RESUMO
Apiculate yeasts belonging to the genus Hanseniaspora are predominant on grapes and other fruits. While some species, such as Hanseniaspora uvarum, are well known for their abundant presence in fruits, they are generally characterized by their detrimental effect on fermentation quality because the excessive production of acetic acid. However, the species Hanseniaspora vineae is adapted to fermentation and currently is considered as an enhancer of positive flavour and sensory complexity in foods. Since 2002, we have been isolating strains from this species and conducting winemaking processes with them. In parallel, we also characterized this species from genes to metabolites. In 2013, we sequenced the genomes of two H. vineae strains, being these the first apiculate yeast genomes determined. In the last 10 years, it has become possible to understand its biology, discovering very peculiar features compared to the conventional Saccharomyces yeasts, such as a natural and unique G2 cell cycle arrest or the elucidation of the mandelate pathway for benzenoids synthesis. All these characteristics contribute to phenotypes with proved interest from the biotechnological point of view for winemaking and the production of other foods.
Assuntos
Hanseniaspora , Vinho , Hanseniaspora/genética , Fermentação , Vinho/análise , Leveduras/genética , BiologiaRESUMO
In the budding yeast Saccharomyces cerevisiae, the FUN-LOV (FUNgal Light Oxygen and Voltage) optogenetic switch enables high levels of light-activated gene expression in a reversible and tunable fashion. The FUN-LOV components, under identical promoter and terminator sequences, are encoded in two different plasmids, which limits its future applications in wild and industrial yeast strains. In this work, we aim to expand the molecular versatility of the FUN-LOV switch to increase its biotechnological applications. Initially, we generated new variants of this system by replacing the promoter and terminator sequences and by cloning the system in a single plasmid (FUN-LOVSP). In a second step, we included the nourseothricin (Nat) or hygromycin (Hph) antibiotic resistances genes in the new FUN-LOVSP plasmid, generating two new variants (FUN-LOVSP-Nat and FUN-LOVSP-Hph), to allow selection after genome integration. Then, we compared the levels of light-activated expression for each FUN-LOV variants using the luciferase reporter gene in the BY4741 yeast strain. The results indicate that FUN-LOVSP-Nat and FUN-LOVSP-Hph, either episomally or genome integrated, reached higher levels of luciferase expression upon blue-light stimulation compared the original FUN-LOV system. Finally, we demonstrated the functionality of FUN-LOVSP-Hph in the 59A-EC1118 wine yeast strain, showing similar levels of reporter gene induction under blue-light respect to the laboratory strain, and with lower luciferase expression background in darkness condition. Altogether, the new FUN-LOV variants described here are functional in different yeast strains, expanding the biotechnological applications of this optogenetic tool.
RESUMO
Global warming has a significant impact on different viticultural parameters, including grape maturation. An increment of photosynthetic activity generates a rapid accumulation of sugars in the berry, followed by a dehydration process which leads to a higher concentration of soluble solids. This effect is exacerbated by current viticultural practices which favor the harvest of very mature grapes to obtain wines with sweet tannins. Considering the initial hyperosmotic stress conditions and the high ethanol concentration of the produced wine, fermentation of grape musts with high sugar content could be problematic for yeast starters. In the present study, we were able to obtain by classical hybridization and spore dissection methods one hybrid and one monosporic wine yeast strain with a combined ethanol and osmotolerant phenotype. The improved yeasts were tested in vinification trials with high sugar concentration and displayed excellent fermentation performance. Importantly, the obtained wines also showed good organoleptic properties during sensory analysis. Based on our results, we believed our improved hybrid and monosporic strains can be considered good alternatives to be used as yeast starters for fermentations with high sugar content.
Assuntos
Vitis , Vinho , Carboidratos/análise , Etanol/análise , Saccharomyces cerevisiae/genética , Esporos/química , Açúcares/análise , Taninos , Vinho/análiseRESUMO
Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.
Assuntos
Derivados de Benzeno/metabolismo , Vias Biossintéticas/genética , Hanseniaspora/genética , Piruvato Descarboxilase/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma , Benzaldeídos/metabolismo , Álcool Benzílico/metabolismo , Fermentação , Hanseniaspora/metabolismoRESUMO
Vale dos Vinhedos appellation of origin has a very recent history as industrial wine making region. In this study we investigated the genetic and phenotypic variability of Saccharomyces cerevisiae strains isolated from South-Brazilian vineyards in order to evaluate strain fermentation aptitude and copper and sulphites tolerance. Merlot grape bunches were collected from three vineyards and yeast isolation was performed after single bunch fermentation. High genotypic variability was found and most of the genotypes revealed to be vine-specific. No industrial strain dissemination was present in the sampled vineyards, although it has been wildly reported in traditional winemaking countries. From the phenotypic traits analysis these Brazilian native strains showed good fermentation performances, good tolerance to sulphites and, in particular, a high copper tolerance level. Copper is the most important metal in the formulation of fungicides against downy mildew (Plasmopara viticola), one of the most harmful disease of the vines, and other fungal pests. The high tolerance to copper suggests an environmental adaptation to the strong use of copper-based fungicides, requested by the wet subtropical climate.
