21ENGBIO - High-Throughput Design of Novel Sensors to Help Address the Impending Phosphate Crisis

It has been predicted that over the next generation, if current trends are maintained, there will be a global shortage of phosphate, a nutrient that is critical for modern agricultural practices. This will have a severe impact on food security worldwide and has been dubbed the impending "Phosphate Crisis". Phosphate is a nutrient that is required for plant growth. It is naturally found in soil and absorbed through the roots of plants, but over time it is depleted. In agricultural settings, phosphate is added back to the soil using fertilizers. However, the phosphorus used in most fertilizers originates from finite mineral sources. To further compound this, phosphorus sources are not evenly distributed geographically, which is likely to lead to an increase in geopolitical tension as the global supply decreases. Beyond the risks posed by the limited availability of phosphate, the application of phosphate fertilizers can also lead to the pollution of water, which results in eutrophication and thus excessive algal or plant growth. This "nutrient pollution" can have a devastating impact on biodiversity and lead to significant economic costs.

In order to avert the "Phosphate Crisis" and reduce the impacts of nutrient pollution, we must change our agricultural practices to optimise the use of phosphate fertilizers. The first step to doing this is to gain a better understanding of how plants utilise phosphate. The processes involved in managing phosphate usage are poorly understood due in part to a lack of sensors that can be used for real-time monitoring of phosphate levels in living plants. These sensors would illuminate how plants manage their phosphate stores and distribute it throughout the plant. Developing sensors for use in whole plants is a challenging due to the diversity of conditions in which the sensor must operate. Current sensors have limited sensitivity and are non-functional in the vacuole, which is a key subcellular phosphate reservoir.

We are taking a novel approach to developing phosphate sensors, using cutting-edge protein-design methods to create and test our sensors in computer simulations before constructing the most promising candidates in the lab. We will combine known phosphate-binding proteins with sensing regions known as circularly-permuted fluorescent proteins, which will create sensors that produce light and change brightness when phosphate is present. We will construct these sensor proteins at scale, using the Edinburgh Genome Foundry, a state-of-the-art facility that can automate the construction and testing of complex biological molecules.

To test our sensors and determine how they would function in plants, we will create a screen that accurately captures the cellular environments where the sensors will be applied. To do this, we will analyse the cellular composition of crop plants supplemented with various levels of phosphate and use this information to recreate these conditions in a simple screen that can be scaled up to test our sensors. We will thoroughly characterise our designed sensors and profile how they behave in a range of conditions in order to create a toolkit of sensors.

All methods and data generated while designing the sensors will be made publicly available. While the sensors will have clear and important utility for studying fundamental phosphate biochemistry, they could also have important biotechnological applications. For example, they could be used to create novel plant strains that can be used to monitor phosphate in the field. This would enable farmers to apply phosphate fertilizers more efficiently, reducing phosphate usage and making sustainable sources of phosphate more viable.

Phosphate is a critical nutrient for plant growth and is administered to crops in fertilizer. However, overuse of phosphate fertilizers can lead to water pollution and mineral sources of phosphorus are finite, which has severe implications for food security. Fertilizer usage must be optimised to mitigate these risks, but this is challenging as fundamental information regarding phosphate regulation is poorly understood, due in part to a lack of robust sensors that can be used to monitored phosphate levels plants. Phosphate sensors have been developed previously, but these function over a narrow range of concentrations and are not functional in the vacuole, which is the major subcellular reservoir of phosphate. To truly understand how phosphate concentrations at the roots correspond with total levels of phosphate in the plant, we must develop novel sensors that function in a range of conditions.
We propose to design and characterise a toolkit of phosphate sensors that operate in a range of conditions and a range of phosphate sensitivities. To do this, we will identify phosphate-binding domains that are suitable candidates for the insertion of circularly-permuted fluorescent proteins (cpFP) in order to create novel phosphate sensors. These putative sensors will be modelled and screened in silico to identify the most promising designs, which will be created in the Edinburgh Genome Foundry (EGF) using combinatorial assembly of binding and sensing domains. To test the sensors, we will develop a novel cell-free screen that recreates ionic conditions found in plants and use it to thoroughly characterise the phosphate sensors.
The sensors generated in the proposal will be useful studying fundamental phosphate biochemistry that could inform basic farming practices in order to minimise the use of phosphate fertilizers. Furthermore, these sensors and the design strategy could form the core enabling technology to create sentinel plants to monitor phosphate levels in the field.