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1.
Yeast ; 40(1): 32-41, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36536407

RESUMO

Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiae genome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressing guide RNAs (gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.


Assuntos
Sistemas CRISPR-Cas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Edição de Genes/métodos , Plasmídeos/genética , RNA Guia de Sistemas CRISPR-Cas
2.
Yeast ; 39(1-2): 4-24, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35146791

RESUMO

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat.


Assuntos
Ecossistema , Árvores , Biodiversidade , Florestas , Leveduras/genética
3.
BMC Genomics ; 22(1): 110, 2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563210

RESUMO

BACKGROUND: The accumulation of intracellular fat depots is a polygenic trait. Therefore, the extent of lipid storage in the individuals of a species covers a broad range and is determined by many genetic factors. Quantitative trait loci analysis can be used to identify those genetic differences between two strains of the same species that are responsible for the differences in a given phenotype. We used this method and complementary approaches to identify genes in the yeast Saccharomyces cerevisiae that are involved in neutral lipid storage. RESULTS: We selected two yeast strains, the laboratory strain BY4741 and the wine yeast AWRI1631, with a more than two-fold difference in neutral lipid content. After crossing, sporulation and germination, we used fluorescence activated cell sorting to isolate a subpopulation of cells with the highest neutral lipid content from the pool of segregants. Whole genome sequencing of this subpopulation and of the unsorted pool of segregants implicated several loci that are involved in lipid accumulation. Three of the identified genes, PIG1, PHO23 and RML2, were investigated in more detail. Deletions of these genes and the exchange of the alleles between the two parental strains confirmed that the encoded proteins contribute to neutral lipid storage in S. cerevisiae and that PIG1, PHO23 and RML2 are the major causative genes. Backcrossing of one of the segregants with the parental strains for seven generations revealed additional regions in the genomes of both strains with potential causative genes for the high lipid accumulation phenotype. CONCLUSIONS: We identified several genes that contribute to the phenotype of lipid accumulation in an allele-specific manner. Surprisingly, no allelic variations of genes with known functions in lipid metabolism were found, indicating that the level of storage lipid accumulation is determined by many cellular processes that are not directly related to lipid metabolism.


Assuntos
Proteínas de Saccharomyces cerevisiae , Vinho , Mapeamento Cromossômico , Humanos , Proteínas Nucleares , Locos de Características Quantitativas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
4.
Microb Cell Fact ; 20(1): 147, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315498

RESUMO

BACKGROUND: Fatty acid-based substances play an important role in many products, from food supplements to pharmaceutical products and biofuels. The production of fatty acids, mainly in their esterified form as triacylglycerol (TAG), has been intensively studied in oleaginous yeasts, whereas much less effort has been invested into non-oleaginous species. In the present work, we engineered the model yeast Saccharomyces cerevisiae, which is commonly regarded as non-oleaginous, for the storage of high amounts of TAG, comparable to the contents achieved in oleaginous yeasts. RESULTS: We investigated the effects of several mutations with regard to increased TAG accumulation and identified six of them as important for this phenotype: a point mutation in the acetyl-CoA carboxylase Acc1p, overexpression of the diacylglycerol acyltransferase Dga1p, deletions of genes coding for enzymes involved in the competing pathways glycogen and steryl ester synthesis and TAG hydrolysis, and a deletion of CKB1, the gene coding for one of the regulatory subunits of casein kinase 2. With the combination of these mutations in a S. cerevisiae strain with a relatively high neutral lipid level already in the non-engineered state, we achieved a TAG content of 65% in the dry biomass. High TAG levels were not only obtained under conditions that favor lipid accumulation, but also in defined standard carbon-limited media. CONCLUSIONS: Baker's yeast, which is usually regarded as inefficient in the storage of TAG, can be converted into a highly oleaginous strain that could be useful in processes aiming at the synthesis of fatty acid-based products. This work emphasizes the importance of strain selection in combination with metabolic engineering to obtain high product levels.


