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1.
Food Chem ; 398: 133863, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35961173

RESUMO

Beer quality generally diminishes over time as staling compounds accumulate through various oxidation reactions. Here, we show that refermentation, a traditional practice where Saccharomyces cerevisiae cells are added to beer prior to bottling, diminishes the accumulation of staling aldehydes. However, commonly used beer yeasts only show a limited lifespan in beer. Using high-throughput screening and breeding, we were able to generate novel S. cerevisiae hybrids that survive for over a year in beer. Extensive chemical and sensory analyses of the two most promising hybrids showed that they slow down the accumulation of staling aldehydes, such as furfural and trans-2-nonenal and significantly increased beer flavor stability for up to 12 months. Moreover, the strains did not change the original flavor of the beer, highlighting their potential to be integrated in existing products. Together, these results demonstrate the ability to breed novel microbes that function as natural and sustainable anti-oxidative food preservatives.


Assuntos
Cerveja , Saccharomyces cerevisiae , Aldeídos/análise , Cerveja/análise , Fermentação , Melhoramento Vegetal , Saccharomyces cerevisiae/genética
2.
Food Chem ; 398: 133860, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35964563

RESUMO

Although prime compounds in yeast metabolism, vitamins in oenology have remained mostly unexplored for decades. Here, a premier characterization of the vitamers in white grape musts has been drawn. A RP-HPLC method has therefore been developed for their direct analysis in musts, allowing for the determination of 19 different vitamers from 8 water-soluble vitaminic groups, including thiamine forms T, TMP and TPP, with LODs between 0.1 and 45.9 µg.L-1 and LOQs between 0.4 and 137.8 µg.L-1. A resulting characterization of 85 grape musts has been drawn from their vitaminic composition. Plus, the use of neither sulfites nor filtration affects the must vitamin content. The method stands as a useful tool for the later determination of yeast requirements, or impact of winemaking products on vitamins. The method has, overall, proven as practical and sensitive, for rapid identification of vitamins and vitamers in musts.


Assuntos
Vitaminas , Vitis , Cromatografia Líquida de Alta Pressão/métodos , Saccharomyces cerevisiae , Tiamina/análise , Vitamina A/análise , Vitamina K/análise , Vitaminas/análise
3.
Methods Mol Biol ; 2564: 213-222, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107344

RESUMO

Fluorescent proteins within fluorescent fusions have been reported to affect cellular growth fitness via altering native protein function and intracellular localization. Here we report in detail a procedure to analyze the growth characteristics of yeast cells expressing such fusions in comparison to unmodified parental strain. This approach can serve as an initial step in fluorescent protein characterization in vivo.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Corantes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
4.
Methods Mol Biol ; 2564: 269-286, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107348

RESUMO

The expression of plant cysteine oxidase (PCO) enzyme in Saccharomyces cerevisiae enables the Arg/Cys N-degron pathway (Cys-NDP) for selective protein degradation that, in plants, functions as direct oxygen perception mechanism. A synthetic construct based on the plant Cys-NDP substrate related to apetala 2.12 (RAP2.12), the dual luciferase oxygen reporter (DLOR), exploits the N-terminal Cys of RAP2.12, and its oxygen-dependent degradation through the Cys-NDP. The luminescent output of DLOR can be used as a proxy for intracellular oxygen dynamics in budding yeast. Replacement of the luciferase reporter of the DLOR with fluorescent proteins would furthermore facilitate the imaging of reporter dynamics in living cells. In this chapter, we describe the methods for delivering the DLOR synthetic construct to yeast and calibrating its output by means of oxygen quantification in the culture with a physical oxygen sensor. We explain the setup needed to carry out hypoxic treatments with several colonies as replicates. We also describe the method to measure oxygen concentration in the culture, the closest indication of intracellular oxygen levels, as a way that would serve to calibrate the DLOR output. Finally, we propose a strategy to replace the luminescent reporters in the DLOR with fluorescent proteins to visualize oxygen dynamics in vivo.


