The genes encoding GST, SulE, and PnbA were synthesized (Generalbial, China), codon-optimized for E. coli, and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards and the cloning requirements of the pET28a(m) vector, after which the fragments were inserted into the pET28a(a) backbone using NdeI and XhoI restriction sites. Competent E. coli BL21(DE3) (D1009S) and DH5α (D0351) cells were purchased from Beyotime (China), and the recombinant plasmids were transformed into E. coli DH5α via heat shock at 42 °C for 1 min, followed by selection on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin, with positive clones verified by sequencing (Qingke, Beijing) to obtain DH5α recombinant strains. Plasmids were extracted using a plasmid extraction kit (DP103, Tiangen) and subsequently transformed into E. coli BL21(DE3) to generate engineered expression strains, which were cryopreserved at –80 °C with 25% (v/v) glycerol as a cryoprotectant. The engineered strains were cultured at 37 °C, 150 rpm in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin for inoculation and scale-up.

The engineered strains BL21-GST, BL21-SulE, and BL21-PnbA were inoculated into LB medium supplemented with 100 μg/mL kanamycin at a 1:100 ratio and activated overnight at 37 °C, 150 rpm. Subsequently, 100 μL of the overnight culture was transferred into 20 mL of LB medium containing 100 μg/mL kanamycin in a sterile 50 mL centrifuge tube and cultured at 37 °C, 180 rpm for 3–4 h until the OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG (ST098-1g, Beyotime) and lowering the incubation temperature to 16 °C for an additional 16 h. Cells were harvested by centrifugation at 10,000 × g for 5 min, and the pellet was resuspended in 5 mL of pre-chilled PBS (pH 7.2) at 4 °C. Cell disruption was performed on ice using an ultrasonic cell disruptor (Jingxin, Shanghai, China) under the conditions of 70 W power, 1 s on/3 s off, for a total of 20 min. The lysate was centrifuged at 13,000 × g for 20 min at 4 °C, and the supernatant was collected as the crude enzyme extract. The supernatant was filtered through a 0.22 μm PES hydrophilic membrane (BS-PES-45, Biosharp), then concentrated to ~1 mL using a 10 kDa ultrafiltration tube (BS-UFC-150-010, Biosharp) by centrifugation at 2,500 × g for 30–40 min at 4 °C. Protein concentration was determined at 595 nm using the Bradford assay (P0006, Beyotime) with BSA as the standard. Finally, the crude enzyme extract was aliquoted in 100 μL portions and stored at –80 °C for further use.

The crude enzyme extract was diluted in PBS to a final concentration of 1 μg/mL, and 20 μg/mL chlorimuron-ethyl (C109992, Aladdin) was added. After incubation at 25 °C for 10 min, a 50 μL sample was taken, diluted 10-fold, and analyzed using a chlorimuron-ethyl ELISA kit (ml103804, mlbio) to determine the residual chlorimuron-ethyl content. A standard curve was generated by plotting the log₁₀-transformed chlorimuron-ethyl concentration as the independent variable against the absorbance at 450 nm (A₄₅₀) as the dependent variable through regression analysis. Based on the standard curve and the measured A₄₅₀ values, the remaining chlorimuron-ethyl concentration and degradation rate (%) were calculated. One unit of enzyme activity (U) was defined as the amount of enzyme required to degrade 1 μmol of chlorimuron-ethyl per minute at 25 °C, and the specific activity was calculated according to the change in chlorimuron-ethyl concentration and its molecular weight (357.76 g/mol). To determine the optimal pH, PBS was adjusted with 1 M NaOH or 1 M HCl, and enzyme activity was measured under different conditions. Similarly, the optimal temperature was determined by incubating the reactions at varying constant temperatures. Relative enzyme activity was calculated by normalizing each condition to the maximum specific activity, which was set as 100%.

The INP-PnbA gene was synthesized and codon-optimized to comply with RFC#10 standards, then cloned into the pET28a(m) vector via NdeI and XhoI restriction sites. A total of 100 μg of the recombinant plasmid was transformed into E. coli BL21(DE3) (D1009S, Beyotime) using the heat-shock method. Transformants were selected on LB agar plates containing 100 μg/mL kanamycin, and positive clones were verified by colony PCR. Correct clones were subsequently inoculated into 5 mL of LB medium containing 100 μg/mL kanamycin and cultured overnight at 37 °C, 180 rpm. For cryopreservation, 1 mL of the BL21-INP-PnbA culture was mixed with 1 mL of 50% (v/v) glycerol and stored at –80 °C for future use.

Engineered strains and control strains were inoculated into LB medium containing 100 μg/mL kanamycin at a 1:100 ratio and cultured overnight at 37 °C, 150 rpm. Subsequently, 100 μL of the overnight culture was transferred into 5 mL of LB medium containing 100 μg/mL kanamycin in a sterile 50 mL centrifuge tube and incubated at 37 °C, 180 rpm for 3–4 h until the OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG (ST098-1g, Beyotime) and lowering the temperature to 16 °C for an additional 16 h. Cells were harvested by centrifugation at 10,000 × g for 5 min, washed three times with PBS (pH 7.2), and resuspended in 1 mL PBS, adjusting the OD600 to 1. Chlorimuron-ethyl (C109992, Aladdin) was added to a final concentration of 20 μg/mL. The reaction was carried out at 25 °C for 30 min, after which samples were centrifuged at 10,000 × g for 1 min. The pellet was discarded, and 50 μL of the supernatant was collected for analysis. Chlorimuron-ethyl concentration was determined using a chlorimuron-ethyl ELISA kit (ml103804, mlbio), and the residual concentration was calculated based on the standard curve and absorbance at 450 nm (A₄₅₀).

