The antioxidant defence of Dekkera bruxellensis against hydrogen peroxide and its relationship to nitrate metabolism.

AIMS: The yeast Dekkera bruxellensis is a Crabtree-positive yeast that tends towards the oxidative/respiratory metabolism in aerobiosis. However, it is more sensitive to H2O2 than Saccharomyces cerevisiae. In order to investigate this metabolic paradox, the present work aimed to uncover the biological defence mechanism used by this yeast to tolerate the presence of exogenous H2O2. METHODS AND RESULTS: Growth curves and spot tests were performed to establish the values of minimal inhibitory concentration and minimal biocidal concentration of H2O2 for different combinations of carbon and nitrogen sources. Cells in exponential growth phase in different culture conditions were used to measure superoxide and thiols [protein (PT) and non-PT], enzyme activities and gene expression. CONCLUSIONS: The combination of glutathione peroxidase (Gpx) and sulfhydryl-containing PT formed the preferred defence mechanism against H2O2, which was more efficiently active under respiratory metabolism. However, the action of this mechanism was suppressed when the cells were metabolizing nitrate (NO3). SIGNIFICANCE AND IMPACT OF STUDY: These results were relevant to figure out the fitness of D. bruxellensis to metabolize industrial substrates containing oxidant molecules, such as molasses and plant hydrolysates, in the presence of a cheaper nitrogen source such as NO3.

Metabolic and biotechnological insights on the analysis of the Pdh bypass and acetate production in the yeast Dekkera bruxellensis.

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.

The biotechnological potential of the yeast Dekkera bruxellensis.

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.

Biological diversity of carbon assimilation among isolates of the yeast Dekkera bruxellensis from wine and fuel-ethanol industrial processes.

Dekkera bruxellensis is considered a spoilage yeast in winemaking, brewing and fuel-ethanol production. However, there is growing evidence in the literature of its biotechnological potential. In this work, we surveyed 29 D. bruxellensis isolates from three countries and two different industrial origins (winemaking and fuel-ethanol production) for the metabolization of industrially relevant sugars. The isolates were characterized by the determination of their maximum specific growth rates, and by testing their ability to grow in the presence of 2-deoxy-d-glucose and antimycin A. Great diversity was observed among the isolates, with fuel-ethanol isolates showing overall higher specific growth rates than wine isolates. Preferences for galactose (three wine isolates) and for cellobiose or lactose (some fuel-ethanol isolates) were observed. Fuel-ethanol isolates were less sensitive than wine isolates to glucose catabolite repression (GCR) induction by 2-deoxy-d-glucose. In strictly anaerobic conditions, isolates selected for having high aerobic growth rates were able to ferment glucose, sucrose and cellobiose at fairly high rates without supplementation of casamino acids or yeast extract in the culture medium. The phenotypic diversity found among wine and fuel-ethanol isolates suggests adaptation to these environments. A possible application of some of the GCR-insensitive, fast-growing isolates in industrial processes requiring co-assimilation of different sugars is considered.

4-Ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in red wines: Microbial formation, prevention, remediation and overview of analytical approaches.

The presence of 4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in red wines affect negatively their aroma conferring horsy, barnyard, smoky and medicinal aromatic notes. These volatile phenols formed from free hydroxycinnamic acids and their ethyl esters by Dekkera/Brettanomyces yeasts, can contaminate wines. Their formation can cause serious negative economic impact to the wine industry worldwide as consumers tend to reject these wines. For these reasons various preventive and remedial treatments have been studied. This review summarises the wine microbial volatile phenols formation, preventive measures during winemaking and remedial treatments in finished wines along with their advantages and limitations for dealing with this sensory defect and impact on wine quality. Also it is important to control the levels of volatile phenols in wines using fast and convenient analytical methods namely with a detection limit below their olfactory perception threshold. The analytical methods available for quality control and performance characteristics as well their advantages and disadvantages when dealing with a complex matrix like wine are discussed in detail.

Dekkera bruxellensis, a beer yeast that specifically bioconverts mogroside extracts into the intense natural sweetener siamenoside I.

In response to growing concerns about the consumption of artificial sweeteners, the demand for natural sweeteners has recently increased. Mogroside V is a common natural sweetener extracted from the fruit of Siraitia grosvenorii, but its taste should be improved for marketability. Here, we screened various microbes for the ability to perform selective hydrolysis of glycosidic bonds in mogroside V, converting it to siamenoside I, which has a higher sweetening power and better taste than other mogrosides. Dekkera bruxellensis showed the most promising results in the screen, and the Exg1 gene (coding for a ß-glucosidase) of D. bruxellensis was cloned and purified. We then used HPLC-MS/MS to assess the ß-glucosidase activity of purified enzymes on p-nitrophenyl ß-glucoside and mogroside V. The results demonstrated that D. bruxellensis had a unique enzyme that can selectively hydrolyze mogrol glycosides and promote the conversion of the natural sweetener mogroside V to siamenoside I.

