We hypothesize that both metabolic (redox) and genetic (knockout) assays can distinguish specific changes due to the unique combinations of direct IR damage, indirect oxidative damage, and reduced gravity incurred by exposure to the lunar environment, low Earth orbit (LEO), interplanetary space, and ground-based simulations even before differential survival becomes apparent. These environmental perturbations are known to result in subtle changes in cell growth and activity, but the resulting data is lacking pathway and molecular specificity. By leveraging a metabolic modeling framework and a series of engineered strains, our primary goal is to perform the ground testing necessary to validate strains that yield distinct responses to specific stressors, both laying the groundwork for a potential (and successful) lunar mission to characterize the relative importance of these changes within the capabilities of the BioSensor instrument and improving the usefulness of the alamarBlue assay for past and future missions.

To test our hypothesis, we will pursue the following Specific Aims:

Aim 1: Derive metabolic pathways usage via modeling of a colorimetric assay. The BioSentinel platform uses a redox dye to detect changes in viability and metabolic activity. AlamarBlue changes color in the presence of cell activity and creates intricate time course data that varies as a function of environmental stress, genetic background, and metabolic activity. To date, the rich information in this data has not been connected explicitly to underlying cellular processes. We will combine the data with metabolic modeling to develop a computational tool that extracts detailed metabolic information. This predictive model will not only enable analysis of our experimental design but can be applied to detect changes caused by lunar-like conditions or any autonomous mission that utilizes this reporter dye.

Aim 2: Explore biological effects of a lunar-like environment using DNA repair and stress response defective mutants. Both IR and reduced gravity induce the production of reactive oxygen species (ROS), which play a key role in the generation of cellular and DNA damage, in addition to direct IR damage (e.g., DNA breaks, membrane damage). We will generate yeast mutants defective in DNA repair and stress response pathways, including oxidative damage repair, homologous recombination, and excision repair. Given the short mission duration and the expected low cumulative IR dose, we will also generate mutants containing multiple gene knockouts to increase sensitivity. We will then perform alamarBlue assays after IR exposure in both dry form and in liquid suspension, to study their response.

Aim 3: Engineer endogenous ROS scavenging capabilities in yeast. An indirect effect of IR and cellular stress is the buildup of ROS. While S. cerevisiae has several strategies for ROS removal, we will engineer ROS scavenging mechanisms and evaluate if they can mitigate the unique damage caused by spaceflight stress and IR. We will over-express a native ROS scavenging enzyme and engineer a mannitol biosynthetic pathway, which specifically targets an ROS produced by IR. This aim demonstrates a potential mitigation for future spaceflight missions, enabling insight into how engineered scavenging mechanisms could improve crew safety.

Aim 2: Explore biological effects of a lunar-like environment using DNA repair and stress response defective mutants. Last year, we initiated a variety of experiments with 40+ yeast strains, including mutants defective in DNA damage repair and stress response. So far, we have completed 17 months of long-term desiccation and gamma radiation experiments and have down-selected to approx. 12 candidate strains as part of the Lunar Explorer Instrument for Space Biology Applications (LEIA) PRISM mission to the lunar surface. The next step is to initiate biocompatibility experiments using flight-like hardware (fluidic cards, optical ground support equipment, etc.). We will also test their sensitivity to simulated galactic cosmic radiation (GCRsim) at the NASA Space Radiation Laboratory in the Spring 2024. This Aim will continue as part of the LEIA PRISM project.

Aim 3: Engineer endogenous reactive oxygen species (ROS) scavenging capabilities in yeast. Like Aim 2, we have generated a series of strains containing genetic countermeasures to improve long-term desiccation and viability, including expression of genes involved in ROS mitigation and exogenous genes from tardigrades. We are also testing better methods to improve long-term desiccation and viability by modifying our current protocols. Even though expression of tardigrade genes was not originally proposed in this study, it is one strategy we are using in support of the LEIA lunar mission, and we are already testing them for desiccation and ionizing radiation tolerance.

We hypothesize that both metabolic (redox) and genetic (knockout) assays can distinguish specific changes due to the unique combinations of direct IR damage, indirect oxidative damage, and reduced gravity incurred by exposure to the lunar environment, low Earth orbit (LEO), interplanetary space, and ground-based simulations even before differential survival becomes apparent. These environmental perturbations are known to result in subtle changes in cell growth and activity, but the resulting data is lacking pathway and molecular specificity. By leveraging a metabolic modeling framework and a series of engineered strains, our primary goal is to perform the ground testing necessary to validate strains that yield distinct responses to specific stressors, both laying the groundwork for a potential (and successful) lunar mission to characterize the relative importance of these changes within the capabilities of the BioSensor instrument and improving the usefulness of the alamarBlue assay for past and future missions.

