RESUMEN
YEASTRACT+ (http://yeastract-plus.org/) is a tool for the analysis, prediction and modelling of transcription regulatory data at the gene and genomic levels in yeasts. It incorporates three integrated databases: YEASTRACT (http://yeastract-plus.org/yeastract/), PathoYeastract (http://yeastract-plus.org/pathoyeastract/) and NCYeastract (http://yeastract-plus.org/ncyeastract/), focused on Saccharomyces cerevisiae, pathogenic yeasts of the Candida genus, and non-conventional yeasts of biotechnological relevance. In this release, YEASTRACT+ offers upgraded information on transcription regulation for the ten previously incorporated yeast species, while extending the database to another pathogenic yeast, Candida auris. Since the last release of YEASTRACT+ (January 2020), a fourth database has been integrated. CommunityYeastract (http://yeastract-plus.org/community/) offers a platform for the creation, use, and future update of YEASTRACT-like databases for any yeast of the users' choice. CommunityYeastract currently provides information for two Saccharomyces boulardii strains, Rhodotorula toruloides NP11 oleaginous yeast, and Schizosaccharomyces pombe 972h-. In addition, YEASTRACT+ portal currently gathers 304 547 documented regulatory associations between transcription factors (TF) and target genes and 480 DNA binding sites, considering 2771 TFs from 11 yeast species. A new set of tools, currently implemented for S. cerevisiae and C. albicans, is further offered, combining regulatory information with genome-scale metabolic models to provide predictions on the most promising transcription factors to be exploited in cell factory optimisation or to be used as novel drug targets. The expansion of these new tools to the remaining YEASTRACT+ species is ongoing.
Asunto(s)
Programas Informáticos , Transcripción Genética , Levaduras , Bases de Datos Genéticas , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Levaduras/genéticaRESUMEN
The species Blastobotrys navarrensis Sesma and Ramirez was delineated based on the description of the single strain CBS 139.77T. Based on its phenotypic similarities to Blastobotrys proliferans, B. navarrensis CBS 139.77T was later considered a synonym of B. proliferans. In the present study, we isolated the yeast strain IST 508 (=PYCC 8784=CBS 16671) from the soil surrounding an olive tree in Ferreira do Alentejo, Portugal. The phylogenetic analysis of D1/D2 domain and ITS sequences from strain IST 508 indicates that is closely related to B. navarrensis and B. proliferans. Although strain IST 508 differs from B. navarrensis CBS 139.77T by 14 substitutions and 20 indels (6.6 % divergence) in the ITS sequence, no divergence was detected at the level of D1/D2 domain, mitochondrial small subunit rDNA, and cytochrome oxidase II sequences. On the other hand, strains IST 508 and CBS 139.77 differ from B. proliferans NRRL Y-17577T by eight substitutions (1.4 % divergence) in the D1/D2 domain sequence, by 16 substitutions (2.7 % divergence) in the cytochrome oxidase II sequence, and by 16 substitutions (3.7 % divergence) in the mitochondrial small subunit rDNA sequence. Due to the high number of variable phenotypic tests in B. proliferans and B. navarrensis, strains from the two species are difficult to distinguish. Contrasting with what is described for other Blastobotrys species, no differences were detected at the level of micromorphology between the two species. Nevertheless, based on the molecular differences between the two strains, CBS 139.77 and IST 508, and B. proliferans NRRL Y-17577T and their phylogenetic analysis, strains CBS 139.77 and IST 508 are from B. navarrensis and this species should be considered as an independent species and not a later synonym of B. proliferans. We propose an emended description of B. navarrensis.
