Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Construction of Plasmids and Escherichia coli Cell-Based Biosensors
2.3. Biosensor Assay
2.4. In Silico Docking Experiments
2.5. Application of Escherichia coli Cell-Based Biosensors in Quercetin and Resveratrol Quantification
2.6. Statistical Analysis
3. Results
3.1. Sensing Mechanism of Escherichia coli Cell-Based Biosensors Employing the ttg Operon System
3.2. Characteristics of Escherichia coli Cell-Based Biosensors Employing the TtgR Wild-Type
3.3. Genetic Engineering of TtgR
3.4. Effects of Engineered TtgRs on the Biosensors
3.4.1. Biosensor-TtgRs with Mutated Asn110
3.4.2. Biosensor-TtgRs with Mutated His114
3.4.3. Biosensor-TtgRs with Mutated Val96 and Ile141
3.4.4. Biosensor-TtgRs with Phe168 and Double Mutations
3.5. Computational Analysis of TtgR–Ligand Interactions
3.5.1. Interaction Between the TtgR Wild-Type and Ligands
3.5.2. Engineered TtgRs with Asn110 Mutations
3.5.3. Engineered TtgRs with His114 and Phe168 Mutations
3.6. Quantification of Quercetin and Resveratrol Using the TtgR-Based Biosensors
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Description | Reference | |
---|---|---|---|
Plasmids | pET-21a(+) | pBR322 ori, Amp r | Novagen |
pCDF-Duet | CloDE13 ori, Str r | Novagen | |
pTtg-eGFP | pET-21a(+) carrying PttgABC:egfp | This study | |
pCDF-TtgR WT | pCDF-Duet carrying ttgR WT | This study | |
pCDF-TtgR mut | pCDF-Duet carrying ttgR mutants: N110F, N110L, N110Y, H114N, H114A, V96S, I141S, I141L, F168W, N110Y/F168W, V96S/H114N | This study | |
E. coli strains | E. coli BL21 (DE3) | F− ompT hsdSB(rB−mB−)gal dcm lon (DE3) | Stratagene |
biosensor-TtgR WT | E. coli BL21 carrying pCDF-TtgR WT and pTtg-eGFP | This study | |
biosensor-TtgR muts | E. coli BL21 carrying pCDF-TtgR muts and pTtg-eGFP | This study |
Abbreviation | Full Name | 3 | 5 | 6 | 7 | 8 | 2′ | 3′ | 4′ | 5′ | 6′ | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Flavone | Lut | Luteolin | H | OH | H | OH | H | H | OH | OH | H | H |
Api | Apigenin | H | OH | H | OH | H | H | H | OH | H | H | |
Que | Quercetin | OH | OH | H | OH | H | H | OH | OH | H | H | |
Kae | Kaempferol | OH | OH | H | OH | H | H | H | OH | H | H | |
Myr | Myricetin | OH | OH | H | OH | H | H | OH | OH | OH | H | |
Gal | Galangin | OH | OH | H | OH | H | H | H | H | H | H | |
4′-HFV | 4′-hydroxyflavone | H | H | H | H | H | H | H | OH | H | H | |
3′-HFV | 3′-hydroxyflavone | H | H | H | H | H | H | OH | H | H | H | |
2′-HFV | 2′-hydroxyflavone | H | H | H | H | H | OH | H | H | H | H | |
5-HFV | 5-hydroxyflavone | H | OH | H | H | H | H | H | H | H | H | |
7-HFV | 7-hydroxyflavone | H | H | H | OH | H | H | H | H | H | H | |
7,3′-DHFV | 7,3′-dihydroxyflavone | H | H | H | OH | H | H | OH | H | H | H | |
Flavanone | Nar | Naringenin | H | OH | H | OH | H | H | H | OH | H | H |
Eri | Eriodictyol | H | OH | H | OH | H | H | OH | OH | H | H | |
Liq | Liquiritigenin | H | H | H | OH | H | H | H | OH | H | H | |
Pin | Pinocembrin | H | OH | H | OH | H | H | H | H | H | H | |
3′-HFVA | 3′-hydroxyflavanone | H | H | H | H | H | H | OH | H | H | H | |
2′-HFVA | 2′-hydroxyflavanone | H | H | H | H | H | OH | H | H | H | H | |
7-HFVA | 7-hydroxyflavanone | H | H | H | OH | H | H | H | H | H | H | |
Stilbene | Res | Resveratrol |