Assuntos
Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/biossíntese , Biocombustíveis , Biomassa , Meios de Cultura/metabolismo , Diacilglicerol O-Aciltransferase/genética , Ácidos Graxos , Glicogênio/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triglicerídeos/análise
5.
Food Microbiol ; 90: 103483, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32336374

RESUMO

Sulfur dioxide is generally used as an antimicrobial in wine to counteract the activity of spoilage yeasts, including Brettanomyces bruxellensis. However, this chemical does not exert the same effectiveness on different B. bruxellensis yeasts since some strains can proliferate in the final product leading to a negative sensory profile due to 4-ethylguaiacol and 4-ethylphenol. Thus, the capability of deciphering the general molecular mechanisms characterizing this yeast species' response in presence of SO2 stress could be considered strategic for a better management of SO2 in winemaking. A RNA-Seq approach was used to investigate the gene expression of two strains of B. bruxellensis, AWRI 1499 and CBS 2499 having different genetic backgrounds, when exposed to a SO2 pulse. Results revealed that sulphites affected yeast culturability and metabolism, but not volatile phenol production suggesting that a phenotypical heterogeneity could be involved for the SO2 cell adaptation. The transcriptomics variation in response to SO2 stress confirmed the strain-related response in B. bruxellensis and the GO analysis of common differentially expressed genes showed that the detoxification process carried out by SSU1 gene can be considered as the principal specific adaptive response to counteract the SO2 presence. However, nonspecific mechanisms can be exploited by cells to assist the SO2 tolerance; namely, the metabolisms related to sugar alcohol (polyols) and oxidative stress, and structural compounds.


Assuntos
Brettanomyces/genética , Brettanomyces/metabolismo , Fermentação , Estresse Fisiológico , Dióxido de Enxofre/metabolismo , Vinho/microbiologia , Microbiologia de Alimentos , Perfilação da Expressão Gênica , RNA-Seq , Transcriptoma
6.
J Biol Chem ; 293(28): 10870-10883, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29866881

RESUMO

Cardiolipin (CL) is a signature phospholipid of the mitochondria required for the formation of mitochondrial respiratory chain (MRC) supercomplexes. The destabilization of MRC supercomplexes is the proximal cause of the pathology associated with the depletion of CL in patients with Barth syndrome. Thus, promoting supercomplex formation could ameliorate mitochondrial dysfunction associated with CL depletion. However, to date, physiologically relevant small-molecule regulators of supercomplex formation have not been identified. Here, we report that ethanolamine (Etn) supplementation rescues the MRC defects by promoting supercomplex assembly in a yeast model of Barth syndrome. We discovered this novel role of Etn while testing the hypothesis that elevating mitochondrial phosphatidylethanolamine (PE), a phospholipid suggested to overlap in function with CL, could compensate for CL deficiency. We found that the Etn supplementation rescues the respiratory growth of CL-deficient Saccharomyces cerevisiae cells in a dose-dependent manner but independently of its incorporation into PE. The rescue was specifically dependent on Etn but not choline or serine, the other phospholipid precursors. Etn improved mitochondrial function by restoring the expression of MRC proteins and promoting supercomplex assembly in CL-deficient cells. Consistent with this mechanism, overexpression of Cox4, the MRC complex IV subunit, was sufficient to promote supercomplex formation in CL-deficient cells. Taken together, our work identifies a novel role of a ubiquitous metabolite, Etn, in attenuating mitochondrial dysfunction caused by CL deficiency.


Assuntos
Cardiolipinas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Etanolaminas/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte de Elétrons , Mitocôndrias/patologia , Saccharomyces cerevisiae/efeitos dos fármacos
7.
BMC Microbiol ; 17(1): 181, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28830344

RESUMO

BACKGROUND: The only hitherto known biological role of yeast Saccharomyces cerevisiae Tum1 protein is in the tRNA thiolation pathway. The mammalian homologue of the yeast TUM1 gene, the thiosulfate sulfurtransferase (a.k.a. rhodanese) Tst, has been proposed as an obesity-resistance and antidiabetic gene. To assess the role of Tum1 in cell metabolism and the putative functional connection between lipid metabolism and tRNA modification, we analysed evolutionary conservation of the rhodanese protein superfamily, investigated the role of Tum1 in lipid metabolism, and examined the phenotype of yeast strains expressing the mouse homologue of Tum1, TST. RESULTS: We analysed evolutionary relationships in the rhodanese superfamily and established that its members are widespread in bacteria, archaea and in all major eukaryotic groups. We found that the amount of sterol esters was significantly higher in the deletion strain tum1Δ than in the wild-type strain. Expression of the mouse TST protein in the deletion strain did not rescue this phenotype. Moreover, although Tum1 deficiency in the thiolation pathway was complemented by re-introducing TUM1, it was not complemented by the introduction of the mouse homologue Tst. We further showed that the tRNA thiolation pathway is not involved in the regulation of sterol ester content in S. cerevisiae, as overexpression of the tEUUC, tKUUU and tQUUG tRNAs did not rescue the lipid phenotype in the tum1Δ deletion strain, and, additionally, deletion of the key gene for the tRNA thiolation pathway, UBA4, did not affect sterol ester content. CONCLUSIONS: The rhodanese superfamily of proteins is widespread in all organisms, and yeast TUM1 is a bona fide orthologue of mammalian Tst thiosulfate sulfurtransferase gene. However, the mouse TST protein cannot functionally replace yeast Tum1 protein, neither in its lipid metabolism-related function, nor in the tRNA thiolation pathway. We show here that Tum1 protein is involved in lipid metabolism by decreasing the sterol ester content in yeast cells, and that this function of Tum1 is not exerted through the tRNA thiolation pathway, but through another, currently unknown pathway.


