This project applied engineering principles to develop Escherichia coli strains for optimized phosphate recovery through two iterative cycles. In Cycle 1, phosphate-binding protein (PBP) was anchored to the cell surface via ice nucleation protein (INP). The Design phase involved creating a strategy to display PBP on the cell membrane, while the Build phase focused on codon optimization and plasmid construction to achieve surface display. Testing confirmed that surface-displayed PBP significantly improved phosphate adsorption, with optimal conditions identified at pH 7 and 35°C. Phosphate desorption was most efficient under extreme pH (3 and 10) and elevated temperatures (45°C). Through Learning, phosphate starvation was found to further enhance adsorption capacity. In Cycle 2, PBP was secreted extracellularly using the PelB signal peptide. The Design targeted extracellular secretion, while Build facilitated PBP production and secretion. Testing of immobilized PBP on NHS-activated beads demonstrated superior adsorption and desorption performance under extreme conditions. Learning emphasized the need to optimize secretion efficiency and strain stability. These cycles of design, building, testing, and iterative learning provide a robust foundation for scalable phosphate recovery systems.

In Cycle 1, the goal was to design an E. coli strain that could efficiently recover phosphate by anchoring the phosphate-binding protein (PBP) on the cell surface. The core concept was to use ice nucleation protein (INP) as a membrane anchor, allowing PBP to be displayed on the cell membrane and thus exposed to the external environment for direct phosphate adsorption.

Once the design strategy was established, the next step was to build the INP-PBP system. The following steps were executed to construct the engineered strain:

Gene synthesis and codon optimization: The coding sequence for PstS (phosphate-binding protein) was synthesized. Additionally, a truncated version of the ice nucleation protein (INP) was used to ensure efficient membrane anchoring. Both sequences were codon-optimized for E. coli to enhance expression efficiency.

Plasmid construction: The PBP gene was fused with the INP sequence at the N-terminus to create a single fusion protein. The genes were inserted into the pET23b vector via NdeI and XhoI restriction sites, under the control of a T7 promoter. This allowed for constitutive expression of the INP-PBP fusion protein in E. coli .

Transformation and expression: The recombinant plasmid was transformed into E. coli DH5α for plasmid storage and then into E. coli BL21 for protein expression. Both strains were grown in LB medium containing ampicillin (50 μg/mL) at 37°C. E. coli DH5α was used solely for plasmid storage, while BL21 was utilized for expression due to its high protein production capability.

Verification of protein expression: To confirm the successful construction and expression of the INP-PBP protein, gel electrophoresis was performed after protein induction. This step verified that the fusion protein had the correct molecular weight, ensuring that the INP-PBP system was properly built.

Several tests were conducted to evaluate the performance of the INP-PBP system. The goal was to verify the phosphate adsorption capacity of the engineered bacteria under different conditions.

Objective: To compare the phosphate adsorption efficiency between the engineered INP-PBP strain and a control strain expressing PBP intracellularly.

Method:

Both the experimental group (INP-PBP) and the control group (intracellular PBP) were cultured in LB medium containing 50 μg/mL ampicillin at 37°C and 250 rpm for 12 hours.

After culturing, the cell density was adjusted to OD600 = 1. Phosphate (KH₂PO₄) was added at a concentration of 10 mg/L, and the mixture was incubated at room temperature for 3 hours with shaking.

The remaining phosphate concentration in the solution was measured using the Malachite Green Phosphate Detection Kit. This kit forms a green phosphomolybdic acid complex when inorganic phosphate is present, and the absorbance was measured at 630 nm.

Principle of Malachite Green Phosphate Detection Kit:

Malachite Green Phosphate Detection Kit combines Malachite Green and Molybdate with free inorganic phosphate in solution. The green phosphomolybdic acid complex is formed. This complex has a characteristic absorbance in the wavelength range 620-640 nm, and the intensity of the absorbance is proportional to the phosphate concentration in the solution. By measuring the absorbance at 630 nm, the residual phosphate concentration in the solution can be quantitatively analyzed.

Results:

The engineered INP-PBP strain significantly reduced the phosphate concentration in the solution, indicating that the surface-displayed PBP effectively adsorbed phosphate.In contrast, the control strain with intracellular PBP showed no significant reduction in phosphate concentration, confirming that surface display is necessary for effective adsorption.

PBP must be displayed on the cell surface to efficiently adsorb phosphate. Intracellular expression alone is insufficient for effective phosphate removal. This result highlights the importance of surface-displayed PBP for developing a high-efficiency phosphate recovery system.

Objective: To determine how phosphate starvation impacts the phosphate adsorption performance of the INP-PBP strain.

Method:

Two groups of INP-PBP bacteria were used: one subjected to phosphate starvation and the other without starvation. Both groups were then tested for phosphate adsorption using the Malachite Green method.

Results:

The phosphate-starved group exhibited a significantly higher adsorption capacity compared to the non-starved group.

Phosphate starvation further enhanced the adsorption capacity of INP-PBP engineered bacteria, demonstrating the effectiveness of genetic modification and starvation treatment in optimizing phosphate recovery.

