Radiation-resistant yeast to support space exploration
Space exploration has always attracted humans, feeding our curiosity and the desire for discovery. From the times of Hipparchus to the modern age, we strived to look farther into our vast universe. However, the inconceivable size of the universe and the harsh environments of space limit our capabilities for further exploration and confine the establishment of extraterrestrial life.
Ionizing radiation (IR) is one of the dangers in open space. IR causes detrimental effects on cells by damaging biological molecules, including DNA. Exposure to excessive amount of IR can result in severe damage, like chromosomal aberrations and genome instability, that the cellular DNA repair machinery cannot fix. This can lead to serious consequences such as acute radiation cell death or tumorigenesis in multicellular organisms. Importantly, tumorigenesis is one of the key concerns in long-term space missions (Furukawa et al., 2020).
In addition to affecting humans, IR is also detrimental to microbes and plants, which are critical sources of nutrients and other compounds during long-lasting space expeditions. Ecological life support systems are being designed to support life in space. Previous projects include the controlled ecological life support system of NASA or the European Space Agency’s MELiSSA. The use of Bioregenerative Life Support Systems (BLSSs), which are artificial Earth-like environments that contain plants, animals, and microorganisms, also contributes to this aim (De Pascale et al., 2021). The main purpose of BLSSs is to regenerate and provide oxygen, water, and nutrient sources for astronauts as well as to recycle waste (Liu et al., 2021). Considering the extremely expensive transport of equipment and consumables from Earth, rational resource utilization is crucial for space expeditions. The use of sustainable bioreactors and biological processing technology would be highly beneficial.
Yeast Saccharomyces cerevisiae has been used by humankind for thousands of years for food production, and in more recent years, its importance as biofactories for fuels, pharmaceuticals, and chemicals has emerged (Nielsen, 2019). Interestingly, it was shown that some yeasts are great candidates for sustaining IR conditions (Dadachova & Casadevall, 2008). This inspired us to explore the possibility of engineering S. cerevisiae to be radiation-resistant, so that it could be used for synthetic biology applications either in space or in high radiation environments on Earth.
Yeast in space
Several species of fungi have already been sent to space to study the molecular changes they undergo in such an environment. These experiments help to shed light on the possible consequences of long-term exposure to elevated IR, the mechanisms of cancer development, and the adaptive abilities of cells in space. Yeast are among the favored organisms for scientists to send to space because they are well-studied single-cellular eukaryotic organisms that are easy to maintain and can survive extreme drying. Yeast was first launched to space with the Apollo 16 mission in 1972 (Taylor et al., 1975).
Artemis 1 mission, expected to launch in November 2022, is set to carry a library of 6,000 gene deletion mutant yeast strains. The mission will last 42 days and include orbiting around the moon. During this expedition, yeasts will be exposed to radiation 10 to 20 times higher than the intensity for any terrestrial exposure (K.R. Wahl et al., 2021; Zea et al., 2022).
Synthetic biology in space opens up new possibilities
In addition to high radiation levels, the space environment also has microgravity. Interestingly, while microgravity also poses risks to living organisms that have adapted to Earth’s gravity conditions, progress in recent years has revealed that microgravity also opens up many new possibilities in biotechnology. These include the ability to assemble picoparticles that can be used for drug delivery and are much smaller than nanoparticles, forming various gels, microfluidics devices and even protein crystals (Giget et al., 1989; Jessup et al., 1993; McPherson & Delucas, 2015; Moraes Neves et al., 2019; Nijhuis et al., 2022; Pashazadeh-Panahi & Hasanzadeh, 2019). Therefore, preparing S. cerevisiae for use in space conditions could open up new synthetic biology opportunities in the future that we cannot even imagine at the moment due to our limited knowledge about biological processes under microgravity.
Melanin to protect cells from radiation
To survive under the harsh space conditions for a long time, yeast cells need extra protection against IR. In our project, we propose to use melanin as a shield that absorbs radiation. With its unique physicochemical properties, melanin is a prevalent pigment among various species. It has been found in high quantities in microorganisms that inhabit areas contaminated with radioactivity, such as the area around the damaged Chernobyl nuclear power plant.
Melanin has been analyzed in respect to its capabilities to guard against high-dose radiation of up to 1.5 kGy in melanized fungi Cryptococcus neoformans and Cryomyces antarcticus. (Pacelli et al., 2017). In this study, melanized microfungi were blasted with deuterons and X-Rays, and it was found that melanin is critical for the radioprotection of the cells and, therefore, it could be potentially used as a biological radioprotector.
Melanin can reduce the harmful effects of IR by several mechanisms. First, stable free radicals in melanin structure scavenge the free radicals generated by IR that could otherwise damage DNA. Secondly, recoil electrons can interact with melanin, resulting in the scattering of their excessive energy. This phenomenon is known as Compton scattering. Thus, melanin molecules provide shielding against the detrimental effects of radiation to protect DNA and other sensitive molecules in the cell from being irreversibly damaged (Eisenman and Casadevall, 2012). In our iGEM project, we set out to engineer yeast that synthesizes and accumulates melanin in three different ways: in the cell cytoplasm, in nanoparticles formed inside the cell, and outside the cell in the cell wall.
Several other iGEM teams have explored the prospects of melanin production. For example, the teams Cambridge 2009, PITT 2014, Stanford-Brown 2016, Sao Carlos-Brazil 2019, and Shanghai SFLS SPBS 2020 have all worked with melanin in their projects. While Cambridge, Stanford-Brown and Shanghai SFLS SPBS worked with melanin in Escherichia coli and team PITT with Propionibacterium acnes, only team Sao Carlos-Brazil worked with S. cerevisiae. Sao Carlos-Brazil has specifically shown success in using the Aga2 yeast display system for recruiting melanin on the outer cell wall. A similar construct was used in one of our experimental approaches described in our ENGINEERING page. However, while the strain from the Sao Carlos-Brazil team required the addition of melanin to the culture medium, we, among other approaches, designed our strain to synthesize melanin itself in the cell wall.
One step further: switching radiation from a hazard to an energy source
While radiation is a source of danger for most organisms, some fungi have the potential to use radioisotopes as a source of energy. For instance, C. cladosporioides, as well as other melanotic fungi present around the Chernobyl nuclear reactors, have been shown to grow towards radioactive nuclides in the environment. This phenomenon of melanized organisms is called radiotropism. Interestingly, melanized C. neoformans cells showed considerably enhanced growth in the presence of radiation (Dadachova et al., 2007). This has led to the question of whether melanin has a role in converting the radiation energy to a form usable by the cells.
There have been numerous studies on measuring and observing the change of the melanin’s structure when it is exposed to IR to answer this question. A study has found that when melanin is exposed to gamma radiation, an electric current is produced (Turick et al., 2011). Moreover, a 4-fold increase in the capacity of irradiated melanin to reduce NADH compared to non-irradiated melanin was observed. It indicates the ability of melanin to convert high-level detrimental energy to metabolically useful reducing power (Dadachova et al., 2007).
S. cerevisiae is the most widely used eukaryotic species for cell factories. If the ability to use radiation as an energy source could be transferred to this yeast, it would open up vast new possibilities to create efficient cell factories that could support a sustainable economy. The first step towards this goal is engineering a melanin-synthesizing strain of S. cereivisiae, the central aim of our iGEM project.
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