The overarching objective of the IMPAS flight experiment is to test if microgravity allows silent fungal metabolite pathways to become activated. Microgravity might generate a unique stress not achievable with ground-based cell culture studies. The research team studies the effects of microgravity on the production of fungal metabolites, and tests the hypothesis that spaceflight alters fungal gene expression, protein production, and overall physiological responses. The IMPAS project seeks to answer the follow hypothetical question: Since Aspergillus nidulans exhibited production of antibiotics on Earth, do these fungal cells yield useful biotherapeutic secondary metabolites when grown in stressed microgravity conditions? Metabolomic characterization of both flight and ground control specimens will answer this question, which may lead to the discovery of potentially useful biotherapeutic compounds.
The broad, long-term goal of this project is to study the changes encountered in various aspects of fungal “omics” under microgravity, as well as the production of fungal secondary metabolites. Natural products discovered from traditional discovery programs, such as lovastatin (a polyketide by Aspergillus terreus) and penicillin (a nonribosomally synthesized peptide by Penicillium chrysogenum), have demonstrated that filamentous fungi are a rich source of chemotherapeutic agents against a variety of diseases. Genome sequencing of various natural product-producing organisms have shown that, even in well-characterized organisms like A. nidulans and A. terreus, the biosynthesis pathways of the vast majority of natural products are still unknown, largely because they are silent until specific conditions trigger their expression. Research has shown that in filamentous fungi secondary metabolite production is highly sensitive to growth conditions. In addition, the IMPAS team has shown that many secondary metabolism pathways are triggered specifically in harsh or stressful conditions.
Fungal samples are grown and optimized for flight in hardware provided by BioServe. Approximately 3 days before launch, spores of the fungi (1 x 107 spores per sample) are aseptically (free from pathogens) spotted on yeast agar glucose (YAG) solid media in Omnitray plates. A total of six Omnitray plates are placed in each Plate Habitat (PHAB), a temperature conducting containment device for the experiment. PHABs are incubated inside a Space Automated Bioproduct Laboratory (SABL) on the International Space Station (ISS).
The experiment can be entirely performed merely by modulating the temperature of the cultures within the PHABs. Samples are stored at 4°C during transport and “activated” and grown at 37°C once on board ISS in the SABL. Two separate SABLs (each containing 4 PHABs with 6 sample Omnitray plates) are used to establish a time course for the experiment; one time point is 4 days, and other 7 days. All samples are “terminated” by cooling to 4°C once the time point is reached. Samples are transported back to Earth at 4°C. Fungal cells remain viable for many weeks dormant at 4°C. From each flight experiment time point (4-days and 7-days), half of the samples are processed immediately upon return to Earth, and the other half are used to regrow the ISS-generated fungal cells under Earth-normal conditions. Metabolomic, proteomic and genomic/transcriptomic analyses are performed. Ground controls are produced separately once data from SABL flight conditions (e.g., temperature change rates) are obtained.
The influence of International Space Station (ISS) conditions on fungal ‘omics’ is reported. The investigation included the A. nidulans wild-type and 3 mutant strains, two of which were genetically engineered to enhance secondary metabolite (SM) production. Whole genome sequencing (WGS) revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the SM global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and SM biosynthesis was observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments and present many economic benefits. Additional analysis and results are available in the NASA GeneLab repository, https://genelab.nasa.gov/.
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