Assuntos
Proteínas de Transporte/metabolismo , Ésteres/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismo , Animais , Proteínas de Transporte/genética , Deleção de Genes , Metabolismo dos Lipídeos , Lipídeos/análise , Camundongos , Fenótipo , Filogenia , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Tiossulfato Sulfurtransferase/classificação , Tiossulfato Sulfurtransferase/genética , Tiossulfato Sulfurtransferase/metabolismo , Vertebrados
8.
Yeast ; 32(9): 583-93, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26108577

RESUMO

Economic growth depends strongly on the availability and price of fuels. There are various reasons in different parts of the world for efforts to decrease the consumption of fossil fuels, but biofuels are one of the main solutions considered towards achieving this aim globally. As the major bioethanol producer, the yeast Saccharomyces cerevisiae has a central position among biofuel-producing organisms. However, unprecedented challenges for yeast biotechnology lie ahead, as future biofuels will have to be produced on a large scale from sustainable feedstocks that do not interfere with food production, and which are generally not the traditional carbon source for S. cerevisiae. Additionally, the current trend in the development of biofuels is to synthesize molecules that can be used as drop-in fuels for existing engines. Their properties should therefore be more similar to those of oil-derived fuels than those of ethanol. Recent developments and challenges lying ahead for cost-effective production of such designed biofuels, using S. cerevisiae-based cell factories, are presented in this review.


Assuntos
Biocombustíveis/análise , Etanol/metabolismo , Microbiologia Industrial , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética
9.
Yeast ; 37(1): 3, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31943346
10.
Microb Cell Fact ; 14: 94, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26122609

RESUMO

The yeast Saccharomyces cerevisiae is one of the oldest and most frequently used microorganisms in biotechnology with successful applications in the production of both bulk and fine chemicals. Yet, yeast researchers are faced with the challenge to further its transition from the old workhorse to a modern cell factory, fulfilling the requirements for next generation bioprocesses. Many of the principles and tools that are applied for this development originate from the field of synthetic biology and the engineered strains will indeed be synthetic organisms. We provide an overview of the most important aspects of this transition and highlight achievements in recent years as well as trends in which yeast currently lags behind. These aspects include: the enhancement of the substrate spectrum of yeast, with the focus on the efficient utilization of renewable feedstocks, the enhancement of the product spectrum through generation of independent circuits for the maintenance of redox balances and biosynthesis of common carbon building blocks, the requirement for accurate pathway control with improved genome editing and through orthogonal promoters, and improvement of the tolerance of yeast for specific stress conditions. The causative genetic elements for the required traits of the future yeast cell factories will be assembled into genetic modules for fast transfer between strains. These developments will benefit from progress in bio-computational methods, which allow for the integration of different kinds of data sets and algorithms, and from rapid advancement in genome editing, which will enable multiplexed targeted integration of whole heterologous pathways. The overall goal will be to provide a collection of modules and circuits that work independently and can be combined at will, depending on the individual conditions, and will result in an optimal synthetic host for a given production process.


Assuntos
Microbiologia Industrial/tendências , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica
11.
Sci Rep ; 14(1): 14233, 2024 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-38902520

RESUMO

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.


