Construction of bacterial cells with an active transport system for

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Construction of bacterial cells with an active transport system for unnatural amino acids Wooseok Ko, Rahul Kumar, Sanggil Kim, and Hyun Soo Lee ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00076 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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ACS Synthetic Biology

Construction of bacterial cells with an active transport system for unnatural amino acids

Wooseok Ko, Rahul Kumar, Sanggil Kim, Hyun Soo Lee* Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea

ABSTRACT Engineered organisms with an expanded genetic code have attracted much attention in chemical and synthetic biology research. In this work, engineered bacterial organisms with enhanced unnatural amino acid (UAA) uptake abilities were developed by screening periplasmic binding protein (PBP) mutants for recognition of UAAs. A FRET-based assay was used to identify a mutant PBP (LBP-AEL) with excellent binding affinity (Kd ≈ 500 nM) to multiple UAAs from 37 mutants. Bacterial cells expressing LBP-AEL showed up to 5-fold enhanced uptake of UAAs, which was determined by genetic incorporation of UAAs into a green fluorescent protein and measuring UAA concentration in cell lysates. To the best of our knowledge, this work is the first report of engineering cellular uptake of UAAs and could provide an impetus for designing advanced unnatural organisms with an expanded genetic code, which function with the efficiency comparable to that of natural organisms. The system would be useful to increase mutant protein yield from lower concentrations of UAAs for industrial and large-scale applications. In addition, the techniques used in this report such as the sensor design and the measurement of UAA concentration in cell lysates could be useful for other biochemical applications.

KEYWORDS. Amino acids, active transport, periplasmic binding proteins, genetic code expansion, protein engineering, FRET sensors

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Construction of engineered organisms with an expanded genetic code has been an attractive research topic in chemical and synthetic biology, because these organisms are able to perform novel functions using additional building blocks. A landmark contribution to this field was made by constructing an engineered Escherichia coli (E. coli) in which an evolved aminoacyltRNACUA and aminoacyl-tRNA synthetase pair (aa-tRNACUA/aa-RS pairs) was used to encode an additional amino acid (AA).1 This system has been extensively used to incorporate unnatural amino acids (UAAs) with attractive biochemical properties.2−9 Although this has important advantages including technical simplicity, high protein yield, and applicability to various proteins and host cells, the system still requires technical improvements for higher efficiency in protein synthesis with minimal perturbation in endogenous translational machineries. For engineered organisms with an expanded genetic code to function properly and efficiently, additional genetic components including exogenous codons, aa-tRNA/aa-RS pairs, and UAAs should be orthogonal to endogenous translational components and compatible with endogenous translational machineries, such as ribosomes and elongation factor Tu. Much effort has been made to achieve this orthogonality and compatibility.8,10−17 As an additional codon, the amber codon (TAG) is most frequently used because it does not encode any AA and its usage is rare. However, the amber codon is still used for a stop signal for endogenous protein synthesis and is recognized by a release factor. This incompatibility causes cytotoxicity and a decrease in efficiency of UAA incorporation. To solve these problems, bacterial hosts have been engineered with knock-outs of release factor 1 and/or the replacement of all amber codons in the chromosome with the other stop codons.10−12 Compatibility of aa-tRNA with endogenous translational machineries has also been engineered for better interaction with ribosomes13,14 and elongation factor Tu15,16. Bacterial cells are able to synthesize all 20 canonical AAs and have AA transporters for their efficient uptake. Likewise, an engineered organism with the ability to synthesize and/or transport an UAA would be an advanced unnatural organism for expansion of the natural genetic repertoire. A few reports have shown that direct incorporation of biosynthesized UAAs is possible and, in some cases, more efficient than the incorporation using UAAs.18−20 In this study, we engineered a periplasmic binding protein (PBP) to recognize UAAs, and identified a mutant PBP from 37 mutants, which recognizes multiple UAAs with high binding affinity. When the mutant PBP was expressed, we observed significantly improved incorporation of UAAs resulting from improved uptake (Figure 1). The engineered organism with an expanded


