The coding gene sequence of phosphate-binding protein (PBPs) PstS was synthesized, and the ice-crystal nucleoprotein truncated sequence (INP) was synthesized and placed upstream of PstS. The above genes were codon optimized for E. coli, and then cloned into pET23b plasmid through NdeI and XhoI restriction sites to obtain the recombinant plasmid (Genewiz, USA). The recombinant plasmid expresses downstream coding genes through T7 promoter composition. After the sequencing was verified correctly (Cytology, China), the recombinant plasmid was extracted using the plasmid extraction kit (Tiangen, China). Next, the recombinant plasmid was transformed into E.coli DH5α and BL21. E.coli DH5α was used to store the plasmid and E.coli BL21 was used to express the plasmid. The engineered strains were cultured in LB medium containing ampicillin (Amp)(50 μg/mL) at 37°C.

The engineered strains were cultured in Luria-Bertani (LB) medium containing ampicillin (final concentration 50 μg/mL). The culture conditions were 250 rpm and 37°C. After 12 hours of culture, 1 mL bacterial solution was taken, centrifuged (5000 rpm, 10 min), the supernatant was abandoned, and the bacterial precipitate was suspended with Tris-HCl buffer (pH 7.4), and the cell density (OD600) was adjusted to 1. Add 10 mg/L KH2PO4. Reaction at room temperature at 250 rpm for 3 hours. The concentration of Phosphate in solution was analyzed using the Malachite Green Phosphate Detection Kit (S0196M, Beyotime).

To analyze the effect of phosphorus starvation on the adsorption capacity of engineered bacteria. Before the adsorption experiment, the engineered bacteria (OD600 = 1) were suspended in Tris-HCl buffer (pH 7) and reacted at 250 rpm at room temperature for 3 hours, so that the cells were placed under low phosphorus conditions, and then the adsorption experiment was carried out. In addition, the engineered bacteria (OD600 = 1) were re-suspended in buffers of different pH and treated under the same conditions for 3 hours. Where pH 3 and 5 are acetic acid buffers, pH 7 is PBS buffers, and pH 8 and 10 are Tris-HCl buffers. Then the phosphate adsorption experiment was carried out. To analyze the effect of temperature, adjust the reaction temperature at 25°C, 35°C, or 45°C during the adsorption reaction.

Bacteria were cultured overnight in LB medium containing a high concentration of Pi medium (150 mg/L KH2PO4). Centrifuge to collect bacterial precipitates. Use a buffer (10 mM Tris-HCl, 1 mM MgCl₂, pH 7.5) to wash the bacterial precipitate twice to remove residual media. It is then re-suspended into the buffer and the bacteria OD600 = 1 is adjusted. Reaction at room temperature at 250 rpm for 3 hours. The concentration of Phosphate in solution was analyzed using the Malachite Green Phosphate Detection Kit (S0196M, Beyotime). The effect of pH on desorption was assessed using buffers of different pH values. The effect of temperature on desorption was evaluated using different reaction temperatures.

50 mL bacteria were cultured overnight, centrifuged (10000 rpm, 1 min) and then re-suspended with 10 ml PBS. Ultrasonic crushing (150W, ultrasonic 1 second, interval 3 seconds, a total of 20 minutes). Then centrifuge at 5000 rpm for 10min. The supernatant was collected and centrifuged at 39,000 rpm for 1h. The supernatant was collected as the cytoplasmic component. 2 mL PBS was used as the cell membrane component. The adsorption of phosphate by different components of engineering bacteria was determined according to the phosphate adsorption experiment process.

The coding gene sequence of phosphate-binding protein (PBPs) PstS is synthesized, and the secretory tag (PelB) sequence is synthesized, which is placed upstream of PstS. The above genes were codon optimized for E. coli, and then cloned into pET23b plasmid through NdeI and XhoI restriction sites to obtain the recombinant plasmid (Genewiz, USA). The recombinant plasmid expresses downstream coding genes through T7 promoter composition. After the sequencing was verified correctly (Cytology, China), the recombinant plasmid was extracted using the plasmid extraction kit (Tiangen, China). Next, the recombinant plasmid was transformed into E.coli DH5α and BL21. E.coli DH5α was used to store the plasmid and E.coli BL21 was used to express the plasmid. The engineered strains were cultured in LB medium containing ampicillin (Amp)(50 μg/mL) at 37°C.

The engineered strains were cultured in Luria-Bertani (LB) medium containing ampicillin (final concentration 50 μg/mL). The culture conditions were 250 rpm and 37°C. After 12 hours of culture, 1 mL of bacterial solution was taken, centrifuged (5000 rpm, 10 min), the supernatant was abandoned, and the bacterial precipitation was re-suspended with Tris-HCl buffer (pH 7.4), and the cell density (OD600) was adjusted to 1. Add 10 mg/L KH2PO4. Reaction at room temperature at 250 rpm for 3 hours. The concentration of Phosphate in solution was analyzed using the Malachite Green Phosphate Detection Kit (S0196M, Beyotime).

