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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502345

RESUMO

Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This "metabolic flux probe" (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.


Assuntos
6-Fitase/metabolismo , Isótopos de Carbono/análise , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Marcação por Isótopo/métodos , Análise do Fluxo Metabólico/métodos , Saccharomycetales/metabolismo , Isótopos de Carbono/metabolismo , Saccharomycetales/crescimento & desenvolvimento
2.
Molecules ; 26(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34443298

RESUMO

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.


Assuntos
Antracenos/farmacologia , Corantes/farmacologia , Perileno/análogos & derivados , Fármacos Fotossensibilizantes/farmacologia , Plâncton/crescimento & desenvolvimento , Quinonas/farmacologia , Saccharomycetales/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fluorescência , Morfogênese/efeitos dos fármacos , Perileno/farmacologia , Plâncton/efeitos dos fármacos , Saccharomycetales/citologia , Saccharomycetales/efeitos dos fármacos
3.
Elife ; 102021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34013887

RESUMO

Recent results comparing the temporal program of genome replication of yeast species belonging to the Lachancea clade support the scenario that the evolution of the replication timing program could be mainly driven by correlated acquisition and loss events of active replication origins. Using these results as a benchmark, we develop an evolutionary model defined as birth-death process for replication origins and use it to identify the evolutionary biases that shape the replication timing profiles. Comparing different evolutionary models with data, we find that replication origin birth and death events are mainly driven by two evolutionary pressures, the first imposes that events leading to higher double-stall probability of replication forks are penalized, while the second makes less efficient origins more prone to evolutionary loss. This analysis provides an empirically grounded predictive framework for quantitative evolutionary studies of the replication timing program.


Assuntos
Replicação do DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , Evolução Molecular , Genoma Fúngico , Modelos Genéticos , Saccharomycetales/genética , Simulação por Computador , Período de Replicação do DNA , Regulação Fúngica da Expressão Gênica , Filogenia , Origem de Replicação , Saccharomycetales/classificação , Saccharomycetales/crescimento & desenvolvimento
4.
Arch Microbiol ; 203(6): 3707-3714, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33938972

RESUMO

Under the influence of electromagnetic waves of millimeter range with the frequency of 51.8 GHz, changes in the morphology, growth parameters and mitotic activity of yeasts C. guilliermondii NP-4 are revealed. Filamentous and giant cells appeared in a population of exposed yeasts. The sigmoid shape of the growth curve remained but the lag phase duration was increased by 2 h in comparison with non-exposed yeasts; accordingly, the log and stationary phases followed 2 h later. The specific growth rate in the log growth phase and colony-forming ability of exposed yeasts was decreased. It is suggested that yeasts have some response mechanisms to 51.8-GHz frequency electromagnetic waves. The results can be used to understand the response mechanisms of microorganisms to non-ionizing radiation, as well as to develop approaches to protect living organisms from it. The effect of electromagnetic waves of 51.8-GHz frequency to suppress yeasts can be applied in biotechnology and medicine.


Assuntos
Radiação Eletromagnética , Saccharomycetales/efeitos da radiação , Cinética , Saccharomycetales/citologia , Saccharomycetales/crescimento & desenvolvimento
5.
Methods Mol Biol ; 2302: 1-20, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877619

RESUMO

Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1 mg of pure PARL per liter of yeast culture.


Assuntos
Metaloproteases/isolamento & purificação , Proteínas Mitocondriais/isolamento & purificação , Saccharomycetales/crescimento & desenvolvimento , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Metaloproteases/genética , Proteínas Mitocondriais/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , Transformação Genética
6.
World J Microbiol Biotechnol ; 37(5): 88, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33881636

RESUMO

In this work we explored the potential of several strains of Kazachstania unispora to be used as non-conventional yeasts in sourdough fermentation. Properties such as carbohydrate source utilization, tolerance to different environmental factors and the performance in fermentation were evaluated. The K. unispora strains are characterized by rather restricted substrate utilization: only glucose and fructose supported the growth of the strains. However, the growth in presence of fructose was higher compared to a Saccharomyces cerevisiae commercial strain. Moreover, the inability to ferment maltose can be considered a positive characteristic in sourdoughs, where the yeasts can form a nutritional mutualism with maltose-positive Lactic Acid Bacteria. Tolerance assays showed that K. unispora strains are adapted to a sourdough environment: they were able to grow in conditions of high osmolarity, high acidity and in presence of organic acids, ethanol and salt. Finally, the performance in fermentation was comparable with the S. cerevisiae commercial strain. Moreover, the growth was more efficient, which is an advantage in obtaining the biomass in an industrial scale. Our data show that K. unispora strains have positive properties that should be explored further in bakery sector.