Assuntos
Cisteína Dioxigenase , Saccharomyces cerevisiae , Cisteína/metabolismo , Cisteína Dioxigenase/metabolismo , Luciferases/metabolismo , Oxigênio/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Braz. j. biol ; 83: e253009, 2023. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1339373

RESUMO

Abstract Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.


Resumo Hoje, o foco global da pesquisa é explorar a solução da crise energética e da poluição ambiental. Como em outros países agrícolas, grandes quantidades de cascas de melancia (WMP) são descartadas como resíduos no meio ambiente no Paquistão, mas a gestão adequada desses resíduos é a mais recente solução para salvar o meio ambiente da poluição. O trabalho enfatiza o papel das leveduras etanologênicas para utilizar açúcares significativos presentes no WMP para fermentação de bioetanol de baixo custo. A hidrólise de ácido clorídrico diluído de WMP foi realizada em condições otimizadas empregando RSM (metodologia de superfície de resposta) e seguindo o projeto de composto central (CCD). Este projeto experimental é baseado na otimização da etanologenesis envolvendo alguns parâmetros independentes importantes, como hidrolisado de WMP e razão de meio sintético (X1), temperatura de incubação (X2) e temperatura de incubação (X3) para rendimento máximo de etanol explorando o padrão (Saccharomyces cerevisiae K7) também como leveduras experimentais (Metchnikowia cibodasensis Y34). Os resultados revelaram que os rendimentos máximos de etanol obtidos a partir de S. cerevisiae K7 foi de 0,36 ± 0,02 g / g de açúcares redutores, enquanto M. cibodasensis Y34 rendeu 0,40 ± 0,01 g de etanol / g de açúcares redutores. O isolado de levedura M. cibodasensis Y34 apareceu como um etanologeno promissor e incorpora um potencial prospectivo para a valorização fermentativa de WMP em bioetanol.


Assuntos
Cucurbitaceae , Etanol , Saccharomyces cerevisiae , Água , Biotransformação , Estudos Prospectivos , Fermentação
6.
Microb Cell Fact ; 21(1): 180, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064410

RESUMO

BACKGROUND: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. RESULTS: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. CONCLUSIONS: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.


Assuntos
COVID-19 , Humanos , Fator de Acasalamento , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Saccharomyces cerevisiae/metabolismo , Saccharomycetales
7.
Curr Protoc ; 2(9): e525, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36069669

RESUMO

Unicellular eukaryotic organisms such as yeast and protozoa serve as useful models for studying the impact of chemicals on cell physiology, cellular growth, and genome duplication. The yeast Saccharomyces cerevisiae has been widely used to assess apoptosis induced by chemicals due to its genetic tractability, ease of evaluation, and readily available impact assessment tools. Apoptosis in S. cerevisiae is characterized by many features, including increased cell death, loss of membrane integrity, release of caspases, chromatin condensation, and nuclear fragmentation, which are similar to the ones observed in mammalian cells. Current methods of apoptosis assessment typically require specialized equipment and reagents, which limits wide adoption. Here, we describe a rapid, inexpensive, and easy-to-perform assay in yeast for the analysis of late-stage apoptotic features in cells treated with a chemical. We describe a protocol for assessing loss of cell survival and changes in the nucleus. We demonstrate the approach by using acetic acid and hydrogen peroxide as test chemicals. This assay for the study of late-stage apoptotic features in S. cerevisiae can be performed reliably and rapidly by any laboratory with basic equipment and may be extended for studying apoptosis in similar single-cell organisms after treatment with toxicological agents. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culture of Saccharomyces cerevisiae, treatment with acetic acid or hydrogen peroxide, and semi-quantitative growth assay Basic Protocol 2: DAPI staining and fluorescence microscopy for the assessment of change in nucleus-to-cytoplasm ratio and nuclear integrity.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácido Acético/metabolismo , Animais , Apoptose/fisiologia , Caspases/metabolismo , Peróxido de Hidrogênio/metabolismo , Mamíferos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
Cell Rep ; 40(10): 111316, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36070694

RESUMO

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Microscopia Crioeletrônica , Poli T , RNA Polimerase III/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Terminadoras Genéticas
9.
Am J Hum Genet ; 109(9): 1692-1712, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36055214

RESUMO

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.


