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In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own can't ferment maltose and maltotriose, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles.
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Cerveza , Fermentación , Hanseniaspora , Odorantes , Saccharomyces cerevisiae , Cerveza/microbiología , Cerveza/análisis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Hanseniaspora/metabolismo , Hanseniaspora/crecimiento & desarrollo , Odorantes/análisis , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/análisis , Compuestos Orgánicos Volátiles/química , Etanol/metabolismo , Aromatizantes/metabolismo , Aromatizantes/química , Acetatos/metabolismo , Técnicas de Cocultivo , Alcohol Feniletílico/análogos & derivadosRESUMEN
More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.
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Antifúngicos , Aspergillus fumigatus , Quinasas DyrK , Proteínas Fúngicas , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Aspergillus fumigatus/genética , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/enzimología , Animales , Antifúngicos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Ratones , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Azoles/farmacología , Aspergilosis/microbiología , Aspergilosis/tratamiento farmacológico , Pulmón/microbiología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética , FemeninoRESUMEN
In silico tools such as genome-scale metabolic models have shown to be powerful for metabolic engineering of microorganisms. Saccharomyces pastorianus is a complex aneuploid hybrid between the mesophilic Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. This species is of biotechnological importance because it is the primary yeast used in lager beer fermentation and is also a key model for studying the evolution of hybrid genomes, including expression pattern of ortholog genes, composition of protein complexes, and phenotypic plasticity. Here, we created the iSP_1513 GSMM for S. pastorianus CBS1513 to allow top-down computational approaches to predict the evolution of metabolic pathways and to aid strain optimization in production processes. The iSP_1513 comprises 4,062 reactions, 1,808 alleles, and 2,747 metabolites, and takes into account the functional redundancy in the gene-protein-reaction rule caused by the presence of orthologous genes. Moreover, a universal algorithm to constrain GSMM reactions using transcriptome data was developed as a python library and enabled the integration of temperature as parameter. Essentiality data sets, growth data on various carbohydrates and volatile metabolites secretion were used to validate the model and showed the potential of media engineering to improve specific flavor compounds. The iSP_1513 also highlighted the different contributions of the parental sub-genomes to the oxidative and non-oxidative parts of the pentose phosphate pathway. Overall, the iSP_1513 GSMM represent an important step toward understanding the metabolic capabilities, evolutionary trajectories, and adaptation potential of S. pastorianus in different industrial settings. IMPORTANCE: Genome-scale metabolic models (GSMM) have been successfully applied to predict cellular behavior and design cell factories in several model organisms, but no models to date are currently available for hybrid species due to their more complex genetics and general lack of molecular data. In this study, we generated a bespoke GSMM, iSP_1513, for this industrial aneuploid hybrid Saccharomyces pastorianus, which takes into account the aneuploidy and functional redundancy from orthologous parental alleles. This model will (i) help understand the metabolic capabilities and adaptive potential of S. pastorianus (domestication processes), (ii) aid top-down predictions for strain development (industrial biotechnology), and (iii) allow predictions of evolutionary trajectories of metabolic pathways in aneuploid hybrids (evolutionary genetics).
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Genoma Fúngico , Redes y Vías Metabólicas , Saccharomyces , Saccharomyces/genética , Saccharomyces/metabolismo , Redes y Vías Metabólicas/genética , Genoma Fúngico/genética , Modelos Biológicos , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Evolución Molecular , Microbiología Industrial/métodosRESUMEN
Kazachstania bulderi is a non-conventional yeast species able to grow efficiently on glucose and δ-gluconolactone at low pH. These unique traits make K. bulderi an ideal candidate for use in sustainable biotechnology processes including low pH fermentations and the production of green chemicals including organic acids. To accelerate strain development with this species, detailed information of its genetics is needed. Here, by employing long read sequencing we report a high-quality phased genome assembly for three strains of K. bulderi species, including the type strain. The sequences were assembled into 12 chromosomes with a total length of 14 Mb, and the genome was fully annotated at structural and functional levels, including allelic and structural variants, ribosomal array and mating type locus. This high-quality reference genome provides a resource to advance our fundamental knowledge of biotechnologically relevant non-conventional yeasts and to support the development of genetic tools for manipulating such strains towards their use as production hosts in biotechnological processes.
