This study successfully constructed an engineered probiotic system capable of targeted synthesis and in situ release of neohesperidin, integrating a pH-responsive targeting control with a dual biosecurity safeguard mechanism for live treatment of colorectal cancer (CRC).
Figure 1: Schematic illustration of the engineered probiotic system for neohesperidin production and biosecurity control.
We constructed and iteratively optimized four generations of the neohesperidin production system.
To establish and validate a biosynthetic pathway for producing neohesperidin from exogenously supplied hesperetin, and to evaluate the synergistic catalytic capability of key enzymes UGT73B2, VvRHM-NRS, and Cm1,2RhaT in E. coli.
The recombinant plasmid pET28a-VvRHM-UGT73B2-Cm1,2RhaT was transformed into E. coli BL21(DE3) to construct the engineered strain BL21-Neohes. The strain was inoculated in LB medium containing 50 μg/mL kanamycin and cultured at 37 °C and 150 rpm until OD₆₀₀ ≈ 0.6. The temperature was then lowered to 25 °C, and 3 g/L hesperetin was added as substrate for 48 h of fermentation. A 0.5 mL aliquot of culture was mixed thoroughly with 0.5 mL methanol, centrifuged at 13,500 r/min for 5 min, and the supernatant was filtered through a 0.22 μm PES hydrophilic membrane before HPLC analysis. Samples were analyzed by HPLC equipped with an Angilent C18 column (4.6 mm × 250 mm, 5 μm) at 290 nm. The mobile phases were: A, ultrapure water containing 0.1% trifluoroacetic acid; B, methanol containing 0.1% trifluoroacetic acid. Chromatographic conditions: injection volume 10 μL, flow rate 1 mL/min, column temperature maintained at 35 °C. Gradient elution program: 0–0.1 min, 10% B; 0.1–10.0 min, linear increase from 10% to 40% B; 10.0–20.0 min, linear increase from 40% to 60% B; 20.0–22.0 min, linear decrease from 60% to 10% B; 22.0–25.0 min, 10% B.
Figure 2. Neohesperidin synthesis system. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of UGT73B2, Cm1,2RhaT, VvRHM; Figure E: electrophoresis of gene circuit diagram.
HPLC analysis showed a significant peak at the same retention time as the neohesperidin standard in the engineered strain BL21-Neohes, which was absent in wild-type BL21. Quantitative analysis revealed a neohesperidin yield of
Figure 3. Engineered strain synthesizing neohesperidin, with protein expression induced by 0.5 mM IPTG and 3 g/L hesperetin added as substrate. Figure A: HPLC chromatogram of neohesperidin produced by the engineered strain; Figure B: neohesperidin content in wild-type BL21 and engineered strain after 48 h fermentation at 25 °C; Figure C: time-course curve of neohesperidin production at 25 °C for 48 h; Hes: hesperetin; Hes7Og: hesperetin-7-O-glucoside; Neohes: neohesperidin; Figure D: comparison of neohesperidin content produced by the engineered strain after 24 h fermentation at different temperatures.
A neohesperidin synthesis system using hesperetin as substrate was successfully constructed, confirming that UGT73B2, VvRHM-NRS, and Cm1,2RhaT can efficiently and synergistically catalyze neohesperidin production in E. coli, laying the foundation for subsequent pathway optimization.
To enable specific neohesperidin synthesis by engineered bacteria within the acidic tumor microenvironment of colorectal cancer (pH ≈ 5.8–6.5), we constructed a pH-responsive synthesis system based on the endogenous acid-sensing promoter pcadBA. This system follows a "validate-then-apply" research paradigm: first, a pcadBA-mRFP fluorescent reporter system was constructed to validate promoter responsiveness under different pH conditions; subsequently, a functional system driving expression of the key enzyme Cm1,2RhaT via pcadBA was built to achieve targeted neohesperidin synthesis under acidic conditions.
To validate the induction capacity and specificity of the endogenous acid-sensing promoter pcadBA from E. coli under low pH conditions, and to evaluate its feasibility as a tumor microenvironment-responsive element.