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Complejo IV de Transporte de Electrones , Saccharomycetales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , ADN de Hongos/genética , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Complejo IV de Transporte de Electrones/genética , Ácidos Grasos/química , Técnicas de Tipificación Micológica , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The YEASTRACT+ information system (http://YEASTRACT-PLUS.org/) is a wide-scope tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in yeasts of biotechnological or human health relevance. YEASTRACT+ is a new portal that integrates the previously existing YEASTRACT (http://www.yeastract.com/) and PathoYeastract (http://pathoyeastract.org/) databases and introduces the NCYeastract (Non-Conventional Yeastract) database (http://ncyeastract.org/), focused on the so-called non-conventional yeasts. The information in the YEASTRACT database, focused on Saccharomyces cerevisiae, was updated. PathoYeastract was extended to include two additional pathogenic yeast species: Candida parapsilosis and Candida tropicalis. Furthermore, the NCYeastract database was created, including five biotechnologically relevant yeast species: Zygosaccharomyces baillii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica and Komagataella phaffii. The YEASTRACT+ portal gathers 289 706 unique documented regulatory associations between transcription factors (TF) and target genes and 420 DNA binding sites, considering 247 TFs from 10 yeast species. YEASTRACT+ continues to make available tools for the prediction of the TFs involved in the regulation of gene/genomic expression. In this release, these tools were upgraded to enable predictions based on orthologous regulatory associations described for other yeast species, including two new tools for cross-species transcription regulation comparison, based on multi-species promoter and TF regulatory network analyses.
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Biología Computacional/métodos , Bases de Datos Genéticas , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Genómica , Levaduras/genética , Sitios de Unión , Candida tropicalis/genética , Redes Reguladoras de Genes , Kluyveromyces/genética , Filogenia , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Programas Informáticos , Especificidad de la Especie , Factores de Transcripción/genética , Transcripción Genética , Yarrowia/genética , Zygosaccharomyces/genéticaRESUMEN
Responding to the recent interest of the yeast research community in non-Saccharomyces cerevisiae species of biotechnological relevance, the N.C.Yeastract (http://yeastract-plus.org/ncyeastract/) was associated to YEASTRACT + (http://yeastract-plus.org/). The YEASTRACT + portal is a curated repository of known regulatory associations between transcription factors (TFs) and target genes in yeasts. N.C.Yeastract gathers all published regulatory associations and TF-binding sites for Komagataellaphaffii (formerly Pichia pastoris), the oleaginous yeast Yarrowia lipolytica, the lactose fermenting species Kluyveromyces lactis and Kluyveromyces marxianus, and the remarkably weak acid-tolerant food spoilage yeast Zygosaccharomyces bailii. The objective of this review paper is to advertise the update of the existing information since the release of N.C.Yeastract in 2019, and to raise awareness in the community about its potential to help the day-to-day work on these species, exploring all the information available in the global YEASTRACT + portal. Using simple and widely used examples, a guided exploitation is offered for several tools: (i) inference of orthologous genes; (ii) search for putative TF binding sites and (iii) inter-species comparison of transcription regulatory networks and prediction of TF-regulated networks based on documented regulatory associations available in YEASTRACT + for well-studied species. The usage potentialities of the new CommunityYeastract platform by the yeast community are also discussed.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Yarrowia , Bases de Datos Genéticas , Genómica , Saccharomyces cerevisiae , Levaduras/genéticaRESUMEN
Beer production is predominantly carried out by Saccharomyces species, such as S. cerevisiae and S. pastorianus. However, the introduction of non-Saccharomyces yeasts in the brewing process is now seen as a promising strategy to improve and differentiate the organoleptic profile of beer. In this study, 17 non-Saccharomyces strains of 12 distinct species were isolated and submitted to a preliminary sensory evaluation to determine their potential for beer bioflavouring. Hanseniaspora guilliermondii IST315 and H. opuntiae IST408 aroma profiles presented the highest acceptability and were described as having 'fruity' and 'toffee' notes, respectively. Their presence in mixed-culture fermentations with S. cerevisiae US-05 did not influence attenuation and ethanol concentration of beer but had a significant impact in its volatile composition. Notably, while both strains reduced the total amount of ethyl esters, H. guilliermondii IST315 greatly increased the concentration of acetate esters, especially when sequentially inoculated, leading to an 8.2-fold increase in phenylethyl acetate ('rose', 'honey' aroma) in the final beverage. These findings highlight the importance of non-Saccharomyces yeasts in shaping the aroma profile of beer and suggest a role for Hanseniaspora spp. in improving it.