Assuntos
Pão/microbiologia , Frutose/metabolismo , Glucose/metabolismo , Saccharomycetales/fisiologia , Técnicas Bacteriológicas , Fermentação , Lactobacillales/fisiologia , Concentração Osmolar , Saccharomyces cerevisiae/fisiologia , Saccharomycetales/crescimento & desenvolvimento
7.
J Biol Chem ; 296: 100654, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33845046

RESUMO

Vitamin B12 (cobalamin) is an essential micronutrient for human health, and mutation and dysregulation of cobalamin metabolism are associated with serious diseases, such as methylmalonic aciduria and homocystinuria. Mutations in ABCD4 or LMBRD1, which encode the ABC transporter ABCD4 and lysosomal membrane protein LMBD1, respectively, lead to errors in cobalamin metabolism, with the phenotype of a failure to release cobalamin from lysosomes. However, the mechanism of transport of cobalamin across the lysosomal membrane remains unknown. We previously demonstrated that LMBD1 is required for the translocation of ABCD4 from the endoplasmic reticulum to lysosomes. This suggests that ABCD4 performs an important function in lysosomal membrane cobalamin transport. In this study, we expressed human ABCD4 and LMBD1 in methylotrophic yeast and purified them. We prepared ABCD4 and/or LMBD1 containing liposomes loaded with cobalamin and then quantified the release of cobalamin from the liposomes by reverse-phase HPLC. We observed that ABCD4 was able to transport cobalamin from the inside to the outside of liposomes dependent on its ATPase activity and that LMBD1 exhibited no cobalamin transport activity. These results suggest that ABCD4 may be capable of transporting cobalamin from the lysosomal lumen to the cytosol. Furthermore, we examined a series of ABCD4 missense mutations to understand how these alterations impair cobalamin transport. Our findings give insight into the molecular mechanism of cobalamin transport by which ABCD4 involves and its importance in cobalamin deficiency.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membranas Intracelulares/metabolismo , Lipossomos/metabolismo , Mutação , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Saccharomycetales/metabolismo , Vitamina B 12/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Humanos , Proteínas de Transporte Nucleocitoplasmático/genética , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento
8.
Appl Biochem Biotechnol ; 193(7): 2182-2197, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33682050

RESUMO

During the pretreatment and hydrolysis of lignocellulosic biomass to obtain a hydrolysate rich in fermentable sugars, furaldehydes (furfural and hydroxymethylfurfural), phenolic compounds, and organic acids are formed and released. These compounds inhibit yeast metabolism, reducing fermentation yields and productivity. This study initially confirmed the ability of Spathaspora passalidarum to ferment xylose and demonstrated its sensibility to the inhibitors present in the hemicellulosic sugarcane bagasse hydrolysate. Then, an adaptive laboratory evolution, with progressive increments of hydrolysate concentration, was employed to select a strain more resistant to hydrolysate inhibitors. Afterward, a central composite design was performed to maximize ethanol production using hydrolysate as substrate. At optimized conditions (initial cell concentration of 30 g/L), S. passalidarum was able to produce 19.4 g/L of ethanol with productivity, yield, and xylose consumption rate of 0.8 g/L.h and 0.4 g/g, respectively, in a sugarcane bagasse hemicellulosic hydrolysate. A kinetic model was developed to describe the inhibition of fermentation by substrate and product. The values obtained for substrate saturation and inhibition constant were Ks = 120.4 g/L and Ki = 1293.4 g/L. Ethanol concentration that stops cell growth was 30.1 g/L. There was an agreement between simulated and experimental results, with a residual standard deviation lower than 6%.