Objective: To identify the optimal pH and temperature conditions for phosphate adsorption by INP-PBP bacteria.

Method:

The engineered bacteria were incubated in solutions with varying pH values (3, 5, 7, 8, 10) and temperatures (25°C, 35°C, 45°C). After incubation, the remaining phosphate concentration in each solution was measured using the Malachite Green method.

Results:

The highest adsorption efficiency was observed at pH 7 and 35°C.

Optimal phosphate adsorption by INP-PBP bacteria occurred at pH 7 and 35°C, making these ideal conditions for the phosphate recovery system.

Objective: The phosphate desorption process is a key step in the whole phosphate recovery system. By adjusting the pH value and temperature of the environment, the engineered bacteria can release the phosphate that has been adsorbed, thus achieving the recovery of phosphate. Therefore, verifying the desorption capacity of engineered bacteria at different pH and temperature conditions is crucial for optimizing phosphate recovery systems.

Method:The INP-PBP bacteria were subjected to different pH (3, 5, 7, 8, 10) and temperature (25°C, 35°C, 45°C) conditions, and the amount of phosphate released from the bacteria was measured.

Results:

Desorption was most efficient under acidic (pH 3) and alkaline (pH 10) conditions, and at 45°C. Strong acidic and alkaline conditions, along with high temperatures, promote effective phosphate desorption, providing crucial parameters for optimizing phosphate recovery systems.

Objective：This test aimed to verify if INP successfully anchored PBP to the cell membrane by comparing the phosphate adsorption capacities of cell membrane and cytoplasmic fractions.

Experimental Setup：

• Bacterial Culture: INP-PBP bacteria were cultured overnight in LB medium (50 mL) at 37°C.

• Cell Lysis: After harvesting the cells, they were resuspended in PBS and lysed using ultrasonication (150W, 1-second pulse, 3-second interval, 20 minutes).

• Fraction Separation: The lysate was first centrifuged at 5,000 rpm for 10 minutes. The supernatant was then ultracentrifuged at 39,000 rpm for 1 hour, separating the cytoplasmic fraction (supernatant) and cell membrane fraction (pellet).

• Resuspension: The cell membrane pellet was resuspended in 2 mL PBS for adsorption experiments.

Results

The cell membrane fraction exhibited significantly higher phosphate adsorption than the cytoplasmic fraction, indicating that PBP was primarily located on the membrane.This confirmed that INP successfully anchored PBP to the cell membrane, enhancing phosphate adsorption.

The results validated the INP anchoring strategy, showing that PBP was effectively localized on the cell membrane, improving the bacteria’s phosphate adsorption efficiency.

Through the series of tests conducted in Cycle 1, the INP-PBP system demonstrated effective phosphate adsorption, particularly when PBP was anchored on the cell surface. However, several challenges and limitations were identified, which highlighted the need for further improvements to enhance the practical applicability of the system:

Survival in Complex Environments: While the INP-PBP bacteria effectively adsorbed phosphate under controlled lab conditions, their survival rate in real-world environments, such as complex wastewater, was low. The outer membrane pressure in such environments appears to strain the engineered bacteria, reducing their viability and overall system efficiency.

High Maintenance Costs: Maintaining the activity and stability of the engineered bacteria was costly. Continuous operation of the system requires careful control of environmental conditions to prevent protein inactivation. This introduces additional complexity and raises the operational costs, making large-scale implementation more challenging.

Limited Adsorption Capacity: Although the surface display of PBP was effective, the overall phosphate adsorption capacity remained limited. Since PBP could only be displayed on the cell surface, the amount of protein available for phosphate binding was restricted by the surface area of the bacteria. This limitation makes it difficult to meet the demands of large-scale phosphate removal systems, especially in environments with high phosphate concentrations.

These challenges suggest that while the PhosLock 1.0 system validates the feasibility of phosphate recovery, it needs significant optimization. Future iterations should focus on improving bacterial robustness in complex environments, reducing operational costs by enhancing strain stability, and increasing adsorption capacity through alternative strategies, such as extracellular secretion or immobilization techniques. These insights directly informed the design of Cycle 2, where extracellular secretion of PBP was explored to overcome the limitations observed in this phase.

In Cycle 2, the design strategy shifted from displaying PBP on the cell surface to secreting it extracellularly. The primary goal was to increase the phosphate-binding protein (PBP) available for adsorption by releasing it into the extracellular space, allowing for more flexible application in large-scale systems. This strategy also aimed to address the limitations of Cycle 1, including the restricted adsorption capacity and the challenges of maintaining the engineered bacteria in harsh environments.

Key design considerations included:

Extracellular Secretion: To achieve efficient secretion, the PelB signal peptide was chosen to direct the PBP (PstS) to the periplasmic space, where it could be secreted into the extracellular medium.

Protein Immobilization: Once secreted, PBP would be immobilized on NHS-activated beads to increase its phosphate adsorption capacity and stability, ensuring efficient use in recovery systems.

Scalability and Stability: The design sought to overcome the survival challenges faced by engineered bacteria in complex wastewater environments by decoupling phosphate adsorption from bacterial viability. Immobilizing secreted PBP would also reduce the need for live bacteria to remain active throughout the process.