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Cerveza/análisis , Hanseniaspora/metabolismo , Saccharomyces cerevisiae/metabolismo , Cerveza/microbiología , Técnicas de Cocultivo , Etanol/metabolismo , Fermentación , Aromatizantes/análisis , Aromatizantes/metabolismo , Humanos , Odorantes/análisis , Gusto , Compuestos Orgánicos Volátiles/análisis , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT-www.yeastract.com) information system has been, for 11 years, a key tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in Saccharomyces cerevisiae. Since its last update in June 2017, YEASTRACT includes approximately 163000 regulatory associations between transcription factors (TF) and target genes in S. cerevisiae, based on more than 1600 bibliographic references; it also includes 247 specific DNA binding consensus recognized by 113 TFs. This release of the YEASTRACT database provides new visualization tools to visualize each regulatory network in an interactive fashion, enabling the user to select and observe subsets of the network such as: (i) considering only DNA binding evidence or both DNA binding and expression evidence; (ii) considering only either positive or negative regulatory associations; or (iii) considering only one set of related environmental conditions. A further tool to observe TF regulons is also offered, enabling a clear-cut understanding of the exact meaning of the available data. We believe that with this new version, YEASTRACT will improve its role as an open web resource instrumental for Yeast Biologists and Systems Biology researchers.
Asunto(s)
Bases de Datos Genéticas , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Transcripción Genética , Regulón , Factores de Transcripción/metabolismoRESUMEN
Zygosaccharomyces bailii and two closely related species, Z. parabailii and Z. pseudobailii ("Z. bailii species complex", "Z. bailii sensu lato" or simply "Z. bailii (s.l.)"), are frequently implicated in the spoilage of acidified preserved foods and beverages due to their tolerance to very high concentrations of weak acids used as food preservatives. The recent sequencing and annotation of these species' genomes have clarified their genomic organization and phylogenetic relationship, which includes events of interspecies hybridization. Mechanistic insights into their adaptation and tolerance to weak acids (e.g., acetic and lactic acids) are also being revealed. Moreover, the potential of Z. bailii (s.l.) to be used in industrial biotechnological processes as interesting cell factories for the production of organic acids, reduction of the ethanol content, increase of alcoholic beverages aroma complexity, as well as of genetic source for increasing weak acid resistance in yeast, is currently being considered. This chapter includes taxonomical, ecological, physiological, and biochemical aspects of Z. bailii (s.l.). The focus is on the exploitation of physiological genomics approaches that are providing the indispensable holistic knowledge to guide the effective design of strategies to overcome food spoilage or the rational exploitation of these yeasts as promising cell factories.
Asunto(s)
Ácidos/metabolismo , Genómica , Zygosaccharomyces/genética , Zygosaccharomyces/metabolismo , Ácidos/farmacología , Filogenia , Zygosaccharomyces/clasificación , Zygosaccharomyces/efectos de los fármacosRESUMEN
BACKGROUND: The food spoilage yeast species Zygosaccharomyces bailii exhibits an extraordinary capacity to tolerate weak acids, in particular acetic acid. In Saccharomyces cerevisiae, the transcription factor Haa1 (ScHaa1) is considered the main player in genomic expression reprogramming in response to acetic acid stress, but the role of its homologue in Z. bailii (ZbHaa1) is unknown. RESULTS: In this study it is demonstrated that ZbHaa1 is a ScHaa1 functional homologue by rescuing the acetic acid susceptibility phenotype of S. cerevisiae haa1Δ. The disruption of ZbHAA1 in Z. bailii IST302 and the expression of an extra ZbHAA1 copy confirmed ZbHAA1 as a determinant of acetic acid tolerance. ZbHaa1 was found to be required for acetic acid stress-induced transcriptional activation of Z. bailii genes homologous to ScHaa1-target genes. An evolutionary analysis of the Haa1 homologues identified in 28 Saccharomycetaceae species genome sequences, including Z bailii, was carried out using phylogenetic and gene neighbourhood approaches. Consistent with previous studies, this analysis revealed a group containing pre-whole genome duplication species Haa1/Cup2 single orthologues, including ZbHaa1, and two groups containing either Haa1 or Cup2 orthologues from post-whole genome duplication species. S. cerevisiae Cup2 (alias Ace1) is a transcription factor involved in response and tolerance to copper stress. Taken together, these observations led us to hypothesize and demonstrate that ZbHaa1 is also involved in copper-induced transcriptional regulation and copper tolerance. CONCLUSIONS: The transcription factor ZbHaa1 is required for adaptive response and tolerance to both acetic acid and copper stresses. The subfunctionalization of the single ancestral Haa1/Cup2 orthologue that originated Haa1 and Cup2 paralogues after whole genome duplication is proposed.