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Saccharomycetales , Saccharomycetales/genética , Alelos , Biotecnología , Concentración de Iones de HidrógenoRESUMEN
More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.
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The initial growth rate of a yeast strain is a key parameter in the production of fermented beverages. Fast growth is linked with higher fermentative capacity and results in less slow and stuck fermentations unable to reach the expected final gravity. As concentrations of metabolites are in a constant state of flux, quantitative data on how growth rate affects the production of aromatic compounds becomes an important factor for brewers. Chemostats allow to set and keep a specific dilution rate throughout the fermentation and are ideal system to study the effect of growth on aroma production. In this study, we ran chemostats alongside batch and fed-batch cultures, compared volatile profiles detected at different growth rates, and identified those affected by the different feeding profiles. Specifically, we quantified six abundant aroma compounds produced in anaerobic glucose-limited continuous cultivations of S. cerevisiae at different dilution rates. We found that volatile production was affected by the growth rate in four out of six compounds assayed, with higher alcohols and esters following opposite trends. Batch and fed-batch fermentations were devised to study the extent by which the final concentration of volatile compounds is influenced by glucose availability. Compared with the batch system, fed-batch fermentations, where the yeast growth was artificially limited by a slow constant release of nutrients in the media, resulted in a significant increase in concentration of higher alcohols, mirroring the results obtained in continuous fermentations. This study paves the way to further process development optimization for the production of fermented beverages. IMPORTANCE The production of fermentation beverages will need to quickly adapt to changes in both the climate and customer demands, requiring the development of new strains and processes. Breakthroughs in the field are hindered by the limited knowledge on the interplay between physiology and aroma compound production in yeast. No quantitative data on how growth rate affects aroma profile is available in the literature to guide optimization of the complex flavors in fermented beverages. In this study, we exploited the chemostat system, alongside with batch and fed-batch cultures, to compare volatile profiles at different growth rates. We identified the aromatic compounds affected by the different feeding profiles and nutrient limitations. Moreover, we uncovered the correlation between yeast growth, esters, and higher alcohols production. This study showcases the potential of the application of feeding profiles for the manipulation of aroma in the craft beverage industry.
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Saccharomyces cerevisiae , Compuestos Orgánicos Volátiles , Saccharomyces cerevisiae/metabolismo , Odorantes , Fermentación , Técnicas de Cultivo Celular por Lotes , Alcoholes/metabolismo , Glucosa/metabolismo , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
The pathogenic fungus Aspergillus fumigatus is a major etiological agent of fungal invasive and chronic diseases affecting tens of millions of individuals worldwide. Draft genome sequences of two clinical isolates (Af293 and A1163) are commonly used as reference genomes for analyses of clinical and environmental strains. However, the reference sequences lack coverage of centromeres, an accurate sequence for ribosomal repeats, and a comprehensive annotation of chromosomal rearrangements such as translocations and inversions. Here, we used PacBio Single Molecule Real-Time (SMRT), Oxford Nanopore and Illumina HiSeq sequencing for de novo genome assembly and polishing of two laboratory reference strains of A. fumigatus, CEA10 (parental isolate of A1163) and its descendant A1160. We generated full length chromosome assemblies and a comprehensive telomere-to-telomere coverage for CEA10 and near complete assembly of A1160 including ribosomal repeats and the sequences of centromeres, which we discovered to be composed of long transposon elements. We envision these high-quality reference genomes will become fundamental resources to study A. fumigatus biology, pathogenicity and virulence, and to discover more effective treatments against diseases caused by this fungus.
