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Results for “Saccharomyces cerevisiae (yeast)”

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At least 19 records
All sourcesNASA NTRSDOE OSTI
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↗
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↗
NASA NTRSDate not supplied

Investigating Biological Responses to Space-like Radiation using the yeast Saccharomyces cerevisiae

As we plan crewed missions to the Moon, Mars, and beyond, it is essential to understand how persistent exposure to space radiation affects biology. Unlike on the International Space Station, where crew support and sample return are possible, experiments for long-duration missions require autonomous systems with no sample return. Human cells would be ideal biosensors, but limitations in culture methods, extended prelaunch storage, and long flight durations make it very difficult to keep human cells alive. Unlike other model systems, yeast can survive the constraints of long-duration spaceflight. Despite a billion years of evolution separating yeast from humans, we share homology in hundreds of genes important for basic cell function, including responses to DNA damage. Thus, yeast are excellent biosensors for detecting types/extent of damage induced by space radiation. BioSentinel is NASA’s latest biological CubeSat, and first interplanetary space bioscience mission. BioSentinel is manifested on Artemis 1, the first test flight of NASA’s Space Launch System, in the coming year. The BioSensor payload within BioSentinel contains two yeast strains. The wild type serves as a control for health and “normal” DNA damage repair (DDR). The rad51 deletion mutant is defective for DDR and will undergo alterations to growth and metabolism as it accumulates radiation damage. Changes in growth and metabolic activity will be measured using a 3-color LED detection system and the metabolic redox dye alamarBlue®. Preliminary tests indicate a significant change in alamarBlue response to space-like, low-dose ionizing radiation. We will discuss these findings in four parts – Introduction to biological CubeSats and the BioSentinel mission (presented by Sergio Santa Maria), preliminary responses to space-like ionizing radiation (presented here), a deeper dive into tracking metabolic changes after exposure to ionizing radiation (presented by Diana Gentry), and a look into methods for correcting flight optical data (presented by Abbey Kim). This work is funded by NASA’s Advanced Exploration Systems.

CubeSat↗
NASA NTRS2020

BioSentinel ISS Control Experiment Investigation Summary Form

The main science objective of BioSentinel is to develop biosensors to study the effects of the space radiation environment on biological organisms. Given its similarities to human cells, we decided to use the budding yeast Saccharomyces cerevisiae. Yeast cells share homology to human cells, particularly in the response to DNA damage caused by ionizing radiation. Most importantly, yeast cells can be desiccated and survive in that form for long periods of time. Thus, budding yeast is the ideal model organism for long-term experiments in preparation for future manned missions to deep space. In addition to the ISS mission, our biosensors will fly in a nanosatellite designed for deep space, which is manifested to fly as a secondary payload onboard NASA’s Artemis-1 rocket.

BioSentinel↗
NASA NTRS1979

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

Klein, H. P.↗
DOE OSTI2020

The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels

Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.

09 BIOMASS FUELS↗
DOE OSTI2022

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae

Past work from our lab, focused on engineering the budding yeast Saccharomyces cerevisiae for fermentation of the non-native pentose sugar xylose, discovered that hyperactivation of the RAS/Protein Kinase A (PKA) pathway was needed for rapid anaerobic xylose fermentation. Interestingly, the mechanism of PKA hyperactivation has a dramatic impact on growth and metabolism on xylose; deletion of the RAS inhibitor IRA2 permits rapid growth and fermentation, while deletion of the PKA regulatory subunit BCY1 allows for fermentation without growth on xylose. To understand how a single deletion in the PKA pathway can decouple growth and metabolism, we performed transcriptomic analysis of these strains, predicting that altered PKA activity would impact global gene expression and identify pathways important for growth and metabolism coordination.

Gasch, Audrey↗
DOE OSTI2024

A deep learning-guided automated workflow in LipidOz for detailed characterization of fungal fatty acid unsaturation by ozonolysis

