Team:Aachen/Description
Project Description and Motivation
M.A.R.S. – Energy generation through light-driven protocells, and a bioreactor that purifies them using magnetism
In January we started with a large brainstorming session to find a project for the competition. By splitting up, researching for individual topics, and presenting the
results to each other, a pre-selection of suitable projects could be made early on. The decision-making process was supported by multiple consultations with our supervisors,
and so we finally decided on March 17, 2020, for “M.A.R.S.” as our project for this year’s iGEM competition.
Energy as, in the truest sense of the word, the driving force of all life and being, is the most limiting factor in the industrial implementation of scientific discoveries. Sustainable and efficient energy supply thus plays a decisive factor in actual up-scaling. With our project, we want to drive current and future ideas from scientists all over the world and thus contribute to their implementation.
The specific goal of our project “M.A.R.S.”, short for Magnetic ATP Recycling System, is to use already successful and easily reproducible research to establish an innovative way for powering enzymatic reactions of all kinds. To do so, we want to create light-powered, mitochondrion-like protocells, and a bioreactor that will recycle those cells by magnetism.
Energy as, in the truest sense of the word, the driving force of all life and being, is the most limiting factor in the industrial implementation of scientific discoveries. Sustainable and efficient energy supply thus plays a decisive factor in actual up-scaling. With our project, we want to drive current and future ideas from scientists all over the world and thus contribute to their implementation.
The specific goal of our project “M.A.R.S.”, short for Magnetic ATP Recycling System, is to use already successful and easily reproducible research to establish an innovative way for powering enzymatic reactions of all kinds. To do so, we want to create light-powered, mitochondrion-like protocells, and a bioreactor that will recycle those cells by magnetism.
"So, you want to create ATP. But what is ATP and why is it so important?"
A lot of chemical reactions would not be possible in a sufficient time without
enzymes catalyzing them. However, also these enzymes often do not work without the help of certain
molecules known as cofactors. A very important cofactor is adenosine triphosphate (ATP). Its highly
energetic phosphate bonds provide the necessary driving force for enzymatic conversion reactions inside
the cell. This is the reason why ATP is often referred to as the energy currency of cells. It is a
necessary component of all life.
"Great! So, we want to have as much ATP as we can get. But there is a
problem, right?"
Basically, the more ATP the better! But the energy carried by an ATP molecule
must be invested during its regeneration from adenosine diphosphate (ADP) and phosphate. In living organisms,
this creation takes place during cell respiration which is a very complex process that cannot be emulated
artificially. Thus, ATP needed in industrial processes and science must be created differently. Currently
used methods consume a lot of resources and chemical synthesis causes avoidable pollution. So, the industrial
production of ATP is expensive and environmentally harmful so far.
more information about currently used methods for ATP regeneration
"And what can be done to improve the current situation?"
This is the point where our project M.A.R.S. enters the stage. We want to
improve the current situation by developing a bioreactor capable of cyclic ATP creation. The energy
needed for this is taken from sunlight, and all resources involved are reused in the next cycle. This
makes ATP production with M.A.R.S. environmentally friendly and much cheaper than previous
methods.
"How do you want to achieve your goal?"
We use a previously found approach for ATP regeneration, where
Liposomes are equipped with Bacteriorhodopsin and ATP synthase to create a chassis-protocell.
Together those membrane proteins are able to store the energy of sunlight as chemical energy in
the form of ATP. In M.A.R.S., this ATP regeneration step is followed by magnetic immobilization
of the chassis carrying Bacteriorhodopsin and ATP synthase. Those chassis are Liposomes in the
original approach. However, in M.A.R.S. we use Polymersomes. Immobilizing them after the ATP
regeneration step enables harvesting the ATP while the chassis remain functioning. As the
immobilization is induced by a magnetic field it can be reversed by turning off this field.
So, turning off the magnetic field enables another cycle of ATP regeneration.
As you can imagine, we would not be able to create M.A.R.S. without hard-working allies. In the following they want to introduce themselves in more detail:
Hi, I am Bacteriorhodopsin and, originally,
I come from Halobacterium salinarum where I work together with my colleague ATP synthase.
We use light to provide the Halobacteria with energy in times of lack of oxygen.
In the course of this, I love to build up proton gradients on membranes.
My name is ATP synthase. As my colleague Bacteriorhodopsin already told you,
I am involved in the process of transforming light into chemical energy in H. salinarum. After Bacteriorhodopsin
has built up the proton gradient using light, I, in turn, use the proton gradient to covalently bind
ADP and phosphate to ATP. However, although we originally work together in H. salinarum the version of
me that is found in M.A.R.S. is isolated from Saccharomyces cerevisiae.
I am a Polymersome. Many of my kind can be found in the bioreactor M.A.R.S.
created by iGEM Aachen. We serve as basic chassis carrying Bacteriorhodopsin and ATP synthase around,
which enables their work. We are made of a PETOz-PDMS-PETOz triblock polymer, 100-500nm large in diameter,
and resemble the structure of lipid vesicles. However, we are preferred over Liposomes as we are more stable.
