Team:Sorbonne U Paris/Design

IGEM

Design

Introduction

One of the main challenges of long-duration space travel today is the intensity and duration of exposure to short-wave ionizing radiations such as UVB and UVC radiation or X-rays and gamma rays. During an expedition to Mars, the doses of cosmic radiation would be between 0.210 +/- 0.040 mGy/Day [1], which can represent a danger for the members of the crew, and represents one of the main constraints to manned flights with the change of gravitational forces (mainly weightlessness) and the psychological risks linked to the isolation [2].

In addition to these health constraints, there are purely logistical constraints: how to guarantee access to the equipment necessary for human life for a long duration flight for which no refuelling will be possible?

Two scenarios are thus envisaged for long duration flights, including a long-term mission and a short-term mission, with a respective duration of 1000 days (including 525 days on Mars) and 500 days (including 30 days on Mars) [3]. In the case of a 500 days stay on the red planet, the production of food resources and the recycling of the crew's waste become a crucial element for the success of the mission, and the development of a reliable life support system is one of the main issues of the exobiology research.

Bioreactors made of bacteria and algae could reduce the mass of consumables to be carried on board the spacecraft by nearly 90% by allowing the recycling of urine and air by mimicking terrestrial ecosystems [3][4]. The impact of changing gravitational forces and radiation exposure on these organisms, however, has been less studied than on humans, and their viability for such long flights may be questioned, especially since the biochemistry of photosynthetic cells already generates a relatively high number of Reactive Oxygen Species (ROS), a phenomenon that could be packaged during exposure to cosmic radiation [5][6][7][8].

The stresses undergone by photosynthetic organisms would thus include acute (eruption of Solar Particle Events or SPEs) and chronic stresses that have not been studied on board the international space station because of the protection conferred by the earth's magnetic field [3].

Our project therefore consisted in providing protection from ionizing radiation to a model photosynthetic organism in synthetic biology, the alga Chlamydomonas reinhardtii, by attempting to respect the constraints related to life-support systems and by taking into consideration the specific challenges related to the conservation of photosynthetic activity during these exposures.

State of the art

Reliable life support systems

In this part, we will focus on Biological Life Support Systems (BLSS) which include all the systems based on living organisms and ensuring the breathing, the treatment of waste, the detoxification of the environment and the feeding of the crews during long flights. Beyond their simple efficiency, these systems must be reliable (long-lasting efficiency, viability of the organisms constituting them), not require too much work from the crew mobilized on other missions, not require supplies (self-sufficiency), not generate excess waste, and above all, because of the very nature of long-duration flights, not have too much mass [9].

Our team focused on the question of oxygen within the vessels: as it cannot be replenished, it is necessary to form a loop circuit allowing it to be regenerated throughout the flight.

As the crew cannot do without oxygen for more than a few minutes, particular attention is paid to the reliability of the equipment: within the framework of a mission lasting 500 days, the biological systems must have a Time Before Failure (TBF) higher than 5E+6 days and a failure rate lower than 2E-7 per day [10].

Every parameter that can damage living organisms must be taken into account, and we decided to focus our project on this issue, as most iGEM teams have focused in the past on nutrient production or bioreactor design. The stability of organisms over time, and in particular that of photosynthetic organisms confronted with cosmic radiation, has not been addressed.

All space agencies have a program to develop loop systems that convert part of the waste produced by the crew into reusable raw materials. The European Space Agency (ESA) has set up the MELiSSA (Micro-ecological life-support system) project, a loop system comprising four compartments: the first one allowing the degradation of fibers from astronauts' waste and containing thermophilic anaerobic bacteria, the second compartment allowing a primary production of oxygen and food resources with photoheterotrophic bacteria, the third one an oxidation of the ammonia present with the help of nitrifying bacteria and the last one with higher plants and other photoautotrophic bacteria in order to produce water and food resources [4].