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genetic code is able to efficiently uptake an additional AA and use it for protein synthesis in a similar fashion as endogenous AA incorporation. The improved uptake of UAAs was also confirmed by determining their concentration in cell lysates. RESULTS AND DISCUSSION A UAA is an essential component for genetic incorporation, which is supplied in growth medium, and is expected to diffuse into the cytoplasm via passive transport. Although few studies have been published regarding UAA uptake, it has been suggested that UAA uptake significantly affects the efficiency of UAA incorporation.21,22 In gram-negative bacteria such as Escherichia coli (E. coli), natural AAs can be imported by diffusion and/or AA transport systems.23,24 Among bacterial AA transport systems, the ATP-binding cassette (ABC) transporters have been extensively studied and are involved in active transport of AAs.25,26 In the ABC-transport systems, PBPs have a critical role in the efficiency and specificity of AA uptake.24 PBPs are well-studied proteins and have been engineered to develop protein sensors for their target ligands.27−38 In some cases, PBPs have been engineered to modulate their binding affinity for both cognate and non-cognate ligands.34−38 To engineer a PBP for improving UAA uptake, the leucine-binding protein (LBP) from E. coli was chosen, because it was functionally and structurally well-characterized and had been previously engineered to modulate its binding affinity.36,39−43 In addition, LBP recognizes other natural AAs including Phe and has a space in its AA binding site for engineering.39,43 To engineer LBP, an analytical method was required so that mutant LBPs could be evaluated for their desired binding properties. Previously, we developed AA-sensing LBPs by using a fluorescent UAA encoded by genetic code expansion and yellow fluorescence protein (YFP) fusion, and the sensor proteins produced significant FRET change upon ligand binding.36 The sensor proteins have YFP at the N-terminus and L-(7-hydroxycoumarin-4-yl)ethylglycine (CouA) at the G178 position in LBP. This protein design was used for mutant LBP screening. In the X-ray crystal structure43 of LBP complexed with Phe, the phenyl ring of Phe is located between W18 and Y276 (Figure 2A). Based on the structure, W18 and Y276 were selected for mutation. The residues are close to the bound Phe and were expected to produce a large space when replaced with smaller residues. The fusion gene for YFP-LBP tagged with His6 at the C-terminus was prepared by overlap extension polymerase chain reaction (PCR), and an amber mutation (TAG) for CouA



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incorporation was introduced into position 178 of LBP by site-directed mutagenesis to create the YFP-LBP-G178TAG gene. To engineer the sensor protein for UAA binding, W18 of LBP was mutated to all other AAs except Cys, Phe, Tyr, and Trp, and each mutant gene (YFP-LBPG178TAG-W18X, 16 mutants) was expressed by the genetic code expansion method expressing the engineered aa-tRNA/aa-RS (CouRS) pair in the presence of 1 mM CouA.36,44 Each mutant protein was purified by affinity purification and analyzed by SDS-PAGE followed by fluorescence gel imaging and Coomassie staining (Figure S1). The analyses showed clear fluorescence from genetically incorporated CouA at position 178 of LBP and the correct protein size (64 kDa) for the YFP-LBP fusion proteins. Each mutant was evaluated for its binding ability to UAAs (1−8) containing aromatic rings (Figure 2B) by measuring fluorescence intensity at 468 nm (ICouA) and 537 nm (IYFP) upon excitation at 360 nm and calculating FRET ratio (R, IYFP/ICouA) changes before and after ligand binding (Figure 3A and Figure S2). FRET ratio changes were calculated by using the following equation: FRET ratio change = ΔR / R0 = (Rx − R0) / R0

(1)

