A Cell–Cell Communication-Based Screening System for Novel

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A Cell−Cell Communication-Based Screening System for Novel Microbes with Target Enzyme Activities Haseong Kim,†,# Eugene Rha,†,# Wonjae Seong,†,¶ Soo-Jin Yeom,† Dae-Hee Lee,†,¶ and Seung-Goo Lee*,†,¶ †

Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea ¶ Biosystems and Bioengineering Program, University of Science and Technology, 217 Gajung-ro, Yuseong-gu, Daejeon, South Korea S Supporting Information *

ABSTRACT: The development of synthetic biological devices has increased rapidly in recent years and the practical benefits of such biological devices are becoming increasingly clear. Here, we further improved the design of a previously reported high-throughput genetic enzyme screening system by investigating device-compatible biological components and phenol-mediated cell−cell communication, both of which increased the efficiency and practicality of the screening device without requiring the use of flow cytometry analysis. A sensor cell was designed to detect novel microbes with target enzyme activities on solid media by forming clear, circular colonies with fluorescence around the unknown microbes producing target enzymes. This mechanism of detection was enabled by the combination of pre-effector phenolic substrate treatment in the presence of target enzyme-producing microbes and control of the growth and fluorescence of remote sensor cells via phenol-mediated cell−cell communication. The sensor cells were applied to screen soil bacteria with phosphatase activity using phenyl phosphate as phenolic substrates. The sensor cells facilitated successful visualization of phosphatase activity in unknown microbes, which were identified by 16S rRNA analysis. Enzyme activity assays confirmed that the proposed screening technique was able to find 23 positive clones out of 33 selected colonies. Since many natural enzymatic reactions produce phenolic compounds from phenol-derived substrates, we anticipate that the proposed technique may have broad applications in the assessment and screening of novel microbes with target enzymes of interest. This method also can provide insights into the identification of novel enzymes for which screening assays are not yet available. KEYWORDS: metagenome, microbiome, high-throughput screening, enzyme, cell−cell communication, phenol, genetic circuit products that induce fluorescent protein expression results in a high false-positive rate in the screening procedure. In particular, phenol, as one of the cytosolic products, diffuses freely among cells, and phenol treatment is even known to increase membrane permeability in Escherichia coli.10 Here, we present an advanced version of GESS8 by investigating additional functional layers of phenotypic reporters and cell−cell communication in order to enhance the efficiency of the screening technique. Antibiotic-resistance genes and auxotrophic genes were employed, and the compatibility of these genes as phenotypic reporters in the GESS was explored by inserting the genes upstream of the fluorescent reporters (Figure 1A,C); thus, the screening could be performed on solid medium without requiring flow cytometry sorting. In addition, nonspecifically diffusible phenol molecules were used as mediators of cell−cell communication

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n recent years, advances in synthetic biology have facilitated the use of synthetic approaches in a wide range of applications, including molecular biosynthesis in bioreactors,1 bioremediation,2 diagnostics,3 and numerous biosensors4,5 for monitoring cell metabolites.6 Transcription factor-based biosensors are one of the most actively studied genetic circuits and have been reliably used for the screening and engineering of novel enzymes by direct monitoring of enzyme activity in a high-throughput (HT) and quantitative manner.5,7 We recently developed an HT genetic enzyme screening system (GESS) that could be applied to screen more than 200 different enzymes by choosing an appropriate substrate that produces phenol molecules8,9 (Figure 1A,B). This technique combines a genetic circuit that expresses a fluorescent reporter through a transcriptional regulator induced by phenol with a flow cytometry sorter to screen more than 106 variants per day. Despite the simplicity of screening and the quantitative fluorescence response of the GESS approach, the dependence of the technique on flow cytometry analysis, which is expensive and time-consuming, is considered a limitation of the system. Moreover, in cell culture broth, the diffusibility of the cytosolic © XXXX American Chemical Society

