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1.
Sci Rep ; 10(1): 21779, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311634

RESUMO

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.


Assuntos
/química , Glicoproteína da Espícula de Coronavírus/química , Humanos , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomycetales/química , Saccharomycetales/genética , Saccharomycetales/metabolismo , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/genética
2.
PLoS Biol ; 18(11): e3000917, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33180788

RESUMO

The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell-cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit.


Assuntos
Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Divisão do Núcleo Celular/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mitose/fisiologia , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomycetales/genética , Saccharomycetales/metabolismo , Fuso Acromático/fisiologia
3.
Sci Rep ; 10(1): 16314, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004914

RESUMO

Lachancea kluyveri, a weak Crabtree positive yeast, has been extensively studied for its unique URC pyrimidine catabolism pathway. It produces more biomass than Saccharomyces cerevisiae due to the underlying weak Crabtree effect and resorts to fermentation only in oxygen limiting conditions that renders it as a suitable industrial host. The yeast also produces ethyl acetate as a major overflow metabolite in aerobic conditions. Here, we report the first genome-scale metabolic model, iPN730, of L. kluyveri comprising of 1235 reactions, 1179 metabolites, and 730 genes distributed in 8 compartments. The in silico viability in different media conditions and the growth characteristics in various carbon sources show good agreement with experimental data. Dynamic flux balance analysis describes the growth dynamics, substrate utilization and product formation kinetics in various oxygen-limited conditions. We have also demonstrated the effect of switching carbon sources on the production of ethyl acetate under varying oxygen uptake rates. A phenotypic phase plane analysis described the energetic cost penalty of ethyl acetate and ethanol production on the specific growth rate of L. kluyveri. We generated the context specific models of L. kluyveri growing on uracil or ammonium salts as the sole nitrogen source. Differential flux calculated using flux variability analysis helped us in highlighting pathways like purine, histidine, riboflavin and pyrimidine metabolism associated with uracil degradation. The genome-scale metabolic construction of L. kluyveri will provide a better understanding of metabolism behind ethyl acetate production as well as uracil catabolism (pyrimidine degradation) pathway. iPN730 is an addition to genome-scale metabolic models of non-conventional yeasts that will facilitate system-wide omics analysis to understand fungal metabolic diversity.


Assuntos
Genoma Fúngico/genética , Saccharomycetales/genética , Acetatos/metabolismo , Simulação por Computador , Metabolismo Energético , Etanol/metabolismo , Genes Fúngicos/genética , Redes e Vias Metabólicas/genética , Modelos Biológicos , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo
4.
PLoS One ; 15(9): e0239432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946508

RESUMO

Only quite recently, we have shown that yeast strains Clavispora lusitaniae 146 and Pichia fermentans 27 can act as efficient biocontrol agents for combating postharvest fungal diseases in lemons. During postharvest and storage conditions, microorganisms are subject to different stress factors that could affect both their survival and their protective capacity. Understanding the tolerance of yeasts to environmental stress factors could support the future development and commercial application of biological control formulations based on such organisms. Thus, the impact of different stressors on the viability and protection efficiency of C. lusitaniae strain 146 and P. fermentans strain 27 was evaluated, and the yeasts were subjected to oxidative stress, thermal treatments, exposure to NaOCl, osmotic stress, and ultraviolet irradiation. Candida oleophila strain O served as the reference control. C. lusitaniae 146 was more resistant to H2O2 in plate assays; however, in liquid media there was no significant difference to the other strains. Strain 146 was less affected by NaOCl, being able to survive with 300 ppm. P. fermentans 27 was the strain most heavily affected by osmotic pressure, while strains 146 and strain O showed a similar adaptation. UV-B irradiation severely affected C. oleophila and P. fermentans, while C. lusitaniae was the most resistant. Strains 146 and 27 were similarly tolerant to thermal shocks, compared to the reference strain, which was less viable. In in vivo tests, exposure to 10 mM H2O2, 45°C or 200 ppm NaOCl prior to fruit inoculation, reduced the antagonistic activity against the pathogen Penicillium digitatum. However, in no case was the biocontrol efficiency reduced to less than 50%. As C. lusitaniae 146 demonstrated a great potential to combat P. digitatum under a wide range of conditions, the organism is a promising candidate as an effective and valuable alternative to toxic fungicides.


