Salim Atakhanov • Kim Büttgen • Selma Busch • Daniel Costard • Maximilian Dreimann • Raphael Egging • Pia Ergezinger • Julia Gehrmann • Florian Kroh • Sven Leisen • Vladyslav Los • Lina Schmidt • Shlok Szatkowski • Marcel Wittmund
Prof. Dr. Lars Blank • Prof. Dr. Ulrich Schwaneberg • Prof. Dr. Wolfgang Wiechert
Dr. Yu Ji • Volkan Besirlioglu
Dr. Verena Resch
The complex regeneration of biochemical energy sources represents a cost-intensive hurdle for many production and research processes. With M.A.R.S., we want to establish an innovative strategy to create light-powered, mitochondrion-like protocells and a bioreactor that will recycle those cells by magnetism. Through the design of our reusable recycling system it will be able to power every ATP-driven enzyme cascade, making M.A.R.S. universally applicable. By extracting Bacteriorhodopsin out of Halobacterium salinarum, a phototrophic archaea species, and combining it with an ATP synthase from Saccharomyces cerevisiae in self-produced polymersomes and liposomes, we get simple but effective chassis, which make it possible to cover the energy requirement of any enzyme reaction cascade. Binding those chassis to magnet particles via anchor peptides enables the reuse of the entire protocell system within the reactor by means of magnetic purification, whereby they can be fed directly into enzyme cascades, without depending on living cells.
There are many enzyme systems which use adenosine triphosphate as a cofactor, but there is no efficient ATP regeneration method yet. Acetyl phosphate, phosphoenolpyruvate and creatine phosphate are expensive phosphate donors used for conventional ATP regeneration systems, thus not ideal for industrial application . A common method is using polyphosphate to regenerate ATP, but this can slow down some reactions . What is missing is a method to regenerate ATP like cells do, without using expensive metabolites or lowering the efficiency of the main reaction.
After figuring out the basic design, we wanted to optimize it by making it reusable. Doing so was possible by connecting these powerplants with magnetic particles using anchor peptides. After the use of M.A.R.S, our system can be separated from the product and can be used again.
We need two anchor peptides and a spacer domain, forming one complex. The first anchor peptide binds to the polymersome; the second anchor peptide binds polystyrene coated magnetic particles. A spacer domain connects both anchor peptides, which enables the whole construct to link the ATP factories to the magnetic particle.
We also wanted to provide an easy to assemble and cheap photobioreactor, with the functionality to recycle the chassis by using magnets.
We used two different approaches: Mathematical modeling and molecular dynamics simulation
In our mathematical model, we simulated the implementation of our complete system in the context of a real industrial application. We could show that a set-up of our concept exceeds the efficiency of existing ATP-Regeneration systems and can compete with the conversion rates of whole cell approaches while preventing any formation of secondary metabolites. We ran several in silico experiments with our mathematical model, compared thousands of different sets of parameters, to find the optimal parameters for M.A.R.S. like the mean chassis diameter or the initial CAR, ADP and Pi concentration.
With our extensive molecular dynamics simulations, we went much further than just rendering our models in simple solvents and gathering data plots with the standard GROMACS workflow. We assisted the wet lab team by creating valuable predictions in silico, like the integration behavior of our proteins into the different lipid batches. Furthermore, we used molecular dynamics to create comprehensive molecular movies of the components of M.A.R.S. like the main ATP Recycling-Mechanism of our chassis as well as the coupling to magnetic particles.
We choose a lipid mixture of POPC, DOTAP and DOPC to have slightly positively charged liposomes for better bacteriorhodopsin integration.
For the liposome formation, we decided to use the film rehydration technique as a proven method . It consists of the rehydration of a dried lipid film with a buffer solution which creates small unilamellar vesicles. To make the liposomes easier to detect under the microscope, we want to form giant unilamellar vesicles. This was achieved by creating an oil-surfactant mix where an aqueous solution which contains the lipid batch is added.
The desired mixture of 30% DOTAP, 35% DOPC, and 35% POPC did not work; there were no liposomes visible. We then tried liposomes consisting of only POPC first. POPC worked with different buffers. DOTAP and DOPC also worked for themselves. After that, we tried a mixture of 80% POPC and 20% DOTAP to get closer to the desired positively charged composition, which also worked.