Assuntos
Proteínas de Ligação ao Cálcio , Doenças Mitocondriais , Proteínas de Ligação ao Cálcio/genética , Homeostase/genética , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Sistema Nervoso/metabolismo , Saccharomyces cerevisiae/metabolismo
10.
Environ Monit Assess ; 194(10): 728, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064812

RESUMO

Previous studies have shown that the soil microbial population and soil enzyme activity are seriously affected by fracturing flow-back fluid (FFBF) from the shale gas mining process. However, the toxic effect of FFBF on specific bacteria, fungi, and plankton has not been systematically confirmed in detail. In this paper, a toxic effect evaluation of FFBF was conducted using the representative toxicity test organisms Vibrio fischeri, Daphnia, Aspergillus niger, and S. cerevisiae, indicating that FFBF can significantly decrease the survival rate of these species. The results also showed that there was a significant negative correlation between the concentration of some inorganic toxicity factors and the survival rate when Daphnia was used as the test organism, indicating that the toxicity degree order for these inorganic toxicity factors is Ba2+ > Li+ > As3+ > Cl- > Cu2+ > Rb2+ > Ga2+ > V2+ > Na+. In addition, other toxic factors, including polycyclic aromatic hydrocarbons (PAHs), were also determined, and the order of toxic effects with a negative correlation to the Daphnia survival rate was confirmed. These results showed that the biological toxicity of FFBF was caused not only by inorganic toxicity factors such as heavy metals but also by organic compounds such as PAHs. The results not only provide a significant reference value for the systematic assessment of biological toxicity by FFBF, but they also have great significance for developing approaches to appropriate FFBF treatment.


Assuntos
Aliivibrio fischeri , Hidrocarbonetos Policíclicos Aromáticos , Animais , Aspergillus , Aspergillus niger , Daphnia , Monitoramento Ambiental , Saccharomyces cerevisiae
11.
Zhongguo Zhong Yao Za Zhi ; 47(15): 4066-4073, 2022 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-36046896

RESUMO

CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.


Assuntos
Produtos Biológicos , Saccharomyces cerevisiae , Sistemas CRISPR-Cas , Carotenoides/metabolismo , Edição de Genes/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Oxid Med Cell Longev ; 2022: 8368717, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36082083

RESUMO

Nowadays, the environmentally friendly approach to everyday life routines including body supplementation with pharma-, nutraceuticals and dietary supplements gains popularity. This trend is implemented in pharmaceutical as well as cosmetic and antiageing industries by adopting a newly developed green chemistry approach. Following this trend, a new type of solvents has been created, called Natural Deep Eutectic Solvents (NADES), which are produced by plant primary metabolites. These solvents are becoming a much better alternative to the already established organic solvents like ethanol and ionic liquids by being nontoxic, biodegradable, and easy to make. An interesting fact about NADES is that they enhance the biological activities of the extracted biological compounds. Here, we present our results that investigate the potential antiageing effect of CiAPD14 as a NADES solvent and three plant extracts with it. The tested NADES extracts are from propolis and two well-known medicinal plants-Sideritis scardica and Plantago major. Together with the solvent, their antiageing properties have been tested during the chronological lifespan of four Saccharomyces cerevisiae yeast strains-a wild type and three chromatin mutants. The chromatin mutants have been previously proven to exhibit characteristics of premature ageing. Our results demonstrate the potential antiageing activity of these NADES extracts, which was exhibited through their ability to confer the premature ageing phenotypes in the mutant cells by ameliorating their cellular growth and cell cycle, as well as by influencing the activity of some stress-responsive genes. Moreover, we have classified their antiageing activity concerning the strength of the observed bioactivities.