where R0 is the FRET ratio at [UAA] = 0, and Rx is the FRET ratio at [UAA] = x. The result showed that wild type (WT) LBP was not able to recognize any of the eight UAAs tested. However, the W18G and W18A mutants showed moderate affinity to multiple UAAs. Interestingly, the W18G mutant had higher binding affinity to five UAAs than L-Leu, and the W18L mutant had moderate affinity to the bulky UAA (7)45 containing 8-hydroxyquinoline. Unfortunately, the response of all mutant sensors to UAA 6 and 8 was minimal due to the large size or negative charge of the side chain. Before we further characterized the W18X mutants, more mutations were introduced into selected W18X mutants (W18G, W18A, W18V, and W18L). In the structure of LBP complexed with Phe,43 the Y276 residue is located close to the phenyl group of Phe (Figure 2A), and mutation of Y276 to a smaller residue was expected to make more space to accommodate the aromatic UAAs. Five mutations (Y276G, Y276A, Y276V, Y276I, and Y276L) were introduced into the four W18X mutants, respectively, to afford twenty mutant YFP-LBPs. Each mutant was expressed by genetic code expansion to incorporate CouA, purified by affinity purification (Figure S3), and evaluated for UAA binding by the FRET assay (Figure 3B and Figure S4). Among the five Y276 mutations, the Y276G and Y276A mutations showed a negative effect on binding affinity to all tested AAs including L-Leu. However, the other mutations increased binding affinity for most of the tested UAAs with a dependency on


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W18 mutations. Importantly, combinations of W18G and W18A mutations with Y276V, Y276I, and Y276L mutations showed higher affinity to UAAs than L-Leu, and two mutants with W18G-Y276L and W18A-Y276L mutations had excellent binding affinity to multiple UAAs with significantly lower affinity to L-Leu. These results showed that engineering LBP coupled with the FRET-based assay using the genetic code expansion technique made it possible to produce UAA-sensing proteins with higher binding affinity to UAAs than the natural ligand, L-Leu. Recently, genetic incorporation of pyrrolysine (Pyl) derivatives has drawn much attention owing to the substrate promiscuity of Pyl-RS and its applicability to both prokaryotic and eukaryotic cells.46 For selected LBP mutants, their affinity to Pyl derivatives was tested. Seven Pyl derivatives (Figure 2C) were selected and tested with six LBP mutants (W18A-Y276L, W18A-Y276I, W18A-Y276V, W18G-Y276L, W18V-Y276L, and W18L-Y276L) (Figure 4 and Figure S5). UAA 9 containing an azide group showed the highest affinity among the tested Pyl derivatives, and the W18A-Y276L mutant showed broad binding affinity to the Pyl derivatives as well as the aromatic UAAs. Notably, the ΔR/R0 of the W18A-Y276L mutant for UAA 9 had the largest values for all tested UAAs including the aromatic UAAs. In the previous study36, an M21E mutation in LBP was found to stabilize the ligand-bound conformation of the protein and increase binding affinity. This finding was applied to the W18A-Y276L mutant to generate an additional increase in its binding affinity to UAAs. The M21E mutation was introduced into the YFP-LBP-G178TAG-W18A-Y276L gene, and the mutant gene was expressed by genetic code expansion for CouA incorporation. The target protein was prepared and evaluated for UAA binding. As expected, for all tested AAs, the mutant sensor showed increased ΔR/R0 values upon ligand binding, proving that the mutation increased binding affinity (Figure S6). By introducing the M21E mutation, a further increase in binding affinity could be achieved. Next, the binding properties of the optimized mutant, YFP-LBP-G178CouA-W18A-M21EY276L (YFP-LBP-CouA-AEL), were evaluated by measuring dissociation constants for selected UAAs. The protein was titrated with each UAA and fluorescence spectra were recorded (Figure S7). From these experiments, ΔR/R0 was plotted against UAA concentration, and dissociation constants were calculated (Table 1). All dissociation constants were less than 10 μM, and the constants for UAAs 2, 5, and 9 were less than 1 μM, which were comparable with the affinity (Kd = 0.41 μM)36 of WT LBP to L-Leu. YFP-LBP-CouA-AEL was also



