Project Description

The Problem

Synthetic biology is at the forefront of sustainable resource management, supplying novel approaches to addressing pressing global challenges by repurposing molecular biological systems into tools at our disposal. Our venture is based on the convergence of this emerging field and the urgent need for long-term phosphate recovery - an intricate problem with far-reaching implications for agriculture, the planet, and resource logistics.

Phosphorus is an essential nutrient for agriculture and the overall ecosystem. However, its current manufacturing and utilisation patterns have resulted in enormous waste and environmental harm. The world is facing an urgent issue due to the rapid loss of phosphate mineral reserves, referred to as "Phosphogeddon". Phosphate, a non-renewable resource, is essential for agriculture, but its production and widespread use have serious environmental consequences, including toxic waste and geopolitical tensions over limited supplies. With the cost of phosphate rising due to these factors, we must look to long-term alternatives that can address these global issues. Current phosphate applications, particularly in fertilisers, are inefficient, with crops utilising only 10-25% of the applied phosphate. This causes significant phosphate runoff, contributing to negative environmental effects such as algal blooms and eutrophication, highlighting the need for more sustainable and effective phosphate recycling from alternative sources.

Despite the broad applicability of synthetic biology in resource recovery, we have specifically not addressed biomining or biochemical extraction of phosphate rock. These provide a partial solution, but they have their own challenges, including the presence of harmful substances such as heavy metals and micropollutants, as well as the production of nitrous gases. Furthermore, these methods rely on extraction processes, which are not viable long-term solutions given the limited availability of phosphorus resources. Instead, we focus on improving bio-reclamation procedures and rendering them more efficient and economically feasible.

Motivated by the pressing need for sustainable phosphate management solutions and inspired by recent breakthroughs in synthetic biology, our team has opted to investigate the novel usage of phosphatases from the Flavobacterium family and phosphate-binding proteins (PBPs) for phosphate recovery. These proteins can be designed to have better phosphate affinity and selectivity. We are particularly interested in potential modifications that can allow these proteins to distinguish phosphates from similar molecules such as arsenate, which poses a significant challenge in current recovery methods but, if overcome, can pave the way for a new era of improved and efficient recovery.

The Solution

Our project proposes using modern synthetic biology methods to improve the properties of PafA. We predict that by incorporating these genetically altered organisms into a tailored bioprocess, we will be able to further enhance phosphate recovery efficiency, with the added benefits of lowering costs and environmental implications compared to traditional approaches.

Inspiration and Sources

This project was inspired by academic papers, real-world challenges, and previous iGEM teams' precedents. For example, Venkiteshwaran, Wells, and Mayer's (2020) work on using PBPs for phosphate recovery highlighted both the potential and challenges of current methods, directing our attention to improving the functionality of these proteins through synthetic biology.

Previous iGEM projects, such as the University of Bath’s 2022 project PhoBac, demonstrated bioengineered solutions' potential in addressing environmental concerns. Furthermore, the broader research community, such as Hutchison et al.'s (2021) findings on protein immobilisation techniques, provided a solid scientific foundation for our approach, which is used to reduce the loss of proteins to increase economic viability.

The Method

diagram of the method

Characterisation of Wild-Type PafA

Measuring Enzyme Activity

To measure enzyme activity, we will test the concentration of pNP at a given time after the start of enzyme catalysis of PNPP. After the maximum catalytic rate is deduced, the substrate concentration at half of this maximum rate will provide the Km value for PafA. Kcat can also be calculated by determining the concentration of inorganic phosphate produced in a given time through measuring the absorbance of the product due to pNP being yellow.. Product inhibition can be measured by plotting a graph of inorganic phosphate concentration against enzyme catalytic activity. Only monoesterase activity will be measured as PafA only acts on PME molecules.

Measuring Thermostability

To measure its thermostability, PafA will be exposed to PNPP at various temperatures, with its maximum catalytic efficiency measured at each temperature; then, this data can be plotted to determine the ideal temperature for maximum efficiency. To determine the unfolding temperature for PafA, circular dichroism (CD) spectroscopy can be used to determine its thermal stability and, thus, which temperatures it remains folded at, which will also align with the temperatures at which it exhibits the highest catalytic activity. The optimum temperature is likely to fall within the range of 20-45°C due to the mesophilic nature of the enzyme.

Measuring pH Stability

PafA is known to have an optimum pH of 8.5. Still, to test this in the laboratory, we will conduct multiple experiments by mixing PafA and PNPP at varying pHs to determine which pH shows the greatest formation of ES complexes and, thus, the greatest catalytic activity. This data will be plotted on a curve with pH (x-axis) and enzyme activity (y-axis) to display the results clearly.

Improving the Expression & Stability of PafA

Our goal for this section of the project is to use AI protein redesign tools to improve the expression, thermostability and pH stability of PafA.

