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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Aim18p and Aim46p are chalcone isomerase domain–containing mitochondrial hemoproteins in Saccharomyces cerevisiae

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid–binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. Furthermore, this study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, β-1,3- and β-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of β-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3 –10 μm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ~25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the β-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Comparison of stress tolerance mechanisms between Saccharomyces cerevisiae and the multistress-tolerant Pichia kudriavzevii

Yeasts play a vital role in both research and industrial biomanufacturing. Saccharomyces cerevisiae has been extensively utilized as a model system. However, its application is often constrained by limited tolerance to the diverse stress conditions encountered in bioprocesses. These challenges have driven increasing interest in nonconventional, multistress-tolerant yeasts as alternative biomanufacturing hosts. This review highlights Pichia kudriavzevii as a promising nonconventional yeast for industrial applications. Unlike S. cerevisiae, P. kudriavzevii exhibits exceptional tolerance to high temperatures, elevated concentrations of furanic and phenolic inhibitors, osmotic stress, salinity, and extreme pH. These traits make it an attractive candidate for industrial processes without requiring extensive genetic modifications to enhance stress resistance. As a result, P. kudriavzevii has emerged as a flagship species for advancing bioeconomy. Despite its industrial potential, the molecular mechanisms underlying P. kudriavzevii's superior stress tolerance remain poorly understood. This review compiles current knowledge on P. kudriavzevii and compares its stress tolerance mechanisms with those of S. cerevisiae, providing insights into its innate resilience. By expanding our understanding of nonconventional yeasts, this review aims to facilitate their broader adoption as robust microbial platforms for industrial biomanufacturing.

Frousnoon, Thasneem Banu↗
DOE OSTI2026

Engineering of 2‐ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae

Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.

2-ketoacid decarboxylase↗
DOE OSTI2020

Production of xylose enriched hydrolysate from bioenergy sorghum and its conversion to β-carotene using an engineered Saccharomyces cerevisiae

A new bioprocess has been created that allows for producing β-carotene from the xylose portion of bioenergy sorghum. Bioenergy sorghum was pretreated in a pilot-scale continuous hydrothermal reactor followed by disc refining. Xylose was extracted using low-severity dilute acid hydrolysis. A xylose yield of 64.9% (17.4 g/L) was obtained by hydrolyzing at 120 °C for 5 min with 2% sulfuric acid. The xylose-enriched syrup was separated and concentrated to either 32 g xylose/L (medium-concentrated hydrolysate, MCB) or 66 g xylose/L (high-concentrated hydrolysate, HCB). The non- (NCB), medium-, and high-concentrated xylose syrup were neutralized and fermented to β-carotene using Saccharomyces cerevisiae strain SR8B, which had been engineered for xylose utilization and β-carotene production. In HCB, MCB, and NCB cultures, the yeast produced β-carotene titers of 114.50 mg/L, 93.56 mg/L, and 82.50 mg/L, which corresponds to specific yeast biomass productions of 7.32 mg/g DCW, 8.10 mg/g DCW, and 8.29 mg/g DCW, respectively.

09 BIOMASS FUELS↗
NASA NTRS1979

Oxygen as a factor in eukaryote evolution - Some effects of low levels of oxygen on Saccharomyces cerevisiae

A comparative study of the effects of varying levels of oxygen on some of the metabolic functions of the primitive eukaryote, Saccharomyces cerevisiae, has shown that these cells are responsive to very low levels of oxygen: the level of palmitoyl-Co A desaturase was greatly enhanced by only 0.03 vol % oxygen. Similarly, an acetyl-CoA synthetase associated predominantly with anaerobic growth was stimulated by as little as 0.1% oxygen, while an isoenzyme correlated with aerobic growth was maximally active at much higher oxygen levels (greater than 1%). Closely following this latter pattern were three mitochondrial enzymes that attained maximal activity only under atmospheric levels of oxygen.

Jahnke, L.↗
NASA NTRS2005

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

NASA Discipline Cell Biology↗
DOE OSTI2020

Data from Enhanced 2′-Fucosyllactose Production by Engineered Saccharomyces cerevisiae using Xylose as a Co-Substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli , Saccharomyces cerevisiae , Corynebacterium glutamicum , and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from E. coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

Conversion↗
NASA NTRS1999

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

Non-NASA Center↗
DOE OSTI2022

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for L-Malic Acid Production from Xylose by Engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae . To enable malic acid production in a xylose-assimilating S. cerevisiae , we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol- and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L‒1) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L‒1). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L‒1 of malic acid was produced, with a productivity of 0.32 g L‒1 h. These results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae .

Conversion↗
DOE OSTI2021

A novel d-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar d-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize d-xylose through expression of heterologous d-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new d-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on d-xylose media. The d-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L –1 ) appeared not to inhibit d-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for d-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Data for High-Level β-Carotene Production from Xylose by Engineered Saccharomyces cerevisiae without Overexpression of a Truncated HMG1 (t HMG1 )

β‐Carotene is a natural pigment and health‐promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β‐carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β‐carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose‐fermenting S. cerevisiae . The resulting strain produced β‐carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of t HMG1 , which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and β‐carotene production in yeast when glucose is used, did not further improve the production of β‐carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for β‐carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl‐CoA pool, and an upregulated expression level of rate‐limiting genes in the β‐carotene‐producing pathway, including ACS1 and HMG1 . As a result, 772.8 mg/L of β‐carotene was obtained in a fed‐batch bioreactor culture with xylose feeding. Considering the inevitable large scale production of xylose when cellulosic biomass‐based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae .

Conversion↗
NASA NTRS2016

The Effects of Spaceflight on Cellular Aging in Saccharomyces cerevisiae

The conditions encountered during spaceflight place unique stresses on physiological processes that oftentimes lead to deleterious effects. Identifying these effects and better understanding their molecular mechanisms will be essential in enabling long-duration space travel by humans. Studies in Saccharomyces cerevisiae suggest an aging model that involves the accumulation of toxic components, such as excess extrachromosomal rDNA and damaged mitochondria. This build-up then limits the replicative lifespan (the number of times a mother cell can form a new daughter cell). Remarkably, each new daughter cell emerges completely renewed from the senescing mother cell through an asymmetric distribution of aging determinants via mechanisms that are intricately linked to the budding process. When exposed to simulated microgravity, S. cerevisiae undergoes an altered budding process characterized by a breakdown in bud scar polarity. Because the budding process is critical to replicative aging, we hypothesize that the replicative lifespan may be affected by microgravity as well. To measure relative replicative aging rates, we will construct a strain of yeast in which daughter cells are inviable. In this strain, the Cre recombinase will be expressed under the control of the daughter cell specific promoter, pSCW11, and LoxP sites will be inserted at both flanks of two essential genes involved in the cell cycle, UBC9 and CDC20, using a CRISPRCas9 system. Thus, UBC9 and CDC20 will be excised from daughter cells, leading to cell-cycle arrest and eventual death. To mimic the low shear conditions encountered in microgravity, this strain will be grown in rotating wall vessels. The number of viable mother cells will be monitored over time, and this rate will be compared to cells growing in standard conditions. Because asymmetric division also occurs in mammalian cells (e.g. in neural stem cells), this study will provide insight into how cellular aging rates may change in mammals and will help empower humans to thrive in space for extended and even indefinite periods of time.

aging↗
NASA NTRS1998

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Non-NASA Center↗
DOE OSTI2021

Quorum-sensing molecules increase ethanol yield from Saccharomyces cerevisiae

ABSTRACT One strategy to increase the yield of desired fermentation products is to redirect substrate carbon from biomass synthesis. Nongenetic approaches to alter metabolism may have advantages of general applicability and simple control. The goal of this study was to identify and evaluate chemicals for their ability to inhibit the growth of Saccharomyces cerevisiae while allowing ethanol production with higher yields. Eight potential growth-inhibitory chemicals were screened for their ability to reduce cell growth in 24-well plates. Effective chemicals were then evaluated in cultivations to identify those that simultaneously reduced biomass yield and increased ethanol yield. The yeast quorum-sensing molecules 2-phenylethanol, tryptophol and tyrosol were found to increase the ethanol yield of S. cerevisiae JAY 270. These molecules were tested with seven other yeast strains and ethanol yields of up to 15% higher were observed. The effects of 2-phenylethanol and tryptophol were also studied in bioreactor fermentations. These findings demonstrate for the first time that the ethanol yield can be improved by adding yeast quorum-sensing molecules to reduce the cell growth of S. cerevisiae, suggesting a strategy to improve the yield of ethanol and other yeast fermentation products by manipulating native biological control systems.

Huang, Xing-Feng (ORCID:0000000251165177)↗
NASA NTRSDate not supplied

Single-Cell Analysis of Yeast (Saccharomyces cerevisiae) Using Hydrogel Encapsulation

Space radiation poses a major health risk to astronauts. To fulfill NASA’s mission of exploration beyond Earth, the biological effects of Galactic Cosmic Radiation and gamma radiation must be investigated to elucidate cellular damage mechanisms and inform countermeasure protocols to safely bring humans beyond Earth’s magnetosphere. Budding yeast (Saccharomyces cerevisiae) are commonly used in experiments as a model organism for studying the effects of radiation on eukaryotes. Radiobiology of yeast at the single cell level is poorly understood, yet crucial for informing models to aid in the design and interpretation of experiments. We are using a novel method of microencapsulation in hydrogel particles (PicoShells) to enable analysis of the distribution of radiation-induced damage among yeast cells at the single-cell level, in high throughput. Here we describe the development of methods for culturing, visualization, and quantification of encapsulated yeast. The encapsulated yeast are cultured in Yeast extract-Peptone-Dextrose (YPD) medium, fixed in formaldehyde or ethanol, and stained with DAPI or propidium iodide, then visualized using microscopy or enumerated using flow cytometry, with the aim of developing a protocol to enumerate the distribution of viable cells in each PicoShell. This will allow us to quantify how different forms of radiation can generate different distributions of damage across a population of cells, ultimately providing insight into the biological effects of space-relevant ionizing radiation.

yeast↗