This study systematically engineered and validated multiple strains along two axes: chlorimuron-ethyl degradation and indole-3-acetic acid (IAA) biosynthesis. We retrieved the gene sequences of three degradative enzymes—SulE, PnbA, and GST—from the literature and NCBI, designed the gene circuits and plasmid maps, and cloned them into the pET28a(m) vector before transforming E. coli BL21(DE3) to obtain herbicide-degrading strains. Western blot (WB) and ELISA confirmed successful expression and catalytic activity of all three enzymes; PnbA showed the best performance for chlorimuron-ethyl degradation. We further optimized reaction temperature and pH and achieved cell-surface display of PnbA to enhance whole-cell catalysis. For IAA biosynthesis, we constructed a dual-enzyme strain (iaaM + iaaH) and quantified IAA via ELISA, showing stable production within 12–24 h. A wheat germination assay verified the growth-promoting effects of IAA. To address biosafety, we built a cold-inducible promoter system and demonstrated its feasibility at 16 °C, indicating potential for environmental containment in field applications.

Construction of Gene Circuits

To engineer strains capable of degrading chlorimuron-ethyl, we selected three enzymes: SulE (esterase) from Hansschlegelia zhihuaiae, PnbA (carboxylesterase) from strain CHL1, and GST (glutathione S-transferase) from Klebsiella jilinsis 2N3. We retrieved the corresponding gene sequences from NCBI and created the gene circuit diagrams and plasmid maps for each (see Figure 1).

Figure 1. Gene circuit designs and plasmid maps for the three chlorimuron-ethyl–degrading enzymes.

Overexpression Strains for Degradative Enzymes

We synthesized GST, SulE, and PnbA coding sequences (Generalbial, China), performed codon optimization for E. coli, and removed EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI sites to comply with RFC#10 and pET28a(m) cloning requirements. Inserts were cloned into pET28a(m) via NdeI/XhoI. After PCR amplification, target bands were verified (Figure 2), and the recombinant plasmids were transformed into E. coli DH5α by heat shock (42 °C, 1 min). Kanamycin (100 μg/mL) LB agar (1.5% agar) was used to select positive colonies, which were sequence-verified (Tsingke, Beijing). Plasmids were purified (DP103, Tiangen) and transformed into E. coli BL21(DE3) to generate the expression strains.

Figure 2. Agarose gel electrophoresis confirming insert amplification

Expression and Assay of Degradative Enzymes

To confirm expression, IPTG induction was performed for all three strains, crude lysates were prepared, and Western blot (WB) verified protein production. Results indicated successful expression of SulE, PnbA, and GST (see Figure 3).

Figure 3. Western blot verification of the three degradative enzymes

Functional Validation: Standard Curve

We first established a standard curve for chlorimuron-ethyl by regressing A450 values against the log10 concentration of the herbicide (Figure 4).

Figure 4. Chlorimuron-ethyl calibration: (A) Concentration vs. A₄₅₀; (B) log₁₀(concentration) vs. A₄₅₀.

To compare catalytic activities, 1 mL of crude enzyme (1 μg/mL) was incubated with 20 μg/mL chlorimuron-ethyl at 25 °C for 10 min. Samples (50 μL) were 10× diluted and analyzed using a competitive ELISA kit (ml103804, mlbio). Student’s t-test was used (two groups; p < 0.05 considered significant), with ≥3 biological replicates. PnbA performed best, yielding A450 = 0.57, versus 0.39 for SulE and 0.40 for GST (see Figure 5).

Figure 5. ELISA readouts after treatment with the three enzymes

Using the calibration curve, residual herbicide and degradation rates were calculated from A450 values. After 10 min at 25 °C, PnbA achieved 61.2% degradation, versus 29.8% (SulE) and 32.3% (GST) (Figure 6).

Figure 6. Degradation percentages after 10 min at 25 °C

Specific activity (U/μg) was defined as enzyme required to degrade 1 μmol of chlorimuron-ethyl per minute at 25 °C (MW = 357.76 g/mol). Measured values: PnbA = 0.0038 U/μg, SulE = 0.0019 U/μg, GST = 0.0021 U/μg (Figure 7).

