4. Single-cell analysis of GFP expression by flow cytometry. Population of E. coli BL21(DE3) cells harboring pAR-mLacI and pMH-GFP (A) and those containing pARmLacI/pMW-GFP (B). Cells were induced with 0.7 mM IPTG and various concentrations of L-rhamnose. The inset graphs show average GFP fluorescence at various concentrations of Lrhamnose. E. coli BL21(DE3) was used as control. Figure 5. GFP expression and growth of Lemo21(DE3) cells containing pMW-GFP at various concentrations of L-rhamnose with 0.7 mM IPTG. BL21(DE3) was used as control. Figure 6. Titrated expression of the CodB-GFP fusion protein from E. coli K-12 MG1655 harboring pAR-mLacI and pMH-CodB-GFP. L-Rhamnose dose-dependent kinetics of cell growth (A) and CodB-GFP production (B). Flow cytometry analysis of CodB-GFP production (C) and cell size (D). Fluorescence microscopy analysis of filamentous cells (E) or normal cells (F) expressing the CodB-GFP fusion protein with 4 mM L-rhamnose. E. coli K-12 MG1655 was used as control.


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Figure 2


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Figure 4


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Figure 6


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Tunable Control of an Escherichia coli Expression System for the

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