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，在下一代，全球将出现磷酸盐短缺，磷酸盐是一种对现代农业实践至关重要的营养物质。这将对全球粮食安全产生严重影响，并被称为迫在眉睫的“磷酸盐危机”。磷酸盐是植物生长所必需的营养物质。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，它会被耗尽。在农业环境中，磷酸盐通过肥料被添加回土壤中。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，磷源在地理上的分布并不均匀，随着全球供应的减少，这可能会导致地缘政治紧张局势的加剧。除了磷酸盐供应有限所带来的风险之外，磷肥的施用还可能导致水的污染，从而导致富营养化，从而导致藻类或植物过度生长。这种“营养物污染”会对生物多样性造成毁灭性影响，并导致巨大的经济成本。为了避免“磷酸盐危机”和减少营养污染的影响，我们必须改变我们的农业实践，优化磷肥的使用。要做到这一点，第一步是更好地了解植物是如何利用磷酸盐的。管理磷酸盐使用的过程鲜为人知，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它们的磷酸盐储存并将其分配到整个植物中。由于传感器必须在多种条件下工作，因此开发用于整个工厂的传感器是一项挑战。电流传感器灵敏度有限，并且在液泡中不起作用，液泡是关键的亚细胞磷酸盐储存库。我们正在采用一种新颖的方法来开发磷酸盐传感器，使用尖端的蛋白质设计方法在计算机模拟中创建和测试我们的传感器，然后在实验室中构建最有希望的候选传感器。我们将把已知的磷酸盐结合蛋白与被称为循环排列荧光蛋白的传感区域结合起来，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，这是一种可以自动构建和测试复杂生物分子的先进设备。为了测试我们的传感器并确定它们如何在植物中发挥作用，我们将创建一个屏幕，准确捕捉传感器将被应用的细胞环境。为了做到这一点，我们将分析补充了不同水平磷酸盐的作物植物的细胞组成，并利用这些信息在一个简单的屏幕上重现这些条件，这个屏幕可以按比例放大以测试我们的传感器。我们将彻底描述我们设计的传感器，并描述它们在一系列条件下的行为，以创建传感器工具包。在设计传感器时产生的所有方法和数据都将公开。这些传感器在研究基础磷酸盐生物化学方面具有明确而重要的用途，同时也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品种，用于监测田间的磷酸盐。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.