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

据预测，如果目前的趋势保持下去，下一代将出现全球磷酸盐短缺，而磷酸盐是一种对现代农业实践至关重要的营养素。这将对全球粮食安全产生严重影响，并被称为即将到来的“磷酸盐危机”。磷酸盐是植物生长所需的营养物质。它自然存在于土壤中，并通过植物的根部吸收，但随着时间的推移，它会耗尽。在农业环境中，使用肥料将磷酸盐添加回土壤。然而，大多数肥料中使用的磷来自有限的矿物来源。更糟糕的是，磷的来源在地理上分布不均匀，随着全球供应量的减少，这可能导致地缘政治紧张局势加剧。除了磷酸盐供应有限造成的风险外，施用磷肥还可能导致水污染，导致富营养化，从而导致藻类或植物过度生长。这种“养分污染”会对生物多样性造成破坏性影响，并导致巨大的经济成本。为了避免“磷酸盐危机”，减少养分污染的影响，我们必须改变农业实践，优化磷肥的使用。这样做的第一步是更好地了解植物如何利用磷酸盐。对磷酸盐使用管理过程了解甚少，部分原因是缺乏可用于实时监测活植物中磷酸盐水平的传感器。这些传感器将阐明植物如何管理其磷酸盐储存并将其分布在整个植物中。开发用于整个工厂的传感器是一项挑战，因为传感器必须在多种条件下工作。电流传感器的灵敏度有限，而且在液泡中不起作用，而液泡是亚细胞磷酸盐的关键储存库。我们正在采取一种新的方法来开发磷酸盐传感器，使用尖端的蛋白质设计方法来创建和测试我们的传感器，然后在实验室中构建最有前途的候选人。我们将联合收割机结合已知的磷酸盐结合蛋白和称为环状排列荧光蛋白的传感区域，这将创造出当磷酸盐存在时产生光并改变亮度的传感器。我们将使用爱丁堡基因组铸造厂（Edinburgh Genome Foundry）大规模构建这些传感器蛋白质。爱丁堡基因组铸造厂是一个最先进的设施，可以自动构建和测试复杂的生物分子。为了测试我们的传感器并确定它们在植物中的功能，我们将创建一个屏幕，准确捕捉传感器应用的细胞环境。为此，我们将分析补充了不同水平磷酸盐的作物的细胞组成，并利用这些信息在一个简单的屏幕上重现这些条件，该屏幕可以放大以测试我们的传感器。我们将彻底验证我们设计的传感器，并描述它们在一系列条件下的行为，以创建传感器工具包。设计传感器时生成的所有方法和数据将公开提供。虽然传感器将在研究基础磷酸盐生物化学方面具有明确和重要的实用性，但它们也可能具有重要的生物技术应用。例如，它们可以用来创造新的植物菌株，可用于监测实地的磷酸盐。这将使农民能够更有效地施用磷肥，减少磷酸盐的使用，使可持续的磷酸盐来源更加可行。

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.