Assuntos
Plantago , Própole , Sideritis , Cromatina , Longevidade , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Própole/farmacologia , Saccharomyces cerevisiae , Solventes/química
13.
Nat Commun ; 13(1): 5273, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36071116

RESUMO

Binding to binding site clusters has yet to be characterized in depth, and the functional relevance of low-affinity clusters remains uncertain. We characterized transcription factor binding to low-affinity clusters in vitro and found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, small clusters with binding sites an order of magnitude lower in affinity give rise to high mean occupancies at physiologically-relevant transcription factor concentrations. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters in a synthetic and native gene regulatory network in S. cerevisiae. In both systems, clusters of low-affinity binding sites generated transcriptional output comparable to single or even multiple consensus sites. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.


Assuntos
Saccharomyces cerevisiae , Fatores de Transcrição , Sequência de Bases , Sítios de Ligação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
PLoS Pathog ; 18(9): e1010784, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36074792

RESUMO

Malassezia, a lipophilic and lipid-dependent yeast, is a microorganism of current interest to mycobiologists because of its role as a commensal or pathogen in health conditions such as dermatological diseases, fungemia, and, as discovered recently, cancer and certain neurological disorders. Various novel approaches in the study of Malassezia have led to increased knowledge of the cellular and molecular mechanisms of this yeast. However, additional efforts are needed for more comprehensive understanding of the behavior of Malassezia in interactions with the host. This article reviews advances useful in the experimental field for Malassezia.


Assuntos
Malassezia , Saccharomyces cerevisiae
15.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076919

RESUMO

Arbuscular mycorrhizal fungi (AMF) are important symbiotic microorganisms in soil that engage in symbiotic relationships with legumes, resulting in mycorrhizal symbiosis. Establishment of strong symbiotic relationships between AMF and legumes promotes the absorption of nitrogen by plants. Ammonium nitrogen can be directly utilised by plants following ammonium transport, but there are few reports on ammonium transporters (AMTs) promoting ammonium nitrogen transport during AM symbiosis. Lotus japonicus is a typical legume model plant that hosts AMF. In this study, we analysed the characteristics of the Lotus japonicus ammonium transporter LjAMT2;2, and found that it is a typical ammonium transporter with mycorrhizal-induced and ammonium nitrogen transport-related cis-acting elements in its promoter region. LjAMT2;2 facilitated ammonium transfer in yeast mutant supplement experiments. In the presence of different nitrogen concentrations, the LjAMT2;2 gene was significantly upregulated following inoculation with AMF, and induced by low nitrogen. Overexpression of LjAMT2;2 increased the absorption of ammonium nitrogen, resulting in doubling of nitrogen content in leaves and roots, thus alleviating nitrogen stress and promoting plant growth.


Assuntos
Compostos de Amônio , Lotus , Micorrizas , Fungos , Micorrizas/genética , Nitrogênio , Proteínas de Plantas/genética , Raízes de Plantas/genética , Saccharomyces cerevisiae/genética , Simbiose/genética
16.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076954

RESUMO

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.


Assuntos
Autofagossomos , Proteínas de Saccharomyces cerevisiae , Animais , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Endopeptidases/metabolismo , Mamíferos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
17.
Molecules ; 27(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36080396

RESUMO

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.


Assuntos
Citocromos c , Dimetil Sulfóxido , Citocromos c/metabolismo , Dimetil Sulfóxido/química , Cinética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Solventes , Termodinâmica , Água
18.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077205

RESUMO

Ripened oriental melon (Cucumis melo) with orange-colored flesh is rich in ß-carotene. Lycopene ß-cyclase (LCYB) is the synthetic enzyme that directly controls the massive accumulation of ß-carotene. However, the regulatory mechanism underlying the CmLCYB-mediated ß-carotene accumulation in oriental melon is fairly unknown. Here, we screened and identified a transcription factor, CmNAC34, by combining bioinformatics analysis and yeast one-hybrid screen with CmLCYB promoter. CmNAC34 was located in the nucleus and acted as a transcriptional activator. The expression profile of CmNAC34 was consistent with that of CmLCYB during the fruit ripening. Additionally, the transient overexpression of CmNAC34 in oriental melon fruit promoted the expression of CmLCYB and enhanced ß-carotene concentration, while transient silence of CmNAC34 in fruit was an opposite trend, which indicated CmNAC34 could modulate CmLCYB-mediated ß-carotene biosynthesis in oriental melon. Finally, the yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), ß-glucuronidase (GUS) analysis assay, and luciferase reporter (LUC) assay indicated that CmNAC34 could bind to the promoter of CmLCYB and positively regulated the CmLCYB transcription level. These findings suggested that CmNAC34 acted as an activator to regulate ß-carotene accumulation by directly binding the promoter of CmLCYB, which provides new insight into the regulatory mechanism of carotenoid metabolism during the development and ripening of oriental melon.