Assuntos
Citrus/microbiologia , Viabilidade Microbiana , Saccharomycetales/fisiologia , Citrus/crescimento & desenvolvimento , Estresse Oxidativo , Controle Biológico de Vetores , Saccharomycetales/metabolismo , Temperatura
5.
Nucleic Acids Res ; 48(15): 8243-8254, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32720681

RESUMO

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes - pho1, pho84, and tgp1 - that are repressed under phosphate-replete conditions by 5' flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor 'precocious' lncRNA 3'-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Longo não Codificante/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Transcrição Genética , Homeostase , Nutrientes/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , RNA Mensageiro , Regulon , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
6.
Nat Commun ; 11(1): 3494, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661402

RESUMO

Cellular processes are inherently noisy, and the selection for accurate responses in presence of noise has likely shaped signalling networks. Here, we investigate the trade-off between accuracy of information transmission and its energetic cost for a mitogen-activated protein kinase (MAPK) signalling cascade. Our analysis of the pheromone response pathway of budding yeast suggests that dose-dependent induction of the negative transcriptional feedbacks in this network maximizes the information per unit energetic cost, rather than the information transmission capacity itself. We further demonstrate that futile cycling of MAPK phosphorylation and dephosphorylation has a measurable effect on growth fitness, with energy dissipation within the signalling cascade thus likely being subject to evolutionary selection. Considering optimization of accuracy versus the energetic cost of information processing, a concept well established in physics and engineering, may thus offer a general framework to understand the regulatory design of cellular signalling systems.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
7.
Nat Commun ; 11(1): 2952, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528053

RESUMO

The formation and maintenance of subcellular structures and organelles with a well-defined size is a key requirement for cell function, yet our understanding of the underlying size control mechanisms is limited. While budding yeast cell polarization and subsequent assembly of a septin ring at the site of bud formation has been successfully used as a model for biological self-assembly processes, the mechanisms that set the size of the septin ring at the bud neck are unknown. Here, we use live-cell imaging and genetic manipulation of cell volume to show that the septin ring diameter increases with cell volume. This cell-volume-dependence largely accounts for modulations of ring size due to changes in ploidy and genetic manipulation of cell polarization. Our findings suggest that the ring diameter is set through the dynamic interplay of septin recruitment and Cdc42 polarization, establishing it as a model for size homeostasis of self-assembling organelles.


Assuntos
Saccharomycetales/citologia , Saccharomycetales/metabolismo , Biologia Celular , Divisão Celular/fisiologia , Crescimento Celular , Polaridade Celular/fisiologia , Tamanho Celular
8.
Food Microbiol ; 91: 103497, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32539965

RESUMO

In this work, Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, and Wickerhanomyces anomalous Y12, all of them previously isolated from fermented table olive biofilms, were used (alone or in combination) as multifunctional starters for Manzanilla Spanish-style green table olive fermentations. Their performances were evaluated through the changes in the key physico-chemical and microbiological parameters, correlation between AI-2 production and biofilm formation, inoculum imposition, metataxonomic analysis and sensory characteristics of the finished products. Inoculation only with lactic acid bacteria (LAB) strains led to higher titratable acidities and lower pH values than the spontaneous fermentation (non-inoculated control), mainly during the first steps of processing. However, the sequential inoculation of the yeast and then the combination of the 3 LAB strains showed the most favourable evolution. LPG1 strain and, particularly Lp13, were excellent biofilms former and showed the major imposition on the fruit epidermis, as corroborated by rep-PCR analysis. Production of AI-2 was lower in the treatment inoculated exclusively with yeast Y12 but had the highest presence in the sequential yeast-LAB inoculum, with its maximum concentration and maximum LAB population on fruits (19th days) strongly related. Metataxonomic analysis of the biofilms at the end of the fermentation revealed, in addition to Lactobacillus, high proportions of sequences from genera Marinilactobacillus, Alkalibacterium, Halolactobacillus, and low levels of Halomonas and Aerococcus. Compositional data analysis of the omics data revealed that Lpl15 was scarcely efficient for controlling the spontaneous microbiota since its treatment presented the highest proportions of Aerococcus genus. Finally, the sensory analysis showed similar characteristics for the treatment inoculated with LPG1 and the spontaneous process, with olives inoculated with the yeast (alone or in combination with Lactobacillus strains) showing attractive scores. Then, inoculation of Spanish-style table olive fermentations with a sequential yeast and LAB combination could be an advisable practice.