Nitrate boosts anaerobic ethanol production in an acetate-dependent manner in the yeast Dekkera bruxellensis.

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.

Volatile phenols are produced by strains of Dekkera bruxellensis under Brazilian fuel ethanol industry-like conditions.

Dekkera bruxellensis is a spoilage yeast in wine and fuel ethanol fermentations able to produce volatile phenols from hydroxycinnamic acids by the action of the enzymes cinnamate decarboxylase (CD) and vinyphenol reductase (VR) in wine. However, there is no information about this ability in the bioethanol industry. This work evaluated CD and VR activities and 4-ethylphenol production from p-coumaric acid by three strains of D. bruxellensis and PE-2, an industrial Saccharomyces cerevisiae strain. Single and multiple-cycle batch fermentations in molasses and sugarcane juice were carried out. Dekkera bruxellensis strains showed similar CD activity but differences in VR activity. No production of 4-ethylphenol by S. cerevisiae in any fermentation system or media was observed. The concentrations of 4-ethylphenol peaked during active growth of D. bruxellensis in single-cycle fermentation but they were lower than in multiple-cycle fermentation. Higher concentrations were observed in molasses with molar conversion (p-coumaric acid to 4-ethylphenol) ranging from 45% to 85%. As the first report on 4-ethylphenol production in sugarcane musts by D. bruxellensis in industry-like conditions, it opens up a new avenue to investigate its effect on the viability and fermentative capacity of S. cerevisiae as well as to understand the interaction between the yeasts in the bioethanol industry.

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation.

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.

Interaction of Saccharomyces cerevisiae-Lactobacillus fermentum-Dekkera bruxellensis and feedstock on fuel ethanol fermentation.

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.

Characterization of an extracellular ß-glucosidase from Dekkera bruxellensis for resveratrol production.

Polygonum cuspidatum is a widely grown crop with a rich source of polydatin (also called piceid) for resveratrol production. Resveratrol is produced from piceid via enzymatic cleavage of the sugar moiety of piceid. In this study, Dekkera bruxellensis mutants were selected based on their high p-nitrophenyl-ß-d-glucopyranoside and piceid conversion activities. The enzyme responsible for piceid conversion was a heterodimeric protein complex that was predominantly secreted to the extracellular medium and consisted of two subunits at an equal ratio with molecular masses of 30.5 kDa and 48.3 kDa. The two subunits were identified as SCW4p and glucan-ß-glucosidase precursor in D. bruxellensis. Both proteins were individually expressed in Saccharomyces cerevisiae exg1Δ mutants, which lack extracellular ß-glucosidase activity, to confirm each protein's enzymatic activities. Only the glucan-ß-glucosidase precursor was shown to be a secretory protein with piceid deglycosylation activity. Our pilot experiments of piceid bioconversion demonstrate the possible industrial applications for this glucan-ß-glucosidase precursor in the future.

Osmotolerance of Dekkera bruxellensis and the role of two Stl glycerol-proton symporters.

Dekkera bruxellensis is important for lambic beer fermentation but is considered a spoilage yeast in wine fermentation. We compared two D. bruxellensis strains isolated from wine and found that they differ in some basic properties, including osmotolerance. The genomes of both strains contain two highly similar copies of genes encoding putative glycerol-proton symporters from the STL family that are important for yeast osmotolerance. Cloning of the two DbSTL genes and their expression in suitable osmosensitive Saccharomyces cerevisiae mutants revealed that both identified genes encode functional glycerol uptake systems, but only DbStl2 has the capacity to improve the osmotolerance of S. cerevisiae cells.

On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes.

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.

Reducing the negative sensory impact of volatile phenols in red wine with different chitosans: Effect of structure on efficiency.

"Brett character" is a negative sensory attribute acquired by red wines when contaminating Dekkera/Brettanomyces yeasts produce 4-ethylphenol and 4-ethylguaiacol, known as volatile phenols (VPs), from cinnamic acid precursors. In this study, chitins and chitosans with different structural features, namely deacetylation degree (5-91%) and molecular weight (24-466kDa) were used for the reduction of this sensory defect. Chitins and chitosans decreased 7-26% of the headspace abundance of VPs without changing their amounts in wines. The efficiency of reduction increased with the deacetylation degree and applied dose. Reduction of headspace abundance of VPs by chitosans enabled significant decreases in the negative phenolic and bitterness attributes and increased positive fruity and floral attributes. Results show that chitosan with high deacetylation degrees, including fungal chitosan, which is already approved for use in wines, is an efficient approach for reducing the negative sensory impact of VPs in red wines.

New potentially antihypertensive peptides liberated in milk during fermentation with selected lactic acid bacteria and kombucha cultures.

Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography-mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health.

Parallel Evolution of Chromatin Structure Underlying Metabolic Adaptation.