Bacteriorhodopsin ATP synthase
After cultivation, we used osmotic shock for cell lysis and ran five rounds of centrifugation at 50,000 g and resuspension, finishing with sucrose-density gradient centrifugation at 280,000 g for eight hours. After these purification steps, we aimed to have three different fractions of which fractions one and two should have been the ones with the highest purple membrane purity. Due to complications with the rotor of the ultracentrifuge, the last step was not possible to implement.
ATP synthase was produced by the commonly known „baker’s yeast“ Saccharomyces cerevisiae. First, we isolated mitochondria in which the ATP synthase is located. For a higher yield and easier purification, we decided to use a specific inhibitor protein for the ATP synthase called IF1. The IF1 protein inhibits the hydrolyzation of ATP by blocking the rotation of the F1-subunit in one direction . IF1 was modified with a His-tag for easier purification and expressed in Escherichia coli.
Before continuing with further purification, we lysed the mitochondria, following it up with a gel-electrophoresis and a western blot, including antibody immunodetection, to ensure that we purified mitochondria.
After successful implementation, we wanted to continue with the inhibition of the ATP synthase and further purification with affinity chromatography. However, because of time issues, we were not able to complete this final step.
Bacteriorhodopsin and ATP synthase integrated into the membrane
Higher fluorescence means more anchors are bound to the magnetic particle´s surface. Within the first test with the magnetic particles, we identified MBP-1 to have the best binding. Under the same conditions, MBP-1 had the highest fluorescence. eGFP worked as a negative control and showed no fluorescence, which means no binding.
eGFP before 2nd washing step eGFP after 2nd washing step
LCI before 2nd washing step LCI after 2nd washing step
MBP1 before 2nd washing step MBP1 after 2nd washing step
Cg-Def before 2nd washing step Cg-Def after 2nd washing step
TA2 before 2nd washing step TA2 after 2nd washing step
The tests with the liposomes were more difficult. There is no easy way of separating the liposomes from unbound anchors. We first tried centrifugation, which did not work because the liposomes broke. The next try was to use a gentler method for separation, dialysis. Due to the bigger size of the anchors, we tried a molecular cut off 50 kDa. The liposomes were stable this time, but the anchors did not get through the dialysis tube. Next tries should be performed with a molecular cut off 100 kDa and maybe even 300 kDa.
Because we could not get bigger dialysis tubes fast enough, we tried out our binding candidates with low concentrations. The aim was to see whether the liposomes or the whole background shows fluorescence first. With LCI the liposomes showed a fluorescence first. Therefore, more tests with LCI should be performed.
POPC with Tris 30mM buffer, POPC with Tris 30mM buffer,
0.2 µM eGFP, Brightfield mode 0.2 µM eGFP, eGFP mode
POPC with Tris 30mM buffer, POPC with Tris 30mM buffer,
0.25 µM LcI, eGFP mode 0.25 µM LcI, eGFP mode with zoom:
With these first results, we concluded that a possible biadhesive peptide would be a fusion protein of MBP-1 and LCI. Both anchor peptides are connected through a stiff spacer Domain Z. This construct is able to connect polystyrene coated magnetic particles to POPC liposomes. Further tests should be performed with different lipid compositions, especially with the desired slightly positively charged lipids. The whole fusion protein can then be expressed and binding of both complexes can be checked. This could be done through magnetical separation to see if the liposomes would also be separated.
We approached the design and construction of the bioreactor holistically. Therefore, we wanted to mitigate several pain points with current solutions, mainly the reliance of human labour between the tasks.
The main part of the reactor is the orbital shaker. It must mix the reactants reliably. Furthermore, it has to be easy to manufacture and assemble as well as be comparatively cheap. We used Erlenmeyer flasks to change as little of the laboratory parameters as possible. We designed a linker which transforms the rotation of the electric motor into an eccentric motion. The timing belt allows for the seamless translation of torque to the other shafts. Under each flask there is a compartment for specialized equipment. In our project, we use electric magnets to immobilize the magnetic particles at the bottom of the flask.
To automate the process of pumping out the products as much as possible, we designed an automatic arm with a mechanical guide for the tubes to drive them into the Erlenmeyer flasks. Therefore, the tubes do not interfere with the shaking motion.
Lastly, we began to work on an IoT Platform, so that the user can decide on shaking intensity, duration, etc. and the process begins autonomously, automating the process as much as possible.
