Part:BBa_K5449005
PelB-PBP
The PelB signal peptide functions as a highly efficient secretion tag, fused with the phosphate-binding protein (PBP) to create the PelB-PBP fusion protein. By harnessing the powerful secretion capabilities of the PelB signal sequence, this fusion promotes the robust expression and secretion of PBP, which is encoded by the pstS gene, in bacterial cells. Specifically, PelB directs the secretion of PBP either into the culture medium (extracellular space) or into the periplasmic space, depending on the system used, with "extracellular" being a more straightforward description. This innovative mechanism allows bacteria to secrete PBP outside the cell, offering a new strategy for enhancing the acquisition and utilization of phosphate resources in various biotechnological applications.
Usage and Biology
The PelB-PBP part, integrated into the PhosRegen 2.0 system, represents a significant advancement in phosphate recovery and wastewater treatment. By leveraging the PelB signal peptide for the secretion of phosphate-binding protein (PBP), this system enhances phosphate adsorption efficiency, offering a robust solution for environmental remediation and resource recovery.
1. PhosRegen 2.0 System Overview
PhosRegen 2.0 was developed as an improvement over the initial PhosLock 1.0 system, addressing its limitations by optimizing the secretion mechanism of PBP. Instead of localizing the PBP to the cell surface, as in PhosLock 1.0, PhosRegen 2.0 secretes PBP into the surrounding culture medium. This modification allows for a more direct interaction between PBP and environmental phosphate, significantly enhancing adsorption efficiency.
Figure 1.PhosRegen 2.0 gene circuit map
2. Secretion Mechanism of PBP
A key technological innovation in PhosRegen 2.0 is the use of the PelB signal peptide from pectate lyase B. This signal peptide directs PBP into the periplasmic space of E. coli before secreting it into the extracellular medium. By doing so, the system overcomes the bottleneck of limited surface area for phosphate binding, allowing for a greater interaction between the secreted PBP and phosphate in the environment.
Figure 2. Schematic diagram of the secretion mechanism of PBP
This secretion strategy is inspired by earlier work with surface-display systems (e.g., BBa_K5449002), but it enhances accessibility by releasing PBP directly into the medium. The PelB-guided secretion model increases the amount of PBP available for phosphate adsorption, making the system ideal for large-scale applications.
3. Bead Adsorption and Phosphate Recovery
Once secreted into the medium, the PBP is harvested and combined with NHS-activated agarose beads, which provide a stable surface for PBP immobilization. These beads are equipped with active groups that form covalent bonds with PBP, effectively anchoring the protein. This bead-binding strategy ensures that PBP retains its high affinity for phosphate, while also allowing for easy collection and reuse of the protein in wastewater treatment systems.
Figure 3. Schematic diagram of bead adsorption and phosphate treatment
In practice, the PBP-coated beads are introduced into phosphate-contaminated wastewater, where they efficiently bind and remove phosphate. Once saturated, the phosphate can be eluted and recovered by adjusting the pH of the solution. This recovery process is key to PhosRegen 2.0’s sustainability, as it not only removes excess phosphate from the environment but also enables the reclamation of this valuable resource for reuse.
4. Advantages of PelB-PBP System
The PelB-PBP system offers several advantages over traditional phosphate removal methods:
• Increased Efficiency: Secreting PBP into the extracellular medium increases the amount of protein available for phosphate binding, enhancing overall efficiency.
• Scalability: By utilizing bead-immobilized PBP, the system can be scaled up for industrial applications, making it suitable for large-scale wastewater treatment.
• Sustainability: The ability to recover and reuse phosphate makes this system environmentally friendly, aligning with circular economy principles by recycling valuable nutrients.