Assuntos
Alimentos e Bebidas Fermentados/microbiologia , Lactobacillus/metabolismo , Olea/microbiologia , Saccharomycetales/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Técnicas de Cocultura , Fermentação , Alimentos e Bebidas Fermentados/análise , Microbiologia de Alimentos , Frutas/microbiologia , Homosserina/análogos & derivados , Homosserina/análise , Homosserina/biossíntese , Lactobacillus/classificação , Lactobacillus/crescimento & desenvolvimento , Lactonas/análise , Microbiota/genética , Saccharomycetales/crescimento & desenvolvimento
9.
J Ind Microbiol Biotechnol ; 47(4-5): 437-448, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32377991

RESUMO

Sophorolipids (SLs) are surface-active molecules produced by the non-pathogenic yeast Starmerella bombicola CGMCC 1576. Several genes involved in the synthesis of SLs have been identified. However, the regulation mechanism of the synthesis pathway for SLs has not been investigated. We recently discovered a protein in S. bombicola, which is structurally related to Yarrowia lipolytica YlBro1. To identify the function of the protein SbBro1 in S. bombicola, the deletion, overexpression, and complementary mutant strains were constructed. We found that the deletion mutant no longer produced SLs. Transcriptome analysis indicated that the expression levels of the key enzyme genes of SLs biosynthetic pathway were significantly down-regulated in the Δbro1, especially the expression level of cyp52m1 encoding the first rate-limiting enzyme in SL synthesis pathway was down-regulated 13-folds and the expression of fatty acid ß-oxidation-related enzymes was also down-regulated. This study can give insight into the regulation of SL synthesis.


Assuntos
Proteínas Fúngicas/metabolismo , Ácidos Oleicos/biossíntese , Saccharomycetales/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Saccharomycetales/genética , Transcriptoma
10.
Nat Commun ; 11(1): 2267, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385287

RESUMO

To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in 20-40% of unperturbed yeast cells, DNA synthesis continues during anaphase, late in mitosis. High cyclin-Cdk activity inhibits DNA synthesis in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA synthesis to finish at subtelomeric and some difficult-to-replicate regions. DNA synthesis during late mitosis correlates with elevated mutation rates at subtelomeric regions, including copy number variation. Thus, yeast cells temporally overlap DNA synthesis and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.


Assuntos
Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , DNA Fúngico/metabolismo , Saccharomycetales/metabolismo , Anáfase/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Genes Fúngicos , Metáfase , Mitose , Taxa de Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Telômero/metabolismo
11.
Appl Environ Microbiol ; 86(15)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32471916

RESUMO

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Glucose/metabolismo , Saccharomycetales/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Oxirredução , Pichia/enzimologia , Pichia/metabolismo , Saccharomycetales/enzimologia
12.
Nat Commun ; 11(1): 2144, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358542

RESUMO

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.


Assuntos
Saccharomycetales/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Biotecnologia/métodos , Genoma Fúngico/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Sci Rep ; 10(1): 6073, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269331

RESUMO

As a complex microbial ecosystem, wine is a particularly interesting model for studying interactions between microorganisms as fermentation can be done by microbial consortia, a unique strain or mixed culture. The effect of a specific yeast strain on its environments is unique and characterized by its metabolites and their concentration. With its great resolution and excellent mass accuracy, ultrahigh resolution mass spectrometry (uHRMS) is the perfect tool to analyze the yeast metabolome at the end of alcoholic fermentation. This work reports the change in wine chemical composition from pure and mixed culture fermentation with Lachancea thermotolerans, Starmerella bacillaris, Metschnikowia pulcherrima and S. cerevisiae. We could clearly differentiate wines according to the yeast strain used in single cultures and markers, which reflect important differences between the yeast species, were extracted and annotated. Moreover, uHRMS revealed underlining intra species metabolomics differences, showing differences at the strain level between the two Starmerella bacillaris. Non volatile metabolomics analysis of single and sequential fermentations confirmed that mixed fermentations lead to a different composition. Distinct metabolites appeared in wines from sequential fermentation compared to single fermentation. This suggests that interactions between yeasts are not neutral.


