Will de Barros Pita

The ability to use nitrate confers advantage to Dekkera bruxellensis over S. cerevisiae and can explain its adaptation to industrial fermentation processes

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.

5-Hydroxymethylfurfural induces ADH7 and ARI1 expression in tolerant industrial Saccharomyces cerevisiae strain P6H9 during bioethanol production

The aims of this work were to obtain, by evolutionary engineering, an industrial strain of Saccharomyces cerevisiae tolerant to high concentrations of HMF and to determine the expression levels of genes previously described as responsible for this tolerance. Cells were grown under anaerobic and oxygen limited conditions, in the presence of glucose or sucrose as carbon sources. P6H9 strain presented high expression levels for genes ADH7 and ARI1 in presence of HMF. This tolerant strain also showed higher ethanol productivity, biomass formation and alcohol dehydrogenase activity comparing to sensitive strains. Results suggest that S. cerevisiae P6H9 strain presents potential to be used for second-generation ethanol production.

The influence of nitrate on the physiology of the yeast Dekkera bruxellensis grown under oxygen limitation.

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. Copyright

Physiology and gene expression profiles of Dekkera bruxellensis in response to carbon and nitrogen availability

The assimilation of nitrate, a nitrogenous compound, was previously described as an important factor favoring Dekkera bruxellensis in the competition with Saccharomyces cerevisiae for the industrial sugarcane substrate. In this substrate, nitrogen sources are limited and diverse, and a recent report showed that amino acids enable D. bruxellensis to grow anaerobically. Thus, understanding the regulation of nitrogen metabolism is one fundamental aspect to comprehend the competiveness of D. bruxellensis in the fermentation environment. In the present study, we evaluated the physiological and transcriptional profiles of D. bruxellensis in response to different carbon and nitrogen supplies to determine their influence on growth, sugar consumption, and ethanol production. Besides, the expression of genes coding for nitrogen permeases and enzymes involved in the biosynthesis of glutamate and energetic metabolism were investigated under these conditions. Our data revealed that genes related to nitrogen uptake in D. bruxellensis are under the control of nitrogen catabolite repression. Moreover, we provide indications that glutamate dehydrogenase and glutamate synthase may switch roles as the major pathway for glutamate biosynthesis in D. bruxellensis. Finally, our data showed that in nonoptimal growth conditions, D. bruxellensis leans toward the respiratory metabolism. The results presented herein show that D. bruxellensis and S. cerevisiae share similar regulation of GDH–GOGAT pathway, while D. bruxellensis converts less glucose to ethanol than S. cerevisiae do when nitrogen is limited. The consequence of this particularity to the industrial process is discussed.

Adaptation of Dekkera bruxellensis to lignocellulose-based substrate

Adaptation of Dekkera bruxellensis to lignocellulose hydrolysate was investigated. Cells of D. bruxellensis were grown for 72 and 192 H in batch and continuous culture, respectively (adapted cells). Cultivations in semisynthetic medium were run as controls (nonadapted cells). To test the adaptation, cells from these cultures were reinoculated in the lignocellulose medium, and growth and ethanol production characteristics were monitored. Cells adapted to lignocellulose hydrolysate had a shorter lag phase, grew faster, and produced a higher ethanol concentration as compared with nonadapted cells. A stability test showed that after cultivation in rich medium, cells partially lost the adapted phenotype but still showed faster growth and higher ethanol production as compared with nonadapted cells. Because alcohol dehydrogenase genes have been described to be involved in the adaptation to furfural in Saccharomyces cerevisiae, an analogous mechanism of adaptation to lignocelluloses hydrolysate of D. bruxellensis was hypothesized. However, gene expression analysis showed that genes homologous to S. cerevisiae ADH1 were not involved in the adaptation to lignocelluloses hydrolysate in D. bruxellensis.

Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça

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. Copyright

Transcriptomic response of Saccharomyces cerevisiae for its adaptation to sulphuric acid-induced stress

AbstractIn 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.

Magnesium ions in yeast: setting free the metabolism from glucose catabolite repression

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.

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.

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