Assuntos
Celulose/química , Etanol/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharum/química , Xilose , Xilose/química , Xilose/metabolismo
9.
Food Microbiol ; 97: 103750, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33653523

RESUMO

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.


Assuntos
Enterococcaceae/crescimento & desenvolvimento , Etanol/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Enterococcaceae/química , Enterococcaceae/genética , Enterococcaceae/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Saccharomycetales/química , Saccharomycetales/genética , Propriedades de Superfície
10.
Methods Mol Biol ; 2280: 231-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751439

RESUMO

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Saccharomycetales/crescimento & desenvolvimento , Oxirredutases do Álcool/metabolismo , Técnicas de Cultura Celular por Lotes , Técnicas Biossensoriais , Cromatografia por Troca Iônica , Clonagem Molecular , Formaldeído/análise , Formaldeído/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Metanol/análise , Metanol/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/genética
11.
Methods Mol Biol ; 2280: 249-260, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751440

RESUMO

Flavocytochrome b2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b2) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b2, under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b2 a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.


Assuntos
L-Lactato Desidrogenase (Citocromo)/metabolismo , Engenharia de Proteínas/métodos , Saccharomycetales/crescimento & desenvolvimento , Técnicas Biossensoriais , Cromatografia de Afinidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , L-Lactato Desidrogenase (Citocromo)/genética , Ácido Láctico/análise , Plasmídeos/genética , Regiões Promotoras Genéticas , Saccharomycetales/genética , Saccharomycetales/metabolismo , Transformação Genética
12.
Food Microbiol ; 96: 103712, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33494893

RESUMO

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.


Assuntos
Ácido Láctico/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Aminoácidos/metabolismo , Técnicas de Cocultura , Fermentação , Frutas/metabolismo , Frutas/microbiologia , Oenococcus/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/crescimento & desenvolvimento , Vitis/metabolismo , Vitis/microbiologia
13.
Microb Cell Fact ; 20(1): 4, 2021 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-33413399

RESUMO

BACKGROUND: Nerol (C10H18O), an acyclic monoterpene, naturally presents in plant essential oils, and is used widely in food, cosmetics and pharmaceuticals as the valuable fragrance. Meanwhile, chemical synthesis is the only strategy for large-scale production of nerol, and the disadvantages of chemical synthesis greatly limit the production and its application. These defects drive the interests of researchers shift to the production of nerol by eco-friendly methods known as biosynthesis methods. However, the main technical bottleneck restricting the biosynthesis of nerol is the lacking of corresponding natural aroma-producing microorganisms. RESULTS: In this study, a novel multi-stress-tolerant probiotics Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified by whole genome sequencing and metabolomics technology. GXDK6 showed a broad pH tolerance in the range of 2.5-10.0. The species also showed salt tolerance with up to 12% NaCl and up to 18% of KCl or MgCl2. GXDK6 exhibited heavy-metal Mn2+ tolerance of up to 5494 ppm. GXDK6 could also ferment with a total of 21 kinds of single organic matter as the carbon source, and produce abundant aromatic metabolites. Results from the gas chromatography-mass spectrometry indicated the production of 8-14 types of aromatic metabolites (isopentanol, nerol, geraniol, phenylethanol, isobutanol, etc.) when GXDK6 was fermented up to 72 h with glucose, sucrose, fructose, or xylose as the single carbon source. Among them, nerol was found to be a novel aromatic metabolite from GXDK6 fermentation, and its biosynthesis mechanism had also been further revealed. CONCLUSION: A novel aroma-producing M. guilliermondii GXDK6 was identified successfully by whole genome sequencing and metabolomics technology. GXDK6 showed high multi-stress-tolerant properties with acid-base, salty, and heavy-metal environments. The aroma-producing mechanism of nerol in GXDK6 had also been revealed. These findings indicated the aroma-producing M. guilliermondii GXDK6 with multi-stress-tolerant properties has great potential value in the fermentation industry.