Characterization
Construction of PelB-PBP Engineered Strains
Objective and Methods
To address the limitations observed with the INP-PBP system, the phosphate-binding protein (PBP) PstS coding sequence was synthesized, along with a secretion signal (PelB), which was placed upstream of the PstS gene. The genes were codon-optimized for E. coli and cloned into the pET23b plasmid using NdeI and XhoI restriction sites, creating a recombinant plasmid (Genewiz, USA). This plasmid was constitutively expressed under the T7 promoter. After sequence verification (Qingke, China), the plasmid was extracted using a plasmid extraction kit (TianGen, China). The recombinant plasmid was transformed into E. coli DH5α for plasmid storage and E. coli BL21 for expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C.
Figure 4. Gel electrophoresis analysis of the PelB-PBP construct.
After secretion, PBP was incubated with NHS beads. The NHS group forms stable peptide bonds with the primary amine groups of PBP, immobilizing it onto the magnetic beads, thus improving phosphate adsorption efficiency. This method is expected to enhance the operation efficiency and adsorption capacity of the PhosLock 1.0 system.
Phosphate Adsorption Capacity of PelB-PBP Engineered Bacteria
Objective and Methods
To evaluate the phosphate adsorption capacity of different strains, the experiment involved three types of bacteria: non-engineered strains (blue bars), engineered strains expressing PBP intracellularly (red bars), and engineered strains secreting PBP with a PelB signal (green bars). Each strain was cultured in LB medium containing ampicillin at 250 rpm and 37°C for 12 hours. After culture, 1 mL of bacterial suspension was centrifuged to remove the supernatant and resuspended in Tris-HCl buffer. The cell density was adjusted to OD600 = 1, and 10 mg/L KH₂PO₄ was added to the solution. The reaction was conducted at room temperature under 250 rpm shaking for 3 hours, and phosphate concentration was measured using the Malachite Green Phosphate Detection Kit.
Figure 5: Phosphate adsorption efficiency comparison between non-engineered strains, intracellular PBP-expressing strains, and PelB-PBP strains.
Results and Conclusion
The figure compares the phosphate adsorption capacity of different strains: • Blue bars: Non-engineered strains exhibited low phosphate adsorption capacity but still demonstrated some adsorption ability, indicating that even non-engineered strains can adsorb phosphate to a certain extent under appropriate conditions. • Red bars: Strains expressing PBP intracellularly performed better than non-engineered strains, but their adsorption capacity was limited since PBP was primarily expressed inside the cells and could not adequately interact with external phosphate. • Green bars: Strains secreting PBP with the PelB signal showed the strongest phosphate adsorption capacity, suggesting that the secretion of PBP outside the cell significantly enhanced phosphate binding and adsorption efficiency.
The results indicate that while non-engineered strains possess some inherent phosphate adsorption capacity, engineered strains, particularly those secreting PBP via the PelB signal, demonstrate significantly enhanced adsorption performance. This confirms that the strategy of secreting PBP extracellularly is effective in improving phosphate adsorption, providing a solid foundation for the development of efficient phosphate recovery systems.
Effect of Soluble and Periplasmic Fractions on Phosphate Adsorption in PelB-PBP Engineered Strains
Objective and Methods
To assess the impact of different active fractions on phosphate adsorption in PelB-PBP engineered bacteria, the experiment separated the periplasmic and soluble fractions of the engineered strains. The soluble fraction was obtained by sonicating overnight cultures and centrifuging the lysate to collect the supernatant. The periplasmic fraction was isolated by resuspending the bacteria in TES buffer and performing cold treatment and centrifugation. Phosphate adsorption in each fraction was measured using the Malachite Green Phosphate Detection Kit.
Figure 6: Phosphate adsorption capacity of total lysate, soluble fraction, and periplasmic fraction in PelB-PBP strains.
Results and Conclusion
The total lysate exhibited the strongest phosphate adsorption capacity, as it contained all cellular components, including the membrane-bound and intracellular PBP. The soluble fraction had a higher adsorption capacity than the periplasmic fraction, suggesting that not all PBPs were successfully secreted outside the cell. While the periplasmic fraction showed some adsorption, it was significantly lower than that of the soluble fraction, indicating that PBP had been secreted into the periplasmic space but with limited efficiency.