evaluated for specificity to the L-form of UAAs 3, 5, 9, and 10 over their D-forms (Figure S8). (The other AAs could not be tested due to the commercial inaccessibility and synthetic difficulty of their D-forms.) The FRET ratio change was minimal with the tested D-AAs, showing that the mutant LBP had specific binding properties to L-form AAs. From the multi-step engineering of LBP, YFP-LBP-CouA-AEL was identified and characterized. The mutant protein was then applied for active transport of UAAs. To test if the mutant LBP was able to recognize and actively transport UAAs in cells, the protein was expressed in bacterial cells with an expanded genetic code. The YFP gene in YFP-LBP-CouAAEL was removed, and the amber codon at position 178 was replaced with the original codon for Gly. The LBP gene with the three mutations (W18A, M21E, and Y276L) was coexpressed with the corresponding aa-tRNA and aa-RS pair for each UAA tested. An emerald GFP gene (emGFP-F39TAG) containing an amber codon at position 39 was also expressed to evaluate the efficiency of the incorporation of each UAA by fluorescence (Figure S9). These engineered organisms with an expanded genetic code and the novel uptake system grew in the presence of each UAA listed in Table 1 at varied concentrations, and fluorescence from the expressed emGFP was measured. Control cells expressing WT LBP were also tested for comparison. emGFP fluorescence in the engineered organisms was significantly higher than that in control cells at the same concentration of all tested UAAs (Figure 5). For the aromatic UAAs (1, 2, 3, 5, and 7), emGFP fluorescence from the organisms expressing the mutant LBP (LBP-AEL) was saturated at 2 to 5-fold lower concentrations of UAAs, meaning that the organisms could produce the same amount of the emGFP mutant with a 2 to 5-fold lower concentration of UAAs. For the Pyl derivatives (9 and 10), the same effect was observed although the improvement was not as high as it was for the other UAAs. For UAAs 2, 5, and 9, the same assay was applied to a special bacterial strain (B-95.ΔAΔfabR)11 derived from the BL21 strain in which endogenous TAG codons were replaced with the other stop codons along with deletion of release factor 1, and the accompanying toxic genetic variations were fixed. In this bacterial strain, the effect of increased uptake by LBP-AEL was similar to that shown in Figure 5 (Figure 6A). In addition, the same experiments were carried out with three TAG codons (positions 39, 90, and 151) in the emGFP gene, and 3 to 5-fold lower UAA concentration for saturation and 20 to 50% higher maximum fluorescence were observed (Figure 6B). Overall, the organisms with an expanded genetic code showed saturated emGFP expression with a 5-fold lower concentration of UAAs, and increased maximum expression of emGFP when LBP-AEL was expressed, proving that LBP-AEL increased UAA concentration via the engineered uptake


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system. Interestingly, the B-95.ΔAΔfabR strain expressing LBP-AEL has both the TAG codon assigned for UAAs and the active transport system for UAAs, making it an advanced organism with an expanded genetic code that has not been achieved before. To confirm that the increase of emGFP fluorescence caused by expression of LBP-AEL originated from increased cellular concentrations of each UAA, their concentration in the tested cells expressing LBP-AEL was measured in comparison with that in the control cells expressing LBP-WT. This experiment was performed for UAAs 2, 5, and 9 which had Kd < 1 μM. Due to the limited examples for measurement of cellular concentration of UAAs,22 no appropriate assay for this experiment was found from the literature. Therefore, a method for measuring cellular concentration of UAAs was developed using the FRET sensor protein, YFPLBP-CouA-AEL. Initially, cell lysate from E. coli BL21 were prepared, and the cell lysate was mixed with YFP-LBP-CouA-AEL (100 nM). The lysate containing the sensor protein was titrated with each UAA, and fluorescence spectra were recorded (Figure S10). ΔR/R0 values were calculated and plotted against UAA concentration. For the linear part (< 10-6 M) of the plots, regression equations were obtained and used to determine UAA concentration. Then, E. coli BL21 containing pET-LBP-WT or pET-LBP-AEL were grown in the presence of each UAA (1 mM), cell lysates were prepared, and fluorescence spectra from the sensor protein were obtained. By using the fitting equations (Figure S10) for each UAA, their concentrations in each cell lysate were determined (Figure 7). The results showed a clear increase in UAA concentration for the cell lysates prepared from cells expressing LBP-AEL. The increase was approximately 5-fold for UAA 9 and 2 to 3-fold for UAAs 2 and 5. These results reveal that the increase in emGFP expression shown in the uptake experiments originated from the increase in cellular uptake of each UAA caused by the novel active transport system consisting of the engineered LBP. Although it does not measure the exact concentration of UAAs in cells, this assay provides a good estimation for relative cellular concentration of UAAs. Based on the dissociation constants listed in Table 1, UAAs, 2, 5, and 9, had the strongest affinity to LBP-AEL, and were expected to be most effective in their uptake increase via the expression of LBP-AEL. However, the results from uptake experiments showed no correlation between the amount of increased uptake and binding affinity. This phenomenon can be explained by two factors. In the uptake experiments in Figure 5 and 6, uptake increase was measured by the fluorescence increase from emGFP expression via incorporation of UAAs, however, the affinity of each aa-RS to the corresponding UAA could also affect uptake increase.