Figure 7. Specific activities of PnbA, SulE, and GST

Optimal Conditions for PnbA

First, we investigated the catalytic capacity of PnbA at different temperatures by adjusting the incubator to fixed setpoints and testing the crude enzyme for its optimal reaction temperature. Taking the highest specific activity as 100%, we calculated the relative activity of each group. The results showed that PnbA exhibited optimal catalytic performance at 30 °C (Figure 8).

Figure 8. Effect of temperature on PnbA activity

Next, to investigate the effect of pH on the catalytic efficiency of PnbA, we adjusted the pH of PBS using 1 M NaOH and 1 M HCl, and tested the crude enzyme for its optimal pH. The experimental results demonstrated that PnbA showed the highest catalytic activity at pH = 7 (Figure 9).

Figure 9. Effect of pH on PnbA activity

Analysis of Chlorimuron-ethyl Degradation by Whole-cell Catalysts

To evaluate whole-cell catalytic degradation of chlorimuron-ethyl, we used ice nucleation protein (INP) as an anchoring carrier to express PnbA on the bacterial cell surface. Specifically, we introduced the INP sequence into the BL21-PnbA strain to obtain the BL21-INP-PnbA engineered strain, and collected the cultured cell pellets.

A 20 μg/mL chlorimuron-ethyl solution (C109992, Aladdin) was added, and the reaction was carried out at 25 °C for 30 minutes. The competitive ELISA assay was then performed to measure the degradation capacity of wild-type BL21, BL21-PnbA, and BL21-INP-PnbA.

The results showed that BL21 and BL21-PnbA exhibited negligible degradation, whereas BL21-INP-PnbA effectively degraded 28.1 μg/mL of chlorimuron-ethyl within 30 minutes (Figure 10).

Figure 10. Analysis of chlorimuron-ethyl degradation by different whole-cell catalysts.(A) Genetic circuit diagram of PnbA surface display; (B) PCR verification of the INP fragment;(C) Whole-cell catalytic degradation of chlorimuron-ethyl after 30 minutes of reaction

Construction of IAA-producing Strains

First, we designed the genetic circuit for IAA biosynthesis and obtained the iaaM and iaaH genes—key components of the IAA pathway—from the NCBI database. These genes were codon-optimized for E. coli and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with the RFC#10 assembly standard and pET28a(m) cloning requirements. The optimized gene fragments were cloned into the pET28a(m) vector via the NdeI and XhoI restriction sites. Finally, the verified constructs were transformed into E. coli BL21 (DE3) to obtain the engineered IAA-producing strains (Figure 11).

Figure 11. Schematic diagram of the IAA biosynthetic pathway and related genes

Evaluation of IAA Production in Engineered Strains

First, the standard curve of indole-3-acetic acid (IAA) was established using a commercial ELISA assay kit by measuring absorbance at 450 nm (Figure 12).

Figure 12. Standard curve of indole-3-acetic acid (IAA). (A) Relationship between IAA concentration and A₄₅₀ absorbance in ELISA assay.(B) Linear regression of log₁₀(IAA concentration) versus A₄₅₀ absorbance

Next, the engineered strain was cultured in 5 mL of LB medium supplemented with kanamycin. When the OD₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce expression, and the culture was shaken at 180 rpm for 12 hours. After induction, 1.5 mL of bacterial culture was centrifuged, and the supernatant was analyzed using the IAA ELISA kit, measuring absorbance at 450 nm. IAA concentrations were calculated by comparison with the standard curve. The results showed that the IAA-producing engineered strain synthesized 14.9 μM of IAA after 12 hours of induction at 30°C, whereas wild-type BL21 and the strain expressing only IaaM produced negligible amounts of IAA (Figure 13).

Figure 13. Comparison of IAA concentrations between engineered strains and wild-type BL21 after 12-hour induction at 30°C

Finally, we quantified the IAA production over a 24-hour period, and the results demonstrated that the engineered strain continuously produced IAA, reaching a final concentration of 15.09 μM within 24 hours (Figure 14).