However, we chose to use the model of Chlamydomonas reinhardtii, an edible green alga because it is non-toxic [12], which has been very well characterized genetically [13] and is especially a model organism in synthetic biology [14], as a chassis. Since our project focuses on oxygen production, however, we had to take into account the mixotrophic nature of Chlamydomonas reinhardtii metabolism [15].

Risks associated with exposure to ionizing radiation in photosynthetic organisms

Exposure to ionizing radiation results in direct and indirect effects on cell viability. For UV radiation alone (the least energetic radiation tested in our study) the direct effects include the formation of Cyclobutane pyrimidine dimer (CPD) and the production of 6-4 photoproducts while the indirect effects are the production of 8-oxo-7,8-dihydroguanine and significant oxidative stress.

This oxidative stress is itself a consequence of the radiolysis of water molecules, which leads to the formation of reactive oxygen species (here abbreviated ROS) that oxidize purine and pyrimidine bases, proteins, as well as the desoxyribose backbone of DNA. All of the above damages lead to the formation of single or double-strand breaks (SSB/DSB) in the DNA.

Another notable consequence of UV radiation exposure is the C5 methylation of cytosines, which is an epigenetic marker for the regulation of certain genes involved in the conformation of the DNA double helix in eukaryotes. However, since exposure to UV radiation can be natural, there are generally several repair mechanisms, the most famous of which are light repair, dark repair, global genome repair and transcription coupled repair [16][17].

Most of the repair and radiation protection mechanisms studied in the early 2000s were thus linked to the protection of the cell's genetic information. This paradigm was overturned in 2007 by Michael J. Daly's team, who demonstrated that the model organism for the study of radioresistance, Deinococcus radiodurans, as well as other organisms exposed to radiation, had as a common trait a higher Mn/Fe ratio than that of sensitive bacteria.

The high intracellular concentration of Mn(II) ions is then presented by the team as a mechanism for protecting proteins against oxidative stress and the protein carbonylation that this involves [18][19]. Protein carbonylation is indeed a particular oxidative damage of proteins during an exposure to reactive oxygen species. Direct oxidation can thus lead to the formation of ketones or aldehydes on amino acids such as lysine, proline or arginine, thus modifying their chemical and structural properties. However, these reactions are not always direct because oxidation phenomena can also occur on lipids, leading to the formation of cytotoxic products such as acrolein, 4-hydroxy-trans-2-nonenal (unsaturated α-β acids), 4-oxo-trans-2-nonenal (keto-aldehyde), or malondialdehyde (di-aldehyde), which can subsequently transfer their aldehyde group to proteins [20].

Michael J. Daly's theory gradually became established as a result of comparing proteome and genome damage in susceptible and resistant organisms: while resistant organisms do indeed develop elaborate repair mechanisms and often have multiple copies of their genome, one of the most striking differences between the two types of organisms is the low protein carbonylation in resistant organisms. This allows DNA repair proteins to remain operational longer and basic cellular mechanisms to be maintained, whereas susceptible organisms suffer high mortality due to metabolic arrest [19]. The protection of the proteome is thus the top priority of a living cell during radiation exposure, and the role of ROS scavengers of small metabolites or of certain enzymes is preponderant in reducing cell death, well before the DNA repair mechanisms.

In photosynthetic organisms, the production of ROS is generally linked to the mechanisms of respiration and photosynthesis. For example, ferredoxin and carrier electrons of photosystem I have a high electrochemical potential and regularly generate superoxide anions. Generally, the anion formed is protonated into hydrogen peroxide, which is less dangerous for the cell and can be taken over by catalase. Excess superoxide anions can also be dismuted by superoxide dismutase.

Signaling pathways are thus directly allocated to ROS in order to regulate the balance between the reactive species produced and those reduced by the cell [21]. An imbalance in this balance can be dangerous for photosystems.