To test our hypothesis, we will pursue the following Specific Aims:

Aim 1: Derive metabolic pathways usage via modeling of a colorimetric assay. The BioSentinel platform uses a redox dye to detect changes in viability and metabolic activity. AlamarBlue changes color in the presence of cell activity and creates intricate time course data that varies as a function of environmental stress, genetic background, and metabolic activity. To date, the rich information in this data has not been connected explicitly to underlying cellular processes. We will combine the data with metabolic modeling to develop a computational tool that extracts detailed metabolic information. This predictive model will not only enable analysis of our experimental design but can be applied to detect changes caused by lunar-like conditions or any autonomous mission that utilizes this reporter dye.

Aim 2: Explore biological effects of a lunar-like environment using DNA repair and stress response defective mutants. Both IR and reduced gravity induce the production of reactive oxygen species (ROS), which play a key role in the generation of cellular and DNA damage, in addition to direct IR damage (e.g., DNA breaks, membrane damage). We will generate yeast mutants defective in DNA repair and stress response pathways, including oxidative damage repair, homologous recombination, and excision repair. Given the short mission duration and the expected low cumulative IR dose, we will also generate mutants containing multiple gene knockouts to increase sensitivity. We will then perform alamarBlue assays after IR exposure in both dry form and in liquid suspension, to study their response.

Aim 3: Engineer endogenous ROS scavenging capabilities in yeast. An indirect effect of IR and cellular stress is the buildup of ROS. While S. cerevisiae has several strategies for ROS removal, we will engineer ROS scavenging mechanisms and evaluate if they can mitigate the unique damage caused by spaceflight stress and IR. We will over-express a native ROS scavenging enzyme and engineer a mannitol biosynthetic pathway, which specifically targets an ROS produced by IR. This aim demonstrates a potential mitigation for future spaceflight missions, enabling insight into how engineered scavenging mechanisms could improve crew safety.

We have initiated metabolic modeling experiments by testing cell growth and metabolic activity in yeast cells using minimal media. We have also started measuring parameters important for metabolism, like glucose uptake and oxygen consumption over time.

New instrumentation to measure a series of metabolic parameters (oxygen consumption, pH, conductivity, optical absorbance) is being developed by the team. The goal is to use these devices to measure the response to ionizing radiation in a series of yeast strains.

We hypothesize that both metabolic (redox) and genetic (knockout) assays can distinguish specific changes due to the unique combinations of direct IR damage, indirect oxidative damage, and reduced gravity incurred by exposure to the lunar environment, low Earth orbit (LEO), interplanetary space, and ground-based simulations even before differential survival becomes apparent. These environmental perturbations are known to result in subtle changes in cell growth and activity, but the resulting data is lacking pathway and molecular specificity. By leveraging a metabolic modeling framework and a series of engineered strains, our primary goal is to perform the ground testing necessary to validate strains that yield distinct responses to specific stressors, both laying the groundwork for a potential (and successful) lunar mission to characterize the relative importance of these changes within the capabilities of the BioSensor instrument and improving the usefulness of the alamarBlue assay for past and future missions.

To test our hypothesis, we will pursue the following Specific Aims:

Aim 1: Derive metabolic pathways usage via modeling of a colorimetric assay. The BioSentinel platform uses a redox dye to detect changes in viability and metabolic activity. AlamarBlue changes color in the presence of cell activity and creates intricate time course data that varies as a function of environmental stress, genetic background, and metabolic activity. To date, the rich information in this data has not been connected explicitly to underlying cellular processes. We will combine the data with metabolic modeling to develop a computational tool that extracts detailed metabolic information. This predictive model will not only enable analysis of our experimental design but can be applied to detect changes caused by lunar-like conditions or any autonomous mission that utilizes this reporter dye.

Aim 2: Explore biological effects of a lunar-like environment using DNA repair and stress response defective mutants. Both IR and reduced gravity induce the production of reactive oxygen species (ROS), which play a key role in the generation of cellular and DNA damage, in addition to direct IR damage (e.g., DNA breaks, membrane damage). We will generate yeast mutants defective in DNA repair and stress response pathways, including oxidative damage repair, homologous recombination, and excision repair. Given the short mission duration and the expected low cumulative IR dose, we will also generate mutants containing multiple gene knockouts to increase sensitivity. We will then perform alamarBlue assays after IR exposure in both dry form and in liquid suspension, to study their response.

Aim 3: Engineer endogenous ROS scavenging capabilities in yeast. An indirect effect of IR and cellular stress is the buildup of ROS. While S. cerevisiae has several strategies for ROS removal, we will engineer ROS scavenging mechanisms and evaluate if they can mitigate the unique damage caused by spaceflight stress and IR. We will over-express a native ROS scavenging enzyme and engineer a mannitol biosynthetic pathway, which specifically targets an ROS produced by IR. This aim demonstrates a potential mitigation for future spaceflight missions, enabling insight into how engineered scavenging mechanisms could improve crew safety.