Figure 14. Time-course of IAA production in engineered E. coli within 24 hours

IIAA Promotion of Plant Growth

Table 1. Effect of IAA on the growth and development of wheat seedlings

To verify whether IAA promotes plant growth, we selected healthy and intact wheat seeds, disinfected them with 75% ethanol for 1 minute, and rinsed them three times with sterile water. The seeds were then soaked in 0 μM IAA solution (control) and 10 μM IAA solution (treatment) for 4–6 hours. After soaking, the seeds were evenly placed on moist filter paper in Petri dishes, which were maintained at approximately 25°C in a constant-temperature incubator. The filter paper was kept moist throughout the experiment. Germination and growth were observed daily, and root length and plant height were measured on days 5, 10, and 15 to assess the growth-promoting effect of IAA (Table 1).

Figure 15. Growth comparison of control and IAA-treated wheat seedlings on day 15

Experimental results showed that:

• On day 5, the average plant height and root length of the treatment group were 4.17 cm and 2.43 cm, compared to 3.87 cm and 2.10 cm in the control group.
• On day 10, the treatment group averaged 10.40 cm in height and 9.27 cm in root length, while the control group measured 8.90 cm and 5.07 cm, respectively.
• On day 15, the treatment group reached 17.47 cm in height and 15.9 cm in root length, compared to 14.83 cm and 8.23 cm in the control.

Overall, IAA-treated wheat seedlings exhibited significantly enhanced growth, confirming that IAA effectively promotes seed germination and seedling development (Figure 15).

Validation of the Cold-Inducible Promoter Function

Given that our application scenario occurs in September, we selected a cold-inducible promoter to prevent potential release of the engineered bacteria into the environment. To evaluate its function, we constructed an mRFP reporter system driven by the PcspA promoter (Figure 16A, B). Experimental results showed that at 16°C, the engineered bacteria expressed red fluorescent protein (RFP) efficiently, whereas expression at 37°C (the optimal growth temperature for E. coli) was significantly lower (Figure 16C).

Figure 16. Verification of the cold-inducible promoter system. (A) Design of the PcspA–mRFP genetic circuit. (B) Verification of PcspA and mRFP fragments. (C) Fluorescence analysis under low-temperature induction.

Construction and Verification of the Suicide System

Next, we cloned T4 phage-derived lysis genes—T4 holin and T4 lysozyme—into plasmids to test their functionality. At 37°C, both wild-type and engineered strains showed similar growth curves, with no observable cell lysis. However, when the temperature was reduced to 16°C, the engineered strain exhibited progressive cell death, confirming the successful activation and function of the suicide gene system (Figure 17).

Figure 17. Verification of the temperature-dependent suicide system. (A) Design of the suicide gene circuit. (B) Validation of T4 holin and T4 lysozyme fragments. (C) Growth curve at 37°C (no induction). (D) Growth curve at 16°C, showing temperature-triggered self-lysis.

This study successfully constructed and validated multiple functional engineered strains, achieving the following:

• Chlorimuron-ethyl–degrading strain:All three enzymes showed degradation activity, with PnbA performing best (61.2% degradation rate, specific activity 0.0038 U/μg). Under optimized conditions, PnbA displayed excellent catalytic efficiency, and surface expression via INP further enhanced its degradation performance.
• IAA-producing strain: The engineered strain stably synthesized 14.9–15.09 μM IAA within 12–24 hours, effectively promoting wheat germination and growth.
• Cold-inducible biosafety system: The PcspA promoter successfully triggered gene expression at 16°C, demonstrating potential for environmental containment and biosafety assurance.

In summary, this work establishes an efficient microbial catalytic system for chlorimuron-ethyl degradation, constructs an IAA-producing strain that stimulates plant growth, and integrates a cold-inducible safety mechanism for biocontainment.

Together, these advances provide a feasible and controllable synthetic biological strategy for agricultural pollutant remediation and crop growth enhancement.