Chlorimuron-ethyl is a sulfonylurea herbicide widely used in the soybean–wheat rotation system. It inhibits acetohydroxyacid synthase (AHAS/ALS) to block the synthesis of branched-chain amino acids, thereby suppressing crop growth. However, its strong soil persistence may cause rooting disorders in subsequent crops, soil microbiome imbalance, and water pollution, posing risks to agricultural production and the ecosystem.

To address this, we designed and constructed a chlorimuron-ethyl degradation system. We screened three degradative enzymes from different sources—SulE (esterase) from H. zhihuaiae, PnbA (carboxylesterase) from strain CHL1, and GST (glutathione S-transferase) from K. jilinsis 2N3—and achieved heterologous expression and validation in E. coli. ELISA quantification showed that PnbA performed best, degrading 61.2% of chlorimuron-ethyl within 10 min, with optimal conditions at 30 ℃ and pH 7.

We further applied a cell surface display strategy to anchor PnbA on the host surface, enabling whole-cell catalysis and improving degradation efficiency. Meanwhile, to ensure controllability and biosafety in environmental applications, we designed an cold-inducible suicide system, allowing on-demand self-lysis to mitigate risks of gene and strain release. Altogether, from enzyme validation and whole-cell catalysis to safety control, we provide a complete degradation solution, offering a new avenue for bioremediation of chlorimuron-ethyl in farmland.

Cycle 1-1: Validation of Degradative Enzymes

Design

Chlorimuron-ethyl, a sulfonylurea herbicide commonly used in soybean–wheat rotations for its low cost and rapid action, inhibits AHAS/ALS, thereby blocking the synthesis of valine, leucine, and isoleucine. This disrupts protein/enzyme production, inhibits cell division in meristematic tissues, and ultimately causes plant death.

However, due to its long soil persistence (≥6 months), chronic use impairs rooting and growth of subsequent crops, disrupts soil microbial communities and enzyme activities, and reduces soil fertility. Its high water solubility facilitates entry into aquatic systems, interfering with aquatic plant growth and potentially causing DNA damage in aquatic/amphibian species—threatening ecosystems and biodiversity. It also suppresses chlorophyll and superoxide dismutase, reducing photosynthesis and nutrient uptake.

Therefore, we selected three degradative enzymes: SulE from H. zhihuaiae, PnbA from strain CHL1, and GST from K. jilinsis 2N3.

Figure 1. Principle of chlorimuron-ethyl–degrading enzymes.

Build

To obtain strains capable of efficiently degrading chlorimuron-ethyl, we designed a dedicated genetic circuit comprising the chlorimuron-ethyl–degrading enzymes. We selected E. coli BL21 (DE3) as the chassis organism and, using standard molecular cloning procedures, inserted the coding sequences of SulE (esterase from H. zhihuaiae), PnbA (carboxylesterase from strain CHL1), and GST (glutathione S-transferase from K. jilinsis 2N3) into the pET28a(m) expression vector. The resulting recombinant plasmids were then transformed into E. coli BL21 (DE3) to establish the engineered degradation strains (see Figure 2).

Figure 2. Gene circuit and plasmid maps for the degradation system.

Testing

Coding sequences for SulE, PnbA, and GST were obtained from NCBI, codon-optimized for E. coli, and edited to remove EcoRI, XbaI, SpeI, PstI, NdeI, XhoI to meet RFC#10 and pET28a(m) requirements, then synthesized (Generalbial, China). Inserts were cloned via NdeI/XhoI into pET28a(m). After PCR amplification and gel verification (Figure 3), heat-shock transformation (42 ℃, 1 min) into E. coli DH5α was performed. Kan (100 μg/mL) LB agar (1.5% agar) was used to select positives, which were sequenced (Tsingke, Beijing). Plasmids were purified (DP103, Tiangen) and transformed into E. coli BL21(DE3) to yield expression strains.

Figure 3. Agarose gel confirming insert amplification.

For expression, strains were IPTG-induced, crude lysates prepared, and Western blot (WB) confirmed protein production for all three enzymes (Figure 4).

Figure 4. Western blot validation of SulE, PnbA, and GST expression.

To evaluate the enzyme activity and degradation efficiency, we first established a standard calibration curve for chlorimuron-ethyl. The log10 of chlorimuron-ethyl concentration was used as the independent variable, and the absorbance at 450 nm (A450) measured by ELISA served as the dependent variable.

A regression analysis was performed to generate the standard curve correlating concentration and absorbance (Figure 5).

Figure 5. Standard curve of chlorimuron-ethyl. (A) Relationship between chlorimuron-ethyl concentration and A₄₅₀ absorbance determined by ELISA.(B) Linear correlation between log₁₀ (chlorimuron-ethyl concentration) and A₄₅₀ absorbance.