Assuntos
Ácidos Graxos Voláteis , Fermentação , Ácidos Graxos Voláteis/metabolismo , Leveduras/metabolismo , Leveduras/crescimento & desenvolvimento , Yarrowia/metabolismo , Yarrowia/crescimento & desenvolvimento , Ensaios de Triagem em Larga Escala/métodos , Biomassa , Biocombustíveis/microbiologia , Ácidos Carboxílicos/metabolismo , Pichia/metabolismo , Pichia/crescimento & desenvolvimento
12.
Nat Ecol Evol ; 6(4): 448-460, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35210580

RESUMO

Domestication of plants and animals is the foundation for feeding the world human population but can profoundly alter the biology of the domesticated species. Here we investigated the effect of domestication on one of our prime model organisms, the yeast Saccharomyces cerevisiae, at a species-wide level. We tracked the capacity for sexual and asexual reproduction and the chronological life span across a global collection of 1,011 genome-sequenced yeast isolates and found a remarkable dichotomy between domesticated and wild strains. Domestication had systematically enhanced fermentative and reduced respiratory asexual growth, altered the tolerance to many stresses and abolished or impaired the sexual life cycle. The chronological life span remained largely unaffected by domestication and was instead dictated by clade-specific evolution. We traced the genetic origins of the yeast domestication syndrome using genome-wide association analysis and genetic engineering and disclosed causative effects of aneuploidy, gene presence/absence variations, copy number variations and single-nucleotide polymorphisms. Overall, we propose domestication to be the most dramatic event in budding yeast evolution, raising questions about how much domestication has distorted our understanding of the natural biology of this key model species.


Assuntos
Domesticação , Saccharomycetales , Animais , Variações do Número de Cópias de DNA , Estudo de Associação Genômica Ampla , Estágios do Ciclo de Vida , Saccharomyces cerevisiae/genética , Saccharomycetales/genética
13.
Nat Commun ; 10(1): 4551, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591416

RESUMO

Analysis of biomedical images requires computational expertize that are uncommon among biomedical scientists. Deep learning approaches for image analysis provide an opportunity to develop user-friendly tools for exploratory data analysis. Here, we use the visual programming toolbox Orange ( http://orange.biolab.si ) to simplify image analysis by integrating deep-learning embedding, machine learning procedures, and data visualization. Orange supports the construction of data analysis workflows by assembling components for data preprocessing, visualization, and modeling. We equipped Orange with components that use pre-trained deep convolutional networks to profile images with vectors of features. These vectors are used in image clustering and classification in a framework that enables mining of image sets for both novel and experienced users. We demonstrate the utility of the tool in image analysis of progenitor cells in mouse bone healing, identification of developmental competence in mouse oocytes, subcellular protein localization in yeast, and developmental morphology of social amoebae.


Assuntos
Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Redes Neurais de Computação , Animais , Dictyostelium/citologia , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Internet , Estágios do Ciclo de Vida , Camundongos Transgênicos , Oócitos/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Biochem J ; 391(Pt 2): 383-8, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16008522

RESUMO

Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.


Assuntos
Fase G2 , Fosfolipases A/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Fase G2/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio , Mutação , Fosfolipases A/química , Fosfolipases A2 , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
G3 (Bethesda) ; 6(9): 3003-14, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27371952

RESUMO

The capacity to map traits over large cohorts of individuals-phenomics-lags far behind the explosive development in genomics. For microbes, the estimation of growth is the key phenotype because of its link to fitness. We introduce an automated microbial phenomics framework that delivers accurate, precise, and highly resolved growth phenotypes at an unprecedented scale. Advancements were achieved through the introduction of transmissive scanning hardware and software technology, frequent acquisition of exact colony population size measurements, extraction of population growth rates from growth curves, and removal of spatial bias by reference-surface normalization. Our prototype arrangement automatically records and analyzes close to 100,000 growth curves in parallel. We demonstrate the power of the approach by extending and nuancing the known salt-defense biology in baker's yeast. The introduced framework represents a major advance in microbial phenomics by providing high-quality data for extensive cohorts of individuals and generating well-populated and standardized phenomics databases.


Assuntos
Genômica/métodos , Saccharomyces cerevisiae/genética , Software , Bases de Dados Genéticas , Aptidão Genética , Humanos , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento
16.
Biotechnol Biofuels ; 9: 5, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26740819