Another factor that could affect UAA uptake is the concentration of each UAA in the periplasm, since LBP is a periplasmic protein and thus is able to bind to AAs in the periplasm. Therefore, the results in Figure 5, 6 and 7 could not be directly correlated with the binding affinity of LBPAEL to each UAA. CONCLUSION Bacterial cells are able to actively transport 20 proteinogenic AAs to generate higher cellular concentrations of the essential nutrients. In this report, engineered bacterial cells with an expanded genetic code and enhanced ability of UAA uptake were constructed by evolving a natural AA binding protein. The engineering of the AA binding protein was carried out by using the FRET-based assay system with a fluorescent UAA and YFP, and 37 mutant proteins were purified and tested for binding affinity to 15 UAAs. The FRET-based assay allowed facile characterization of mutant proteins, resulting in the identification of a mutant sensor protein which had comparable binding affinity to multiple UAAs with that of the WT protein to L-Leu. When the mutant LBP was expressed in bacterial cells, saturation of GFP expression by the genetic incorporation of UAAs was achieved with 2−5-fold lower concentrations of UAAs. In addition, the increased uptake via LBP-AEL was confirmed by measuring the concentration of UAAs in cell lysates utilizing the same FRET-based sensor protein. By expressing the UAA binding protein in B95.ΔAΔfabR, advanced unnatural organisms with an expanded genetic code composed of reassigned codons and an active transport system for UAAs were constructed. This work could provide an impetus for the design of advanced unnatural organisms that can function with the same efficiency as their natural counterparts. Additionally, the sensor design and the method for measuring UAA concentration in cell lysates could be useful for other biochemical applications. Finally, this work could be expanded to identify mutant PBPs to actively transport biochemically interesting UAAs such as UAA 8 which is known to have poor uptake due to its negatively charged side chain. METHODS General. All chemicals and DNA oligomers were obtained from commercial sources and used without further purification. All fluorescence spectra were measured by Hitachi F-7000 Fluorescence Spectrophotometer. - Expression and purification of YFP-LBP fusion proteins


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The LBP gene was amplified from E. coli DH10βTM (Invitrogen, California USA), and the YFP gene was obtained from a commercial source by gene synthesis. The YFP-LBP fusion gene with a His6-tag at the C-terminus was synthesized by overlap extension PCR and inserted into the NcoI and KpnI restriction sites in pBAD/Myc-His (Invitrogen) to generate pBAD-YFPLBP-WT. All mutations for LBP engineering were performed by site-directed mutagenesis according to the manufacturer’s protocol (Invitrogen, California USA). Each pBAD plasmid containing YFP-LBP-G178TAG gene with designated mutations was co-transformed with pEvol-CouA-RS47 into the E. coli C321.∆A strain10 (Addgene). Transformed cells were amplified in lysogeny broth (LB) supplemented with ampicillin (100 μg/mL) and chloramphenicol (35 µg/mL), and the start culture (2 mL) was used to inoculate 100 mL LB supplemented with ampicillin (100 µg/mL), chloramphenicol (35 µg/mL), and 1 mM CouA at 37 °C. Protein expression was induced by adding 0.2% L-arabinose when optical density reached 0.8 (550 nm), and the culture was grown overnight at 37 °C. Cells were harvested by centrifugation at 10000 rpm for 5 min at 4 °C, resuspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and pH 8.0), and sonicated. Target proteins were purified by using Ni-NTA affinity chromatography under native conditions according to the manufacturer's protocol (Qiagen, Hilden Germany). Purified proteins were dialyzed against a phosphate buffer (50 mM NaH2PO4, 50 mM NaCl, and pH 9.0) to remove natural amino acids bound to the proteins. - Fluorescence measurements Fluorescence was measured in an assay buffer (50 mM NaH2PO4, 50 mM NaCl, and pH 9.0) containing a YFP-LBP-G178CouA derivative at the designated concentration of an amino acid ligand. Fluorescence intensity for each sample was recorded from 420 nm to 600 nm with excitation at 360 nm, and FRET ratio changes were calculated from each fluorescence spectrum. All values were averaged from three independent measurements. Dissociation constants in Table 1 were calculated using the Origin software. - Uptake assay A gene cassette containing the mutant LBP (LBP-AEL) gene and the emGFP gene with an amber codon at position 39 was synthesized by overlap extension PCR. The gene cassette contains a signal sequence for periplasmic targeting and an additional ribosome-binding site between the genes. The gene cassette was inserted into the NcoI and XhoI restriction sites in