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Aspergillus fumigatus , Hongos , Aspergillus fumigatus/genética , Elementos Transponibles de ADN/genética , Humanos , Análisis de Secuencia de ADN , Telómero/genéticaRESUMEN
The quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has proven difficult, even with the most sophisticated analytical technologies. Here, we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for the quantitation of low abundance proteins, and as proof of principle, we choose two interacting proteins involved in the fission of mitochondria in yeast, Fis1p and Mdv1p. In Saccharomyces cerevisiae, the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We use FCS in single GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures, with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism that controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Saccharomyces yeasts have evolved into an important model system to study mitonuclear incompatibilities, thanks to recent advances in the field of sequencing, yeast hybridisation and multigenerational breeding. Yeast hybrids contain two homologous proteomes but retain only one type of mitochondria allowing studies on the effect of mitochondria on phenotype and gene expression. Here, we discuss the recent developments in the growing field of yeast mitogenomics spanning from the impact that this organelle has in shaping yeast fitness and genome evolution to the dissection of molecular determinants of mitonuclear incompatibilities. Applying the state-of-the-art genetic tools to a broader range of natural yeast species from different environments will help progress the field and untap the mitochondrial potential in strain development.
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Hibridación Genética , Proteoma , Adaptación Fisiológica/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Fenotipo , Proteoma/genéticaRESUMEN
The lager yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and are divided into two broad groups, Group I and II. The two groups evolved from at least one common hybridisation event but have subsequently diverged with Group I strains losing many S. cerevisiae chromosomes while the Group II strains retain both sub-genomes. The complex genomes, containing orthologous alleles from the parental chromosomes, pose interesting questions regarding gene regulation and its impact on the fermentation properties of the strains. Superimposed on the presence of orthologous alleles are complexities of gene dosage due to the aneuploid nature of the genomes. We examined the contribution of the S. cerevisiae and S. eubayanus alleles to the gene expression patterns of representative Group I and II strains during fermentation. We show that the relative expression of S. cerevisiae and S. eubayanus orthologues is positively correlated with gene copy number. Despite the reduced S. cerevisiae content in the Group I strain, S. cerevisiae orthologues contribute to biochemical pathways upregulated during fermentation which may explain the retention of specific chromosomes in the strain. Conversely, S. eubayanus genes are significantly overrepresented in the upregulated gene pool in the Group II strain. Comparison of the transcription profiles of the strains during fermentation identified both common and unique gene expression patterns, with gene copy number being a dominant contributory factor. Thus, the aneuploid genomes create complex patterns of gene expression during fermentation with gene dosage playing a crucial role both within and between strains.
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Saccharomyces cerevisiae , Saccharomyces , Transcriptoma , Aneuploidia , Cerveza , Fermentación , Saccharomyces/genética , Saccharomyces cerevisiae/genética , Transcriptoma/genéticaRESUMEN
Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.
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Hybrids between species can harbor a combination of beneficial traits from each parent and may exhibit hybrid vigor, more readily adapting to new harsher environments. Interspecies hybrids are also sterile and therefore an evolutionary dead end unless fertility is restored, usually via auto-polyploidisation events. In the Saccharomyces genus, hybrids are readily found in nature and in industrial settings, where they have adapted to severe fermentative conditions. Due to their hybrid sterility, the development of new commercial yeast strains has so far been primarily conducted via selection methods rather than via further breeding. In this study, we overcame infertility by creating tetraploid intermediates of Saccharomyces interspecies hybrids to allow continuous multigenerational breeding. We incorporated nuclear and mitochondrial genetic diversity within each parental species, allowing for quantitative genetic analysis of traits exhibited by the hybrids and for nuclear-mitochondrial interactions to be assessed. Using pooled F12 generation segregants of different hybrids with extreme phenotype distributions, we identified quantitative trait loci (QTLs) for tolerance to high and low temperatures, high sugar concentration, high ethanol concentration, and acetic acid levels. We identified QTLs that are species specific, that are shared between species, as well as hybrid specific, in which the variants do not exhibit phenotypic differences in the original parental species. Moreover, we could distinguish between mitochondria-type-dependent and -independent traits. This study tackles the complexity of the genetic interactions and traits in hybrid species, bringing hybrids into the realm of full genetic analysis of diploid species, and paves the road for the biotechnological exploitation of yeast biodiversity.
