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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Genome-wide toxicogenomic study of the lanthanides sheds light on the selective toxicity mechanisms associated with critical materials

Significance The growing use of lanthanides in various industries has increased the potential for human exposure to large concentrations of these heavy metals throughout the life cycle of new technologies, requiring more detailed investigations into their toxicological properties. The eukaryote model organism Saccharomyces cerevisiae is ideal to apply functional toxicogenomics tools at the system level and probe fundamental cellular functions disrupted by rare-earth metals. A comprehensive mechanistic assay was conducted to evaluate toxicity across the entire lanthanide series and provide information about which toxicological mechanisms may be shared by the different metals. We identified distinct characteristic behaviors between early and middle/late lanthanides, highlighting the powerful discrimination capabilities of natural systems and pointing to detailed mechanistic pathways associated with lanthanide exposure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Into the wild: new yeast genomes from natural environments and new tools for their analysis

Genomic studies of yeasts from the wild have increased significantly in the past few years. This revolution has been fueled by advances in high-throughput sequencing technologies and a better understanding of yeast ecology and phylogeography, especially for biotechnologically important species. The present review aims to first introduce new bioinformatic tools available for the generation and analysis of yeast genomes. Furthermore, we assess the accumulated genomic data of wild isolates of industrially relevant species, such as Saccharomyces spp., which provide unique opportunities to further investigate the domestication processes associated with the fermentation industry and opportunistic pathogenesis. The availability of genome sequences of other less conventional yeasts obtained from the wild has also increased substantially, including representatives of the phyla Ascomycota (e.g. Hanseniaspora) and Basidiomycota (e.g. Phaffia). Here, we review salient examples of both fundamental and applied research that demonstrate the importance of continuing to sequence and analyze genomes of wild yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparative chemical genomic profiling across plant-based hydrolysate toxins reveals widespread antagonism in fitness contributions

Abstract The budding yeast Saccharomyces cerevisiae has been used extensively in fermentative industrial processes, including biofuel production from sustainable plant-based hydrolysates. Myriad toxins and stressors found in hydrolysates inhibit microbial metabolism and product formation. Overcoming these stresses requires mitigation strategies that include strain engineering. To identify shared and divergent mechanisms of toxicity and to implicate gene targets for genetic engineering, we used a chemical genomic approach to study fitness effects across a library of S. cerevisiae deletion mutants cultured anaerobically in dozens of individual compounds found in different types of hydrolysates. Relationships in chemical genomic profiles identified classes of toxins that provoked similar cellular responses, spanning inhibitor relationships that were not expected from chemical classification. Our results also revealed widespread antagonistic effects across inhibitors, such that the same gene deletions were beneficial for surviving some toxins but detrimental for others. This work presents a rich dataset relating gene function to chemical compounds, which both expands our understanding of plant-based hydrolysates and provides a useful resource to identify engineering targets.

09 BIOMASS FUELS↗
DOE OSTI2021

Multidimensional genome-wide screening in yeast provides mechanistic insights into europium toxicity

Abstract Europium is a lanthanide metal that is highly valued in optoelectronics. Even though europium is used in many commercial products, its toxicological profile has only been partially characterized, with most studies focusing on identifying lethal doses in different systems or bioaccumulation in vivo. This paper describes a genome-wide toxicogenomic study of europium in Saccharomyces cerevisiae, which shares many biological functions with humans. By using a multidimensional approach and functional and network analyses, we have identified a group of genes and proteins associated with the yeast responses to ameliorate metal toxicity, which include metal discharge paths through vesicle-mediated transport, paths to regulate biologically relevant cations, and processes to reduce metal-induced stress. Furthermore, the analyses indicated that europium promotes yeast toxicity by disrupting the function of chaperones and cochaperones, which have metal-binding sites. Several of the genes and proteins highlighted in our study have human orthologues, suggesting they may participate in europium-induced toxicity in humans. By identifying the endogenous targets of europium as well as the already existing paths that can decrease its toxicity, we can determine specific genes and proteins that may help to develop future therapeutic strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Predictive models of the genetic bases underlying budding yeast fitness in multiple environments

Abstract The ability of organisms to adapt and survive depends on the effects of genes and the environment on fitness. However, the multigenic nature of fitness and genotype-by-environment interactions hinder our understanding of the genetic basis of fitness. Here, we established fitness prediction models for 35 environments using machine learning and existing fitness data and different genetic variant types for a Saccharomyces cerevisiae population. Models revealed that the predictive ability of genetic variants varied across environments, with copy number variants explaining the majority of fitness variation in most cases. Model interpretation showed that different variant types identified distinct gene sets associated with predictive variants. These gene sets were significantly enriched in experimentally validated genes affecting fitness in only a subset of environments, indicating that many genes influencing fitness remain unexplored. Notably, non-experimentally validated genes were more important than validated ones for fitness predictions. Gene contributions to predictions were both isolate- and environment-dependent, pointing to gene-by-gene and gene-by-environment interactions. Furthermore, models uncovered experimentally validated and novel candidate genetic interactions for a well-characterized stress, the fungicide benomyl. These findings highlight the feasibility of identifying the genetic basis of fitness by using different genetic variant types and offer novel targets for future functional analysis.

DNA copy number variations↗
DOE OSTI2021

Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

Abstract Background Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae , O. polymorpha , and K. marxianus in glucose and cellulosic media at different temperatures. Results In this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeasts Kluyveromyces marxianus and Ogataea polymorpha , as well as Saccharomyces cerevisiae , and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. Conclusions The eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.

09 BIOMASS FUELS↗
DOE OSTI2025

Machine learning reveals genes impacting oxidative stress resistance across yeasts

Reactive oxygen species (ROS) are highly reactive molecules encountered by yeasts during routine metabolism and during interactions with other organisms, including host infection. Here, we characterized the variation in resistance to ROS across the ancient yeast subphylum Saccharomycotina and used machine learning (ML) to identify gene families whose sizes were predictive of ROS resistance.

AI↗
DOE OSTI2022

A versatile enhanced freeze-substitution protocol for volume electron microscopy

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues ( Hordeum vulgare ), nematode ( Caenorhabditis elegans ) and yeast ( Saccharomyces cerevisiae ). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

YPK9 and WHI2 Negatively Interact during Oxidative Stress

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson’s disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741 COM ). In contrast, deleting YPK9 from BY4741 COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741 COM and BY4741 COM ypk9Δ contain a novel premature stop codon near the 3' end of WHI2 (WHI2 G1324T ), whereas the collection’s YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2p E442* ) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2 G1324T strain’s genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

Yeast respond to hypotonic shock with a calcium pulse

We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

NASA Discipline Plant Biology↗
NASA NTRS1996

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Non-NASA Center↗
NASA NTRS1993

Diploid yeast cells yield homozygous spontaneous mutations

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

Non-NASA Center↗
NASA NTRS1991

The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

Non-NASA Center↗
NASA NTRS1988

Nonselective enrichment for yeast adenine mutants by flow cytometry

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

Non-NASA Center↗
DOE OSTI2021

Identification and analysis of sugar transporters capable of co–transporting glucose and xylose simultaneously

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Furthermore, our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