Special Issue: Synthetic Biology in Asia Received: December 16, 2015

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Figure 1. Part compatibility for the genetic enzyme screening system. (A) Fluorescence reporter system in which phenol compounds induce dmpR activation, that triggers GFP-encoding or antibiotic resistance (AR) genes. (B) Intracellular target enzymes metabolize a substrate molecule and produce phenol, which activates DmpR and results in expression of the downstream GFP-encoding gene. (C) The D-AAT-encoding gene was added at the N-terminus of EGFP. The system transformed into the D-glutamate auxotrophic host, WM335. (D) Cell−cell communication-based screening system. Phenol molecules were used not only as an enzyme activity indicator but also as a signal transmitter. Phenol turns on the transcription factor (dmpR), whose activation induces the expression of D-AAT- and GFP-encoding genes. Relative viabilities of (E) pGESS-Cm, (F) pGESS-Tc, and (G) pGESS-Km were calculated by dividing the number of colonies in different antibiotics (Cm/Tc/Km concn) and phenol concentrations by the colony number under no antibiotics and no phenol. (H) A representative response pattern of pGESS-DAAT against various phenol concentrations without D-glutamate. The higher is the phenol concentration, the greater is the colony number. The error bars in panels E−H represent standard deviations.

auxotrophic E. coli to survive and fluoresce. In microbiome screening applications, we confirmed that MP-GESS, when used along with an appropriate phenolic substrate (e.g., phenyl phosphate), could be successfully visualized with a fluorescence band around the edge of the target colony. A total of 36 colonies were chosen out of approximately 2000 microbiome colonies in the phosphatase screening plates. Subsequently, 16S rRNA identified most of the selected microbes as well-known environmental microorganisms with target enzyme activity.

in order to enhance the modularity of the two cellular processes: enzymatic activity in unknown microbes and the detection of the enzyme activity in sensor cells (Figure 1D); that is, it was possible to alter the physical and chemical conditions to which the enzyme-producing host cells were exposed and to observe stable reporter signals from the sensor cells independently of the enzyme-producing cells. This modularity enabled the system to bypass issues with heterologous expression in a surrogate host through metagenome screening. For example, Simon et al. reported that only 40% enzyme activity was detected following the random cloning of enzyme-coding genes in E. coli.11 Therefore, rather than constructing a metagenomics DNA library in E. coli cells, simple treatment of environmental microorganisms with an appropriate phenolic substrate and capturing its phenolic product through neighboring sensor cells could maximize the enzyme activity of the original host. Using the GESS-compatible biological components and cell− cell communication, we introduced a novel screening technique, designated the microbe prototyping-based GESS (MP-GESS), which could be used to identify remote microbial enzyme activity via phenol diffusion on solid medium. The genes encoding D-amino acid aminotransferase (D-AAT) and enhanced green fluorescent protein (EGFP) were used together as a dual reporter system that could respond to phenol molecules; the presence of phenol allowed D-glutamate

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RESULTS AND DISCUSSION Compatible Biological Components for the Screening System. To promote the practicality of the GESS, the compatibility of additional biological components was investigated with antibiotic-resistance genes and auxotrophic genes as phenotypic reporters. Antibiotic-resistance genes are wellstudied biological units used in a wide range of microbial research studies.12 We replaced the reporter region of our previous construct, pGESSv4,8 with chloramphenicol (pGESSCm)-, tetracycline (pGESS-Tc)-, and kanamycin (pGESS-Km)resistance genes (Figure 1A, Supplementary Figure 1A,B). Note that pGESSv4 consisted of a transcriptional regulator, dmpR, which responded to phenolic compounds, and its downstream EGFP reporter (Supplementary Figure 1A). After the cells were spread on a plate containing phenol and various concentrations B

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Figure 2. Evaluation of MP-GESS. (A) Soil microbes with a substrate were first spread on a dLB plate. After incubating the plate at a microbe optimal temperature for 12 h, sensor cells were sprayed on the plate, followed by incubation at 37 °C for 12 h. Target colonies with green fluorescence ring were selected via microscope analysis, and sequence analysis can be performed for the identification of the selected hits. (B) TPL active E. coli cells were clearly highlighted by the sensor cells with fluorescence around the edges of the TPL cells (left and center). Because of the rapid growth of C. f reundii cells and strong TPL activity, sensor cells were widely spread across the plate. However, sensor cells were not properly grown and fluoresced on the edge of the plate (arrows with “No cells” labels) where there were no C. f reundii colonies. (C) Sensor cells, microbiome from soil samples, and a substrate were all together spread on a dLB plate. After 12 h incubation, no satellite sensor colonies were observed in the absence of TPL activity in E. coli cells (left). However, E. coli (center) and Citrobacter f reundii (right) cells with TPL activity provided phenol molecules to neighboring sensor cells to survive and exhibited green fluorescence.