Assuntos
Monoterpenos Acíclicos/metabolismo , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Metaboloma , Saccharomycetales/metabolismo , Estresse Fisiológico , Sequenciamento Completo do Genoma/métodos , Proteínas Fúngicas/genética , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento
14.
Int J Food Microbiol ; 341: 109048, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33486390

RESUMO

Yeasts are the leading cause of spoilage in yogurt. Considering the high demand from consumers to use natural products as an alternative to additives, essential oils (EOs) could be a promising solution to guarantee high microbiological standards. The present study highlighted the in vitro antifungal potential of cinnamon, ginger, lemongrass, mandarin, orange, lemon and lime EOs against spoilage yeasts isolated from yogurts prepared with pasteurized buffalo milk. A total of 74 isolates represented by 14 different species of Candida, Rhodotorula, Debaryomyces, Kluyveromyces and Yarrowia genera were subjected to a disc diffusion assay, showing lemongrass EO to have the highest antifungal activity (40.97 ± 9.86 mm), followed by cinnamon (38.46 ± 6.59 mm) and orange (12.00 ± 4.52 mm) EOs. Yarrowia lipolytica was less susceptible to lemongrass EO than Candida sake and Yarrowia deformans isolates. Ginger EO exhibited the lowest efficacy. A minimum inhibitory concentration (MIC) assay showed the ability of lemongrass and cinnamon EOs to inhibit the growth of all selected isolates at concentrations between ≤0.31 and 1.25 µL/mL. Therefore, for the first time, the two best-performing EOs (lemongrass and cinnamon) based on in vitro assays were assessed for their potential roles as preservatives in an in vivo yogurt model prepared at the laboratory scale. Since some limitations, such as the inhibition of lactic acid bacteria by cinnamon EO, consequently leading to fermentation failure as well as species-specific antifungal activity of lemongrass EO, were observed, further studies are needed to explore the possibility of using a slightly higher concentration of lemongrass EO and/or combinations of different EOs and/or their components. Finally, since yogurt spoilage could also be prevented by correct sanitation procedures of the production environment, the sanitizers commonly used in the food industry were tested against all isolates, showing the high efficiency of alcohol-based sanitizers and the ineffectiveness of chlorine-based sanitizers.


Assuntos
Antifúngicos/farmacologia , Cinnamomum zeylanicum/química , Cymbopogon/química , Óleos Voláteis/farmacologia , Leveduras/crescimento & desenvolvimento , Iogurte/microbiologia , Candida/efeitos dos fármacos , Candida/crescimento & desenvolvimento , Candida/isolamento & purificação , Citrus sinensis/química , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Contaminação de Alimentos/análise , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/isolamento & purificação , Leveduras/efeitos dos fármacos , Leveduras/metabolismo
15.
Biotechnol Appl Biochem ; 68(1): 148-156, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32125024

RESUMO

Mathematical modeling represents and predicts biological systems, explains underlying mechanisms, constituting one of the key focus points for fundamental and applied research to improve our understanding and to decrease costs. Organic acids are used in several industries such as monomers for bioplastics, food preservatives and additives, pharmaceuticals, and agriculture. Nonpetrochemical, sustainable production of organic acids is therefore of great interest. An important step in production of organic acids is the determination of growth and acid production dynamics, as the product itself may have direct and indirect inhibitory effects on the host's metabolism. The aim of this study it twofold: (i) to determine the parameters related to energetics of growth and production as growth ( K x ) and nongrowth associated (mATP ) maintenance constants and (ii) to set up and analyze an unstructured, black-box kinetic model to describe the dynamics of the growth and production of citric acid by Candida oleophila ATCC20177 using published batch fermentation data. K x and mATP were found to be 2.3 ± 1.7 and 5.25 ± 2.75, respectively, for the published P/O ratio of 1.45. The parameter sensitivities and correlations are determined using the Monte Carlo approach, and the final model is tested using chemostat data.