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Ácido Acético/metabolismo , Cobre/metabolismo , Proteínas Fúngicas/metabolismo , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Zygosaccharomyces/metabolismo , Adaptación Biológica , Clonación Molecular , Evolución Molecular , Expresión Génica , Filogenia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Zygosaccharomyces/genéticaRESUMEN
Zygosaccharomyces bailii is one of the most problematic spoilage yeast species found in the food and beverage industry particularly in acidic products, due to its exceptional resistance to weak acid stress. This article describes the annotation of the genome sequence of Z. bailii IST302, a strain recently proven to be amenable to genetic manipulations and physiological studies. The work was based on the annotated genomes of strain ISA1307, an interspecies hybrid between Z. bailii and a closely related species, and the Z. bailii reference strain CLIB 213T. The resulting genome sequence of Z. bailii IST302 is distributed through 105 scaffolds, comprising a total of 5142 genes and a size of 10.8 Mb. Contrasting with CLIB 213T, strain IST302 does not form cell aggregates, allowing its manipulation in the laboratory for genetic and physiological studies. Comparative cell cycle analysis with the haploid and diploid Saccharomyces cerevisiae strains BY4741 and BY4743, respectively, suggests that Z. bailii IST302 is haploid. This is an additional trait that makes this strain attractive for the functional analysis of non-essential genes envisaging the elucidation of mechanisms underlying its high tolerance to weak acid food preservatives, or the investigation and exploitation of the potential of this resilient yeast species as cell factory.
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Adaptación Fisiológica/genética , Ingeniería Genética/métodos , Genoma Fúngico , Haploidia , Zygosaccharomyces/genética , Mapeo Cromosómico , Cruzamientos Genéticos , Tecnología de Alimentos , Tamaño del Genoma , Humanos , Concentración de Iones de Hidrógeno , Anotación de Secuencia Molecular , Estrés Fisiológico , Secuenciación Completa del Genoma , Zygosaccharomyces/metabolismoRESUMEN
The YEASTRACT (http://www.yeastract.com) information system is a tool for the analysis and prediction of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in June 2013, this database contains over 200,000 regulatory associations between transcription factors (TFs) and target genes, including 326 DNA binding sites for 113 TFs. All regulatory associations stored in YEASTRACT were revisited and new information was added on the experimental conditions in which those associations take place and on whether the TF is acting on its target genes as activator or repressor. Based on this information, new queries were developed allowing the selection of specific environmental conditions, experimental evidence or positive/negative regulatory effect. This release further offers tools to rank the TFs controlling a gene or genome-wide response by their relative importance, based on (i) the percentage of target genes in the data set; (ii) the enrichment of the TF regulon in the data set when compared with the genome; or (iii) the score computed using the TFRank system, which selects and prioritizes the relevant TFs by walking through the yeast regulatory network. We expect that with the new data and services made available, the system will continue to be instrumental for yeast biologists and systems biology researchers.