(Figure 1H, Supplementary Figure 3). WM335 growth is known to require more than a threshold concentration of Dglutamate;14 therefore, the reason for the lack of colony formation in 1 μM phenol may be because Pseudomonasderived dmpR was not sufficiently sensitive to detect the low concentration of phenol required to satisfy the minimum survival conditions for the D-glutamate concentration in E. coli cells. In metagenome screening, the use of antibiotics may not be appropriate since the presence of unknown metagenomic enzymes with antibiotic resistance activity may induce the formation of sensor cell colonies, which could result in false positives. More importantly, unlike E. coli host-based metagenome screening, it is not possible to grow the nativemicroorganisms on a plate containing antibiotics. Therefore, the D-AAT-encoding gene with the fluorescent reporter, pGESS-DAAT, was only used in further screening applications. Phenol-Mediated Remote Enzyme Activity Detection. Phenol acts as a gratuitous or nonmetabolizable inducer of DmpR activation in E. coli because E. coli cannot grow by metabolizing phenol.15 Additionally, the low cytotoxic effects of the phenol molecules enable the use of phenol as a reliable and quantitative indicator of enzyme activity.8 In the present study, phenol was used as a cell−cell interaction mediator for the detection of remote cellular enzyme activity. Treatment with an appropriate substrate for cells harboring the target enzyme activity of interest will result in an area in which phenol is diffused nonspecifically in the vicinity of the cells. Sensor cells harboring pGESS-DAAT in this area will detect the phenol molecules and will fluoresce next to the enzyme-producing

of antibiotics, the number of colonies on the plate was counted. From this analysis, cells harboring pGESS-Cm and pGESS-Tc exhibited quantitative increases in viability in proportion to the phenol concentration in the medium and showed minimal viability in the absence of phenol (Figure 1E,F). Cells harboring pGESS-Cm showed profiles similar to those of cells harboring pGESS-Tc, whereas pGESS-Km-transformed cells showed the lowest viability among the three tested strains (Figure 1G). In the case of pGESS-Tc, higher fluorescence intensity of colonies was observed as the phenol concentration increased (Supplementary Figure 2). To evaluate the performance of pGESS-Tc, the plasmid was transformed into E. coli cells expressing tyrosine phenol-lyase (TPL), which produces phenol by metabolizing tyrosine. As a result, E. coli cells expressing TPL could survive and form colonies on the plate, whereas cells lacking the TPL gene did not form any visible colonies. The auxotrophic gene encoding D-AAT from thermophilic Bacillus sp. is known to be a well-expressed, thermostable protein that can be used in a screening system with the Dglutamate auxotroph E. coli WM335.13 We developed sensor cells harboring a genetic circuit consisting of the D-AAT, EGFP, and dmpR genes (pGESS-DAAT) and transformed this genetic circuit into a D-glutamate auxotrophic E. coli WM335 mutant as host cells (Figure 1C, Supplementary Figure 1C). The viability of the cells was then examined in the presence of various concentrations of phenol without D-glutamate. Cells harboring pGESS-DAAT formed an increasing number of colonies as the phenol concentration increased; however, no colonies were observed when the phenol concentration was 0 or 1 μM C

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Figure 3. Screening of soil microbes for phosphatase activity. Soil samples from the Gab river in Sintangin-dong, Daejeon, South Korea were spread on a dLB plate with phenyl phosphate as a substrate. After incubation at 20 °C for 12 h, sensor cells were sprayed, followed by an additional 12-h incubation at 37 °C. (A) The catalytic reaction of phosphatases with phenylphosphate produces phenol molecules. Factories are located along the river where the soil sample was obtained. (B) A total of seven candidate colonies were selected as the sensor cells visualized around the candidates. (C) As a negative control, three colonies with autofluorescence were collected from the dLB plate containing no sensor cells. (D) The selected 10 colonies were examined by colony PCR to confirm that the fluorescence cells were sensor cells. The 1,551kb band (Figure S1C) clearly showed that the selected colonies were sensor cells except the negative controls and C7.