The red fluorescent protein mRFP was placed downstream of the pcadBA promoter to construct the recombinant plasmid pSB1A3-pcadBA-mRFP, which was then transformed into E. coli DH5α to obtain the engineered strain. The strain was inoculated in M9 medium (containing 50 μg/mL ampicillin) buffered at pH 5.8 (MOPS buffer) and pH 7.3 (PBS buffer), and cultured at 37 °C and 180 rpm. Samples were collected every 4 h to measure OD₆₀₀ and mRFP fluorescence intensity (Ex: 584 nm, Em: 607 nm), and normalized fluorescence (Fluorescence/OD₆₀₀) was calculated.
Figure 4. Acid-responsive biosensor. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of CadC+RFP, pCadBA, CadC; Figure E: gene circuit diagram.
Under pH 5.8 conditions, normalized fluorescence increased significantly over time, reaching
Figure 5. Functional validation of the acid-responsive promoter pCadBA. Figure A: comparison of relative fluorescence intensity of PcadC-mRFP engineered strain under different pH conditions. Figure B: comparison of OD600 of PcadC-mRFP engineered strain under different pH conditions.
The pcadBA promoter can efficiently activate downstream gene expression under acidic conditions while maintaining low background expression under neutral conditions, demonstrating good pH sensitivity and specificity. It is suitable as a tumor microenvironment-responsive switch, providing a reliable component for constructing targeted drug synthesis systems.
Based on the validation of pcadBA functionality, this system was used to drive expression of the key enzyme Cm1,2RhaT in the neohesperidin synthesis pathway, enabling initiation of the final glycosylation step only under acidic conditions, thereby achieving site-specific drug synthesis in tumor regions.
The pcadBA promoter was placed upstream of the Cm1,2RhaT gene to drive its expression, constructing the recombinant plasmid VvRHM-UGT73B2-pcadBA-Cm1,2RhaT, which was transformed into E. coli BL21(DE3) to obtain the acid-responsive engineered strain. The strain was cultured in M9 medium at pH 5.8 (MOPS buffer) and pH 7.3 (PBS buffer) until OD₆₀₀ ≈ 1.2. Then, 3 g/L hesperetin as substrate and 0.5 mM IPTG to induce expression of UGT73B2 and VvRHM-NRS were added. Fermentation was continued for 48 h, with samples collected every 12 h. After methanol extraction, centrifugation, and membrane filtration, neohesperidin production was analyzed by HPLC (chromatographic conditions as previously described).
Figure 6. Acid-responsive neohesperidin synthesis system. Figure A: plasmid map; Figure B, C: agarose gel electrophoresis of pcadBA, CadC; Figure D: gene circuit diagram.
Under pH 5.8 conditions, neohesperidin production reached
Figure 7. Low pH induction of promoter-driven neohesperidin synthesis: neohesperidin production under different pH conditions. Note: Culture with OD600=1.2 was placed into media of different pH, protein expression was induced by 0.5 mM IPTG, and 3 g/L hesperetin was added as substrate. Neohesperidin synthesis was measured every 12 h within 48 h.
An acid-responsive neohesperidin synthesis system based on the pCadC promoter was successfully constructed, enabling specific initiation and efficient synthesis of the drug under acidic conditions mimicking the tumor microenvironment. This system, through an "environment-triggered" mechanism, significantly enhances therapeutic targeting and biosafety, providing key technical support for precise intervention using intelligent probiotics.
To construct a metabolic pathway from naringenin to hesperetin, addressing the dependency on exogenous precursor supplementation and improving the system's self-sufficiency.
The recombinant plasmid pET28a-ThF3'H-CPR-MpOMT was transformed into E. coli BL21(DE3) to obtain the engineered strain BL21-Hes. The strain was inoculated in LB medium containing 50 μg/mL kanamycin and cultured at 37 °C and 150 rpm until OD₆₀₀ ≈ 0.6. The temperature was then lowered to 25 °C, and 20 g/L naringenin was added as substrate for 24 h of fermentation. Sample processing and HPLC detection methods were the same as described above.
Figure 8. Hesperetin synthesis system. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of ThF3'H, CPR, MpOMT; Figure E: gene circuit diagram.
HPLC results showed a distinct peak at the same retention time as the hesperetin standard in the BL21-Hes strain, which was undetectable in wild-type BL21. Quantitative analysis indicated a hesperetin yield of
Figure 9. Engineered strain synthesizing hesperetin, with 20 g/L naringenin added as substrate. Figure A: HPLC chromatogram of neohesperidin produced by the engineered strain; Figure B: hesperetin content in wild-type BL21 and engineered strain after 24 h fermentation at 25 °C.