Finding catalytic and otherwise important residues

In experiments, it was found that mutations that had the largest impact on PafA were present in first-shell residues, located in the active site and involved in the catalytic function of the enzyme. However, mutations within second-shell residues, which surround the active site but are not directly involved in catalysis significantly impacted the catalytic activity, substrate specificity, product inhibitions, and transition state analogues' binding in PafA (Markin et al., 2021; Chikunova and Ubbink, 2022).

Therefore, when selecting residues that are suitable for redesign, we will identify the residues that are imperative for the protein's function, which we will not mutate but look at identifying residues where mutations can be introduced that could improve the enzyme within the first and second shell of the enzyme.

Fixed backbone redesign

ProteinMPNN (PMPNN) is a fixed backbone redesign program that generates alternative sequences for a given topology and can be used to try to improve the stability of enzymes (Sumida et al., 2024). PMPNN can be programmed to retain the catalytic residues and the top 50% of conserved residues in the protein structure, subsequently allowing the program to predict alternative amino acid sequences for the remaining regions, ensuring that the protein folds into a stable conformation while maintaining the essential catalytic and conserved residues. Experiments showed that flexible regions of the enzyme can be rigidified, and enzyme stability improved using this method (Sumida et al., 2024). We intend to subject PafA to a similar redesign regime with the hope that catalytic activity and stability can be enhanced. It may also be useful to utilise the recently developed Ligand MPNN, which explicitly models small molecule binders in the design process so that enzyme redesign can occur whilst considering substrate binding (Dauparas et al., 2023). To ensure that when we improve the enzyme, it will continue to function, we will use an organophosphate ligand in silico to ensure that the AI design programme considers and recapitulates the active site's overall shape.

Once we have generated some proposed altered sequences of ProteinMPNN, we will utilise AlphaFold3 (AF3) to confirm which sequences fold well enough to for expression and functionality, furthermore, we can use it to ensure that it will bind around an organophosphate ligand in silico. Furthermore, MPNN models often provide energy scores for designed sequences with lower energy scores typically indicating more favourable conformations and potentially higher protein stability (Wang et al., 2024). By combining MPNN output data for sequence design with AF2 predictions for folding propensity, we can make informed selections of the sequences we intend to G-block order.

We will then design overhangs and a plasmid backbone to insert the sequences onto and order the sequences of redesigned PafA that we intend to test to see if they have improved PafA functionality. In addition, we will order the original unedited sequences and a phosphatase that is product-inhibited as a control.

Testing the designs

For us to test the improved designs of PafA, we will conduct an organophosphate assay, comparing it to the unedited pafA sequences to test if we have improved PafA. We intend to do repeats to test this and conduct temperature-controlled experiments to test if the thermostability and the pH stability of PafA have been improved. These experiments will be conducted using the same protocols as those in the “Characterisation of Wild-type PafA” section to ensure we can compare results.

We will use traditional cloning techniques to clone the improved PafA variants into the same expression system as WT PafA. The redesigned PafA sequences will be generated and amplified using PCR, ligating onto the expression vectors. This modified vector can then be transformed into E. coli cells, along with the appropriate resistance markers.

To efficiently test several redesigned candidates in parallel, we will utilise larger well plates to conduct the experiments and automate techniques where possible.

The Column

We intend to trial making a “column” or batch method that will enable PafA and PBPs to be used in combination to simultaneously convert organic phosphates to inorganic, and bind these inorganic phosphates to the device. These bound phosphates will be able to be released using the mechanism of the PBP- often this is via changes in pH triggering a change in the folding of the PBP, allowing release. There’s also an interesting PBP explored via other iGEM teams which releases in response to light, which may be interesting to trial.

Initially, we are trying both a column using sepharose beads to bind both PafA and the PBP (provided by our supervisor) using their 6xHis tag. However, after consultation with Dr Routoula, we will also try using silica/glass beads provided by the bioengineering department. This is as this is a more commonly used tethering mechanism that is less likely to unfold our protein, minimising its effectiveness. The mechanism of how to tether the protein to the column will need to be understood prior to this.

We shall test by using a PNPP assay to test if the immobilised enzymes are able to still function as intended.

References

  • Chowdhury, R.B. et al. (2017) ‘Key sustainability challenges for the global phosphorus resource, their implications for global food security, and options for mitigation’, Journal of Cleaner Production, 140, pp. 945–963. Available at: https://doi.org/10.1016/j.jclepro.2016.07.012.
  • Lidbury, I.D.E.A. et al. (2022) ‘A widely distributed phosphate-insensitive phosphatase presents a route for rapid organophosphorus remineralization in the Biosphere’, Proceedings of the National Academy of Sciences, 119(5). doi:10.1073/pnas.2118122119.
  • Sumida, K.H. et al. (2024) ‘Improving protein expression, stability, and function with proteinmpnn’, Journal of the American Chemical Society, 146(3), pp. 2054–2061. doi:10.1021/jacs.3c10941.s001.