pET28a. The plasmids containing LBP-WT and/or emGFP with three amber codons were prepared using the same method. Each pET plasmid was co-transformed with a pEvol plasmid containing an aatRNA/aaRS pair gene into BL21 (DE3) or B95.ΔAΔfabR strain11. Transformed cells were amplified in lysogeny broth (LB) supplemented with kanamycin (100 μg/mL) and chloramphenicol (35 µg/mL), and the start culture (200 μL) was used to inoculate 10 mL P-5052 medium48 (50 mM Na2HPO4, 50 mM KH2PO4, 25 mM (NH4)2SO4, 2 mM MgSO4, 0.1 % trace metals, 0.5 % glycerol, 0.05 % glucose, 0.2 % arabinose, and 0.2 % lactose) supplemented with kanamycin (100 µg/mL), chloramphenicol (35 µg/mL), and each UAA at an indicated concentration at 37 ℃. After incubation for 18 hours at 37 ℃, cell density was measured using a spectrophotometer (600 nm). The same number (4.0 × 109) of cells was centrifuged, and the cell pellets were resuspended with 100 μL Bugbuster (Novagen) containing a DNAse (benzonase, Sigma-Aldrich), and the mixture was incubated for 1 hour at room temperature. Then, cell debris was removed by centrifugation, and the supernatant was diluted with 100 μL PBS. Fluorescence intensity of emGFP was measured at 509 nm with excitation at 487 nm. - Determination of UAA concentration in cell lysates E. coli BL21 (DE3) was amplified in lysogeny broth (LB), and the start culture (200 μL) was used to inoculate 10 mL P-5052 medium (50 mM Na2HPO4, 50 mM KH2PO4, 25 mM (NH4)2SO4, 2 mM MgSO4, 0.1 % trace metals, 0.5 % glycerol, and 0.05 % glucose). When an optical density was reached at 0.8 (550 nm), lactose (0.2 %) was added (in order to maintain the same culture condition with the following uptake experiments), and the cells were incubated at 37 ℃ for 2 hours. After incubation, the cells were harvested and washed 4 times with PBS at 4 ℃. The cell pellet was resuspended in 200 μL Bugbuster (Novagen) containing a DNAse (benzonase, Sigma-Aldrich), and the mixture was incubated for 1 hour at room temperature. Cell debris was removed by centrifugation, and the supernatant was extracted with 200 μL CHCl3. The resulting aqueous layer (150 μL) was diluted into 450 μL of a FRET assay buffer (50 mM phosphate buffer, 50 mM NaCl, pH 9.0). The mixture was titrated with each UAA in the presence of the sensor protein (YFP-LBP-AEL, 100 nM), and fluorescence spectra were recorded with excitation at 360 nm. FRET ratio changes were calculated, plotted against UAA concentration (up to 10-6 M), and fitted to a line to obtain a fitting equation. Next, E. coli BL21 (DE3) containing pET-LBP-WT or pET-LBP-AEL were amplified in lysogeny broth (LB) supplemented with kanamycin (100 μg/mL), and each start culture (200 μL) was used to


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inoculate 10 mL P-5052 medium (50 mM Na2HPO4, 50 mM KH2PO4, 25 mM (NH4)2SO4, 2 mM MgSO4, 0.1 % trace metals, 0.5 % glycerol, and 0.05 % glucose) supplemented with kanamycin (100 μg/mL). When an optical density was reached at 0.8 (550 nm), UAA (1 mM) and lactose (0.2 %) were added to each culture, and the cells were incubated at 37 °C for 2 hours. Cell lysates were prepared by the same procedure, and fluorescence assay was carried out with the sensor protein. FRET ratio changes were calculated, and UAA concentration in each lysate was determined by using the fitting equation from each titration experiment.