To verify the catalytic efficiency of the three degradation enzymes, we tested the ability of their crude enzyme extracts to degrade chlorimuron-ethyl. A total of 1 mL of enzyme solution (1 μg/mL) was added to 20 μg/mL chlorimuron-ethyl, and the reaction was carried out at 25 ℃ for 10 minutes. Afterward, 50 μL of each sample was diluted tenfold, and the remaining chlorimuron-ethyl concentration was determined using an ELISA kit (ml103804, Mlbio).

For data analysis, Student’s t-test was applied to compare two groups, with p < 0.05 considered statistically significant. All experiments were performed with at least three independent biological replicates (n ≥ 3). The results demonstrated that PnbA showed the highest degradation capacity, with an absorbance at 450 nm (A450) of 0.57, compared to 0.39 for SulE and 0.40 for GST (Figure 6).

Figure 6. ELISA analysis of chlorimuron-ethyl degradation by different enzymes.

Based on the standard curve and the measured A450 values, we calculated the residual chlorimuron-ethyl concentration and corresponding degradation rates (%). After a 10-minute reaction at 25 ℃, PnbA achieved a 61.2% degradation rate, while SulE and GST showed 29.8% and 32.3%, respectively (Figure 7).

Figure 7. Degradation efficiency of SulE, PnbA, and GST after 10-minute reactions at 25 ℃.

Next, we defined one enzyme activity unit (U) as the amount of enzyme required to degrade 1 μmol of chlorimuron-ethyl per minute at 25 ℃. Specific activity (U/μg) was calculated based on the change in chlorimuron-ethyl concentration and its molecular weight (357.76 g/mol). The results showed that PnbA exhibited the highest specific activity of 0.0038 U/μg, followed by GST (0.0021 U/μg) and SulE (0.0019 U/μg) (Figure 8).

Figure 8. Comparison of specific enzyme activities of SulE, PnbA, and GST.

We further investigated the optimal reaction conditions of PnbA. By adjusting the incubation temperature, we determined the optimal temperature based on relative activity, calculated as a percentage of the maximum specific activity (100%). The results indicated that PnbA exhibited its highest catalytic activity at 30 ℃ (Figure 9A). To determine the optimal pH, the pH of PBS buffer was adjusted using 1 M NaOH and 1 M HCl, and enzyme activity was measured accordingly. The results confirmed that PnbA performed best at pH = 7 (Figure 9B).

Figure 9. Optimal reaction conditions for PnbA. (A) Temperature-dependent enzyme activity assay. (B) pH-dependent enzyme activity assay.

Learn

We successfully evaluated the catalytic performance of the three chlorimuron-ethyl–degrading enzymes and identified PnbA as the most effective enzyme.

At 25 ℃ for 10 minutes, PnbA degraded 61.2% of chlorimuron-ethyl with a specific activity of 0.0038 U/μg. Furthermore, the optimal catalytic conditions of PnbA were determined to be 30 ℃ and pH = 7, providing a solid foundation for subsequent whole-cell catalysis experiments.

Cycle 1-2 Cell Surface Display Technology

Design

Cell surface display enables the presentation of peptides or proteins on the surface of microbial cells by fusing them with an anchoring motif. The fusion between the target protein and anchoring protein can occur through N-terminal fusion, C-terminal fusion, or sandwich fusion, depending on the structural requirements. In this project, we selected ice nucleation protein (INP) as the anchoring carrier. INP is a well-characterized outer membrane protein originating from ice-nucleating bacteria associated with plants, known for its ability to induce ice crystal formation in supercooled water. Due to its robust anchoring efficiency, INP has been widely applied in E. coli surface display systems. Therefore, we designed a fusion construct where INP was fused to the N-terminus of PnbA, enabling the anchoring of PnbA on the cell surface to facilitate whole-cell catalysis of chlorimuron-ethyl degradation (Figure 10).

Figure 10. Schematic diagram of PnbA surface display using INP as an anchoring protein.

Build

To enable whole-cell catalysis, we utilized ice nucleation protein (INP) as a carrier for anchoring PnbA to the outer membrane of E. coli. The INP sequence was introduced into the BL21–PnbA strain, generating the engineered strain BL21–INP–PnbA (Figure 11).

Figure 11. Construction of the BL21–INP–PnbA strain for whole-cell degradation of chlorimuron-ethyl.

Test

To evaluate the catalytic performance of the surface-displayed system, we collected the cell pellets from the cultured strains and resuspended them in a 20 μg/mL chlorimuron-ethyl solution (C109992, Aladdin).The mixtures were incubated at 25 ℃ for 30 minutes.