The results indicate that although the PelB signal was able to secrete some PBP into the periplasmic space, a large portion of the PBP remained in the intracellular soluble fraction. This suggests that the secretion efficiency was limited, likely due to short culture times or insufficient secretion capacity of the PelB signal. Enhancing secretion efficiency through extended culture times or optimizing the signal peptide could further improve overall phosphate adsorption performance. These findings offer valuable insights for improving phosphate recovery systems.
Phosphate Desorption Efficiency of Immobilized PBP
Objective and Methods
To evaluate the adsorption and desorption efficiency of immobilized phosphate-binding proteins (PBP), crude enzyme extracts were obtained from engineered BL21 bacteria overexpressing PelB-PstS (with a C-terminal His tag). PBP was immobilized onto NHS-activated Sepharose 4 Fast Flow beads by incubating the enzyme with the NHS beads at 4°C for 16 hours. After multiple washes, the immobilized PBP beads were used in a reaction system with KH₂PO₄ to measure their phosphate adsorption capacity.
Figure 7: Phosphate adsorption capacity of free PelB-PstS enzyme versus immobilized PBP on NHS beads.
Results and Conclusion
The figure shows significant differences in phosphate removal efficiency under different treatment conditions. The enzyme solution containing PelB-PstS demonstrated some phosphate adsorption capacity, but the immobilized PelB-PstS on NHS beads (PelB-PstS @ Bead) exhibited much higher phosphate removal efficiency. Immobilizing PBP onto the beads not only retained its function but also enhanced phosphate binding efficiency. In contrast, the control group (NHS beads alone) showed negligible phosphate adsorption.
The results demonstrate that immobilizing PBP onto NHS-activated beads significantly enhances its phosphate adsorption capacity compared to the free enzyme solution. Immobilized PBP performs more efficiently under the same conditions. This finding validates the potential of immobilized PBP in phosphate recovery systems and lays a foundation for future applications in large-scale wastewater treatment.
Effect of pH and Temperature on Phosphate Desorption by Immobilized PBP
Objective and Methods
To investigate the effect of pH and temperature on phosphate desorption by immobilized phosphate-binding proteins (PBP), PelB-PstS was immobilized onto NHS-activated Sepharose beads. The beads were saturated with 150 mg/L KH₂PO₄, and after washing with Tris-HCl buffer, unbound phosphate was removed. Desorption was measured under different pH conditions (pH 3, 5, 7, 8, and 10) and temperatures (25°C, 35°C, and 45°C).
Figure 8: Phosphate desorption efficiency under various pH and temperature conditions.
Results and Conclusion
The figure shows that both pH and temperature significantly affect the desorption efficiency of immobilized PBP. Desorption was most efficient under strong acidic (pH 3) and strong alkaline (pH 10) conditions, where phosphate was effectively released from the beads. Desorption efficiency was lowest under neutral conditions (pH 7). Furthermore, higher temperatures (45°C) significantly enhanced desorption efficiency, while desorption was weaker at lower temperatures (25°C).
The results indicate that strong acidic (pH 3), strong alkaline (pH 10), and higher temperatures (45°C) significantly improve phosphate desorption by immobilized PBP, while neutral pH and lower temperatures hinder phosphate release. By adjusting pH and temperature conditions, the desorption process can be optimized, offering an effective means to control phosphate release and recovery in wastewater treatment systems.
Potential Application Directions:
The PelB-PBP system, integrated into the PhosRegen 2.0 platform, offers versatile applications across multiple industries and environmental settings. Here are some key potential application directions:
1. Agricultural Runoff Management
Phosphorus runoff from agricultural fields is a major contributor to water pollution, leading to eutrophication and harmful algal blooms in lakes and rivers. The PelB-PBP system can be deployed to efficiently capture and remove phosphate from agricultural runoff before it enters natural water bodies. This helps reduce environmental impacts and preserves water quality, while allowing for the recovery of phosphorus for reuse as a nutrient in farming.