phosphatases into phenol and phosphate. After incubating the soil microbes for 12 h at 20 °C, sensor cells were sprayed on the plate. The sensor cells were prepared in advance by culturing in 10 mL of dLB medium with 10 μM phenol for 2 days, followed by washing cells twice with fresh dLB medium; the optical density at 600 nm (OD600) of the sensor cells was approximately 1 before spraying. The plates were observed using a fluorescence multizoom microscope equipped with a GFP filter. As expected from the TPL results, sensor cells formed circular rings emitting green fluorescence around the edges of candidate colonies with phosphatase activity (Figure 3, Supplementary Figure 4). Seven candidates producing phosphatase (Figure 3A) and three unknown fluorescent colonies (Figure 3B) were selected, and colony polymerase chain reaction (PCR) was performed using the primers (MP-F1 and MP-R1 in Table S1), as shown in Supplementary Figure 1C. With the exception of C7, all colonies were confirmed to contain the 1.5-kbp pGESS-DAAT cassette, which indicated that the fluorescence of the colonies resulted from the sensor cells. The unknown soil microorganisms C8, C9, and C10 were presumed to emit autofluorescence rather than containing the sensor cells (Figure 3C). To confirm whether the phosphatase-positive colonies actually possessed phosphatase activity, 36 out of approximately 4000 colonies were chosen (Figure 4A), and the phosphatase activities of their crude extracts were determined based on the amount of pNP released from pNP-phosphate. Figure 4B represents the specific activities (U/mg) of phosphatase of the selected colonies along with their identification using 16s rRNA analysis. Note that 1 U/mg indicates that 1 mg of the crude

cells. Notably, the sensor cells should be grown in medium with 10 μM phenol and no D-glutamate in a seed culture followed by washing the cells twice to remove phenol and D-glutamate in the medium. To evaluate whether the sensor cells were able to detect the enzymatic activities of their neighboring microorganisms, we used E. coli cells engineered to express the TPL enzyme, which metabolizes tyrosine into pyruvate, ammonia, and phenol. Citrobacter f reundii was also used as a natural producer of TPL (Supplementary Figure 1D). Our protocol involved spreading the TPL-producing cells on a plate, followed by incubation at 37 °C. Then, sensor cells were sprayed after the TPL-producing cells formed colonies on the plate (Figure 2A). After culture of the cells, fluorescent sensor cells were observed next to the TPL active cells for both TPL producers, whereas no sensor cells were detected around E. coli cells that did not exhibit TPL activity (Figure 2B). Our technique effectively visualized the TPL-producing colonies by forming a clear ring of fluorescence around the target colonies (Figure 2C). These results confirmed that the sensor cells properly responded to the phenol molecules produced by the target enzyme activity of the neighboring cells, which enabled the prototyping of environmental microorganisms with an appropriate phenolic substrate. Screening of Microbes with Phosphatase Enzyme Activity. MP-GESS was applied to screen microbiomes having phosphatases that are generally used in molecular cloning and for immunological detection. Soil samples were obtained from a river in Sintangin-dong, Daejeon, South Korea. The microorganisms from the sample were spread on a diluted-LB (dLB) plate containing phenyl-phosphate, which is decomposed by D

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Figure 4. Confirmation of phosphatase activity from 36 hits from the soil samples. (A) Green and bright field images of 36 colonies which were selected using microscope analysis equipped with a GFP filter. N1, N2, and N3 indicate negative controls. (B) Specific enzyme activities of the 36 candidate colonies were measured using their crude extract. 16S rRNA analysis of the 36 candidate colonies identified six types of genus, Shigella, Serratia, Rheinheimera, Pseudomonas, Escherichia, and Aeromonas. Negative controls showed no enzymatic activities while 23 candidates out of 33 (approximately 70%) samples show 10 to 30 U/mg activities for pNP-phosphate. One unit of enzyme is its activity required to produce 1 nmol/min pNP.

organisms,24,25 can directly identify target genes from genetic resources, as they are based on the actual activity of the gene product. Previously, we introduced a generally applicable function-based screening system, GESS, which detects phenol as a quantitative indicator of an intracellular enzyme activity of interest. However, the dependency of this system on flow cytometry analysis limits its practical use. Moreover, in the case of metagenome screening, heterologous expression in the surrogate host demonstrates low efficiency for foreign metagenomic genes.26 The key to this approach is to separate target enzyme-producing host cells and sensor cells via phenolmediated cell−cell communication. Subjecting target enzymeproducing host cells to physical and chemical treatment (e.g., finding thermostable enzymes) for optimal enzyme activity and the use of separated sensor cells that stably provide fluorescence reporting signals of the remote enzyme activity allow for efficient screening and identification. Moreover, this method is applicable to a broad range of different enzymes when appropriate substrates for which the catalytic product is phenol or p-nitrophenol are chosen. Recent advances in synthetic biology have addressed fundamental challenges to human life, such as food and health issues.20,27 The application of these synthetic biology-based techniques, however, is strictly regulated due to biosafety concerns. Specifically, genetically modified organisms may release functional DNA into the environment and affect native species via the transfer of undesirable functional synthetic genes.28 Despite recent approaches to ensure biosafety, such as genetic codon replacement,29 orthogonal nucleic acid sys-