We found an anchor peptide that binds specifically and has a high affinity to the magnetic particles, that possess a polystyrene surface. The anchor peptide MBP-1 showed the most promising binding. While testing multiple other peptides, we found out that LCI is the equivalent to MBP-1 respectively to the liposomes. The investigated anchor peptides were fused to an eGFP Protein to offer a visual confirmation of possible binding abilities.
Magnet LCI + Liposome
The next step would be the expression of an entire protein, which features an additional linker between those two binding domains, to create a bridge between our ATP Factories and magnetic particles.
Apart from the proof of the different compounds of M.A.R.S. we wanted to come up with a specific example were our system can be used.
To not only show that our system might work in Vitro, we aimed to test our system in a real industrial environment. By collecting Data of a specific enzyme reaction, we simulated how M.A.R.S. would act in the context of the following application. The enzyme carboxylate reductase (CAR) needs NADPH and ATP as cofactors. It´s part of very important enzymatic processes and enzyme cascades to produce pharmaceutical precursors such as R-PAC. Additional applications where our system comes into play will be elaborated in the following abstracts of this poster.
As a first reaction of the specific cascade, on which we set a special focus on, the CAR synthetizes a benzaldehyde out of a carbon acid . This substrate is present in second generation feedstock and therefore offers a sustainable alternative to the common production methods. In a series of experiments, we tested the normally used regeneration system, whole cells as cofactor regeneration approach and did some negative controls to prove the dependency on ATP of this specific enzyme. We could represent the activity of the enzyme under the different conditions in form of kinetic curves.
As a result, we saw less main product conversion with the whole cell system that with the normally used regeneration system. The whole cell approach tends to over reduce the wanted product to an alcohol, which compromises the efficiency of this method.
We simulated the enzymatic activity of the CAR in the reaction where our system provides the ATP as a cofactor. With our system cytotoxic products and products in high concentrations can be synthesised. M.A.R.S. enables a comparable activity after 2 hours to that of the whole cell approach, without production of unwanted secondary metabolites. In the long run, significantly higher product conversion can possibly be achieved.
M.A.R.S. enables the industry to produce any specific relevant chemical, without the ineffectiveness of whole cell approaches or the unsustainable implications of purely chemical regeneration systems.
One of the many ATP-dependent enzyme families is Carboxylic Acid Reductases (CARs). CARs can be used to catalyze reactions by themselves, yet they are often part of more sophisticated enzyme cascades. Because of their broad spectrum of catalyzed reactions and their high stereoselectivity, they are an ideal tool to produce precursors or complicated pharmaceuticals that could not be easily synthesized before . One example of these important precursors is cinnamyl alcohol, which can be produced from glucose by the combination of CARs with other reductases . A more recent proposal is the usage of CARs to synthesize (S)-2-aminobutanol, an important chiral precursor for the pharmaceutical compound Ethambutol, which is an antibiotic for tuberculosis treatment  .
ATP’s potential as a vital compound in the production of pharmaceuticals is not purely secondary- there is much more to it.
However, the use of ATP in the pharmaceutical sector is not limited to its activity as a cofactor. The direct application of ATP, for example, in the treatment of mitochondrial disease, is just another of many examples of the relevance of ATP recycling systems in the industry .
There is also a strong dependency on CARs for the production of fine chemicals, for example, in the agrochemical sector, where they find application in simple two-step reaction cascades to synthesize arbitrary secondary amines  
Since, as mentioned above, CARs are ATP-dependent enzymes, secure ATP recycling is also essential in the field of chemical production.
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We want to thank our PIs Prof. Dr. Lars Blank, Prof. Dr. Ulrich Schwaneberg and
Prof. Dr. Wolfgang Wiechert as well as our Team Instructor Dr. Yu Ji and our Team Advisor Volkan Besirlioglu for accompanying us through this project and for always being at our side with help and advice.
In the same way we want to thank Dr. Verena Resch for taking over a large part of the design of this poster.
Last but not least, we want to thank those who have supported us with financial help or by providing rooms and materials:
This project was funded by the "Hans Hermann Voss-Stiftung".
Perhaps, if we manage to let go of comforting familiarity of mitochondrion and embrace seemingly unlimited potential of prokaryotes-inspired energy regeneration, we might one day be able to solve some current energy depending problems and even to energize artiﬁcial life.