The iaaM and iaaH genes of the IAM pathway were synthesized (Generalbial, China) and assembled into a bicistronic sequence with the RBS B0034 between the two genes. The sequences were codon-optimized for E. coli and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards and the cloning requirements of the pET28a(m) vector. The constructs were cloned into the pET28a(a) backbone using NdeI and XhoI restriction sites. After sequence verification, the recombinant plasmids were transformed into E. coli BL21(DE3) (D1009S, Beyotime). Positive clones were selected on LB agar plates (1.5% agar) supplemented with 100 μg/mL kanamycin. Strains were preserved at –80 °C using 25% (v/v) glycerol as a cryoprotectant.

The IAA-producing engineered strains were inoculated into 5 mL of LB medium containing kanamycin and cultured overnight (12–16 h) at 37 °C, 180 rpm in a shaking incubator for activation. A 100 μL aliquot of the activated culture was transferred into 5 mL of fresh LB medium in a 25 mL Erlenmeyer flask and incubated at 37 °C, 180 rpm for 3–4 h. The optical density of the culture was measured at 600 nm (OD₆₀₀) using a microplate reader (FlexStation 3, Molecular Devices, USA). When OD₆₀₀ reached 0.6, protein expression was induced by adding 0.5 mM IPTG, and the incubation temperature was adjusted to 30 °C for an additional 12 h at 180 rpm. Throughout cultivation, the flasks were wrapped in aluminum foil to prevent IAA degradation caused by light exposure. After induction, 1.5 mL of culture was collected into a 1.5 mL microcentrifuge tube and centrifuged at 10,000 × g for 1 min. From the supernatant, 1 mL was collected, and 10 μL was mixed with 900 μL of blank LB medium to prepare a 100-fold diluted sample, which was thoroughly mixed and used for analysis. The IAA concentration was determined using an indole-3-acetic acid ELISA kit (YJ147100, YanjuBio). A standard curve was generated with known IAA concentrations, and absorbance (OD) was measured at 450 nm using a microplate reader (FlexStation 3, Molecular Devices, USA) to quantify IAA levels in the bacterial culture.

Overnight-activated cold-inducible reporter strains were inoculated into LB medium at a 1:100 ratio and cultured at different temperatures for 12 h under 180 rpm shaking conditions. A 200 μL sample of the culture was taken, and fluorescence (excitation wavelength: 584 nm; emission wavelength: 607 nm) as well as OD600 were measured using a microplate reader (Thermo Fisher Scientific, USA). The normalized fluorescence ratio (Fluorescence/OD600) was then calculated.

The cold-inducible promoter PcspA and E. coli lethal genes T4 Holin–T4 Lysozyme were synthesized, with the T4 Holin–T4 Lysozyme operon placed downstream of PcspA. The T4 Holin and T4 Lysozyme sequences were codon-optimized for E. coli, and EcoRI, XbaI, SpeI, and PstI restriction sites were removed to comply with RFC#10 standards. The construct PcspA–T4 Lysis was cloned into the pSB1A3 vector between the XbaI and SpeI sites. Recombinant plasmids were transformed into E. coli BL21, and positive clones were screened on LB agar plates containing 100 μg/mL ampicillin (Amp) and verified by sequencing (Tsingke, China), yielding engineered strains. Engineered strains were cultured at 37 °C in LB broth supplemented with ampicillin, and bacterial growth was monitored by measuring OD600 using a microplate reader (FlexStation 3, Molecular Devices, USA).

Overnight-activated cold-inducible lysis strains were inoculated at a 1:100 ratio into 5 mL of Amp+LB medium and cultured at 16 °C or 37 °C under 180 rpm shaking conditions for 20 h. At regular intervals, 200 μL culture samples were taken, and OD600 was measured using a microplate reader (FlexStation 3, Molecular Devices, USA) to generate bacterial growth curves.

Healthy and intact wheat seeds were surface-sterilized with 75% ethanol for 1 min, followed by three rinses with sterile water. The seeds were then soaked in either 0 μM (control) or 10 μM IAA solution for 4–6 h. After soaking, seeds were evenly placed onto Petri dishes lined with moistened filter paper, which was kept hydrated throughout cultivation at ~25 °C under constant conditions. Seed germination was monitored daily, and root length and shoot height were measured on days 5, 10, and 15 to evaluate the effect of IAA on wheat germination and growth.

Data analysis and graphing were performed using GraphPad Prism software. Results were presented as mean ± standard deviation (SD). For multiple group comparisons, one-way ANOVA followed by Tukey’s post-hoc test was used. For comparisons between two groups, Student’s t-test was applied. A p-value less than 0.05 was considered statistically significant. All experiments were performed with at least three independent biological replicates (n ≥ 3).