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The field of synthetic biology provides a wide range of possible applications. However, there are still challenges that science has not yet overcome completely. Many unicellular eukaryotic organisms are not well understood and are therefore troublesome to transform or to handle in the lab. Transformation of those complex eukaryotes can unlock the treasures hidden in the depths of these enigmatic organisms, for example regarding the fascinating kingdom of fungi. We strived to develop a key to reach their secrets by designing Troygenics, a platform system that can be adapted for the transformation of any eukaryotic cell worldwide.
The world of fungi is, like the human world, host to good as well as evil. Many diseases that plague humans and animals, as well as devastating crop pests, are caused by fungi. Due to multiple levels of specificity and adaptability of our platform system, it can be used to save the world from terrifying threats, while, at the same time, offering the key to the fascinating kingdom of fungi.

Since the earliest beginnings of humanity, the desire to understand the inner workings of the world has been constantly present. With the establishment of synthetic biology several years ago, scientists held a powerful tool in their hands. Synthetic biology allows us to modify, improve and develop new methods for established organisms to greatly boost yield in biotechnological processes, develop novel treatments, and potentially completely change the way we live in the future (Ruder et al., 2011). Furthermore, it also gives us the ability to investigate fascinating microorganisms that have eluded deeper characterization to the present day.

Until today, tapping into the great potential, the marvelous complex diversity of nature and the potential valuable knowledge and components that come with this, remains tricky. One main obstacle solving this task is the fact that many microorganisms, like fungi, kept their fascinating secrets hidden until now, as they are very difficult to handle and to transform in the lab (Case, 1983).

To overcome this issue, that restricts our understanding of the world of complex microorganisms, we developed a platform system called Troygenics, which allows us to transform any eukaryotic microorganism we wish to transform. Our Troygenics are based on the M13 bacteriophage. We modified the major coat protein, so it allows selective endocytosis into the target cells. During this process, the gene of interest - packed up inside the Troygenic - is delivered into the cell of interest, thus transforming the cell of interest transiently at first. The addition of homologous recombination sequences around the gene of interest enable genomic integration and the generation of stable transformations.
Troygenics are easily customizable and specific to their target, as the addition of the cell-specific modification to the major coat protein requires just one easy cloning step.
We disabled the endogenous functionality of the M13 bacteriophage to infect its natural host Escherichia coli by shortening the minor coat protein pIII. Furthermore, to enable precisely targeted endocytosis, we fused target-specific ligands to the major coat protein pVIII. Additionally, to add some further safety mechanisms to our system, we deleted all phage proteins from the DNA that gets packed into our Troygenics, the Application Plasmid, so it only consists of the f1 ori and regions that code for the modified pVIII and the gene of interest, which will specifically be delivered into the target cell. These implemented changes act as two levels of safety mechanism to prevent the Troygenics from reinfecting E. coli and thereby spreading into nature.

The system we developed allows us to transform even the most difficult eukaryotic targets, which will not only simplify many procedures in the lab and in the industry, but also has great potential in basic research. Transforming currently elusive complex microorganisms can open up the possibility for discovery of fascinating functions or silenced gene clusters with unknown products that could benefit medical research, Troygenics can help us unlock the great potential of diversity in nature, as Prof. Zeilinger-Migsich estimated.

Simplifying the transformation of complex eukaryotic microorganisms could help to activate interesting silenced gene clusters.
Prof. Zeilinger-Migsich
Institute for Microbiology, Innsbruck
The Troygenics do not only make lab life easier. There are several different applications of Troygenics in the real world as well. Prof. Dr. Russel Cox told us that, due to the climate change crisis and the fast development of humanity, there is a growing threat caused by eukaryotic pathogens (Fisher et al., 2016). Pathogenic fungi pose a major threat to humans, especially in risk groups, such as elderly people, children, and people with weak immune systems, who often severely suffer and even die from fungal infections (Fisher et al., 2016). In addition, other eukaryotic microorganisms, like the unicellular pathogen Trypanosoma, are on the rise with devastating effects, and trigger serious diseases (Coura & Viñas, 2010).
The climate change crisis has a great impact on the spreading of pathogenic fungi from their original region to all over the world.
Prof. Cox
Leibniz university, Hanover
Even today, entire ecosystems are endangered as eukaryotic pathogens drive whole species to extinction (Scheele et al., 2019). And the worst part is that the common treatments are not effective anymore! The most dangerous pathogens evolve at an alarming pace, making many of them resistant to their conventional combat method already (Meis et al. 2016; Sanglard, 2016). With our Troygenics, we aim to get one step ahead of those most threatening pathogens. By encoding a Cell Death Inducing System (CeDIS) inside the Troygenics, we can selectively transform and fight the pathogens whilst sparing any other non-target organisms nearby.

Apart from the alarming effects on humans and animals, a secondary danger is on the rise as the burden of pathogenic fungi increases. Devastating famines caused by the fungal infection of food products occur more frequently in recent times (Bebber et al., 2014). Plant pathogenic fungi develop resistances against fungicides faster and faster. Some fungal strains, such as Puccinia graminis Ug99, are almost impossible to fight, frequently leading to nearly 100 % harvest loss on infected wheat fields (Newcomb et al., 2016; Pretorius, et al., 2010). Originating in Africa, P. graminis is spreading northward and has already reached Europe. Besides wheat, other staple foods, like potatoes, are also endangered due to fungal infections, as Dr. Patrick Beuters explained to us. With the world population increasing, we cannot withstand the amount of harvest loss we are currently marching towards. Something must be done!
Common staple foods are threatened by a vast range of fungal infections.
Dr. Beuters
Bayer Cropscience
Farmers are already doing their best trying to figure out the necessary fungicides, the right amount and mix to use, and the perfect time to apply it. But now, resistant fungi make their jobs even harder, as farmer Bernd Olligs told us when we visited his farm. Currently, there are two main possible methods that farmers can use to fight plant-pathogens. The first one involves figuring out the perfect treatment for every single plant they own, and taking a risk that the weather, or a newly developed resistance does not thwart their treatment, whereby their entire crop would be ruined. The second method is the application of fungicides on a broad spectrum. Using this method means accepting that it will not only kill the pathogenic fungi, but also many insects, small animals and other organisms that are vitally important for the ecosystem.
Fungal infections in general and fungi resistant to common fungicides make it very complex to figure out how and when to use available fungicides.
Bernd Olligs
Bayer Forward Farm, Rommerskirchen
Fungal species with reported antifungal resistance, by country (modified from (Fisher, Hawkins, Sanglard, & Gurr, 2018)). Increasing color intensity reflects a growing number of reports. The plant maps depict spatiotemporal records of resistance of crop pathogens to azoles (blue scale). The human maps depict spatiotemporal records of resistance of the pathogens A. fumigatus, C. albicans, C. auris, C. glabrata, Cryptococcus gattii, and Cryptococcus neoformans to azoles (red scale).
Our solution, the Troygenics, selectively transform only the targeted pathogen. Cells from every other organism in the vicinity will remain unaffected while the transformed pathogen cells are afterwards destroyed by the payload of our Troygenics, the Cell Death Inducing System. The DNA-encoded CeDIS has two important advantages over conventional methods of delivering toxic substances into pathogen cells: Due to the use of pathogen derived genetic elements the system is highly specific to the target organism. The other advantage is the lowered risk of the development of resistances. Our CeDIS depends on the recognition of several essential genes of the pathogen which decreases the chance of resistance-causing mutations inside the targeted organism.
This makes our Troygenics a platform system that can easily be adapted to solve upcoming issues stemming from complex eukaryotic cells with the help of synthetic biology.

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