Protein

Long-duration space missions, such as those aboard the International Space Station (ISS), demand efficient systems for resource recycling and sustainable food production. Nitrogen recycling through microbial bioconversion offers a promising route to address both waste management and protein deficiency in astronauts. S. cerevisiae naturally prefers ammonium or glutamine as nitrogen sources. In this context, Saccharomyces cerevisiae was engineered to enhance nitrogen assimilation and protein synthesis by:

• Deleting the GAT1 gene (YFL021W).
• Overexpressing key genes involved in nitrogen metabolism: GLN1 and GDH1.

Role of GAT1: GAT1 is a GATA transcription factor regulating Nitrogen Catabolite Repression (NCR). When poor nitrogen sources like urea are present, yeast activates NCR to turn on genes (e.g., DUR1,2) responsible for urea degradation:

• Breaks down urea into ammonia + CO₂.
• Consumes ATP and is energy-intensive.
• Causes nitrogen loss as free ammonia may escape.
• Efficiency varies under fluctuating nitrogen conditions.

Deletion of GAT1 results in:

• Downregulation of urea breakdown genes.
• Reduced energy spent on catabolism.
• Decreased nitrogen loss.
• Redirection of nitrogen flux toward direct ammonium assimilation.

Ammonium Assimilation Pathway:

• GDH1 (YOR375C): Encodes NADP⁺-glutamate dehydrogenase (Gdh1p), catalyzing:
  α-ketoglutarate + NH₄⁺ + NADPH → glutamate + NADP⁺
• GLN1 (YPR035W): Encodes glutamine synthetase (Gln1p), catalyzing:
  glutamate + NH₄⁺ + ATP → glutamine + ADP + Pi

Overexpression of GDH1 and GLN1 increases the flux of nitrogen into glutamate and glutamine, ensuring almost all available ammonium (including that derived from urea) is efficiently assimilated. This boosts amino acid and protein synthesis, increasing single-cell protein yield.

Thus, deletion of GAT1, combined with overexpression of GLN1 and GDH1, is expected to:

• Improve nitrogen uptake.
• Channel nitrogen efficiently into amino acids and proteins.
• Convert nitrogenous waste containing urea into single-cell proteins (SCPs), supporting circular bioeconomy principles and sustainable protein production in space environments.

In the ISS, nitrogenous waste such as urine is:

• Stored and distilled in ECLSS and BLSS systems to separate brine from recyclable water.
• Pre-treated with flushing water and a formula containing chromium trioxide and sulphuric acid to control microbial growth and urea hydrolysis.
• Brine separated during distillation retains a high urea content (5–10 g/L of urine), which can be reutilized using the engineered yeast pathway to produce SCPs . [1]

Radiation Hypothesis

The radiation resistance of microorganisms represents a significant advantage for their potential use in space-based cultivation systems. During one of the Integrated Human Practices (IHP) discussions with Dr. Suresh Naik (Indian Space Research Organization), the importance of considering Galactic Cosmic Radiation (GCRs) while cultivating Saccharomyces cerevisiae in extraterrestrial environments was emphasized.

A comprehensive literature survey revealed several organisms exhibiting remarkable resistance to high doses of radiation, notably cockroaches and tardigrades[3]. While cockroaches are well-known for their resilience, tardigrades presented a particularly intriguing case due to their unique molecular adaptations. A study conducted at the Département AVIV, MNHN, CNRS UMR7196, INSERM U1154 (France) demonstrated that after four hours of irradiation, RNA sequencing of Hypsibius exemplaris revealed significant transcriptional responses: 421 genes were upregulated more than 4-fold, with over 120 genes showing a >16-fold increase. Notably, genes associated with the two major DNA double-strand break (DSB) repair pathways—homologous recombination (HR) and non-homologous end joining (NHEJ)—were strongly induced. Among them, RAD51, a conserved recombinase present in all eukaryotes, was of particular interest. Since RAD51 is also native to yeast[4], its overexpression in S. cerevisiae represents a rational strategy to enhance the efficiency of homologous recombination-based DNA repair. This is particularly relevant because lower eukaryotes preferentially utilize HR, whereas higher eukaryotes, including mammals, rely more heavily on NHEJ for DSB repair.

The mitigation of DNA damage induced by radiation can be broadly classified into two complementary strategies:

• (a) Damage suppression.
• (b) Damage repair.

While RAD51 contributes to efficient repair of single- and double-strand DNA breaks through homologous recombination, additional protection may be provided by the tardigrade-derived protein DSUP (Damage Suppressor Gene)

According to iGEM Registry entry BBa_K2195000, expression of DSUP from Ramazzottius varieornatus in E. coli significantly enhanced survival under UV irradiation. DSUP functions by physically associating with chromatin and mitigating DNA damage from reactive oxygen species and radiation exposure.

Based on these insights, we hypothesize that simultaneous overexpression of RAD51 (for enhanced DNA repair) and DSUP (for DNA damage suppression) in S. cerevisiae will synergistically improve survival under radiation stress, thereby facilitating its cultivation in space environments.

Melanin

A serendipitous discovery of eumelanin production by P. vulgaris in the presence of L-tyrosine via the dopamine biosynthetic pathway inspired the integration of this pigment into our radiation-protection design. Leveraging eumelanin’s strong UV-shielding properties, we coated the bioreactor with a eumelanin+PVA sheet to enhance resistance to space radiation. Polyvinyl alcohol (PVA) was chosen for its lightweight, porous, and chemically inert nature, contributing to payload reduction, heat dissipation, and compatibility with NaOH-based eumelanin processing. The reusable PVA+eumelanin composite thus offers a sustainable and efficient solution for radiation shielding in outer-space environments with the added benefit of biodegradability.