Ten mL of the culture was collected and re-suspended on 1 mL of TES buffer (30mM Tris-HCl, 20% sucrose, and 1 mm EDTA; pH 8.0). The bacterial suspension was incubated at room temperature for 10 min and then centrifuged at 10,000×g and 4°C for 10 min. The precipitate was resuspended in 1 mL of cold 5 mM MgSO4 and incubated on ice for 10 minutes. Centrifuge again at 10,000×g and 4°C for 10 min and collect the supernatant as the periplasmic component. In order to collect the bacterial soluble components and the bacterial lysate, the bacterial lysate cultured overnight was centrifuged, re-suspended in 1 mL PBS, ultrasonic treatment (150W, ultrasonic 30 seconds, 2 times), and the bacterial lysate was collected. Then centrifuge at 10,000×g and 4°C for 10 min. The supernatant was collected as a soluble component. Then, the adsorption capacity of different components of engineering bacteria to phosphate was determined according to the adsorption experiment process.

Firstly, Bradford kit (Biyuntian) was used to determine the protein concentration of different components of engineered bacteria. The protein solution was then mixed with a 5× reduced protein sample buffer in a 4:1 ratio and denatured in a boiling water bath for 15 minutes. Protein was isolated at 120V using 15% SDS-PAGE. Subsequently, the proteins on the SDS-PAGE gel were transferred to the PVDF membrane under cold conditions. The membrane was closed with 5% skim milk (in TBST) at room temperature for 1 hour. Because pET23b carrier carries His label. The membrane was incubated overnight at 4°C with murine-derived His label antibodies (1:1000, AH367, Blue sky). Use TBST washing film three times for 10 minutes each time. They were then incubated at room temperature with horseradish peroxidase labeled Goat anti-mouse IgG (H+L) (1:1000 diluted, A0216, blue sky) for 1 hour. Use TBST washing film three times, after 10 minutes each time, mix the ECL A and B solution in a ratio of 1:1 and set aside. Place the washed PVDF film on absorbent paper and slightly dry the liquid on the surface of the film. The film is then placed on the chemiluminescent instrument rack and covered with the prepared ECL luminescent liquid to ensure that the film is completely immersed. After 1 minute of reaction, absorb the excess liquid at the top with absorbent paper, and put the film into the chemiluminescence instrument to start the chemiluminescence reaction.

PelB-PstS (C-terminal with his tag) overexpressed engineering BL21 was cultured overnight in 100 mL LB medium. Centrifuge (10000 rpm, 1min) to collect bacterial precipitates. The binding buffer (10 mM imidazole, 20 mM Tris-HCl, 10 mM NaCl, pH = 7.4) was used to re-precipitate the suspended bacteria and adjust OD600 = 1. The bacterial solution was broken by ice bath ultrasonic wave. Before ultrasound, anhydrous ethanol was used to clean the ultrasonic broken cylinder head for 5min, and then distilled water was used to clean the cylinder head for 10min. The power was set at 150W, and the ultrasound was 1s at a interval of 2s. Centrifuge at 12,000 rpm at 4℃ for 30min, collect supernatant, discard precipitation, and obtain crude enzyme liquid samples. Subsequently, the PBP was fixed to the NHS-Activated Sepharose 4 Fast Flow (GE 17090601) bead. Specifically, fresh NHS beads (stored in 100% isopropyl alcohol) are washed with a 1 mM HCl solution. Then, 20 mL PBP was added to 20 mL of washed NHS beads and mixed at 30 rpm at 4°C for 16 hours. Beads fixed with PBP (PBP beads) were washed three times successively with 20 mL of 20 mM Tris-HCl (pH 7.5). The control beads are prepared following the same procedure as the PBP beads, but without the addition of PBP.

0.25 mL of PBP beads, control beads or PBP enzyme solution were added to a 2 mL centrifuge tube. 1 mL reaction buffer (10 mM Tris-HCl, 1 mm MgCl2, 10 mg/L KH2PO4, pH 7.4) was added to the tube for 10 min at 25°C. Pi adsorption effect was measured.

The desorption effect of immobilized PBP on Pi was tested

Add 0.25 mL PBP beads to 2 mL centrifuge tube. Then 1 mL phosphate saturation buffer (10 mM Tris-HCl, 1 mM MgCl2, 150 mg/L KH2PO4, pH 7.4) was added to ensure the saturation adsorption of PBP beads on Pi. Mix gently and let stand at 25°C for 10 minutes. The PBP beads were then washed three more times with 1 mL buffer (10 mM Tris-HCl, 1 mM MgCl₂, pH 7.5) to remove the unbound Pi. Then, the Pi content was measured and the influence of different conditions on Pi desorption was evaluated. To assess the effects of temperature. Adjust the reaction temperature to 25°C, 35°C, or 45°C (pH 7.5). To assess the effect of pH, adjust the pH of the reaction buffer to the final values 3, 5, 7, 8, 10 (keep the temperature at 25°C) using HCl or NaOH to assess the effect of pH on Pi.

The data was analyzed and plotted using Graphpad Prism software. Data are presented as mean ± standard deviation (SD). For comparison of multiple sets of data, differences were analyzed using One-way ANOVA and Tukey post hoc tests. For the two sets of data, the two-tailed student's t-test was used for analysis. A P-value of less than 0.05 was considered statistically significant.