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Variación Genética/genética , Sitios de Carácter Cuantitativo/genética , Saccharomyces/genética , Ácido Acético/metabolismo , Frío , Etanol/metabolismo , Fermentación/genética , Genoma Fúngico/genética , Mitocondrias/genética , Fenotipo , Azúcares/metabolismoRESUMEN
Saccharomyces pastorianus is a natural yeast evolved from different hybridization events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% has their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes expresses preferentially one parental allele over the other and that S. eubayanus-like alleles are significantly over-represented among the genes involved in the cold acclimatization. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes investigated in the hybrid are likely to be either exclusively chimeric or unispecific and that the redundancy is discouraged, a scenario that fits well with the gene balance hypothesis. This study offers the first overview of the transcriptional pattern of S. pastorianus and provides a rationalization for its unique industrial traits at the expression level.
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Genoma Fúngico , Saccharomyces cerevisiae , Saccharomyces , Alelos , Cerveza , Fermentación , Saccharomyces/genética , Saccharomyces cerevisiae/genética , TemperaturaRESUMEN
Hybridisation is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces genus, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in unhopped wort and at low temperatures, qualities necessary in good candidates for fermented bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft brewing and beverage industry in general. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while eliminating the hyper-attenuation characteristic and a more complex flavour profile. This study exploits the genetic diversity of yeasts and shows how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.
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Cerveza/análisis , Saccharomyces/genética , Saccharomyces/metabolismo , Cerveza/microbiología , Fermentación , Aromatizantes/análisis , Aromatizantes/metabolismo , Microbiología de Alimentos , Humanos , Hibridación Genética , Odorantes/análisis , Saccharomyces/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , GustoRESUMEN
Non-coding RNAs (ncRNAs), including the more recently identified Stable Unannotated Transcripts (SUTs) and Cryptic Unstable Transcripts (CUTs), are increasingly being shown to play pivotal roles in the transcriptional and post-transcriptional regulation of genes in eukaryotes. Here, we carried out a large-scale screening of ncRNAs in Saccharomyces cerevisiae, and provide evidence for SUT and CUT function. Phenotypic data on 372 ncRNA deletion strains in 23 different growth conditions were collected, identifying ncRNAs responsible for significant cellular fitness changes. Transcriptome profiles were assembled for 18 haploid ncRNA deletion mutants and 2 essential ncRNA heterozygous deletants. Guided by the resulting RNA-seq data we analysed the genome-wide dysregulation of protein coding genes and non-coding transcripts. Novel functional ncRNAs, SUT125, SUT126, SUT035 and SUT532 that act in trans by modulating transcription factors were identified. Furthermore, we described the impact of SUTs and CUTs in modulating coding gene expression in response to different environmental conditions, regulating important biological process such as respiration (SUT125, SUT126, SUT035, SUT432), steroid biosynthesis (CUT494, SUT053, SUT468) or rRNA processing (SUT075 and snR30). Overall, these data capture and integrate the regulatory and phenotypic network of ncRNAs and protein-coding genes, providing genome-wide evidence of the impact of ncRNAs on cellular homeostasis.