Following the design, the construction of the PelB-PBP system proceeded with several critical steps:

Gene Synthesis and Signal Peptide Fusion: The coding sequence for PBP (PstS) was synthesized and fused with the PelB signal peptide, which targets proteins for secretion into the extracellular space. Both sequences were codon-optimized for expression in E. coli .

Plasmid Construction: The PelB-PBP gene construct was inserted into the pET23b vector, with expression driven by the T7 promoter. This allowed for controlled expression of the fusion protein. The plasmid was transformed into E. coli DH5α for storage and BL21 for protein expression.

Verification of Expression: The secreted PBP was confirmed through gel electrophoresis after bacterial culturing and induction with IPTG, ensuring that the PelB signal was correctly guiding PBP secretion.

Immobilization of PBP: Once secreted, the PBP was incubated with NHS-activated Sepharose beads. These beads covalently bind proteins via their primary amine groups, stabilizing the PBP on the bead surface, ready for phosphate adsorption.

To evaluate the performance of the PelB-PBP system, multiple tests were conducted focusing on adsorption capacity, secretion efficiency, and desorption performance.

Objective: To assess the phosphate adsorption capacity of the secreted PBP, compared to non-engineered strains and intracellular PBP expression.

Method:

Three bacterial strains were tested: non-engineered E. coli (negative control), E. coli expressing intracellular PBP (control), and E. coli secreting PBP via PelB.

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.

Results:

The PelB-PBP strain exhibited the highest phosphate adsorption efficiency, significantly surpassing both the non-engineered and intracellular PBP-expressing strains. This confirmed that extracellular secretion enhanced the availability of PBP for phosphate binding.

Objective: To measure the adsorption performance of PelB-PBP immobilized on NHS-activated beads.

Method:

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.

Results:

The immobilized PBP demonstrated significantly higher phosphate adsorption capacity compared to the free PBP in solution, confirming that immobilization not only stabilized the protein but also enhanced its functionality in phosphate recovery.

Objective: To optimize the conditions for phosphate desorption from the immobilized PBP to allow for efficient phosphate recovery.

Method:

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).

Results:

The optimal desorption occurred at pH 3 and pH 10, with higher temperatures (45°C) further enhancing the desorption efficiency. This suggests that extreme pH conditions and elevated temperatures are ideal for releasing phosphate from immobilized PBP, making it more suitable for controlled recovery systems.

Objective: To assess the secretion efficiency of PelB-PBP and compare the relative amounts of PBP in the extracellular, periplasmic, and intracellular fractions.

Method:

After growing the PelB-PBP strain, the periplasmic and extracellular fractions were separated via cold osmotic shock, while the intracellular fraction was isolated by sonication. Phosphate adsorption in each fraction was measured to determine the location of PBP.

Results:

Contrary to expectations, the majority of PBP was retained inside the cell, particularly in the intracellular fraction, indicating that the secretion efficiency of the PelB signal peptide was limited.Only a small portion of PBP was found in the periplasmic and extracellular fractions, suggesting that while the PelB signal peptide did facilitate some secretion, it was not fully effective.

• Limited Secretion Efficiency: The results from Test 4 showed that most of the PBP remained inside the cell, with only a small fraction successfully secreted into the extracellular space. This suggests that the PelB signal peptide’s secretion efficiency needs significant improvement. Future efforts should focus on optimizing the signal peptide or exploring alternative secretion systems to ensure more efficient release of PBP.

• Increased Adsorption Potential with Immobilization: Despite the limited secretion, the small amount of extracellular PBP that was immobilized on NHS-activated beads demonstrated high phosphate adsorption efficiency. This confirmed that immobilization is a valuable strategy, even though the quantity of secreted PBP was lower than expected.

• Decoupling from Bacterial Viability: The immobilization of PBP, though limited by secretion, still offers an approach to decouple phosphate adsorption from the viability of the bacteria. This reduces the dependency on maintaining live bacteria in harsh conditions, which was a challenge in Cycle 1.

• Optimization Needed for Industrial Scale: Although extracellular secretion showed promise, the low secretion efficiency limits the scalability of the system. Improvements in both secretion and immobilization are necessary to make the system viable for large-scale phosphate recovery.

This project successfully developed two engineered phosphate recovery systems through iterative cycles of Design, Build, Test, and Learn. In Cycle 1, PBP was anchored to the cell surface via INP, which improved phosphate adsorption under optimal conditions. However, the limited adsorption capacity and high maintenance requirements led to the system being less suitable for large-scale applications. In Cycle 2, PBP was secreted extracellularly using the PelB signal peptide. Although secretion efficiency was lower than expected, the small amount of secreted PBP that was immobilized on NHS-activated beads demonstrated high adsorption and desorption capacity. This strategy decoupled phosphate recovery from the viability of the bacteria, making the system more adaptable and scalable for industrial use.

Moving forward, the key focus should be on improving secretion efficiency to increase the availability of extracellular PBP, enhancing the immobilization strategy, and optimizing the system for industrial scalability. These improvements will help develop a more effective and practical phosphate recovery solution for real-world applications.

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