ASSOCIATED CONTENT Supporting information is available in the online version of the paper.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]; Tel: +82-2-705-7958; Fax: +82-2-705-7893 Author Contributions W. K. performed experiments and analyzed results, and S. K. helped W. K. do experiments. R. K. synthesized D-amino acids. H. S. L supervised the project and wrote the manuscript. Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS This research was supported by the Global Frontier Research Program (NRF2015M3A6A8065833)

and

the

Basic

Science

Research

Program

(NRF-

2016R1D1A1B03931369 and 2019R1A2C1010665) through the National Research Foundation of Korea (NRF) funded by the Korean government.




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Figure 1. A schematic representation of the engineered organism for active transport of UAAs. A PBP was engineered for binding to UAAs and used to increase UAA uptake. The imported UAAs were then used to synthesize mutant proteins containing UAAs.


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Figure 2. (a) The ligand-binding site of LBP complexed with L-Phe (PDB ID 1USK). Three residues (W18, M21, and Y276) are selected for mutation and shown in stick. (b and c) Chemical structures of UAAs used in this study. (b) Aromatic UAAs; (c) pyrrolysine derivatives.



Figure 3. FRET ratio changes of the sensor proteins with mutations in W18 (A) or in W18 and Y276 (B) for UAA 1−8 and L-Leu. FRET ratios were calculated from ICouA, 468 nm and IYFP, 537


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nm

upon excitation at 360 nm in the presence (50 μM) and the absence of each UAA, and FRET

ratio changes were calculated from equation 1. Each data point represents an average of three independent experiments. Error bars were omitted for clarity, and for all data points, standard deviations were less than ±0.15 (Figure S2 and S4). Assay conditions: 100 nM sensor protein, 50 mM phosphate buffer (pH 9.0), 50 mM NaCl, and 50 μM AA.



Figure 4. FRET ratio changes of the selected sensor protein mutants for Pyl-derivatives and LLeu. FRET ratios were calculated from ICouA, 468 nm and IYFP, 537 nm upon excitation at 360 nm in the presence (50 μM) and the absence of each UAA, and FRET ratio changes were calculated from equation 1. Each data point represents an average of three independent experiments. Error bars were omitted for clarity, and for all data points, standard deviations were less than ±0.15 (Figure S5). Assay conditions: 100 nM sensor protein, 50 mM phosphate buffer (pH 9.0), 50 mM NaCl, and 50 μM AA.


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Table 1. Dissociation constants of YFP-LBP-CouA-AEL for selected UAAs. Dissociation constants were determined from the titration experiments (Figure S7). a From ref. 36.



Figure 5. UAA uptake assays in E. coli BL21(DE3) harboring pET-emGFP-39TAG-LBP-WT (black) or pET-emGFP-39TAG-LBP-AEL (red) and a pEvol plasmid as shown in Figure S9. The emGFP fluorescence from cell lysates prepared from the same number of cells grown at each concentration of UAAs was measured.


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Figure 6. UAA uptake assay in B-95.ΔAΔfabR harboring pET-emGFP-(TAG)n-LBP-WT (black) or pET-emGFP-(TAG)n-LBP-AEL (red) and a pEvol plasmid as shown in Figure S9. The amber codon was introduced at (a) one position (Y39) or (b) three positions (Y39, E90, and Y151). The emGFP fluorescence from cell lysates prepared from the same number of cells grown at each concentration of UAAs was measured.



Figure 7. Determination of UAA concentration in cell lysates prepared from E. coli BL21(DE3) harboring pET-LBP-WT (blue bars) or pET-LBP-AEL (red bars). The cells were grown in the presence of each UAA (1 mM), and UAA concentration in their cell lysates was determined by measuring fluorescence intensity from the sensor protein (YFP-LBP-AEL, 100 nM) and using the regression equations in Figure S10 for each UAA.


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ToC Figure


Construction of bacterial cells with an active transport system for

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