So, M.A.R.S. is more reliable with chassis of my kind.
I am one of many magnetic Polystyrene Beads. Our task in M.A.R.S. is to enable
the magnetic separation of the chassis which makes their reuse in the following cycles possible. Each of us is
bound to multiple Polymersomes because with 3.7μm in diameter we are much larger than them.
We are an Anchor Peptide, a Peptide Spacer, and a Polystyrene Binding Protein forming
one complex. Thus, we also want to introduce ourselves together. The three of us connect Polymersomes with
Polystyrene Beads whereby I, the Anchor Peptide bind to the Polymersome, I, the Polystyrene Binding Protein bind
to the Polystyrene Bead, and I, the Peptide Spacer connect the two others.
And this is how we work together:
ATP - the universal energy carrier
To understand what our project is about and why it is so relevant, let us first take a look at the molecule that our work has been about:Adenosine triphosphate (ATP) is a nucleotide and complex organic molecule, consisting of the nucleic base adenine, the sugar ribose, and a triphosphate unit [1].
It serves as a universal transport and storage material for energy in the cells of all living things and therefore is produced and consumed in large quantities every day. Due to the hydrolytic cleavage of the high-energy anhydride bonds that connect the triphosphate units, it supplies the necessary energy for the work of many enzymes. Either only one of the phosphates can be split off, or both, releasing an energy amount of up to 50 kJ/mol [1].
As a result, however, ATP is converted into one of two less potent energy sources, namely adenosine diphosphate (ADP) or adenosine monophosphate (AMP),
and must be regenerated to make it available again [1].The ATP-ADP cycle, in particular, is a fundamental mechanism of energy exchange in cells [1]. Because of this, the ATP/ADP ratio is strictly regulated and maintained through a rapid conversion of ADP to ATP. The ATP concentration in a regular cell is about ten times greater than that of ADP [2].
Currently used methods for ATP Regeneration
ATP is the most important energy donor in metabolic pathways [3].
Currently, ATP is mainly produced in two different ways, which we believe to be less efficient than in situ ATP recycling with our Magnetic ATP Recycling System. This hypothesis is further evaluated in our proof of concept. On the one hand, ATP can be produced via a whole-cell approach. Industrially, this is mostly achieved with genetically engineered yeast cells [4]. Another popular method is the chemical catalysis utilizing polyphosphate to couple phosphate groups to ADP or AMP.
Both methods, especially the polyphosphate approach, are significantly less sustainable because they can’t be efficiently integrated directly into the systems in need of ATP recycling and often depend on external resources.
Currently, ATP is mainly produced in two different ways, which we believe to be less efficient than in situ ATP recycling with our Magnetic ATP Recycling System. This hypothesis is further evaluated in our proof of concept. On the one hand, ATP can be produced via a whole-cell approach. Industrially, this is mostly achieved with genetically engineered yeast cells [4]. Another popular method is the chemical catalysis utilizing polyphosphate to couple phosphate groups to ADP or AMP.
Both methods, especially the polyphosphate approach, are significantly less sustainable because they can’t be efficiently integrated directly into the systems in need of ATP recycling and often depend on external resources.
ATP Regeneration and Recycling with M.A.R.S.
Now we focus on our project M.A.R.S. First, let us briefly cover the original processes found in the various organisms which inspired us.
The previously mentioned sensitive equilibrium between ATP and ADP within living cells is primarily maintained through a membrane-based recycling process. This process requires an enzyme complex called ATP Synthase, found in the membrane of mitochondria or chloroplasts, for example [2]. It utilizes a proton gradient across the organelle membrane to drive the synthesis of ATP from its components ADP and phosphate (Pi) using phosphorylation [5]. In mitochondria and chloroplasts of eukaryotes, like the yeast Saccharomyces cerevisiae, the proton gradient is built up by an electron transport chain. Different protein complexes in the organelle membrane use the energy of the electron flow to translocate protons from the cytoplasmic to the extracellular side of the membrane. The resulting uneven distribution of protons creates a pH gradient and an electrical transmembrane potential, which ultimately generates the proton motor force required to drive the ATP synthase [6]. Instead of the electron transport chains, some bacteria and archaea use various alternative ways to generate the proton gradient. These include Halobacterium salinarum [7]. Being phototrophic, it can maintain its metabolism with the help of light energy in the event of a lack of oxygen. For that purpose, it produces the purple pigment bacteriorhodopsin (BR) and embeds it in newly synthesized membrane segments, the so-called purple membrane (PM). While absorbing a photon of light, the retinal chromophore of BR is isomerized, which leads to a proton shift by pumping protons from the cytoplasmic to the extracellular side of the protein pore. In this way, it builds up the proton gradient necessary for ATP synthesis across its cell membrane [7].