Asunto(s)
ADN de Hongos/metabolismo , Bases de Datos Genéticas , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Sitios de Unión , Redes Reguladoras de Genes , Genoma Fúngico , Internet , Elementos Reguladores de la Transcripción , Saccharomyces cerevisiae/metabolismo , Programas InformáticosRESUMEN
BACKGROUND: Zygosaccharomyces bailii is considered the most problematic acidic food spoilage yeast species due to its exceptional capacity to tolerate high concentrations of weak acids used as fungistatic preservatives at low pH. However, the mechanisms underlying its intrinsic remarkable tolerance to weak acids remain poorly understood. The identification of genes and mechanisms involved in Z. bailii acetic acid tolerance was on the focus of this study. For this, a genomic library from the highly acetic acid tolerant hybrid strain ISA1307, derived from Z. bailii and a closely related species and isolated from a sparkling wine production plant, was screened for acetic acid tolerance genes. This screen was based on the transformation of an acetic acid susceptible Saccharomyces cerevisiae mutant deleted for the gene encoding the acetic acid resistance determinant transcription factor Haa1. RESULTS: The expression of 31 different DNA inserts from ISA1307 strain genome was found to significantly increase the host cell tolerance to acetic acid. The in silico analysis of these inserts was facilitated by the recently available genome sequence of this strain. In total, 65 complete or truncated ORFs were identified as putative determinants of acetic acid tolerance and an S. cerevisiae gene homologous to most of them was found. These include genes involved in cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription. The role of strong candidates in Z. bailii and S. cerevisiae acetic acid tolerance was confirmed based on homologous and heterologous expression analyses. CONCLUSIONS: ISA1307 genes homologous to S. cerevisiae genes GYP8, WSC4, PMT1, KTR7, RKR1, TIF3, ILV3 and MSN4 are proposed as strong candidate determinants of acetic acid tolerance. The ORF ZBAI_02295 that contains a functional domain associated to the uncharacterised integral membrane proteins of unknown function of the DUP family is also suggested as a relevant tolerance determinant. The genes ZbMSN4 and ZbTIF3, encoding a putative stress response transcription factor and a putative translation initiation factor, were confirmed as determinants of acetic acid tolerance in both Z. bailii and S. cerevisiae. This study provides valuable indications on the cellular components, pathways and processes to be targeted in order to control food spoilage by the highly acetic acid tolerant Z. bailii and Z. bailii-derived strains. Additionally, this information is essential to guide the improvement of yeast cells robustness against acetic acid if the objective is their use as cell factories.
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Ácido Acético/farmacología , Adaptación Biológica/genética , Genes Fúngicos , Zygosaccharomyces/efectos de los fármacos , Zygosaccharomyces/genética , Mapeo Cromosómico , Biología Computacional/métodos , Eliminación de Gen , Expresión Génica , Prueba de Complementación Genética , Biblioteca Genómica , Mutación , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/genéticaRESUMEN
Methanol is a promising feedstock for biomanufacturing, but the efficiency of methanol-based bioprocesses is limited by the low rate of methanol utilization pathways and methanol toxicity. Yeast diversity is an attractive biological resource to develop efficient bioprocesses since any effort with strain improvement is more deserving if applied to innate robust strains with relevant catabolic and biosynthetic potential. The present study is in line with such rational and describes the isolation and molecular identification of seven isolates of the methylotrophic species Candida boidinii from waters derived from the traditional curation of olives, in different years, and from contaminated superficial soil near fuel stations. The yeast microbiota from those habitats was also characterized. The four C. boidinii isolates obtained from the curation of olives' water exhibited significantly higher maximum specific growth rates (range 0.15-0.19 h-1), compared with the three isolates obtained from the fuel contaminated soils (range 0.05-0.06 h-1) when grown on methanol as the sole C-source (1% (v/v), in shake flasks, at 30°C). The isolates exhibit significant robustness towards methanol toxicity that increases as the cultivation temperature decreases from 30°C to 25°C. The better methanol-based growth performance exhibited by C. boidinii isolates from olives´ soaking waters could not be essentially attributed to higher methanol tolerance. These methanol-efficient catabolizing isolates are proposed as a promising platform to develop methanol-based bioprocesses.
