The phnE1 and phnE2 genes from Sinorhizobium meliloti 1021 were synthesized, and codon-optimized for expression in E. coli. These genes were cloned into the pSB1A3 vector as polycistronic units to form a recombinant plasmid. The plasmid was then transformed into E. coli BL21 to create the engineered strain. The strain was grown in LB medium containing 50 µg/mL ampicillin at 37°C.

The engineered strain was cultured overnight in LB medium containing 100 µg/mL ampicillin at 37°C with shaking at 180 rpm. The next day, the culture was diluted 1:100 into LB medium containing 80 mg/L glyphosate (Sigma-Aldrich) and 50 µg/mL ampicillin, then incubated under the same conditions for 3 hours. 1 mL samples were taken, centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was collected. Glyphosate concentration was measured using a Glyphosate ELISA kit (JI-ICHI), following the manufacturer’s protocol. Samples and standards were added to ELISA wells, incubated, and washed. Enzyme conjugates were added, followed by another incubation and washing step. Substrates were added, and after color development, a stop solution was added. Absorbance was measured at 450 nm, and the glyphosate concentration was calculated based on a standard curve.

After overnight activation, the engineered strain was diluted 1:100 into 50 mL LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin and incubated at 37°C with shaking at 180 rpm. 1 mL samples were taken hourly for 5 hours, centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was collected. Glyphosate concentration was measured as described previously, with absorbance at 450 nm.

Construction of phnE1/E2-phnJ Engineered Strain

Building on the phnE1/E2 recombinant plasmid, the J23100-B0034-phnJ sequence from Enterobacter cloacae K7 was synthesized and cloned downstream of the phnE1 and phnE2 genes. The recombinant plasmid was then transformed into E. coli BL21 to form the engineered strain, which was cultured in LB medium containing 50 µg/mL ampicillin at 37°C.

After overnight activation, the engineered strain was diluted 1:100 into 50 mL LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin, then incubated at 37°C with shaking at 180 rpm for 3 hours. Samples were taken at 0 and 3 hours, centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was collected. Glyphosate concentration was measured using the ELISA method at 450 nm. The degradation efficiency of different strains was compared.

The phnO gene from Salmonella enterica LT2 was synthesized and codon-optimized for expression in E. coli. It was cloned into the pET23b vector (using EcoRI and XhoI sites) to form a recombinant plasmid. The plasmid was then transformed into E. coli BL21, and the engineered strain was cultured in LB medium containing 50 µg/mL ampicillin at 37°C.

The phnO-overexpressing strain was inoculated into LB medium containing 50 µg/mL ampicillin and incubated overnight at 37°C with shaking at 200 rpm. The next day, the culture was diluted 1:100 into fresh LB medium and incubated for 2-3 hours until OD600 reached 0.6. Cells were harvested by centrifugation at 10,000 g for 5 minutes and resuspended in pre-cooled 25 mM Tris-HCl buffer (with 0.3 M NaCl, 10 mM imidazole, and 1 mM EDTA, pH 8.0). Cells were lysed by sonication (1s on, 3s off, 250 W, 10 minutes), and the supernatant was collected by centrifugation at 13,000 g for 20 minutes to prepare a crude enzyme extract.

To test phnO activity, 100 µL of crude enzyme extract was added to a 6 mL reaction system containing 1 mM aminomethylphosphonate (AMPA), 3 mM acetyl-CoA, 3 mM magnesium chloride, and 25 mM Tris-HCl buffer. The reaction was incubated at 37°C for 3 hours. phnO acts as an AMPA N-acetyltransferase, catalyzing the reaction between AMPA and acetyl-CoA to produce CoA. A 0.5 mM 44'-dithiodipyridine (DTDP) solution in PBS was freshly prepared, and 1 mL of the reaction mixture was mixed with 100 µL DTDP solution in a 96-well plate and incubated for 30 minutes at room temperature. CoA reacts with DTDP to form thiopyridone, and absorbance at 324 nm was measured to quantify CoA production and assess AMPA degradation.

After overnight activation, the engineered strain was diluted into 50 mL LB medium containing ampicillin and incubated at 37°C with shaking at 180 rpm until OD600 reached 0.6. Cells were harvested, resuspended in Tris-HCl buffer, and lysed by sonication. The crude enzyme extract was prepared as previously described. 100 µL of crude extract was added to a 6 mL AMPA reaction system and incubated at 37°C for 3 hours. CoA production was measured using the DTDP method to assess AMPA degradation.

The engineered strain was cultured as described above, and crude enzyme extract was prepared. The enzyme kinetics were measured by adding 100 µL of crude extract to a 6 mL reaction system containing varying concentrations of AMPA. After a 3-hour incubation at 37°C, CoA production was measured using the DTDP method. One enzyme activity unit was defined as the amount of enzyme that catalyzes the degradation of 1 mM AMPA per minute per mg of protein. The kinetic parameters Km and Vmax were calculated using GraphPad Prism 8.0 software based on the Michaelis-Menten curve.

The cold-inducible PcspA promoter (BBa_K3289001) was synthesized, and the mRFP gene was placed downstream of PcspA. The mRFP gene was codon-optimized for E. coli, with removal of EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The PcspA-mRFP construct was cloned into the pSB1A3 vector (between XbaI and SpeI sites), and the recombinant plasmid was transformed into E. coli BL21. Positive clones were selected on LB plates containing 50 µg/mL ampicillin at 37°C and verified by sequencing. The engineered strain was cultured in LB medium with ampicillin, and optical density at 600 nm (OD600) was measured to monitor growth.

The cold-inducible reporter strain was activated overnight and diluted 1:100 into fresh LB medium. The cultures were incubated at different temperatures (16°C, 25°C, and 37°C) with shaking at 180 rpm for 12 hours. 200 µL samples were taken, and fluorescence intensity (excitation at 584 nm, emission at 607 nm) was measured using a microplate reader. OD600 was also measured, and the normalized fluorescence intensity (Fluorescence/OD600) was calculated.

The mazF toxin gene was cloned downstream of the PcspA promoter. The mazF gene was codon-optimized for E. coli and had EcoRI, XbaI, SpeI, and PstI restriction sites removed to comply with RFC#10 standards. The PcspA-mazF construct was cloned into the pSB1A3 vector and transformed into E. coli BL21.

The cold-inducible suicide strain and control strain were cultured overnight. 100 µL of the culture was inoculated into 5 mL LB medium containing 50 µg/mL ampicillin and incubated at 16°C and 180 rpm for 12 hours. 200 µL samples were taken to measure OD600 to assess cell growth.

All data were analyzed using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). One-way ANOVA followed by Tukey’s post-hoc test was used for multi-group comparisons, while a two-tailed Student’s t-test was used for two-group comparisons. A p-value of <0.05 was considered statistically significant.