Assuntos
Fermentação , Metaboloma , Saccharomycetales/metabolismo , Vinho/microbiologia , Álcoois/metabolismo , Microbiota , Compostos Orgânicos Voláteis/metabolismo , Vinho/normas
14.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118727, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32339526

RESUMO

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Carboxiliases , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/toxicidade , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/toxicidade , Saccharomycetales/genética , Transcriptoma
15.
J Nat Med ; 74(3): 545-549, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32236853

RESUMO

Fungal co-culture is a strategy to induce the production of secondary metabolites by activating cryptic genes. We discovered the production of a new compound, talarodone A (1), along with five known compounds 2-6 in co-culture of Talaromyces pinophilus and Paraphaeosphaeria sp. isolated from soil collected in Miyazaki Prefecture, Japan. Among them, the productions of penicidones C (2) and D (3) were enhanced 27- and sixfold, respectively, by the co-culture. The structure of 3 should be represented as a γ-pyridol form with the reported chemical shifts, but not as a γ-pyridone form, based on DFT calculation.


Assuntos
Piridonas/metabolismo , Saccharomycetales/metabolismo , Talaromyces/metabolismo , Técnicas de Cocultura , Japão , Piridonas/química , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/isolamento & purificação , Microbiologia do Solo , Talaromyces/crescimento & desenvolvimento , Talaromyces/isolamento & purificação
16.
Sci Adv ; 6(15): eaaz3327, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32285001

RESUMO

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , Recombinação Genética , Transdução de Sinais , DNA Helicases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Instabilidade Genômica , Viabilidade Microbiana/genética , Modelos Biológicos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Estresse Fisiológico
17.
Nucleic Acids Res ; 48(8): 4081-4099, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187373

RESUMO

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT's activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Saccharomycetales/genética , Engenharia Celular , Centrômero , Cromatina/química , DNA (Citosina-5-)-Metiltransferases/genética , Técnicas de Introdução de Genes , Genoma Fúngico , Humanos , Redes Neurais de Computação , S-Adenosilmetionina/metabolismo , Saccharomycetales/metabolismo , Estresse Fisiológico/genética , Telômero , Transcrição Genética
18.
Food Microbiol ; 89: 103446, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32138994

RESUMO

Gray mold caused by Botrytis cinerea is a fungal disease that can determine significant economic losses of apple during the storage phase. An alternative to reduce the use of traditional synthetic fungicides is to employ the yeast Starmerella bacillaris as biological control agent (BCA), also with positive effect on apple juice fermentation for the production of cider. Thus, we aimed to evaluate the safety of 16 S. bacillaris strains and their ability to control B. cinerea. In addition, the fermentation performances in apple juice and the volatile organic compounds (VOCs) profile were assessed, both in single-strain and in sequential fermentations with Saccharomyces cerevisiae. The in vitro assays showed that all S. bacillaris strains can be considered safe from the analyzed virulence factors, and were able to significantly constrain the growth of B. cinerea, reducing mycelial growth of 50% in dual-culture and of 90% through VOCs. Moreover, in vivo antagonistic assays revealed a visible decrease of gray mold rot symptoms on apples confirming the potential of S. bacillaris as BCA. GC-MS analysis of the ciders obtained showed increased concentrations in the sequential fermentation of some higher alcohols and terpenes, positively correlated with the cider aromatic quality, and suggested the involvement of benzyl alcohol, known for its antimicrobial action, in the biocontrol efficacy.


Assuntos
Bebidas Alcoólicas/análise , Sucos de Frutas e Vegetais/análise , Odorantes/análise , Saccharomycetales/metabolismo , Compostos Orgânicos Voláteis/química , Fermentação , Armazenamento de Alimentos , Frutas , Malus
19.
NPJ Syst Biol Appl ; 6(1): 7, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221305

RESUMO

The growth and division of eukaryotic cells are regulated by complex, multi-scale networks. In this process, the mechanism of controlling cell-cycle progression has to be robust against inherent noise in the system. In this paper, a hybrid stochastic model is developed to study the effects of noise on the control mechanism of the budding yeast cell cycle. The modeling approach leverages, in a single multi-scale model, the advantages of two regimes: (1) the computational efficiency of a deterministic approach, and (2) the accuracy of stochastic simulations. Our results show that this hybrid stochastic model achieves high computational efficiency while generating simulation results that match very well with published experimental measurements.


Assuntos
Ciclo Celular/fisiologia , Saccharomycetales/metabolismo , Algoritmos , Simulação por Computador , Modelos Biológicos , Processos Estocásticos
20.
Bioengineered ; 11(1): 318-327, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32163000

RESUMO

The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.


Assuntos
Antifúngicos/química , Antifúngicos/metabolismo , Cromogranina A/química , Saccharomycetales/metabolismo , Cromatografia Líquida de Alta Pressão , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Complexo de Golgi/metabolismo , Prognóstico , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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