extract was required to produce 1 nmol of pNP per min. Five strains (Shigella sonnei, Shigella f lexneri, Rheinheimera tangshanensis, Rheinheimera soli, and Escherichia fergusonii) were confirmed to show clear phosphatase activity ranging from 10 to 30 U/mg. Shigella f lexneri is a well-known source of bacterial nonspecific acid phosphatase,16 and this enzyme has been used in the preparation of various phosphorylated products.17 Ryu et al. reported that Rheinheimera soli has weak acid phosphatase activity.18 Additionally, Escherichia fergusonii has also been reported to have acid phosphatase activity.19 However, no studies have reported the phosphatase activities of Shigella sonnei and Rheinheimera tangshanensis, suggesting that further studies of these strains are needed to identify novel phosphatase enzymes. Perspectives on MP-GESS Applications. Improvement of industrial catalytic bioprocesses relies on the identification of novel genetic catalysts via metagenomic analyses or enhancement of the activities of native enzymes.20,21 Highly sensitive and HT screening technologies are essential for the identification of outperformed enzyme activities from diverse genetic resources. Recently, sequence-based screening has attracted considerable attention as a novel HT sequencing technology.22,23 However, sequence-based screening is only applicable when a target DNA sequence is present in the pool of the annotation database, and such screening methods should be followed by additional verification of gene function. In contrast, function-based screening methods, including the observation of a halo or the appearance of color on solid medium and selective separation using auxotrophic microE

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once with M9 liquid medium (Na2HPO4·7H2O, 6.78 g; KH2PO4, 3 g; NaCl, 0.5 g; NH4Cl, 1 g; 2 mM MgSO4; 0.1 mM CaCl2; 0.4% [w/v] glucose; and 0.01% [w/v] thiamine were added to 1 L of liquid medium) and resuspended in the same medium. TPL-producing E. coli or C. freundii, which naturally produces TPL, were spread on a 90 mm plate containing LB solid medium with tyrosine. Then, we mixed the culture medium of pGESS-DAAT vector-containing sensor cells with 0.75% (w/v) agar solution, added the mixture to solid medium on which the TPL-producing cells were growing, and cultured the mixture for 12 h at 37 °C. Finally, the survival and fluorescence of sensor cells in the presence of TPL-producing E. coli or C. f reundii cells were observed using an AZ100 M fluorescence multizoom microscope (Nikon, Tokyo, Japan) equipped with a GFP filter (455−485 nm excitation, 500−545 nm emission). Screening of Soil Microorganisms Having Phosphatase Activity. One gram of soil from the Gab River in Sintangin-dong, Daejeon, South Korea, was collected and added to 50 mL of 1× PBS. After 24 h, 10 mL of the sample supernatant was collected by centrifugation at 1000 rpm for 5 min. Next, 1 mL of the soil sample was spread on 1/10-diluted LB solid medium containing 100 μM phenyl phosphate. WM335 cells harboring pGESS-DAAT were cultured separately in 10 mL of dLB broth with 10 μM phenol at 37 °C for 24 h. Phenol molecules in the cultured cells were washed twice by centrifuging the samples for 15 min at 3000 rpm, followed by the addition of 5 mL of dLB. After this washing step, the OD600 of the sensor cells was approximately 1. Then, the sensor cells were sprayed using a sprayer on the solid plate where the soil microorganisms had formed colonies. After 12 h of cultivation at 37 °C in an incubator, the samples were observed using an AZ100 M fluorescence multizoom microscope (Nikon). Hit colonies were picked with sterile tooth picks and streaked on dLB medium without substrate, phenol, or D-glutamate. During this step, sensor cells were naturally dissipated, and single colonies could be isolated for further 16s rRNA analysis and phosphatase activity assays. Phosphatase Enzyme Assay. The screened cells were harvested and resuspended in 50 mM HEPES buffer (pH 7.5) with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor. The resuspended cells were disrupted by ultrasonication (Fisher Scientific, Pittsburgh, PA, USA) on ice. The cell debris was removed by centrifugation at 15 000g for 20 min at 4 °C, and the supernatant was filtered through a 0.45μm filter. The protein concentration was quantified by the method reported by Bradford.34 The catalytic activities of crude extracts were determined based on the amount of pNP released from 1 mM pNP-phosphate in 0.2 mL of HEPES buffer (pH 7.5) in a round-bottomed 1.5-mL tube. The crude extract enzyme reactions were conducted at 25 °C for 10 min and terminated by adding 1 M Na2CO3. After centrifugation for 15 min at 16 300g to clear the reaction solution, the absorbance change at 420 nm was measured using a Victor V Multilabel Plate Reader (PerkinElmer Life Sciences, Waltham, MA, USA). One unit of enzyme was defined as the activity required to produce 1 nmol of pNP as a product per min under the specified assay conditions.

