Project Selection Process
In January, as the new iGEM Aachen Team 2022, we first brainstormed which topics were interesting for the team. Working in small groups, we researched what topics are suitable for a possible iGEM project. Talking with our mentors and experts from different fields, we discussed with them which project ideas have potential and are realistic to implement. After the meetings, we shortlisted three projects and researched them in detail. Debating the advantages and disadvantages of each project idea with our mentors and within the team, we finally chose MEtaPhos as our project on March 14, 2022.
MEtaPhos aims to use phosphate binding proteins with a molecular optogenetic switch to recover phosphate from wastewater. The idea to use phosphate binding proteins is based on a paper by Hussein and Mayer [1]. The idea of using a light-dependent molecular switch caught our attention. During the process of finding suitable switch candidates and designing the fusion proteins, we got help from our advisors Dr. Drepper and Dr. Krauss from the Forschungszentrum Jülich who are experts in the field of optogenetics. At the same time, the importance and seriousness of the not-yet widely known phosphorus problem motivated us to dedicate our project to this global challenge.
Phosphorus’ Role in Our World
Phosphate is a salt of phosphorus with an anionic entity, often a single $PO_{4}$ tetrahedron called orthophosphate or monophosphate [4]. Phosphorus is one of the six biogenic elements, which means that it is critical and essential to life [5]. The EU Commission has already classified phosphorus as a critical raw material since 2014. On earth, phosphorus does not occur elementally, but only in phosphate compounds. Phosphates are ubiquitous in biology and are often attached to other chemical species for regulation, e.g. phosphate diesters in the DNA backbone [5]. Moreover, it is an industrial source material and irreplaceable product [6, 8]. Currently, phosphate is gained through mining, but the global phosphate reserves are limited [6, 8]. The future main source of phosphate will be wastewater and trash. In figure 1 you can see the current phosphate process arrow. No effective recycling method has been invented yet. If we do not find a way to regain phosphate, we will have difficult challenges to face.
Figure 1: Phosphate process arrow
One challenge is the environmental aspect. Wasting phosphate and unsustainable handling of sewage, fertilizers, and animal manure leads to high phosphate concentrations in groundwater and water bodies, which have an immense impact on the water quality [7, 8]. High concentrations of phosphate can lead to oxygen depletion and eutrophication through the growth of harmful cyanobacteria [7]. It contributes to the formation of harmful algal blooms which have an impact on biodiversity, and invasive species are introduced [7].
Another challenge is the geopolitical and economic aspects. Phosphate supplies are limited because only a few countries hold phosphate resources in considerably large amounts. Europe mostly relies on imported phosphorus from North Africa and Russia, which can threaten access and European food security [6, 8]. On the one hand, phosphorus demand rises due to increasing welfare in many countries and a growing world population [8]. On the other hand, access to phosphorous is under threat due to political unrest and climate change in phosphate mining countries, which can lead to rising prices and difficult export security actions [8].
One of the greatest challenges of the twenty-first century will be food security. Phosphorus has no substitute and is a nonrenewable resource; the use of phosphate as a fertilizer has helped to feed billions of people in the last 50 years [9]. Without phosphate, we will have difficulties harvesting enough food for the growing world population.
The Necessity of Phosphate Recycling
Until now, it was allowed to use clarified sewage sludge as fertilizer. However, from 2029 on an EU law will come into force that will oblige most sewage treatment plants to regain phosphate [10, 11]. The current methods of phosphate removal from wastewater are chemical precipitation, for example with ferric chloride, and biological elimination [12]. The use of heavy metals contributes to soil pollution, which will no longer be tolerated in the EU. Sewage treatment plants must find other solutions to purify wastewater from phosphate. One idea is to build mono-sludge incineration plants where the sewage sludge will burn to ash [10]. The problem is that the phosphate is no longer accessible until a method will be invented to reuse the phosphate in contained in the ashes. MEtaPhos aims to revolutionize the phosphate cycle with a new type of recycling method (see figure 2). Our approach allows the purification of pure phosphate from wastewater through highly phosphate-specific and sensitive proteins. Furthermore, the microbial production of polyphosphate from our purified phosphate generates an even more valuable and versatile product that can be used in industry [13]. There are a variety of applications for polyphosphates. It is used in the food industry, for example as a buffer or to soften meat products [13]. Equally, it can also be found in toothpaste, wall paints and many other areas [13].
Figure 2: Improved phosphate cycle
The use of harmful chemicals is not necessary for the application, and if renewable energy is used for light generation, a sustainable circular process can be established.
Our Idea: A Molecular Optogenetic Switch
MEtaPhos has two approaches for an optogenetic switch. Both approaches work with phosphate binding proteins.
The first approach is an irreversible optogenetic switch, the photosensitizer SOPP3 [3], which stands for singlet oxygen photosensitizing protein 3 and is a photosensitizer from Arabidopsis thaliana. Our goal with SOPP3 was to show the potential of a light switch to control a protein. As a synthetic fusion protein, SOPP3 was linked to the phosphate binding protein 2ABH through a spacer. In figure 3 we illustrated how our SOPP3-2ABH works. Blue light induces the production of reactive oxygen species (ROS). The synthesized ROS lead to the chemical degradation of 2ABH and thus cause the release of the phosphate bound to 2ABH. Upon irradiation, the chemical degradation follows in minutes.
Figure 3: Irreversible approach SOPP3-2ABH
The second approach is a reversible optogenetic switch, the photoreceptor VVD [2], which originated in Neurospora crassa. VVD was our second engineering cycle where we demonstrated that a reversible system can be the next step for improvement. We created another synthetic fusion protein, where VVD was linked to the phosphate affine protein CMI through a linker. In figure 4 we illustrated how our VVD-CMI works. Upon blue light illumination, VVD can dimerize and activate the phosphate binding protein, whereas switching the illumination off leads to the separation of VVD dimers, the inactivation of the phosphate binding protein and consequently, the release of phosphate.
Figure 4: Reversible approach VVD-CMI
In the outlook, we will provide a possible third engineering cycle with other suitable optogenetic switch candidates and matching phosphate binding proteins. We also discuss what can be optimized and what next steps are possible.