Methods | Samples | Tested Conc. (mM) | Measured Conc. (mM) | Accuracy (%) |
---|---|---|---|---|
HPLC | Resveratrol | 10 | 10.12 ± 0.59 | 98.8 |
15 | 15.34 ± 0.48 | 97.8 | ||
Quercetin | 10 | 9.8 ± 0.20 | 98.0 | |
15 | 15.62 ± 0.93 | 96.0 | ||
Biosensor-TtgR WT | Resveratrol | 10 | 9.55 ± 0.64 | 95.5 |
15 | 14.24 ± 0.86 | 94.9 | ||
Biosensor-TtgR N110F | Quercetin | 10 | 9.36 ± 0.25 | 93.6 |
15 | 14.42 ± 0.52 | 96.1 |
TtgRs | I.C of Main Ligands | Detection Limit (mM) | Basal eGFP Signal | Proposed Mechanistic Reasons |
---|---|---|---|---|
WT | Resveratrol (12.4) | 0.0037 | Low | Native binding pocket optimized for resveratrol; strong DNA-binding repression |
2′-HFVA (10.9) | ||||
Quercetin (2.9) | ||||
N110F | Quercetin (11.0) | 0.0063 | Low–Moderate | Aromatic Phe substitution enhances π–π stacking with quercetin; increases conformational flexibility |
Resveratrol (9.1) | ||||
2′-HFVA (8.8) | ||||
N110Y | Resveratrol (9.3) | - | Moderate | Bulkier Tyr may improve aromatic interactions but also cause steric hindrance |
3′-HFVA (8.2) | ||||
Quercetin (2.1) | ||||
H114N/V96S | Resveratrol (4.4) | - | Moderate | Loss of aromaticity reduces π–π interactions; possible altered pocket shape |
3′-HFVA (3.6) | ||||
Quercetin (2.1) | ||||
N110L | 2′-HFVA (8.5) | - | Low | Hydrophobic Leu substitution maintains repression; minimal effect on ligand accommodation |
Resveratrol (7.2) | ||||
Quercetin (3.1) | ||||
N110F/F168W | 2′-HFVA (12.0) | - | Low | Similar polarity to WT; minor structural change |
Quercetin (7.4) | ||||
Resveratrol (5.9) | ||||
H114A | Resveratrol (7.0) | - | Moderate–High | Removal of imidazole side chain disrupts hydrogen bonding |
2′-HFVA (4.0) | ||||
Quercetin (3.1) | ||||
H114N | Resveratrol (11.8) | - | Moderate | Maintains hydrogen bonding potential but alters geometry |
2′-HFVA (8.3) | ||||
Quercetin (4.6) | ||||
V96F | Resveratrol (2.2) | - | Moderate | Bulky aromatic substitution changes hydrophobic packing near ligand site |
2′-HFVA (2.0) | ||||
Quercetin (1.6) | ||||
V96S | Resveratrol (5.8) | - | Moderate–High | Polar Ser alters hydrophobic microenvironment; possible reduced ligand affinity |
2′-HFVA (3.7) | ||||
Quercetin (2.1) | ||||
I141S | Resveratrol (6.8) | - | High | Polar substitution at hydrophobic site destabilizes closed conformation |
Myricetin (5.5) | ||||
Quercetin (3.2) | ||||
F168W | Resveratrol (14.1) | - | Low–Moderate | Larger aromatic Trp modifies π–π stacking geometry and steric profile |
2′-HFVA (12.8) | ||||
Quercetin (6.7) |
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Song, K.; Ji, H.; Lee, J.; Jang, G.; Yoon, Y. Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds. Biosensors 2025, 15, 554. https://doi.org/10.3390/bios15080554
Song K, Ji H, Lee J, Jang G, Yoon Y. Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds. Biosensors. 2025; 15(8):554. https://doi.org/10.3390/bios15080554
Chicago/Turabian StyleSong, Kyeongseok, Haekang Ji, Jiwon Lee, Geupil Jang, and Youngdae Yoon. 2025. "Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds" Biosensors 15, no. 8: 554. https://doi.org/10.3390/bios15080554
APA StyleSong, K., Ji, H., Lee, J., Jang, G., & Yoon, Y. (2025). Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds. Biosensors, 15(8), 554. https://doi.org/10.3390/bios15080554