tems,30 and mutually dependent host-plasmid systems,31 additional improvements must be made to gain wide biotechnical and social acceptance of the safety of synthetic biology-based techniques. However, several enzymes with high efficiency have evolved in nature, and currently, only a tiny fraction of the microbes that can produce these enzymes has been identified.32 Therefore, we anticipate that MP-GESS will contribute to the identification of naturally occurring highperformance enzymes with subjective guidelines for choosing novel microorganisms that can be directly utilized in industrial applications in the field of biotechnology, without concerns for biosafety.

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METHODS pGESS-DAAT Construction. To construct an MP-GESS plasmid (pGESS-DAAT), we used pGESSv4, a DmpR-based GESS plasmid which consisted of a transcriptional regulator, DmpR, and its downstream reporter EGFP.8 The Gibson assembly technique was used for the cloning.33 pGESSv4 was amplified using PCR with the primers vector-F1 and vector-R1 (Table S1) and the Phusion DNA polymerase (NEB, USA). The PCR product (6185 bp) was ligated with the D-AAT gene at the N-terminus of EGFP. For cloning of the D-AAT gene, we used DNA from Bacillus subtilis 168 as the template and amplified 816 bp of the PCR product using the primers DAAT−N-F1 and DAAT-C-R1 (Table S1). Then, with the PCR product as the template, a second PCR was conducted using the primers LP-DAAT-F1 and LP-DAAT-R1 to obtain the final PCR product of 912 bp. The latter primer included the ssrA tag, which controlled the lifetime of the D-AAT protein (protein degradation tag). We reduced the occurrence of nonspecific reactions of pGESS-DAAT by controlling the halflife of D-AAT through the use of the AANDENYALAA amino acid sequence. The pGESS-DAAT construct was then introduced into a transgenic E. coli WM335 strain, a Dglutamate auxotroph that does not grow unless D-glutamate or phenol is added to the culture medium. Construction of pGESS-Cm/Tc/Km and Evaluation of Compatibility. The Cm- and Tc-resistance genes were amplified by PCR from pACYC184 (New England Biolabs, Ipswitch, MA, USA), and the Km-resistance gene was amplified from pET27b (EMD Millipore, Darmstadt, Germany). We used pGESSv4, a DmpR-based GESS plasmid from E. coli,8 as a template and amplified a 6185-bp fragment by PCR with the primers Vector-R1 and Vector-F1 (Table S1). The antibioticresistance genes were then added at the N-terminus of EGFP in pGESSv4 to construct pGESS-Cm, pGESS-Tc, and pGESS-Km using the same cloning protocol as for pGESS-DAAT with appropriate primers. Next, E. coli DH5α cells containing each plasmid were cultured overnight at 37 °C, and the preculture was diluted 106-fold and spread on selection LB-plates containing different combinations of phenol (1−1000 μM) and antibiotics (0, 10, 20, and 30 μg/mL). pGESS-Tc was examined for the detection of tyrosine phenol-lyase. E. coli DH5α cells containing pGESS-Tc were transformed with pHCEIIB-TPL; plated on LB solid medium containing 1 mM tyrosine, 10 μM PLP, and 30 μg/mL tetracycline; and incubated at 30 °C for 48 h. Verification of MP-GESS Performance. A single colony of E. coli WM335 containing the pGESS-DAAT construct was cultured for 24 h at 37 °C with shaking at 200 rpm after the addition of 0.1 mg/mL D-glutamic acid to LB liquid medium, with the E. coli as the seed culture. The seed culture was washed

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ASSOCIATED CONTENT

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.5b00287. F

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Figure S1, vector maps for MP-GESS construction and evaluation; Figure S2, quantitative response of a pGESSTc; Figure S3, colony-forming units (CFU) of pGESSDAAT; Figure S4, two-step protocol-based metagenome screening; and Table S1, primer list (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

H.K. and E.R. contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (20110031944), the Next-Generation Biogreen 21 Program (PJ009524), C1 Gas Refinery Program (2015M3D3A1A01064875), and the KRIBB Research Initiative Program.

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DOI: 10.1021/acssynbio.5b00287 ACS Synth. Biol. XXXX, XXX, XXX−XXX