Parallel evolution occurs when a similar trait emerges in independent evolutionary lineages. Although changes in protein coding and gene transcription have been investigated as underlying mechanisms for parallel evolution, parallel changes in chromatin structure have never been reported. Here, Saccharomyces cerevisiae and a distantly related yeast species, Dekkera bruxellensis, are investigated because both species have independently evolved the capacity of aerobic fermentation. By profiling and comparing genome sequences, transcriptomic landscapes, and chromatin structures, we revealed that parallel changes in nucleosome occupancy in the promoter regions of mitochondria-localized genes led to concerted suppression of mitochondrial functions by glucose, which can explain the metabolic convergence in these two independent yeast species. Further investigation indicated that similar mutational processes in the promoter regions of these genes in the two independent evolutionary lineages underlay the parallel changes in chromatin structure. Our results indicate that, despite several hundred million years of separation, parallel changes in chromatin structure, can be an important adaptation mechanism for different organisms. Due to the important role of chromatin structure changes in regulating gene expression and organism phenotypes, the novel mechanism revealed in this study could be a general phenomenon contributing to parallel adaptation in nature.

Glutamine: a major player in nitrogen catabolite repression in the yeast Dekkera bruxellensis.

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.

Analysis of 100-Year-Old Beer Originated from the Czech Republic.

Three bottles of different beers were found in 2015 during a reconstruction of the brewery of the Raven Trading s.r.o. company in Záhlinice, Czech Republic. Thanks to good storage conditions, it was possible to analyze their original characteristics. All three bottles contained most probably lager type beer. One beer had sulfuric and fecal off-flavors; it was bright with the original extract of 10.3° Plato. The second beer, with an original extract of 7.6° Plato, was dark and very acidic, resembling Lambic. DNA analysis proved the presence of Dekkera bruxellensis, which corresponded to its chemical profile (total acidity, FAN, ethyl acetate, total esters). The third beer contained traces of carbon dioxide bubbles, was light brown and slightly bitter, with an original extract 10.4° Plato. Because it obviously underwent a natural aging process, sweetness, honey, and fruity off-flavors were detected and transformation products of iso-α-acids were found.

Comparative morphological characteristics of three Brettanomyces bruxellensis wine strains in the presence/absence of sulfur dioxide.

The red wine spoilage yeast Brettanomyces bruxellensis has been the subject of numerous investigations. Some of these studies focused on spoilage mechanisms, sulfur dioxide tolerance and nutrient requirements. Pseudomycelium formation, although a striking feature of this species, has however been poorly investigated. Furthermore, literature regarding the induction mechanism of pseudomycelium formation in this yeast is limited and lacks clarity, as results published are contradictory. This study elucidates this phenomenon among strains from geographically different areas. Potential environmental cues were investigated, to attain a better understanding of this mechanism and its role as a survival strategy. SO2 was previously reported to induce this morphological change however results obtained in this study did not support this. Nevertheless, the results obtained using scanning and transmission electron microscopy illustrate, for the first time in this yeast, deformity to the cell membrane and alterations to the fibrillar layers in SO2 treated cells. In addition, the SO2 exposed cultures displayed cell size variations, with cells displaying a decrease in length as well as delayed growth, with a prolonged lag phase. Fluorescence microscopy demonstrated a decrease in metabolic activity and the appearance of inclusion body-like structures in the cells following exposure to SO2.

High intracellular trehalase activity prevents the storage of trehalose in the yeast Dekkera bruxellensis.

UNLABELLED: Dekkera bruxellensis hit the spotlight in the past decade mostly due to its rather high ability to adapt to several different fermentation processes. This yeast relies on different genetic and physiological aspects to achieve and preserve its high industrial fitness and some of these traits are shared with Saccharomyces cerevisiae. We have previously described that D. bruxellensis is unable to make use of accumulating trehalose as a strategy for cell adaptation and survival in the industrial scenario, as opposed to S. cerevisiae. Since trehalose is often involved in mechanisms related to cell protection, we aimed to investigate both cause and effect of the absence of this metabolite in the cell adaptive capacity in the industrial environment. Our results indicate that the major cause for the nonaccumulation of trehalose is the high constitutive activity of neutral trehalase. Therefore, the rate of trehalose degradation could be higher than its rate of synthesis, preventing accumulation. Altogether, our data elucidate the mechanisms involved in the lack of trehalose accumulation in D. bruxellensis as well as evaluates the implications of this feature. SIGNIFICANCE AND IMPACT OF THE STUDY: Dekkera bruxellensis can successfully take advantage of its peculiar physiological and genetic traits in order to adapt and survive in fermentation processes. So far, tolerance to stress has been credited to trehalose synthesis. The data presented in this work provided information on the underlying mechanism that prevents trehalose accumulation and corroborated the recent information that trehalose itself is not implicated in yeast stress tolerance. Second, it showed that D. bruxellensis responds differently to Saccharomyces cerevisiae to excess of sugar, which may explain its preference for respiration (oxidative metabolism) over fermentation (reductive metabolism) even at limited oxygen supply. These findings help to understand the drop on ethanol production in processes overtaken by this yeast.