Assuntos
Cucumis melo , Cucumis melo/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Caroteno/metabolismo
19.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077218

RESUMO

Laser-induced forward transfer (LIFT) is a useful technique for bioprinting using gel-embedded cells. However, little is known about the stresses experienced by cells during LIFT. This paper theoretically and experimentally explores the levels of laser pulse irradiation and pulsed heating experienced by yeast cells during LIFT. It has been found that only 5% of the cells in the gel layer adjacent to the absorbing Ti film should be significantly heated for fractions of microseconds, which was confirmed by the fact that a corresponding population of cells died during LIFT. This was accompanied by the near-complete dimming of intracellular green fluorescent protein, also observed in response to heat shock. It is shown that microorganisms in the gel layer experience laser irradiation with an energy density of ~0.1-6 J/cm2. This level of irradiation had no effect on yeast on its own. We conclude that in a wide range of laser fluences, bioprinting kills only a minority of the cell population. Importantly, we detected a previously unobserved change in membrane permeability in viable cells. Our data provide a wider perspective on the effects of LIFT-based bioprinting on living organisms and might provide new uses for the procedure based on its effects on cell permeability.


Assuntos
Bioimpressão , Bioimpressão/métodos , Contagem de Células , Lasers , Luz , Saccharomyces cerevisiae
20.
Food Res Int ; 160: 111697, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36076400

RESUMO

This work aimed to evaluate the performance of co-cultivation of potential probiotic yeast and lactic acid bacteria (LAB) in producing plant-based fermented beverages. The co-culture comprised LAB Lactiplantibacillus plantarum CCMA0743 with the yeasts Pichia kluyveri CCMA 0615, Pichia guilliermondii CCMA 1753 and Debaryomyces hansenii CCMA 1761 separately. The plant substrate was 75 g oat, 175 g sunflower seeds, and 75 g almonds. The viability of microorganisms in the plant-based matrix was evaluated during fermentation, storage at 4 °C, and under simulated gastrointestinal tract (GIT) conditions. Chemical analysis, antioxidant activity, and sensory profile of the beverages were also determined. The three yeasts and the LAB showed counts greater than 6.0 log CFU/mL after fermentation, and the plant-based matrix protected the yeasts during simulated digestion. P. kluyveri and D. hansenii showed higher survival than P. guilliermondii and L. plantarum after exposure to simulated GIT conditions. The pH of the plant-based matrix reduced from approximately 7 to 3.8. Lactic acid was the main organic acid produced during fermentation. In addition, 113 volatile compounds were detected by gas chromatography-mass spectrometry (GC-MS), including alcohols, aldehydes, alkanes, alkenes, acids, ester, ether, ketones, phenol, and amides. The beverage sensory profile varied with the co-culture. The co-culture D. hansenii and L. plantarum showed higher antioxidant activity than the other co-culture tested, and the homogeneous texture attribute characterized the beverage produced with this combination. Results show the suitability of tested co-cultures to produce a plant-based fermented beverage and indicate more significant potential for D. hansenii and L. plantarum co-culture as a starter for its functionalization.


Assuntos
Lactobacillales , Probióticos , Antioxidantes/análise , Técnicas de Cocultura , Bebidas Fermentadas , Cromatografia Gasosa-Espectrometria de Massas , Probióticos/química , Saccharomyces cerevisiae
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