After incubation, the remaining chlorimuron-ethyl in the supernatant was quantified using a competitive ELISA assay. We compared the degradation capacities of wild-type BL21, BL21–PnbA, and BL21–INP–PnbA. The results showed that BL21 and BL21–PnbA exhibited negligible degradation activity, whereas BL21–INP–PnbA efficiently degraded 28.1 μg/mL of chlorimuron-ethyl within 30 minutes (Figure 12).

Figure 12. Degradation of chlorimuron-ethyl by whole-cell catalysis after 30 minutes of reaction at 25 ℃.The engineered strain BL21–INP–PnbA demonstrated significantly higher degradation efficiency compared to control strains.

Learn

We successfully utilized cell surface display technology to express PnbA on the outer surface of E. coli, enabling whole-cell catalysis. The engineered strain BL21–INP–PnbA effectively degraded 28.1 μg/mL of chlorimuron-ethyl after 30 minutes at 25 ℃, confirming that surface anchoring markedly enhances degradation performance.

Design

To address the issue of soil fertility depletion caused by long-term crop rotation, which limits plant growth and development, our team designed an indole-3-acetic acid (IAA) expression system after consultation with agricultural experts. The goal of this system is to reduce the negative impact of herbicide residues while simultaneously enriching the soil with plant growth–promoting substances, thereby enhancing seed germination and plant development. IAA, a naturally occurring auxin, is synthesized through the tryptophan–indole-3-acetamide (IAM) pathway, which involves two enzymatic reactions. In the first step, tryptophan is converted to IAM by the enzyme tryptophan-2-monooxygenase (IaaM) encoded by the iaaM gene. In the second step, IAM is hydrolyzed to IAA by IAM hydrolase (IaaH) encoded by the iaaH gene (Figure 13).

Figure 13. Schematic diagram of the heterologous IAA biosynthesis pathway in E. coli

Build

To construct the IAA biosynthesis strain, we first designed a gene circuit for IAA synthesis. The genes iaaM and iaaH were retrieved from the NCBI database, codon-optimized for E. coli, and all restriction sites including EcoRI, XbaI, SpeI, and PstI were removed to comply with the RFC#10 standard and pET28a(m) cloning requirements. The optimized genes were cloned into the pET28a(m) expression vector via NdeI and XhoI restriction sites, and the verified constructs were then transformed into E. coli BL21 (Figure 14).

Figure 14. Gene circuit and related genetic elements for IAA biosynthesis in E. coli.

Test

To quantify IAA production, we first established a standard calibration curve using an indole-3-acetic acid ELISA kit, measuring absorbance at 450 nm (Figure 15).

Figure 15. Standard curve of indole-3-acetic acid (IAA).(A) Relationship between IAA concentration and A₄₅₀ absorbance determined by ELISA. (B) Linear correlation between log₁₀ (IAA concentration) and A₄₅₀ absorbance.

The engineered strains were cultured in 5 mL of LB medium containing kanamycin, and when the culture reached OD₆₀₀ = 0.6, 0.5 mM IPTG was added for induction. After 12 hours of shaking incubation at 180 rpm and 30℃, 1.5 mL of culture was centrifuged, and the supernatant was collected for ELISA measurement. Absorbance readings at 450 nm were compared to the standard curve to calculate IAA concentrations. The results showed that the engineered strain produced 14.9 μM of IAA after 12 hours of induction at 30℃, while the wild-type E. coli BL21 and the strain expressing only iaaM exhibited negligible IAA production (Figure 16).

Figure 16. Comparison of IAA concentrations between the engineered strain and wild-type E. coli BL21 after 12 hours of induction at 30℃.

To assess production stability, we measured IAA accumulation over a 24-hour period, and the results demonstrated that the engineered strain could produce 15.09 μM of IAA within 24 hours (Figure 17).

Figure 17. IAA production levels of the engineered strain at different time points within 24 hours.

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

To evaluate the effect of microbially synthesized IAA on plant development, we conducted a wheat seed germination and growth assay. Healthy, intact wheat seeds were surface sterilized with 75% ethanol for 1 minute and rinsed three times with sterile water. The seeds were soaked in either 0 μM IAA (control) or 10 μM IAA (treatment) for 4–6 hours, then placed evenly on moist filter paper in Petri dishes. The dishes were kept at 25℃ under constant humidity, and germination and growth were observed daily. Root length and shoot height were measured on day 5, day 10, and day 15, and the results are summarized in Table 1.

Figure 18. Comparison of wheat seedling growth between the control group (0 μM IAA) and the experimental group (10 μM IAA) on day 15.

On day 5, the treatment group exhibited an average shoot height of 4.17 cm and root length of 2.43 cm, compared to 3.87 cm and 2.10 cm in the control group. On day 10, the treatment group measured 10.40 cm and 9.27 cm, while the control group measured 8.90 cm and 5.07 cm. On day 15, the treated seedlings reached 17.47 cm in height and 15.9 cm in root length, whereas the control group measured 14.83 cm and 8.23 cm, respectively. These results indicate that IAA significantly enhances seed germination and promotes the growth of wheat seedlings (Figure 18).