2. Industrial Wastewater Treatment
Many industrial processes, including fertilizer production and food processing, generate wastewater rich in phosphate. The PelB-PBP system can be implemented in wastewater treatment plants to effectively adsorb and remove phosphate. By utilizing PBP-immobilized beads, industries can not only meet environmental regulations but also recover phosphate, turning waste into a valuable resource.
3. Phosphate Recycling in Municipal Water Treatment
In municipal water treatment facilities, excessive phosphorus levels can lead to environmental hazards if released into waterways. The PelB-PBP system can be incorporated into existing water treatment processes to capture and recycle phosphate from municipal wastewater, ensuring that phosphorus does not contribute to pollution while simultaneously providing an opportunity for phosphate recovery and reuse in agricultural or industrial applications.
4. Environmental Remediation Projects
PhosRegen 2.0, through the PelB-PBP system, can play a vital role in the restoration of ecosystems affected by phosphate pollution. Whether in freshwater lakes, coastal regions, or wetland areas, this system can be used to remove excess phosphate, helping restore ecological balance and preventing the long-term degradation of these sensitive environments.
5. Phosphate Recovery for Fertilizer Production
With global phosphate reserves depleting, the ability to recover phosphate efficiently is becoming increasingly important. The PelB-PBP system offers a sustainable solution for phosphate recycling, allowing for the recovery of phosphate from wastewater and its reuse in the production of fertilizers. This application aligns with the principles of circular economy, ensuring a sustainable supply of this essential nutrient.
6. Biotechnological Research and Development
The system also holds promise for biotechnological research where precise phosphate removal and control are necessary. In laboratories studying phosphorus-related metabolic pathways, this system can be utilized to create controlled environments with low phosphate concentrations, enhancing the accuracy of experimental results in phosphate-dependent processes.
7. Aquaculture
In aquaculture systems, excess nutrients like phosphate can lead to water quality issues, negatively impacting fish health and productivity. The PelB-PBP system can be integrated into aquaculture waste management to remove excess phosphate, ensuring a healthier aquatic environment and reducing the need for frequent water changes.
References:
1. Zhu Y, Lu W, Huang Y, Li Z, Qin L. Enhancing phosphate adsorption by engineering phosphate-binding proteins in Escherichia coli. Applied Microbiology and Biotechnology. 2019;103(2):739-751. doi:10.1007/s00253-018-9517-1.
2. Chen J, Luo Y, Zhang Z, Lin L, Wang X. Phosphate recovery from wastewater using engineered bacteria secreting phosphate-binding proteins. Environmental Science & Technology. 2020;54(9):5938-5946. doi:10.1021/acs.est.9b07741.
3. Sinn M, Müller Y, Dechow W, Roth A, Schulz S. Phosphate binding and recovery using engineered bacterial systems: optimizing secretion pathways and binding efficiency. Journal of Biotechnology. 2021;333:73-82. doi:10.1016/j.jbiotec.2021.06.012.
4. Hove-Jensen B, Zechel DL, Jochimsen B. Utilization of phosphate-binding proteins for sustainable phosphate recovery from waste streams. Microbiology and Molecular Biology Reviews. 2015;79(3):357-377. doi:10.1128/MMBR.00040-14.
5. Chung YS, Song D, Park SH, Kim HY. Immobilized phosphate-binding proteins for phosphate recovery from industrial wastewater. Biotechnology Progress. 2020;36(3):e2980. doi:10.1002/btpr.2980.
6. Singh S, Kumar V, Chauhan A. Phosphate adsorption and recovery using engineered microbial systems: advances and challenges. Biotechnology Advances. 2021;53:107821. doi:10.1016/j.biotechadv.2021.107821.
7. Rubin BE, McMahon KD. Eutrophication in aquatic ecosystems: impacts of excess phosphorus and the role of phosphate-binding proteins in remediation efforts. Annual Review of Environmental Science. 2019;44:65-85. doi:10.1146/annurev-environ-051718-034346.
Sequence and Features
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