RESUMEN
BACKGROUND: The expression and activity of the different Saccharomyces cerevisiae hexose uptake systems (Hxt) and the kinetics of glucose uptake are considered essential to industrial alcoholic fermentation performance. However, the dynamics of glucose uptake kinetics during the different stages of fermentation, depending on glucose and nitrogen availability, is very poorly characterized. The objective of the present work was to examine thoroughly the alterations occurring in glucose uptake kinetics during alcoholic fermentation, by the wine strain S. cerevisiae PYCC 4072, of a synthetic grape juice basal medium with either a limiting or non-limiting initial nitrogen concentration and following nitrogen supplementation of the nitrogen-depleted sluggish fermentation. RESULTS: Independently of the initial concentration of the nitrogen source, glucose transport capacity is maximal during the early stages of fermentation and presumably sustained by the low-affinity and high-capacity glucose transporter Hxt1p. During nitrogen-limited sluggish fermentation, glucose uptake capacity was reduced to approximately 20% of its initial values (Vmax = 4.9 ± 0.8 compared to 21.9 ± 1.2 µmol h⻹ 10â»8 cells), being presumably sustained by the low-affinity glucose transporter Hxt3p (considering the calculated Km = 39.2 ± 8.6 mM). The supplementation of the sluggish fermentation broth with ammonium led to the increase of glucose transport capacity associated to the expression of different glucose uptake systems with low and high affinities for glucose (Km = 58.2 ± 9.1 and 2.7 ± 0.4 mM). A biclustering analysis carried out using microarray data, previously obtained for this yeast strain transcriptional response to equivalent fermentation conditions, indicates that the activation of the expression of genes encoding the glucose transporters Hxt2p (during the transition period to active fermentation) and Hxt3p, Hxt4p, Hxt6p and Hxt7p (during the period of active fermentation) may have a major role in the recovery of glucose uptake rate following ammonium supplementation. These results suggest a general derepression of the glucose-repressible HXT genes and are consistent with the downregulation of Mig1p and Rgt1p. CONCLUSIONS: Although reduced, glucose uptake rate during nitrogen-limited fermentation is not abrogated. Following ammonium supplementation, sluggish fermentation recovery is associated to the increase of glucose uptake capacity, related to the de novo synthesis of glucose transporters with different affinity for glucose and capacity, presumably of Hxt2p, Hxt3p, Hxt4p, Hxt6p and Hxt7p. This study is a contribution to the understanding of yeast response to different stages of alcoholic fermentation at the level of glucose uptake kinetics, in particular under nitrogen limitation or replenish, which is useful knowledge to guide fermentation practices.
Asunto(s)
Etanol/metabolismo , Glucosa/metabolismo , Nitrógeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Fermentación , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Cinética , Compuestos de Amonio Cuaternario/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The ascomycetous yeast Candida membranifaciens has been isolated from diverse habitats, including humans, insects, and environmental sources, exhibiting a remarkable ability to use different carbon sources that include pentoses, melibiose, and inulin. In this study, we isolated four C. membranifaciens strains from soil and investigated their potential to overproduce riboflavin. C. membranifaciens IST 626 was found to produce the highest concentrations of riboflavin. The volumetric production of this vitamin was higher when C. membranifaciens IST 626 cells were cultured in a commercial medium without iron and when xylose was the available carbon source compared to the same basal medium with glucose. Supplementation of the growth medium with 2 g/L glycine favored the metabolization of xylose, leading to biomass increase and consequent enhancement of riboflavin volumetric production that reached 120 mg/L after 216 h of cultivation. To gain new insights into the molecular basis of riboflavin production and carbon source utilization in this species, the first annotated genome sequence of C. membranifaciens is reported in this article, as well as the result of a comparative genomic analysis with other relevant yeast species. A total of 5619 genes were predicted to be present in C. membranifaciens IST 626 genome sequence (11.5 Mbp). Among them are genes involved in riboflavin biosynthesis, iron homeostasis, and sugar uptake and metabolism. This work put forward C. membranifaciens IST 626 as a riboflavin overproducer and provides valuable molecular data for future development of superior producing strains capable of using the wide range of carbon sources, which is a characteristic trait of the species.