In their article Sensitivity of the green algae Chlamydomonas reinhardtii to gamma radiation: Photosynthetic performance and ROS formation, the team of Tânia Gomes [8] highlights the relationship between the dose of gamma radiation received by Chlamydomonas reinhardtii cells, the production of reactive oxygen species and the degradation of the photosystems, a mechanism that leads to a decrease in the efficiency of the photosynthetic activity. The team adds that the formation of these reactive species does not only depend on mechanisms related to photosynthesis but also on mitochondrial metabolism and other various mechanisms [8].

Also, and although some controversy remains about the sensitivity of Chlamydomonas reinhardtii to ionizing radiation [22][23], most of the current experiments only involve short duration and low energy exposures. The study by Tânia Gomes is therefore, to our knowledge, the only reference concerning the effect of short-wave radiation on the biochemistry of the alga Chlamydomonas reinhardtii.

Proposed solution to the problem: protection by a chaperone molecule, manganese

An antioxidant is defined as "natural or synthetic substances that may prevent or delay oxidative cell damage caused by physiological oxidants having distinctly positive reduction potentials, covering reactive oxygen species (ROS)/reactive nitrogen species (RNS) and free radicals (i.e. unstable molecules or ions having unpaired electrons)”(Apak et al., 2018)[24]. ROS scavengers thus behave as antioxidants, preventing protein carbonylation and other cellular damage caused by reactive oxygen species.

However, the term "oxidant", which is a chemical term, should be distinguished from the term "pro-oxidant", which is the biological equivalent and which really denotes a "harmful" capacity to oxidize biological molecules [24]. Within the cell, the damage caused will thus be a function of the kinetics of the oxidation-reduction reactions taking place between the ROS and the biological molecules. The kinetics of the reaction between reactive oxygen species and antioxidants must be faster than those of these same species with biological molecules, in order to divert the harmful effect towards the latter.

As previously mentioned (Risks related to exposure to ionizing radiation in photosynthetic organisms), living organisms are constantly confronted with ROS, and have developed two main mechanisms to protect themselves from the imbalance between their production and elimination.

The first one consists of an enzymatic arsenal allowing to eliminate or to degrade the radicals into less dangerous reactive species. This activity is carried out by enzymes such as catalase (CAT), superoxide dismutase (SOD) or ascorbate peroxidase (APX).

A second strategy is the use of non-enzymatic molecules such as ascorbic acid (AA), flavonoids or certain carotenoid pigments [25].

Recently, many authors have reported the predominant role of "chaperone molecules", made up of simple chemical elements often potentiated by other small molecules through complexation, such as bromide ions [26] or manganese [27]. The central role played by manganese during exposure to ionizing radiation can indeed be demonstrated by a very simple observation: the Mn/Fe balance of radioresistant organisms is unbalanced in favor of Mn, unlike radiosensitive organisms.

The radioresistant cells have up to 300 times more Mn and three times less Fe ions than the sensitive bacteria [18]. This imbalance in favor of Mn has been demonstrated in several organisms such as Bacillus subtilis [28] or Saccharomyces cerevisiae [29], after its observation in Deinococcus radiodurans [27].

Although the mechanism by which Mn(II) catalyzes the reduction of reactive oxygen species was not demonstrated by Michael J. Daly's team, it was nevertheless shown that this redox mechanism leads to the formation of hydrogen peroxide (H2O2) and is influenced by the pH values.

Thus, the higher the pH of the solution containing a bacterial lysate of Deinococcus radiodurans (and therefore basic), the more the redox reaction is inhibited [18]. This observation would mean that the mechanism of action of the antioxidant present in the lysate would be "hydrogen transfer" (two mechanisms of action coexist for antioxidants: hydrogen atom transfer (HAT) and single electron transfer (SET)) (24].

The composition of the bacterial ultrafiltrate consisted of ortophosphate, Mn(II), amino acids and small peptides. For Michael J. Daly's team, these small metabolites would have a catalytic effect on manganese manganese, just like phosphate ions.

Kevin Barnese's team has studied in detail the mechanism by which manganese catalyzes the dismutation of the superoxide ion under physiological conditions. The chemical mechanism is a two-order reaction with two intermediates leading to the formation of hydrogen peroxide (see Figure 1).