Assuntos
Trifosfato de Adenosina/metabolismo , Ácido Cítrico/metabolismo , Modelos Biológicos , Saccharomycetales/crescimento & desenvolvimento , Cinética
16.
Med Mycol ; 59(3): 253-258, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32525988

RESUMO

Candida auris is a serious nosocomial health risk, with widespread outbreaks in hospitals worldwide. Successful management of such outbreaks has depended upon intensive screening of patients to identify those that are colonized and the subsequent isolation or cohorting of affected patients to prevent onward transmission. Here we describe the evaluation of a novel chromogenic agar, CHROMagarTM Candida Plus, for the specific identification of Candida auris isolates from patient samples. Candida auris colonies on CHROMagarTM Candida Plus are pale cream with a distinctive blue halo that diffuses into the surrounding agar. Of over 50 different species of Candida and related genera that were cultured in parallel, only the vanishingly rare species Candida diddensiae gave a similar appearance. Moreover, both the rate of growth and number of colonies of C. auris recovered from swabs of pure and mixed Candida species were substantially increased on CHROMagarTM Candida Plus agar when compared with growth on the traditional mycological isolation medium, Sabouraud dextrose agar. Taken together, the present data suggest that CHROMagarTM Candida Plus agar is an excellent alternative to current conventional mycological media for the screening of patients who are potentially colonized/infected with Candida auris, can be reliably used to identify this emerging fungal pathogen, and should be tested in a clinical setting. LAY ABSTRACT: Candida auris is a novel pathogenic yeast that has been associated with large hospital outbreaks across several continents. Affected patients become colonized, predominantly on the skin, with large quantities of C. auris which they then shed into the hospital environment. Identification of C. auris is challenging using routine laboratory methods, and time consuming when patients are colonized with a mixture of different Candida species. Here we demonstrate that a novel chromogenic agar, CHROMagarTM Candida Plus, permits the rapid differentiation of C. auris from a wide range of other yeast species and is potentially ideally suited to screening of patients that are suspected of being colonized or infected with this medically important yeast.


Assuntos
Ágar/química , Candida/crescimento & desenvolvimento , Candida/isolamento & purificação , Meios de Cultura/química , Ágar/normas , Candidíase/microbiologia , Humanos , Técnicas Microbiológicas , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/isolamento & purificação
17.
J Appl Microbiol ; 131(2): 728-742, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33103297

RESUMO

AIM: Tyrosol, a quorum sensing molecule in yeasts, was reported to reduce lag phase and induces hyphae formation during cell proliferation. However, evidence of any enhancing effect of tyrosol in cellular proliferation within fermentative environment is unclear. In this investigation, selected yeast cells were assessed for their ability to synthesize tyrosol followed by examining the role of the molecule during fermentation. METHODS AND RESULTS: Tyrosols were characterized in four fermentative yeasts viz., Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida glabrata and Candida tropicalis isolated from traditional fermentative cakes of northeast India. All the isolates synthesized tyrosol while C. tropicalis exhibited filamentous growth in response to tyrosols retrieved from other isolates. Purified tyrosols showed protective behaviour in C. tropicalis and S. cerevisiae under ethanol mediated oxidative stress. During fermentation, tyrosol significantly enhanced growth of W. anomalus in starch medium while C. tropicalis exhibited growth enhancement in starch and glucose sources. The chief fermentative yeast S. cerevisiae showed notable enhancement in fermentative capacity in starch medium under the influence of tyrosol con-commitment of ethanol production. CONCLUSION: The study concludes that tyrosol exerts unusual effect in cellular growth and fermentative ability of both Saccharomyces and non-Saccharomyces yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of expression of tyrosol by non-conventional yeasts, where the molecule was found to exert enhancing effect during fermentation, thereby augmenting the process of metabolite production during traditional fermentation.


Assuntos
Fermentação , Álcool Feniletílico/análogos & derivados , Percepção de Quorum , Leveduras/metabolismo , Candida/isolamento & purificação , Candida/metabolismo , Candida glabrata/isolamento & purificação , Candida glabrata/metabolismo , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/crescimento & desenvolvimento , Candida tropicalis/isolamento & purificação , Candida tropicalis/metabolismo , Etanol/metabolismo , Etanol/toxicidade , Índia , Álcool Feniletílico/metabolismo , Saccharomyces/isolamento & purificação , Saccharomyces/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/isolamento & purificação , Saccharomycetales/metabolismo
18.
Food Chem ; 342: 128382, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33092918