For M.A.R.S., we use these two energy regeneration mechanisms, which are ingenious by nature, and combine them into one. This allows us to use only two proteins that are built into the protocells, our so-called chassis.
The minimalistic configuration of the chassis avoids unwanted interactions with enzymes in the environment and thus enables enzyme cascades to be fed directly into the bioreactor. As in H. salinarum, BR generates the
necessary proton gradient with the help of light energy across the membrane of the liposomes or polymersomes, so that the ATP synthase extracted from S. cerevisiae can recycle ATP from ADP and Pi. The big advantage here
is the reverse orientation of the BR, which means that the protons are pumped from the environment into the interior of the protocell. This enables ATP to be recycled directly outside the chassis and avoids an additional
purification process. As a result, this cell-free system can be produced inexpensively and created more easily than a whole electron transport chain. The connection with magnet particles via anchor peptides also enables
purification using magnetism. These properties make M.A.R.S. a reusable and innovative method of ATP regeneration.
The previously mentioned sensitive equilibrium between ATP and ADP within living cells is primarily maintained through a membrane-based recycling process. This process requires an enzyme complex called ATP Synthase, found in the membrane of mitochondria or chloroplasts, for example [2]. It utilizes a proton gradient across the organelle membrane to drive the synthesis of ATP from its components ADP and phosphate (Pi) using phosphorylation [5]. In mitochondria and chloroplasts of eukaryotes, like the yeast Saccharomyces cerevisiae, the proton gradient is built up by an electron transport chain. Different protein complexes in the organelle membrane use the energy of the electron flow to translocate protons from the cytoplasmic to the extracellular side of the membrane. The resulting uneven distribution of protons creates a pH gradient and an electrical transmembrane potential, which ultimately generates the proton motor force required to drive the ATP synthase [6]. Instead of the electron transport chains, some bacteria and archaea use various alternative ways to generate the proton gradient. These include Halobacterium salinarum [7]. Being phototrophic, it can maintain its metabolism with the help of light energy in the event of a lack of oxygen. For that purpose, it produces the purple pigment bacteriorhodopsin (BR) and embeds it in newly synthesized membrane segments, the so-called purple membrane (PM). While absorbing a photon of light, the retinal chromophore of BR is isomerized, which leads to a proton shift by pumping protons from the cytoplasmic to the extracellular side of the protein pore. In this way, it builds up the proton gradient necessary for ATP synthesis across its cell membrane [7].
For M.A.R.S., we use these two energy regeneration mechanisms, which are ingenious by nature, and combine them into one. This allows us to use only two proteins that are built into the protocells, our so-called chassis.
The minimalistic configuration of the chassis avoids unwanted interactions with enzymes in the environment and thus enables enzyme cascades to be fed directly into the bioreactor. As in H. salinarum, BR generates the
necessary proton gradient with the help of light energy across the membrane of the liposomes or polymersomes, so that the ATP synthase extracted from S. cerevisiae can recycle ATP from ADP and Pi. The big advantage here
is the reverse orientation of the BR, which means that the protons are pumped from the environment into the interior of the protocell. This enables ATP to be recycled directly outside the chassis and avoids an additional
purification process. As a result, this cell-free system can be produced inexpensively and created more easily than a whole electron transport chain. The connection with magnet particles via anchor peptides also enables
purification using magnetism. These properties make M.A.R.S. a reusable and innovative method of ATP regeneration.References
[1] Berg, J. M., Tymoczko, J. L., Stryer, L. (2014). Biochemie, Kapitel 15 – Der Stoffwechsel: Konzepte und Grundmuster.
[2] Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2012). Lehrbuch der Molekularen Zellbiologie, Kapitel 14 – Energiegewinnung in Mitochondrien und Chloroplasten.
[3] Jingwen Zhou, Liming Liu, Zhongping Shi, Guocheng Du, Jian Chen, ATP in current biotechnology: Regulation, applications and perspectives, Biotechnology Advances, Volume 27, Issue 1, 2009, Pages 94-101, ISSN 0734-9750, https://doi.org/10.1016/j.biotechadv.2008.10.005.
[4] Sakai, Y., Rogi, T., Yonehara, T. et al. High–Level ATP Production by a Genetically–Engineered Candida Yeast. Nat Biotechnol 12, 291–293 (1994). https://doi.org/10.1038/nbt0394-291
[5] Choi, C. S. (2019). Die mitochondriale Unfolded Protein Response in Saccharomyces cerevisiae.
[6] Berg, J. M., Tymoczko, J. L., Stryer, L. (2014). Biochemie, Kapitel 18 – Die oxidative Phosphorylierung.
[7] Becher, B. M. & Cassim, S. Y. (1975). Improved Isolation Procedures for the Purple Membrane of Halobacterium Halobium. Preparative Biochemistry, 5:2, 161-178, DOI: 10.1080/00327487508061568