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Redes Reguladoras de Genes/genética , ARN no Traducido/genética , Transcripción Genética , Transcriptoma/genética , Regulación Fúngica de la Expresión Génica/genética , Genoma Fúngico , Haploidia , Fenotipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Both Saccharomyces and non-Saccharomyces yeast strains are of great importance for the fermentation industry, especially with the flourishing of craft breweries, which are driving current innovations. Non-conventional yeasts can produce novel beverages with attractive characteristics such as flavour, texture, and reduced alcohol content; however, they have been poorly explored. A new method for screening the fitness of conventional and non-conventional yeast libraries utilising robotic platforms and solidified media representing industrial conditions is proposed. As proof of concept, a library formed of 6 conventional and 17 non-conventional yeast strains was distributed in 96, 384 and 1536 arrays onto a YPD agar medium. Following this, the library was replicated in different conditions mimicking beer and cider fermentation conditions. The colony size was monitored over time, and fitness values measured in maximum pixels/h and maximum biomass were calculated. Significant differences in growth were observed in between the different strains and conditions. As examples, Candida milleri Y-7245 displayed good performance in wort conditions, and Kazachstania yakushimaensis Y-48837 stood out for its performance in apple juice. The method is proposed to be used as a pre-screening step when studying vast yeast libraries. This would enable interested parties to discover potential hits for further study at a low initial cost. Furthermore, this method can be used in other applications where the desired screening media can be solidified.
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The budding yeast has been extensively studied for its physiological performance in fermentative environments and, due to its remarkable plasticity, is used in numerous industrial applications like in brewing, baking and wine fermentations. Furthermore, thanks to its small and relatively simple eukaryotic genome, the molecular mechanisms behind its evolution and domestication are more easily explored. Considerable work has been directed into examining the industrial adaptation processes that shaped the genotypes of species and hybrids belonging to the Saccharomyces group, specifically in relation to beverage fermentation performances. A variety of genetic mechanisms are responsible for the yeast response to stress conditions, such as genome duplication, chromosomal re-arrangements, hybridization and horizontal gene transfer, and these genetic alterations are also contributing to the diversity in the Saccharomyces industrial strains. Here, we review the recent genetic and evolutionary studies exploring domestication and biodiversity of yeast strains.
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Genome-scale computational approaches are opening opportunities to model and predict favorable combination of traits for strain development. However, mining the genome of complex hybrids is not currently an easy task, due to the high level of redundancy and presence of homologous. For example, Saccharomyces pastorianus is an allopolyploid sterile yeast hybrid used in brewing to produce lager-style beers. The development of new yeast strains with valuable industrial traits such as improved maltose utilization or balanced flavor profiles are now a major ambition and challenge in craft brewing and distilling industries. Moreover, no genome annotation for most of these industrial strains have been published. Here, we developed HybridMine, a new user-friendly, open-source tool for functional annotation of hybrid aneuploid genomes of any species by predicting parental alleles including paralogs. Our benchmark studies showed that HybridMine produced biologically accurate results for hybrid genomes compared to other well-established software. As proof of principle, we carried out a comprehensive structural and functional annotation of complex yeast hybrids to enable system biology prediction studies. HybridMine is developed in Python, Perl, and Bash programming languages and is available in GitHub.
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Mitochondrial DNA (mtDNA) in yeast is biparentally inherited, but colonies rapidly lose one type of parental mtDNA, thus becoming homoplasmic. Therefore, hybrids between the yeast species possess two homologous nuclear genomes, but only one type of mitochondrial DNA. We hypothesise that the choice of mtDNA retention is influenced by its contribution to hybrid fitness in different environments, and the allelic expression of the two nuclear sub-genomes is affected by the presence of different mtDNAs in hybrids. Saccharomyces cerevisiae/S. uvarum hybrids preferentially retained S. uvarum mtDNA when formed on rich media at colder temperatures, while S. cerevisiae mtDNA was primarily retained on non-fermentable carbon source, at any temperature. Transcriptome data for hybrids harbouring different mtDNA showed a strong environmentally dependent allele preference, which was more important in respiratory conditions. Co-expression analysis for specific biological functions revealed a clear pattern of concerted allelic transcription within the same allele type, which supports the notion that the hybrid cell works preferentially with one set of parental alleles (or the other) for different cellular functions. Given that the type of mtDNA retained in hybrids affects both nuclear expression and fitness, it might play a role in driving hybrid genome evolution in terms of gene retention and loss.