Learn

In summary, we successfully constructed an IAA-producing E. coli strain capable of synthesizing 14.9 μM of IAA within 12 hours at 30℃. Furthermore, the addition of 10 μM IAA significantly promoted wheat germination and growth, with the experimental group exhibiting greater vigor and development than the control group by day 15. These results demonstrate that our engineered strain can effectively synthesize plant auxin and promote crop growth, providing a potential biological solution for soil fertility enhancement and sustainable agriculture.

Cycle 3-1 Cold-Inducible Promoter Test

Design

To ensure biosafety under field conditions, we designed a temperature-responsive regulatory system tailored to the soybean–wheat crop rotation widely practiced in northern China. This agricultural cycle typically occurs from September to October, when daytime temperatures range from 23℃ to 28℃, and nighttime temperatures drop to 12℃–19℃. Therefore, a cold-inducible promoter was chosen to control gene expression in a temperature-dependent manner—activating degradation genes during the warmer daytime to efficiently break down chlorimuron-ethyl, while triggering self-lysis at lower nighttime temperatures, ensuring that the engineered bacteria do not persist or spread in the environment.

Build

To test the functionality of the cold-inducible promoter, we constructed an mRFP reporter system under the control of the PcspA promoter (Figure 16).

Figure 19. Construction of the cold-inducible promoter validation system, in which the PcspA promoter drives mRFP expression as a visible reporter.

Test

Experimental results demonstrated that after incubation for 12 hours at different temperatures (16℃, 25℃, and 37℃), the measured fluorescence/OD₆₀₀ values were 221.32, 202.08, and 38.92, respectively. These results indicate that the promoter exhibited the strongest activity at 16℃, confirming its low-temperature inducibility (Figure 17).

Figure 20. Performance of the cold-inducible promoter (PcspA) at different temperatures. The fluorescence/OD₆₀₀ ratio is highest at 16℃, indicating maximal induction of mRFP expression.

Learn

We successfully validated the function of the cold-inducible promoter in E. coli, confirming that PcspA can effectively induce the expression of mRFP at 16℃, providing a reliable temperature-sensitive regulatory element for subsequent biosafety control.

Cycle 3-2 Suicide System

Design

To further enhance biosafety, we designed a temperature-dependent suicide system regulated by the CspA promoter, enabling the engineered bacteria to undergo self-lysis when exposed to low temperatures. The system is based on two lytic proteins derived from T4 bacteriophage—T4 Holin and T4 Lysozyme. T4 Holin is a small membrane protein containing two transmembrane domains, with its N-terminus located in the cytoplasm and the C-terminus in the periplasmic space. This structure allows Holin to form pores in the bacterial membrane, disrupting membrane integrity. T4 Lysozyme, a small globular protein consisting of 164 amino acids, binds to the bacterial cell wall, where it cleaves the glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid. This action compromises the cell wall’s structural stability and disrupts osmotic balance, ultimately leading to cell lysis and death.

Build

We constructed a plasmid in which the pCspA promoter drives the co-expression of T4 Holin and T4 Lysozyme, enabling temperature-controlled activation of the suicide system (Figure 18).

Figure 21. Design and fragment verification of the temperature-inducible suicide system driven by the CspA promoter, including T4 Holin and T4 Lysozyme modules.

Test

To evaluate the system, we compared the growth curves of the engineered strain and wild-type E. coli at 37℃ and 16℃. At 37℃, both strains exhibited similar growth patterns, indicating that the suicide system remained inactive under normal cultivation conditions. However, when the temperature was lowered to 16℃, the engineered strain underwent continuous cell death, validating the functional activation of the suicide gene circuit (Figure 19).

Figure 22. Functional validation of the temperature-inducible suicide system at 37℃ and 16℃.

Learn

We successfully constructed and validated the temperature-inducible suicide system, confirming that it can effectively trigger self-lysis at 16℃, while remaining inactive at 37℃. This ensures environmental safety by preventing the uncontrolled proliferation of engineered bacteria outside the intended application context.

In this study, we successfully constructed and validated an integrated chlorimuron-ethyl degradation system with embedded safety controls. Among the three degradation enzymes tested, PnbA was identified as the optimal candidate, exhibiting the highest degradation rate and specific activity. By combining cell surface display technology, the engineered bacteria achieved efficient degradation of chlorimuron-ethyl within a short time frame, significantly enhancing its application potential. Furthermore, the introduction of a cold-inducible suicide system ensures precise control and environmental containment of the engineered strain. Collectively, this work provides an effective biological solution for the remediation of chlorimuron-ethyl pollution, while establishing a biosafe framework for engineered microorganisms in environmental and agricultural applications.