RESUMEN
Agro-industrial residues are low-cost carbon sources (C-sources) for microbial growth and production of value-added bioproducts. Among the agro-industrial residues available, those rich in pectin are generated in high amounts worldwide from the sugar industry or the industrial processing of fruits and vegetables. Sugar beet pulp (SBP) hydrolysates contain predominantly the neutral sugars d-glucose, l-arabinose and d-galactose, and the acidic sugar d-galacturonic acid. Acetic acid is also present at significant concentrations since the d-galacturonic acid residues are acetylated. In this study, we have examined and optimized the performance of a Rhodotorula mucilaginosa strain, isolated from SBP and identified at the molecular level during this work. This study was extended to another oleaginous red yeast species, R. toruloides, envisaging the full utilization of the C-sources from SBP hydrolysate (at pH 5.0). The dual role of acetic acid as a carbon and energy source and as a growth and metabolism inhibitor was examined. Acetic acid prevented the catabolism of d-galacturonic acid and l-arabinose after the complete use of the other C-sources. However, d-glucose and acetic acid were simultaneously and efficiently metabolized, followed by d-galactose. SBP hydrolysate supplementation with amino acids was crucial to allow d-galacturonic acid and l-arabinose catabolism. SBP valorization through the production of lipids and carotenoids by Rhodotorula strains, supported by complete catabolism of the major C-sources present, looks promising for industrial implementation.
RESUMEN
BACKGROUND: Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. RESULTS: The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. CONCLUSIONS: Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are novel candidate genes for genetic engineering to obtain more robust yeast strains against acetic acid toxicity. Among these genes there are number of transcription factors that are documented regulators of a large percentage of the genes found to exert protection against acetic acid thus being considered interesting targets for subsequent genetic engineering. The increase of potassium concentration in the growth medium was found to improve the expression of maximal tolerance to acetic acid, consistent with the idea that the adequate manipulation of nutrient concentration of industrial growth medium can be an interesting strategy to surpass the deleterious effects of this weak acid in yeast cells.
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Ácido Acético/metabolismo , Genoma Fúngico , Estudio de Asociación del Genoma Completo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Etanol/metabolismo , Fermentación , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Quinine has been employed in the treatment of malaria for centuries and is still used against severe Plasmodium falciparum malaria. However, its interactions with the parasite remain poorly understood and subject to debate. In this study, we used the Saccharomyces cerevisiae eukaryotic model to better understand quinine's mode of action and the mechanisms underlying the cell response to the drug. We obtained a transcriptomic profile of the yeast's early response to quinine, evidencing a marked activation of genes involved in the low-glucose response (e.g., CAT8, ADR1, MAL33, MTH1, and SNF3). We used a low inhibitory quinine concentration with no detectable effect on plasma membrane function, consistent with the absence of a general nutrient starvation response and suggesting that quinine-induced glucose limitation is a specific response. We have further shown that transport of [(14)C]glucose is inhibited by quinine, with kinetic data indicating competitive inhibition. Also, tested mutant strains deleted for genes encoding high- and low-affinity hexose transporters (HXT1 to HXT5, HXT8, and HXT10) exhibit resistance phenotypes, correlating with reduced levels of quinine accumulation in the mutants examined. These results suggest that the hexose transporters are facilitators of quinine uptake in S. cerevisiae, possibly through a competitive inhibition mechanism. Interestingly, P. falciparum is highly dependent on glucose uptake, which is mediated by the single-copy transporter PfHT1, a protein with high homology to yeast's hexose transporters. We propose that PfHT1 is an interesting candidate quinine target possibly involved in quinine import in P. falciparum, an uptake mechanism postulated in recent studies to occur through a still-unidentified importer(s).