Efficient conversion from naringenin to hesperetin was successfully achieved, validating the functional activity of ThF3'H, CPR, and MpOMT S142V in E. coli, providing a critical module for constructing a complete pathway synthesis system.
To integrate the hesperetin synthesis pathway with the neohesperidin synthesis pathway, constructing a complete biosynthetic pathway from the single precursor naringenin to neohesperidin, thereby enhancing the system's autonomy and application potential.
The plasmid pET28a-ThF3'H-CPR-MpOMT-VvRHM-UGT73B2-Cm1,2RhaT was constructed and transformed into E. coli BL21(DE3) to generate the engineered strain BL21-Hes-Neohes. The strain was inoculated in kanamycin-containing medium, cultured at 37 °C until OD₆₀₀ ≈ 0.6, cooled to 25 °C, and supplemented with 20 g/L naringenin as the sole precursor for 48 h of fermentation. Samples were extracted and analyzed by HPLC to determine neohesperidin production.
Figure 10. Hesperetin to neohesperidin synthesis. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of ThF3'H, CPR, MpOMT; Figure E: gene circuit diagram.
Quantitative results showed that the engineered strain could synthesize neohesperidin from naringenin, with a yield of
Figure 11. Neohesperidin production by naringenin-to-neohesperidin engineered strains. Note: Comparison of neohesperidin content in wild-type BL21 and engineered strains after 48 h fermentation at 25 °C. Protein expression was induced by 0.5 mM IPTG, with 20 g/L naringenin added as substrate. BL21 is the wild-type E. coli BL21 strain used as negative control; BL21-Hes is E. coli BL21 transformed with the hesperetin production plasmid; BL21-Neohes is E. coli BL21 transformed with the neohesperidin production plasmid; BL21-Hes-Neohes is E. coli BL21 transformed with both the hesperetin and neohesperidin production plasmids.
A complete synthesis pathway from naringenin to neohesperidin was successfully constructed, achieving multi-step cascade catalysis, providing a modular foundation for future de novo synthesis of neohesperidin from glucose.
We constructed a dual biosecurity mechanism comprising "inducible clearance in vivo" and "environmental self-destruction" to prevent uncontrolled proliferation of engineered bacteria within the host or their spread into the environment.
To evaluate the responsiveness and dose dependency of the arabinose-inducible promoter pBAD, a pBAD-mRFP fluorescent reporter system was constructed to verify its expression activity under different L-arabinose concentrations, providing functional basis for subsequent construction of the pBAD-MazF suicide system.
The red fluorescent protein gene mRFP was placed downstream of the pBAD promoter to construct the recombinant plasmid pSB1A3-pBAD-mRFP, which was then transformed into E. coli DH5α to obtain the engineered strain DH5α-pBAD-mRFP. The strain was inoculated in LB medium containing 50 μg/mL ampicillin and supplemented with 0%, 0.05%, 0.2%, or 0.5% L-arabinose, and cultured at 37 °C and 180 rpm. Samples were collected every 2 h to measure OD₆₀₀ and mRFP fluorescence intensity (Ex: 584 nm, Em: 607 nm), and normalized fluorescence (Fluorescence/OD₆₀₀) was calculated.
Figure 12. Arabinose-responsive sensor. Figure A: plasmid map; Figure B: agarose gel electrophoresis of araC-pBAD; Figure C: gene circuit diagram.
In the absence of arabinose, fluorescence remained at low background levels (8 h: 9.90 ± 1.98 AU), indicating minimal leakage expression from the pBAD promoter. With 0.05% arabinose, fluorescence increased to 451.47 ± 139.93 AU at 8 h; with 0.2% and 0.5% arabinose, fluorescence reached 829.13 ± 62.16 AU and 1089.19 ± 108.40 AU, respectively, showing significant dose dependency (p < 0.01). Kinetic analysis revealed a rapid increase in fluorescence within 4 h after induction.
Figure 13. Fluorescent protein expression in engineered strains under different arabinose concentrations.
The pBAD promoter exhibits excellent characteristics of low leakage, high inducibility, and dose-dependent response, making it suitable for constructing controllable gene expression systems, and provides a reliable promoter element for subsequent construction of the pBAD-MazF suicide system.