RESUMO

BACKGROUND: Acetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of second-generation bioethanol. Although several genes have been identified in laboratory yeast strains that are required for tolerance to acetic acid, the genetic basis of the high acetic acid tolerance naturally present in some Saccharomyces cerevisiae strains is unknown. Identification of its polygenic basis may allow improvement of acetic acid tolerance in yeast strains used for second-generation bioethanol production by precise genome editing, minimizing the risk of negatively affecting other industrially important properties of the yeast. RESULTS: Haploid segregants of a strain with unusually high acetic acid tolerance and a reference industrial strain were used as superior and inferior parent strain, respectively. After crossing of the parent strains, QTL mapping using the SNP variant frequency determined by pooled-segregant whole-genome sequence analysis revealed two major QTLs. All F1 segregants were then submitted to multiple rounds of random inbreeding and the superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance in the QTL mapping with the F7 segregants of a major F1 QTL, in which we identified HAA1, a known regulator of high acetic acid tolerance, as a true causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, and a previously identified component, VMA7, were identified as causative alleles in the second major F1 QTL and in three newly appearing F7 QTLs, respectively. The superior HAA1 allele contained a unique single point mutation that significantly improved acetic acid tolerance under industrially relevant conditions when inserted into an industrial yeast strain for second-generation bioethanol production. CONCLUSIONS: This work reveals the polygenic basis of high acetic acid tolerance in S. cerevisiae in unprecedented detail. It also shows for the first time that a single strain can harbor different sets of causative genes able to establish the same polygenic trait. The superior alleles identified can be used successfully for improvement of acetic acid tolerance in industrial yeast strains.

17.
Int J Med Inform ; 74(7-8): 505-17, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15941669

RESUMO

This paper describes a new technique for clustering short time series of gene expression data. The technique is a generalization of the template-based clustering and is based on a qualitative representation of profiles which are labelled using trend Temporal Abstractions (TAs); clusters are then dynamically identified on the basis of this qualitative representation. Clustering is performed in an efficient way at three different levels of aggregation of qualitative labels, each level corresponding to a distinct degree of qualitative representation. The developed TA-clustering algorithm provides an innovative way to cluster gene profiles. We show the developed method to be robust, efficient and to perform better than the standard hierarchical agglomerative clustering approach when dealing with temporal dislocations of time series. Results of the TA-clustering algorithm can be visualized as a three-level hierarchical tree of qualitative representations and as such easy to interpret. We demonstrate the utility of the proposed algorithm on a set of two simulated data sets and on a study of gene expression data from S. cerevisiae.


Assuntos
Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Reconhecimento Automatizado de Padrão , Algoritmos , Biologia Computacional , Análise de Sequência com Séries de Oligonucleotídeos , Saccharomyces cerevisiae/genética
18.
Metallomics ; 7(9): 1338-51, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26067383

RESUMO

The functional link between zinc homeostasis and membrane-related processes, including lipid metabolism regulation, extends from yeast to humans, and has a likely role in the pathogenesis of diabetes. The yeast Izh2 protein has been previously implicated in zinc ion homeostasis and in the regulation of lipid and phosphate metabolism, but its precise molecular function is not known. We performed a chemogenomics experiment to determine the genes conferring resistance or sensitivity to different environmental zinc concentrations. We then determined at normal, depleted and excess zinc concentrations, the genetic interactions of IZH2 at the genome-wide level and measured changes in the transcriptome caused by deletion of IZH2. We found evidence for an important cellular function of the Rim101 pathway in zinc homeostasis in neutral or acidic environments, and observed that phosphatidylinositol is a source of inositol when zinc availability is limited. Comparison of our experimental profiles with published gene expression and genetic interaction profiles revealed pleiotropic functions for Izh2. We propose that Izh2 acts as an integrator of intra- and extracellular signals in providing adequate cellular responses to maintain homeostasis under different external conditions, including - but not limited to - alterations in zinc concentrations.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Membrana/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Zinco/metabolismo , Equilíbrio Ácido-Base , Proteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética
19.
Chemosphere ; 104: 91-6, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24262822

RESUMO

Neonicotinoid insecticides are an important contribution to plant protection products. At the same time, their environmental impact on non-target organisms is often problematic. It has been shown that the toxicity of formulations of neonicotinoid insecticides can originate from non-neonicotinoid additives. In the present study we used chemogenomics to analyse side effects of purified neonicotinoids, additives and formulations on the genome-wide scale. We show that the additives in formulations have more pronounced effects than the active components, and that these effects could explain previously observed negative effects of neonicotinoid insecticides on spermatogenesis in animals. We also demonstrate that cell wall organization and biogenesis in yeast is negatively affected by neonicotinoid substances.


Assuntos
Genoma Fúngico/efeitos dos fármacos , Inseticidas/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Inseticidas/química , Metagenômica/métodos , Saccharomyces cerevisiae/citologia
20.
PLoS One ; 9(7): e102500, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25047918

RESUMO

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.


Assuntos
Autofagia , Cistatina B/análise , Cistatina B/metabolismo , Agregados Proteicos , Dobramento de Proteína , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Clonagem Molecular , Cistatina B/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Estresse Oxidativo , Multimerização Proteica
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