Asunto(s)
Transporte Biológico/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Quinina/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transporte Biológico/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/genética , Modelos Biológicos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The sugar porter family in yeasts encompasses a wide variety of transporters including the hexose transporters and glucose sensors. We analysed a total of 75 members from both groups in nine hemiascomycetous species, with complete and well-annotated genomes: Saccharomyces cerevisiae, Candida glabrata, Zygosaccharomyces rouxii, Kluyveromyces thermotolerans, Saccharomyces kluyverii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii and Yarrowia lipolytica. We present a model for the evolution of the hexose transporters and glucose sensors, supported by two types of complementary evidences: phylogeny and neighbourhood analysis. Five lineages of evolution were identified and discussed according to different mechanisms of gene evolution: lineage A for HXT1, HXT3, HXT4, HXT5, HXT6 and HXT7; lineage B for HXT2 and HXT10; lineage C for HXT8; lineage D for HXT14; and lineage E for SNF3 and RGT2.
Asunto(s)
Ascomicetos/genética , Proteínas de Transporte de Monosacáridos/genética , Filogenia , Receptores de Superficie Celular/genética , Levaduras/genética , Análisis por Conglomerados , Evolución Molecular , Homología de Secuencia de AminoácidoRESUMEN
The non-conventional yeast species Zygosaccharomyces bailii is remarkably tolerant to acetic acid, a highly important microbial inhibitory compound in Food Industry and Biotechnology. ZbHaa1 is the functional homologue of S. cerevisiae Haa1 and a bifunctional transcription factor able to modulate Z. bailii adaptive response to acetic acid and copper stress. In this study, RNA-Seq was used to investigate genomic transcription changes in Z. bailii during early response to sublethal concentrations of acetic acid (140 mM, pH 4.0) or copper (0.08 mM) and uncover the regulatory network activated by these stresses under ZbHaa1 control. Differentially expressed genes in response to acetic acid exposure (297) are mainly related with the tricarboxylic acid cycle, protein folding and stabilization and modulation of plasma membrane composition and cell wall architecture, 17 of which, directly or indirectly, ZbHaa1-dependent. Copper stress induced the differential expression of 190 genes mainly involved in the response to oxidative stress, 15 ZbHaa1-dependent. This study provides valuable mechanistic insights regarding Z. bailii adaptation to acetic acid or copper stress, as well as useful information on transcription regulatory networks in pre-whole genome duplication (WGD) (Z. bailii) and post-WGD (S. cerevisiae) yeast species, contributing to the understanding of transcriptional networks' evolution in yeasts.
Asunto(s)
Ácido Acético/farmacología , Sulfato de Cobre/farmacología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Zygosaccharomyces/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Estrés Fisiológico/efectos de los fármacos , Zygosaccharomyces/genética , Zygosaccharomyces/metabolismoRESUMEN
Acetic acid is an important microbial growth inhibitor in the food industry; it is used as a preservative in foods and beverages and is produced during normal yeast metabolism in biotechnological processes. Acetic acid is also a major inhibitory compound present in lignocellulosic hydrolysates affecting the use of this promising carbon source for sustainable bioprocesses. Although the molecular mechanisms underlying Saccharomyces cerevisiae response and adaptation to acetic acid have been studied for years, only recently they have been examined in more detail in Zygosaccharomyces bailii. However, due to its remarkable tolerance to acetic acid and other weak acids this yeast species is a major threat in the spoilage of acidic foods and beverages and considered as an interesting alternative cell factory in Biotechnology. This review paper emphasizes genome-wide strategies that are providing global insights into the molecular targets, signaling pathways and mechanisms behind S. cerevisiae and Z. bailii tolerance to acetic acid, and extends this information to other weak acids whenever relevant. Such comprehensive perspective and the knowledge gathered in these two yeast species allowed the identification of candidate molecular targets, either for the design of effective strategies to overcome yeast spoilage in acidic foods and beverages, or for the rational genome engineering to construct more robust industrial strains. Examples of successful applications are provided.