Based on validation of pBAD functionality, the toxin gene MazF was substituted for mRFP to construct the pBAD-MazF system, enabling programmed cell death upon arabinose induction for controllable clearance of engineered bacteria within the host.
The MazF gene was placed downstream of the pBAD promoter to construct the recombinant plasmid pSB1A3-pBAD-MazF, which was transformed into E. coli DH5α to obtain the engineered strain. The strain was inoculated in LB medium containing 50 μg/mL ampicillin and cultured at 37 °C and 180 rpm until OD₆₀₀ ≈ 0.6. The experimental group was induced with 0.2% L-arabinose to express MazF, while the control group received no inducer. OD₆₀₀ was measured every 2 h for 24 h.
Figure 14. Arabinose-inducible suicide system. Figure A: plasmid map; Figure B: gene circuit diagram; Figure C, D: agarose gel electrophoresis of araC-pBAD, MazF.
Without induction, the growth curve of the engineered strain was almost identical to that of wild-type DH5α, indicating no leakage expression of MazF and a "shut-off" state of the system. After addition of 0.2% arabinose, OD₆₀₀ rapidly decreased to near zero (0.19 ± 0.16) within 12 h, whereas the control group continued to grow to OD₆₀₀ > 2.31 ± 0.22, showing a significant difference.
Figure 15. Arabinose-inducible suicide system. Figure A: growth of engineered and control strains without arabinose; Figure B: growth of engineered strain under different arabinose concentrations.
The pBAD-MazF system can efficiently initiate programmed cell death upon arabinose induction, enabling rapid and controllable clearance of engineered bacteria. This provides a "user-triggered" safety switch for clinical intervention, significantly enhancing the biosafety of the system.
To evaluate the activity of the cold-shock protein promoter pCspA at different temperatures, a pCspA-mRFP fluorescent reporter system was constructed to verify its specific activation at low temperatures, providing a basis for constructing an environment-responsive suicide system.
The mRFP gene was placed downstream of the pCspA promoter to construct the recombinant plasmid pSB1A3-pCspA-mRFP, which was transformed into E. coli DH5α to obtain the engineered strain. The strain was inoculated in LB medium containing 50 μg/mL ampicillin and cultured at 16 °C, 25 °C, and 37 °C at 180 rpm for 12 h. Samples were collected periodically to measure OD₆₀₀ and mRFP fluorescence intensity, and normalized fluorescence was calculated.
Figure 16. Temperature-responsive biosensor. Figure A: plasmid map; Figure B: agarose gel electrophoresis of pCspA-mRFP; Figure C: gene circuit diagram.
At 16 °C, normalized fluorescence reached
Figure 17. Fluorescent protein expression in temperature-responsive biosensor strains under different temperatures.
The pCspA promoter exhibits the ideal characteristic of "activated at low temperature, shut off at body temperature," making it suitable for constructing environment-responsive gene expression systems. It effectively prevents unintended activation of engineered bacteria within the host, ensuring functional specificity.
Based on validation of pCspA functionality, the mRFP gene was replaced with the toxin gene MazF to construct the pCspA-MazF system, enabling automatic clearance of engineered bacteria in cold external environments to prevent ecological spread.
The recombinant plasmid pSB1A3-pCspA-MazF was constructed and transformed into E. coli DH5α. The experimental group was cultured at 16 °C and 180 rpm, while the control group was cultured at 37 °C. OD₆₀₀ was measured every 2 h for 24 h. The control group consisted of wild-type DH5α.
Figure 18. Cold-inducible suicide system. Figure A: plasmid map; Figure B: agarose gel electrophoresis of MazF; Figure C: gene circuit diagram.
At 16 °C, the engineered strain’s OD₆₀₀ reached only
Figure 19. Cold-inducible suicide system. Figure A: growth of cold-inducible suicide engineered strain after 8 h at different temperatures; Figure B: comparison of growth curves between cold-inducible suicide engineered strain and wild-type strain at 16 °C.
The pCspA-MazF system can automatically initiate MazF expression under low temperature, leading to growth arrest and cell death, achieving an "environmental self-destruction" function. As a second safety barrier, this system effectively prevents survival and spread of engineered bacteria after excretion from the host, establishing an "environment-triggered" biocontainment mechanism.
This study successfully constructed and validated:
