RESUMO

The excess of minerals in the industrial substrates is detrimental for Saccharomyces cerevisiae ethanol fermentation performance. In this work, we sought to understand the effect of some of those minerals on the physiology of Dekkera bruxellensis. Three groups of minerals were classified on the basis of the aerobic growth profiles on glucose: neutrals (K+, Mg2+, P5+ and Zn2+), inducers (Mn2+ and Ca2+) and inhibitors (Al3+, Cu2+ and Fe2+). Cu2+ showed the highest mineral toxicity, and its effect was dependent of the level of medium aeration. On the other hand, copper stimulated respiration by increasing growth on respiratory carbon sources. Most growth inhibitors also hampered glucose fermentation, with changes in carbon distribution to metabolic routes dedicated to anabolic reactions and for alternative reduced co-factors oxidations to maintain cellular homeostasis. The negative effect of Cu2+ on yeast fermentation was partially alleviated by Mg2+ and Mn2+, similar to magnesium antagonism observed for S. cerevisiae. All these results might contribute to understand the action of these minerals in sugarcane substrates on the physiology of D. bruxellensis cells. Therefore, it represents one more step for the consolidation of the industrial use of this yeast in the production of fuel-ethanol as well as other biotechnological goods.

RESUMO

Dekkera bruxellensis has been studied for several aspects of its metabolism over the past years, which has expanded our comprehension on its importance to industrial fermentation processes and uncovered its industrial relevance. Acetate is a metabolite often found in D. bruxellensis aerobic cultivations, whereas its production is linked to decreased ethanol yields. In a previous work, we aimed to understand how acetate metabolism affected the fermentation capacity of D. bruxellensis. In the present work, we evaluated the role of acetate metabolism in respiring cells using ammonium or nitrate as nitrogen sources. Our results showed that galactose is a strictly respiratory sugar and that a relevant part of its carbon is lost, while the remaining is metabolised through the Pdh bypass pathway before being assimilated into biomass. When this pathway was blocked, yeast growth was reduced while more carbon was assimilated to the biomass. In nitrate, more acetate was produced as expected, which increased carbon assimilation, although less galactose was uptaken from the medium. This scenario was not affected by the Pdh bypass inhibition. The confirmation that acetate production was crucial for carbon assimilation was brought by cultivations in pyruvate. All physiological data were connected to the expression patterns of PFK1, PDC1, ADH1, ALD3, ALD5 and ATP1 genes. Other respiring carbon sources could only be properly used by the cells when some external acetate was supplied. Therefore, the results reported herein helped in providing valuable contributions to the understanding of the oxidative metabolism in this potential industrial yeast.

Assuntos

Carbono , Nitratos , Nitratos/metabolismo , Carbono/metabolismo , Galactose , Fermentação , Acetatos

RESUMO

The advancement of knowledge about the physiology of Dekkera bruxellensis has shown its potential for the production of fuel ethanol very close to the conventional fermenting yeast S. cerevisiae. However, some aspects of its metabolism remain uncovered. In the present study, the respiro-fermentative parameters of D. bruxellensis GDB 248 were evaluated under different cultivation conditions. The results showed that sucrose was more efficiently converted to ethanol than glucose, regardless the nitrogen source, which points out for the industrial efficiency of this yeast in sucrose-based substrate. The blockage of the cytosolic acetate production incremented the yeast fermentative efficiency by 27% (in glucose) and 14% (in sucrose). On the other hand, the presence of nitrate as inducer of acetate production reducing the production of ethanol. Altogether, these results settled the hypothesis that acetate metabolism is the main constraint for ethanol production. Besides, this acetate-generating pathway seems to exert some regulatory action on the flux and distribution of the carbon flowing through the central metabolism. These physiological aspects were corroborated by the relative expression analysis of key genes in the crossroad to ethanol, acetate and biomass formation. All the results were discussed in the light of the industrial potential of this yeast.

Assuntos

Dekkera , Saccharomyces cerevisiae , Acetatos/metabolismo , Brettanomyces , Dekkera/genética , Dekkera/metabolismo , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Microbiologia Industrial , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sacarose/metabolismo

RESUMO

The yeast Dekkera bruxellensis is well-known for its adaptation to industrial ethanol fermentation processes, which can be further improved if nitrate is present in the substrate. To date, the assimilation of nitrate has been considered inefficient because of the apparent energy cost imposed on cell metabolism. Recent research, however, has shown that nitrate promotes growth rate and ethanol yield when oxygen is absent from the environment. Given this, the present work aimed to identify the biological mechanisms behind this physiological behaviour. Proteomic analyses comparing four contrasting growth conditions gave some clues on how nitrate could be used as primary nitrogen source by D. bruxellensis GDB 248 (URM 8346) cells in anaerobiosis. The superior anaerobic growth in nitrate seems to be a consequence of increased cell metabolism (glycolytic pathway, production of ATP and NADPH and anaplerotic reactions providing metabolic intermediates) regulated by balanced activation of TORC1 and NCR de-repression mechanisms. On the other hand, the poor growth observed in aerobiosis is likely due to an oxidative stress triggered by nitrate when oxygen is present. These results represent a milestone regarding the knowledge about nitrate metabolism and might be explored for future use of D. bruxellensis as an industrial yeast. KEY POINTS: • Nitrate can be regarded as preferential nitrogen source for D. bruxellensis. • Oxidative stress limits the growth of D. bruxellensis in nitrate in aerobiosis. • Nitrate is a nutrient for novel industrial bioprocesses using D. bruxellensis.

Assuntos

Dekkera , Brettanomyces , Fermentação , Nitratos , Proteômica

RESUMO

The yeast Brettanomyces bruxellensis is able to ferment the main sugars used in first-generation ethanol production. However, its employment in this industry is prohibitive because the ethanol productivity reached is significantly lower than the observed for Saccharomyces cerevisiae. On the other hand, a possible application of B. bruxellensis in the second-generation ethanol production has been suggested because this yeast is also able to use d-xylose and l-arabinose, the major pentoses released from lignocellulosic material. Although the latter application seems to be reasonable, it has been poorly explored. Therefore, we aimed to evaluate whether or not different industrial strains of B. bruxellensis are able to ferment d-xylose and l-arabinose, both in aerobiosis and oxygen-limited conditions. Three out of nine tested strains were able to assimilate those sugars. When in aerobiosis, B. bruxellensis cells exclusively used them to support biomass formation, and no ethanol was produced. Moreover, whereas l-arabinose was not consumed under oxygen limitation, d-xylose was only slightly used, which resulted in low ethanol yield and productivity. In conclusion, our results showed that d-xylose and l-arabinose are not efficiently converted to ethanol by B. bruxellensis, most likely due to a redox imbalance in the assimilatory pathways of these sugars. Therefore, despite presenting other industrially relevant traits, the employment of B. bruxellensis in second-generation ethanol production depends on the development of genetic engineering strategies to overcome this metabolic bottleneck.

Assuntos

Arabinose/metabolismo , Brettanomyces/metabolismo , Etanol/metabolismo , Xilose/metabolismo , Aerobiose , Biomassa , Brettanomyces/genética , Brettanomyces/crescimento & desenvolvimento , Meios de Cultura/metabolismo , Fermentação

RESUMO

Osteoporosis (OP) is a multifactorial disease influenced by genetic factors in more than half of the cases. In spite of the efforts to clarify the relationship among genetic factors and susceptibility to develop OP, many genetic associations need to be further functionally validated. Besides, some limitations as the choice of stably expressed reference genes (RG) should be overcome to ensure the quality and reproducibility of gene expression assays. To our knowledge, a validation study for RG in OP is still missing. We compared the expression levels, using polymerase chain reaction quantitative real time (qPCR) of 10 RG (G6PD, B2M, GUSB, HSP90, EF1A, RPLP0, GAPDH, ACTB, 18 S and HPRT1) to assess their suitability in OP analysis by using GeNorm, Normfinder, BestKeeper and RefFinder programs. A minimal number of two RG was recommended by GeNorm to obtain a reliable normalization. RPLP0 and B2M were identified as the most stable genes in OP studies while ACTB, 18 S and HPRT1 were inadequate for normalization in our data set. Moreover, we showed the dramatic effects of suboptimal RG choice on the quantification of a target gene, highlighting the importance in the identification of the most appropriate reference gene to specific diseases. We suggest the use of RPLP0 and B2M as the most stable reference genes while we do not recommend the use of the least stable reference genes HPRT1, 18 S and ACTB in OP expression assays using PBMC as biological source. Additionally, we emphasize the importance of individualized and careful choice in software and reference genes selection.

Assuntos

Regulação da Expressão Gênica , Predisposição Genética para Doença , Osteoporose Pós-Menopausa/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes

RESUMO

Dekkera bruxellensis is an industrial yeast mainly regarded as a contaminant species in fermentation processes. In winemaking, it is associated with off-flavours that cause wine spoilage, while in bioethanol production this yeast is linked to a reduction of industrial productivity by competing with Saccharomyces cerevisiae for the substrate. In spite of that, this point of view is gradually changing, mostly because D. bruxellensis is also able to produce important metabolites, such as ethanol, acetate, fusel alcohols, esters and others. This dual role is likely due to the fact that this yeast presents a set of metabolic traits that might be either industrially attractive or detrimental, depending on how they are faced and explored. Therefore, a proper industrial application for D. bruxellensis depends on the correct assembly of its central metabolic puzzle. In this sense, researchers have addressed issues regarding the physiological and genetic aspects of D. bruxellensis, which have brought to light much of our current knowledge on this yeast. In this review, we shall outline what is presently understood about the main metabolic features of D. bruxellensis and how they might be managed to improve its current or future industrial applications (except for winemaking, in which it is solely regarded as a contaminant). Moreover, we will discuss the advantages and challenges that must be overcome in order to take advantage of the full biotechnological potential of this yeast.

Assuntos

Dekkera/genética , Dekkera/metabolismo , Microbiologia Industrial , Ácido Acético/metabolismo , Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia

RESUMO

This work describes the response of Lactobacillusvini, a bacterium found as a contaminant in winemaking and fuel ethanol fermentation processes, to acid stress caused by inorganic or weak organic acids. First, we observed for the first time that bacterial cells become resistant to lysis by lysozyme when submitted to acidic stress. Then, the predicted intracellular acidification can be reversed by the presence of arginine, histidine and glutamine. However, these molecules were not able to reverse the effect of resistance to lysis, indicating the independence of these mechanisms. In general, a reduction in the expression of the main genes involved in the synthesis and deposition of material in the cell wall was observed, whereas the genes involved in the reabsorption of this structure showed increased expression. These data suggested that L. vini responds to the acidification of the medium through early entry into the stationary phase, firing two signals for cell wall remodelling and maintenance of intracellular pHin a coordinated way, most probably by alkalization and the proton extrusion process. If this picture is conserved among lactobacilli, it may not only have an impact on research associated with fermentation processes, but also on that associated with probiotic improvement.

Assuntos

Ácidos/metabolismo , Meios de Cultura/química , Lactobacillus/fisiologia , Ácidos/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Meios de Cultura/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Lactobacillus/genética , Lactobacillus/crescimento & desenvolvimento , Estresse Fisiológico

RESUMO

In the past few years, the yeast Dekkera bruxellensis has gained much of attention among the so-called non-conventional yeasts for its potential in the biotechnological scenario, especially in fermentative processes. This yeast has been regarded as an important competitor to Saccharomyces cerevisiae in bioethanol production plants in Brazil and several studies have reported its capacity to produce ethanol. However, our current knowledge concerning D. bruxellensis is restricted to its aerobic metabolism, most likely because wine and beer strains cannot grow in full anaerobiosis. Hence, the present work aimed to fulfil a gap regarding the lack of information on the physiology of Dekkera bruxellensis growing in the complete absence of oxygen and the relationship with assimilation of nitrate as nitrogen source. The ethanol strain GDB 248 was fully capable of growing anaerobically and produces ethanol at the same level of S. cerevisiae. The presence of nitrate in the medium increased this capacity. Moreover, nitrate is consumed faster than ammonium and this increased rate coincided with a higher speed of glucose consumption. The profile of gene expression helped us to figure out that even in anaerobiosis, the presence of nitrate drives the yeast cells to an oxidative metabolism that ultimately incremented both biomass and ethanol production. These results finally provide the clues to explain most of the success of this yeast in industrial processes of ethanol production.

Assuntos

Ácido Acético/metabolismo , Dekkera/efeitos dos fármacos , Etanol/metabolismo , Nitratos/metabolismo , Compostos de Amônio/metabolismo , Anaerobiose , Cerveja/microbiologia , Biomassa , Brasil , Dekkera/metabolismo , Fermentação , Manipulação de Alimentos , Microbiologia de Alimentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Desidrogenase de Glutamato (NADP+)/genética , Desidrogenase de Glutamato (NADP+)/metabolismo , Nitrogênio/metabolismo , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia

RESUMO

Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl-CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re-oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.

Assuntos

Acetatos/metabolismo , Dekkera/metabolismo , Etanol/metabolismo , Fermentação , Acetatos/análise , Aldeído Oxirredutases/antagonistas & inibidores , Carbono/metabolismo , Meios de Cultura , Dekkera/efeitos dos fármacos , Dissulfiram/farmacologia , Glucose/metabolismo , Microbiologia Industrial , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo

RESUMO

In the last years several reports have reported the capacity of the yeast Dekkera (Brettanomyces) bruxellensis to survive and adapt to the industrial process of alcoholic fermentation. Much of this feature seems to relate to the ability to assimilate limiting sources of nutrients, or somehow some that are inaccessible to Saccharomyces cerevisiae, in particular the sources of nitrogen. Among them, amino acids (AA) are relevant in terms of beverage musts, and could also be important for bioethanol. In view of the limited knowledge on the control of AA, the present work combines physiological and genetic studies to understand how it operates in D. bruxellensis in response to oxygen availibility. The results allowed separation of the AA in three groups of preferentiality and showed that glutamine is the preferred AA irrespective of the presence of oxygen. Glutamate and aspartate were also preferred AA in anaerobiosis, as indicated by the physiological data. Gene expression experiments showed that, apart from the conventional nitrogen catabolic repression mechanism that is operating in aerobiosis, there seems to be an oxygen-independent mechanism acting to overexpress key genes like GAP1, GDH1, GDH2 and GLT1 to ensure adequate anaerobic growth even in the presence of non-preferential nitrogen source. This could be of major importance for the industrial fitness of this yeast species.

Assuntos

Aminoácidos/metabolismo , Dekkera/metabolismo , Dekkera/enzimologia , Fermentação , Indústria Alimentícia , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica

RESUMO

In the present work we studied the expression of genes from nitrogen central metabolism in the yeast Dekkera bruxellensis and under regulation by the Nitrogen Catabolite Repression mechanism (NCR). These analyses could shed some light on the biological mechanisms involved in the adaptation and survival of this yeast in the sugarcane fermentation process for ethanol production. Nitrogen sources (N-sources) in the form of ammonium, nitrate, glutamate or glutamine were investigated with or without the addition of methionine sulfoximine, which inhibits the activity of the enzyme glutamine synthetase and releases cells from NCR. The results showed that glutamine might act as an intracellular sensor for nitrogen availability in D. bruxellensis, by activating NCR. Gene expression analyses indicated the existence of two different GATA-dependent NCR pathways, identified as glutamine-dependent and glutamine-independent mechanisms. Moreover, nitrate is sensed as a non-preferential N-source and releases NCR to its higher level. After grouping genes according to their regulation pattern, we showed that genes for ammonium assimilation represent a regulon with almost constitutive expression, while permease encoding genes are mostly affected by the nitrogen sensor mechanism. On the other hand, nitrate assimilation genes constitute a regulon that is primarily subjected to induction by nitrate and, to a lesser extent, to a repressive mechanism by preferential N-sources. This observation explains our previous reports showing that nitrate is co-consumed with ammonium, a trait that enables D. bruxellensis cells to scavenge limiting N-sources in the industrial substrate and, therefore, to compete with Saccharomyces cerevisiae in this environment.

Assuntos

Repressão Catabólica/fisiologia , Dekkera/metabolismo , Regulação Fúngica da Expressão Gênica , Glutamina/metabolismo , Nitrogênio/metabolismo , Compostos de Amônio/metabolismo , Repressão Catabólica/genética , Dekkera/genética , Dekkera/crescimento & desenvolvimento , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/biossíntese , Microbiologia Industrial , Metionina Sulfoximina/metabolismo , Metionina Sulfoximina/toxicidade , Nitratos/metabolismo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Regulon

RESUMO

In a recent work we showed that magnesium (MgII) plays an important role in industrial ethanol production, overcoming the negative effect of the excess of minerals, particularly copper, present in sugarcane juice, with a consequent increase in ethanol yield. This cation has been reported to be involved in several steps of yeast metabolism, acting mainly as a co-factor of several enzymes of fermentation metabolism and protecting yeast cells from stressful conditions. However, despite many physiological investigations, its effect in the molecular mechanisms that control such metabolic activities remains unclear and to date no information concerning its influence on gene expression has been provided. The present work took advantage of the DNA microarray technology to analyse the global gene expression in yeast cells upon fermentation in MgII-supplemented medium. The results of the fermentation parameters confirmed the previous report on the increase in ethanol yield by MgII. Moreover, the gene expression data revealed an unexpected set of up-regulated genes currently assigned as being negatively-regulated by glucose, which belong to respiratory and energy metabolism, the stress response and the glyoxalate cycle. On the other hand, genes involved in ribosome biogenesis were down-regulated. Computational analysis provided evidence for a regulatory network commanded by key transcriptional factors that may be responsible for the biological action of MgII in yeast cells. In this scenario, MgII seems to act by reprogramming the yeast metabolism by releasing many genes from glucose catabolite repression with positive consequences for ethanol production and maintenance of cell viability.

Assuntos

Repressão Catabólica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Magnésio/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Metabolismo Energético/efeitos dos fármacos , Etanol/metabolismo , Fermentação/efeitos dos fármacos , Redes Reguladoras de Genes , Glucose/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transcriptoma

RESUMO

In the present work, we provide biological evidences supporting the participation of NCW2 gene in the mechanism responsible for cell tolerance to polyhexamethylene biguanide (PHMB), an antifungal agent. The growth rate of yeast cells exposed to this agent was significantly reduced in ∆ncw2 strain and the mRNA levels of NCW2 gene in the presence of PHMB showed a 7-fold up-regulation. Moreover, lack of NCW2 gene turns yeast cell more resistant to zymolyase treatment, indicating that alterations in the ß-glucan network do occur when Ncw2p is absent. Computational analysis of the translated protein indicated neither catalytic nor transmembrane sites and reinforced the hypothesis of secretion and anchoring to cell surface. Altogether, these results indicated that NCW2 gene codes for a protein which participates in the cell wall biogenesis in yeasts and that Ncw2p might play a role in the organisation of the ß-glucan assembly.

Assuntos

Antifúngicos/farmacologia , Biguanidas/farmacologia , Parede Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , beta-Glucanas/metabolismo , Parede Celular/química , Parede Celular/genética , Farmacorresistência Fúngica , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , beta-Glucanas/química

RESUMO

In bioethanol production plants, yeast cells are generally recycled between fermentation batches by using a treatment with sulphuric acid at a pH ranging from 2.0 to 2.5. We have previously shown that Saccharomyces cerevisiae cells exposed to sulphuric acid treatment induce the general stress response pathway, fail to activate the protein kinase A signalling cascade and requires the mechanisms of cell wall integrity and high osmolarity glycerol pathways in order to survive in this stressful condition. In the present work, we used transcriptome-wide analysis as well as physiological assays to identify the transient metabolic responses of S. cerevisiae under sulphuric acid treatment. The results presented herein indicate that survival depends on a metabolic reprogramming of the yeast cells in order to assure the yeast cell viability by preventing cell growth under this harmful condition. It involves the differential expression of a subset of genes related to cell wall composition and integrity, oxidation-reduction processes, carbohydrate metabolism, ATP synthesis and iron uptake. These results open prospects for application of this knowledge in the improvement of industrial processes based on metabolic engineering to select yeasts resistant to acid treatment.

Assuntos

Adaptação Biológica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Ácidos Sulfúricos/farmacologia , Transcriptoma , Metabolismo dos Carboidratos , Etanol/metabolismo , Fermentação , Perfilação da Expressão Gênica , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Redes e Vias Metabólicas , Mutação , Estresse Oxidativo , Purinas/biossíntese

RESUMO

The distilled spirit made from sugar cane juice, also known as cachaça, is a traditional Brazilian beverage that in recent years has increased its market share among international distilled beverages. Several volatile compounds produced by yeast cells during the fermentation process are responsible for the unique taste and aroma of this drink. The yeast Dekkera bruxellensis has acquired increasing importance in the fermented beverage production, as the different metabolites produced by this yeast may be either beneficial or harmful to the end-product. Since D. bruxellensis is often found in the fermentation processes carried out in ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile and production of aromatic compounds and to examine whether it is feasible to regard it as a cachaça-producing microorganism. The assays were performed on a small scale and simulated the conditions for the production of handmade cachaça. The results showed that the presence of aromatic and branched-chain amino acids in the medium has a strong influence on the metabolism and production of flavours by D. bruxellensis. The assimilation of these alternative nitrogen sources led to different fermentation yields and the production of flavouring compounds. The influence of the nitrogen source on the metabolism of fusel alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen requirements to obtain the desired level of sensory compounds in the fermentation. Our results suggest that D. bruxellensis has the potential to play a role in the production of cachaça.

Assuntos

Bebidas Alcoólicas/microbiologia , Dekkera/metabolismo , Aromatizantes/metabolismo , Nitrogênio/metabolismo , Saccharum/microbiologia , Brasil , Meios de Cultura/metabolismo , Fermentação , Saccharum/metabolismo

RESUMO

In fuel ethanol production, recycling of yeast biomass includes treatment of cells with diluted sulphuric acid in order to control bacterial population. However, this strategy might lead to a loss of cell viability, with potential negative consequences to the fermentation yield. In a recent paper we showed that the proteins Slt2 and Hog1 are essential for yeast tolerance to sulphuric acid. As a complement of the aforementioned work, we used DNA microarray technology to search for differentially expressed genes in hog1Δ and slt2Δ deletion mutants after treatment with sulphuric acid. Our results show how Slt2p and Hog1p could coordinate the interplay among protein kinase A (PKA), protein kinase C and high-osmolarity glycerol pathways. Moreover, the SSK22 and KDX1 genes may be part of this network, although their proteins were shown to be non-essential for cell growth/survival at low pH. These proteins might work by enhancing the signal which downregulates the PKA pathway leading to cell cycle arrest, in order to regenerate the integrity of yeast cell wall and cell homeostasis under acid shock.

Assuntos

Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ácidos Sulfúricos/farmacologia , Adaptação Biológica , Biocombustíveis , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Epistasia Genética , Etanol/metabolismo , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , MAP Quinase Quinase Quinases/metabolismo , Redes e Vias Metabólicas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteína Quinase C/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo

RESUMO

A previous study showed that the use of nitrate by Dekkera bruxellensis might be an advantageous trait when ammonium is limited in sugarcane substrate for ethanol fermentation. The aim of the present work was to evaluate the influence of nitrate on the yeast physiology during cell growth in different carbon sources under oxygen limitation. If nitrate was the sole source of nitrogen, D. bruxellensis cells presented slower growth, diminished sugar consumption and growth-associated ethanol production, when compared to ammonium. These results were corroborated by the increased expression of genes involved in the pentose phosphate (PP) pathway, the tricarboxylic acid (TCA) cycle and ATP synthesis. The presence of ammonium in the mixed medium restored most parameters to the standard conditions. This work may open up a line of investigation to establish the connection between nitrate assimilation and energetic metabolism in D. bruxellensis and their influence on its fermentative capacity in oxygen-limited or oxygen-depleted conditions.

Assuntos

Dekkera/metabolismo , Nitratos/metabolismo , Oxigênio/metabolismo , Ciclo do Ácido Cítrico , Dekkera/crescimento & desenvolvimento , Etanol/metabolismo , Fermentação , Via de Pentose Fosfato

RESUMO

The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a 2(-ΔΔC(q)) (comparative C(q) method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis.

Assuntos

Dekkera/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Primers do DNA , RNA Mensageiro/genética , Reprodutibilidade dos Testes

RESUMO

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.

Assuntos

Dekkera/metabolismo , Microbiologia Industrial , Nitratos/metabolismo , Saccharomyces cerevisiae/metabolismo , Etanol/metabolismo , Fermentação