Manganese is presented in this study as complexed to a substrate (phosphate or carbonate ion), which reinforces the observations of Michael J. Daly [30]. Of course, the role of manganese in the dismutation of superoxide ion would not be solely direct, and the reaction would run in parallel with cellular enzymatic reactions.

Thus, manganese is a metallic cofactor of superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion into dioxygen and hydrogen peroxide.

Massimiliano Paena's team used a Mn-proteome to identify all the proteins that require manganese as a cofactor or at least interact with it. It appeared that Manganese-dependent superoxide dismutase (sodA) was indeed the most abundant protein in the proteome, followed by enolase whose functions have not been fully elucidated but which is involved in the degradation of carbohydrates. Other proteins would also require the presence of manganese, such as DNA polymerase and topoisomerases, ligases or the Peroxide stress regulator (PerR) [31].

Figure 1: Catalytic mechanism for dismutation of a superoxide ion proposed by Barnese et al. (2012). The L represents phosphate and/or carbonate binding to manganese to form MnHPO4 and/or MnHCO3+ and the n- anion an additional bond between the molecule and these compounds. It is possible to observe that there are two parallel mechanisms: the first purely chemical (left), and the second enzymatic (right). From the article "Chaperone molecules: a radioresistance lead for photosynthetic organisms?", written for the iGEM Proceedings Journal 2021 by Maïa Henry (Sorbonne University team, Paris). [Figure description: illustration of catalytic mechanisms previously developed in the text]

If all these studies confirm the role played by manganese and phosphate in the dismutation of the peroxide ion, the mechanism by which amines or small peptides could intervene in the radioprotection of Deinococcus radiodurans has not yet been elucidated. We therefore felt the need to verify in our study the protective role conferred by peptides inspired by those of Deinococcus radiodurans by means of a proof of concept, failing to have the time to study the chemical functioning of the peptide.

Conclusion

As the peptides involved in the dismutation of the superoxide anion of Deinococcus radiodurans have not yet been identified, our project consisted in using the peptide designed by the team of Gupta et al. [32].

In their study, the team constructed several synthetic peptides based on the amino acids contained in the Deinococcus radiodurans lysate. One of these (DEHGTAVMLK, named MDP), confers a protection equivalent to 60,000 Gy to T4 DNA ligase and 100 Gy to Jurkat T-cells. In-vivo experiments on murine models show that the protection conferred by the peptide is equivalent to 9.5 Gy (LD70/30) [32].

For all these experiments, the medium was supplemented with phosphate and manganese, in accordance with the observations previously made by Michael J. Daly [27].

In order to insert the peptide into our chassis, however, we had to insert a methionine, as this is the initiating amino acid for protein translation. Based on the assumption that methionine would not affect the overall activity of the peptide, this amino acid is in the majority in the peptide fraction of Deinococcus radiodurans, we designed a sequence corresponding to the undecapeptide, a hygromycin resistance gene (AphVII gene) and a strong promoter and terminator from the PSAD gene of Chlamydomonas reinhardtii, which is known for its strong and continuous (constitutive) expression.

We performed this construction based on the Golden Gate [33] technique, itself based on the production of cohesive ends made from type II restriction enzymes, and using the MoClo system made by Pierre Crozet's team [14].

In order to support our approach and to verify the antioxidant activity of our peptide, we decided to perform a large number of proof-of-concept manipulations. To do so, we performed an in vivo test and an in-vitro test to verify the antioxidant activity of our peptide and a test under real conditions by subjecting Chlamydomonas reinhardtii to gamma radiation.

Figure 2: Summary illustration of our project.The idea finally retained by the team is to make our model organism, Chlamydomonas reinhardtii, produce a peptide inspired by Deinococcus radiodurans. [Figure description: On the right, Chlamydomonas reinhardtii is exposed to space radiation. It survives because of the expression of a peptide (on the left) which forms a complex with manganese. This complex allows to neutralize the ROS induced by the radiations.]

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