RESUMO

Supplementation of protein hydrolysate is an important strategy to improve the salt tolerance of soy sauce aroma-producing yeast. In the present study, Tartary buckwheat protein hydrolysates (BPHs) were prepared and separated by ultrafiltration into LM-1 (<1 kDa) and HM-2 (1-300 kDa) fractions. The supplementation of HM-2 fraction could significantly improve cell growth and fermentation of soy sauce aroma-producing yeast Zygosaccharomyces rouxii As2.180 under high salt (12%, w/w) conditions. However, the LM-1 fraction inhibited strain growth and fermentation. The addition of HM-2 promoted yeast cell accumulation of K+, removal of cytosolic Na+ and accumulation of glycerol. Furthermore, the HM-2 fraction improved the cell membrane integrity and mitochondrial membrane and decreased intracellular ROS accumulation of the strain. The above results indicated that the supplementation of BPHs with a molecular weight of 1-300 kDa is a potentially effective and feasible strategy for improving the salt tolerance of soy sauce aroma-producing yeast Z. rouxii.


Assuntos
Fagopyrum/metabolismo , Hidrolisados de Proteína/farmacologia , Saccharomycetales/crescimento & desenvolvimento , Tolerância ao Sal/efeitos dos fármacos , Alimentos de Soja/análise , Fermentação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Peso Molecular , Hidrolisados de Proteína/química , Hidrolisados de Proteína/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/metabolismo , Ultrafiltração , Compostos Orgânicos Voláteis/análise
19.
Appl Biochem Biotechnol ; 193(2): 502-514, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33026615

RESUMO

Ethanol was produced by separate hydrolysis and fermentation using Azolla filiculoides as a biomass. Thermal acid hydrolysis and enzymatic saccharification were used as pretreatment methods to produce monosaccharides from Azolla. The optimal content for thermal acid hydrolysis of 14% (w/v) Azolla weed slurry produced 16.7-g/L monosaccharides by using 200 mM H2SO4 at 121 °C for 60 min. Enzymatic saccharification using 16 U/mL Viscozyme produced 61.6 g/L monosaccharide at 48 h. Ethanol productions with ethanol yield coefficients from Azolla weed hydrolysate using Kluyveromyces marxianus, Candida lusitaniae Saccharomyces cerevisiae, and Pichia stipitis were 26.8 g/L (YEtOH = 0.43), 23.2 g/L (YEtOH = 0.37), 18.2 g/L (YEtOH = 0.29), and 13.7 g/L (YEtOH = 0.22), respectively. Saccharomyces cerevisiae produces the lowest yield as it utilized only glucose. Bioethanol from Azolla weed hydrolysate can be successfully produced by using Kluyveromyces marxianus because it consumed the mixture of glucose and xylose completely within 60 h.


Assuntos
Biomassa , Candida/crescimento & desenvolvimento , Etanol/metabolismo , Gleiquênias/química , Kluyveromyces/crescimento & desenvolvimento , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/crescimento & desenvolvimento
20.
Bioprocess Biosyst Eng ; 44(1): 103-112, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32808048

RESUMO

A number of limitations exist for production of human granulocyte colony-stimulating factor (G-CSF) in Pichia pastoris. In this study, two different specific growth rates (0.015 h-1, 0.01 h-1) were used sequentially in the mixed substrate feeding period during methanol induction phase to enhance the G-CSF titer in the culture broth. Necessary parameters required for implementing the feeding strategy, such as specific product yield on biomass (YP/X) and maintenance coefficient (m) on glycerol, methanol, and sorbitol were estimated using continuous culture technique. Using this strategy, for the same volumetric productivity, about 20% increase in protein titer was achieved over that obtained from the run carried out at a single pre-set value of 0.015 h-1 alone. Thus, implementation of higher specific growth rate (0.015 h-1) set during initial stages of the methanol induction phase followed by a lower specific growth rate (0.01 h-1) helped in achieving increased protein titers.


Assuntos
Técnicas de Cultura Celular por Lotes , Fator Estimulador de Colônias de Granulócitos/biossíntese , Saccharomycetales/crescimento & desenvolvimento , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomycetales/genética
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