Understanding fungal lipid biology and metabolism is critical for antifungal target discovery as lipids play central roles in cellular processes. Nuances in lipid structural differences can significantly impact their functions, making it necessary to characterize lipids in detail to enable and understanding of their roles in these complex systems. In particular, lipid double bond (DB) locations are an important component of lipid structure that can only be determined using a few specialized analytical techniques. Ozone-induced dissociation mass spectrometry (OzID-MS) is one such technique that uses ozone to break lipid DBs, producing pairs of characteristic fragments that allow the determination of DB positions. In this work we apply OzID-MS and LipidOz software to analyze the complex lipids of Saccharomyces cerevisiae yeast strains transfected with different fatty acid desaturases from Histoplasma capsulatum to determine the specific unsaturated lipids produce. The automated data analysis in LipidOz made the determination of DB positions from this large dataset more practical, but manual verification for all targets was still time-consuming. The DL model reduces manual involvement in data analysis, but since it was trained using mammalian lipid extracts, the prediction accuracy on yeast-derived data was reduced. We addressed both shortcomings by retraining the DL model to act as a pre-filter to prioritize targets for automated analysis, providing confident manually verified results but requiring less computational time and manual effort. Our workflow resulted in the determination of novel DB positions and enzymatic specificity.

mass spectrometry, deep learning, Lipidomics, doub↗
DOE OSTI2023

Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets

Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.g., amino acids) that can be screened either directly or by artificial biosensors, with low-throughput targeted validation of the molecule of interest to uncover nonintuitive beneficial metabolic engineering targets and combinations hereof. Using this workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and L -DOPA production from two large 4k gRNA libraries each deregulating 1000 metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library plasmids for individual regulatory targets improving the production of l-tyrosine-derived betaxanthins, identifying 30 targets that increased intracellular betaxanthin content 3.5–5.7 fold. Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased the secreted titer by up to 15%. To investigate whether any of the six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and subjected it to our proposed coupled workflow. The combination of regulating PYC1 and NTH2 simultaneously resulted in the highest (threefold) improvement of the betaxanthin content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a L -DOPA producing strain, identifying 10 targets that increased the secreted titer by up to 89%, further validating our screening by proxy workflow. This coupled approach is useful for strain development in the absence of direct HTP screening assays for products of interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae . A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S . cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S . cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum . Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae . The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S . cerevisiae .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses

To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. Furthermore, these results have implications for the breadth of signaling programs that can give rise to common stress response signatures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

BioSentinel ISS mission: Analysis of ISS Flight Data and Lessons Learned

As we prepare for a future with a human presence on the Moon, Mars, and beyond, the need for countermeasures to protect astronauts against deep space radiation is ever pressing. However, our understanding of how life operates in the space environment, especially past the protection of the Van Allen radiation belts, is critically limited. BioSentinel, a biological CubeSat, aims to further investigate the effects of deep space ionizing radiation, utilizing the budding yeast Saccharomyces cerevisiae to examine the cell’s DNA damage response. The yeast wild type and a rad51Δ mutant strain defective for DNA damage repair will be monitored via the redox dye alamarBlue and a 3-color LED detection system. Selected as a secondary payload on Artemis I, BioSentinel will be the first deep space biological experiment in a half-century and the first biological CubeSat or free-flyer to ever reach a heliocentric orbit. Notably, BioSentinel is the only biological CubeSat to include an ISS control study in addition to a ground control. Comparisons between the ground, ISS, and free-flyer experiments will allow us to isolate the effects of deep space radiation from those of microgravity. The ISS study also provides a unique platform to conduct important technological and biological testing of BioSentinel’s instrumentation in preparation for the deep space mission. In this work, we use a series of data processing tools and scripts to analyze ISS flight data as well as samples exposed to simulated space radiation at Brookhaven National Laboratory, specifically looking at cell growth, metabolic activity, and duplication rates based on optical absorbance and alamarBlue kinetics. These analyses provide a crucial set of controls and a framework for analyzing and interpreting future data sets from the free-flyer, helping us gain further insight into the health risks astronauts will face when exposed to deep space radiation.

Kylie Lauren Lo-Wen Akiyama↗
DOE OSTI2020

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources towards engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, we summarize recent advances in metabolic engineering of yeast to address bottlenecks on xylose assimilation, and to enable simultaneous co-utilization of xylose and other carbon sources in lignocellulosic hydrolysates. We also highlight distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals. Although many challenges remain, recent research investments have facilitated efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding of xylose-induced metabolic rewiring in engineered yeast has encouraged use of xylose as a carbon source for producing various non-ethanol bioproducts. Furthermore, to boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.

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↗
DOE OSTI2020

High–level β–carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (t HMG1 )

Beta-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 beta-carotene from xylose, the sec- ond most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. Here, the resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-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 beta-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 beta-carotene!producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of beta-carotene was ob- tained in a fed-batch bioreactor culture with xylose feeding. Considering the in- evitable 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.

09 BIOMASS FUELS↗