Assuntos
Variação Genética , Genótipo , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Agricultura , Brasil , Cobre/toxicidade , Sulfato de Cobre/toxicidade , DNA Fúngico/genética , Tolerância a Medicamentos , Fazendas , Fermentação , Fungicidas Industriais/química , Fungicidas Industriais/toxicidade , Genes Fúngicos , Mapeamento Geográfico , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Cinética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/isolamento & purificação , Dióxido de Enxofre/toxicidade , Vitis/química , Vitis/microbiologia , Vinho/microbiologiaRESUMO
Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Among the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to that in grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were 20-200 times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in the H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.
Assuntos
Benzaldeídos/metabolismo , Álcoois Benzílicos/metabolismo , Aromatizantes/metabolismo , Hanseniaspora/metabolismo , Vitis/microbiologia , Vinho/análise , Benzaldeídos/análise , Álcoois Benzílicos/análise , Fermentação , Aromatizantes/análise , Hanseniaspora/genética , Vitis/metabolismoRESUMO
In several grape varieties, the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoid-related compounds, such as glycosylated benzyl and 2-phenylethyl alcohol, which contribute to wine with floral and fruity aromas after being hydrolysed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora, are known to predominate during the first stages of alcoholic fermentation. Although non-Saccharomyces yeast strains have a well-recognized genetic diversity, understanding of their impact on wine flavour richness is still emerging. In this study, 11 Hansenisapora vineae strains were used to ferment a chemically defined simil-grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape-derived secondary metabolites. GC-MS analysis was performed to determine volatile compounds in the produced wines. Our results showed that benzyl alcohol, benzyl acetate and 2-phenylethyl acetate are significantly synthesized by H. vineae strains. Levels of these compounds found in fermentations with 11 H. vineae different strains were one or two orders of magnitude higher than those measured in fermentations with a known S. cerevisiae wine strain. The implications for winemaking in response to the negative correlation of benzyl alcohol, benzyl acetate and 2-phenylethyl acetate production with yeast assimilable nitrogen concentrations are discussed. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Fermentação , Hanseniaspora/metabolismo , Nitrogênio/metabolismo , Fenóis/metabolismo , Vinho , Acetatos/metabolismo , Compostos de Amônio/química , Compostos de Amônio/metabolismo , Álcool Benzílico/metabolismo , Compostos de Benzil/metabolismo , Aromatizantes/análise , Aromatizantes/química , Cromatografia Gasosa-Espectrometria de Massas , Nitrogênio/química , Fenóis/análise , Fenóis/química , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Saccharomyces cerevisiae/metabolismo , Vitis/químicaRESUMO
Background Saccharomyces cerevisiae is the main microorganism responsible for alcoholic fermentation. In this process, the consumption of nitrogen is of great importance since it is found in limiting quantities and its deficiency produces sluggish and/or stuck fermentations generating large economic losses in the wine-making industry. In a previous work we compared the transcriptional profiles between genetically related strains with differences in nitrogen consumption, detecting genes with differential expression that could be associated to the differences in the levels of nitrogen consumed. One of the genes identified was ICY1. With the aim of confirming this observation, in the present work we evaluated the consumption of ammonium during the fermentation of strains that have deleted or overexpressed this gene. Results Our results confirm the effect of ICY1 on nitrogen uptake by evaluating its expression in wine yeasts during the first stages of fermentation under low (MS60) and normal (MS300) assimilable nitrogen. Our results show that the mRNA levels of ICY1 diminish when the amount of assimilable nitrogen is low. Furthermore, we constructed strains derived from the industrial strain EC1118 as a null mutant in this gene as well as one that overexpressed it. Conclusions Our results suggest that the expression of ICY1 is regulated by the amount of nitrogen available in the must and it is involved in the consumption of ammonium, given the increase in the consumption of this nitrogen source observed in the null mutant strain.
Assuntos
Saccharomyces cerevisiae/genética , Vinho/microbiologia , Leveduras/genética , Fermentação , Saccharomyces cerevisiae/metabolismo , Leveduras/metabolismo , Expressão Gênica , Clonagem Molecular , Deleção de Genes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , NitrogênioRESUMO
AIMS: The purpose of this study was to select autochthonous yeasts with metabolic ability to degrade L-malic acid for its potential use in young wine deacidification. METHODS AND RESULTS: Fifty seven Patagonian nonSaccharomyces yeast of oenological origin were identified by conventional molecular methods and tested in their capability to grow at the expense of L-malic acid. Only four isolates belonging to Pichia kudriavzevii species showed this property, and one of them was selected to continue with the study. This isolate, named as P. kudriavzevii ÑNI15, was able to degrade L-malic acid in microvinifications, increasing the pH 0·2-0·3 units with a minimal effect on the acid structure of wine. Additionally, this isolate produced low levels of ethanol, important levels of glycerol (10·41 ± 0·48 g l(-1) ) and acceptable amounts of acetic acid (0·86 ± 0·13 g l(-1) ). In addition, it improved the sensorial attributes of wine increasing its fruity aroma. CONCLUSIONS: The selection of yeasts for oenological use among nonSaccharomyces species led to the finding of a yeast strain with novel and interesting oenological characteristics which could have significant implications in the production of well-balanced and more physicochemical and microbiological stable young wines. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of P. kudriavzevii ÑNI15 as mixed starter with S. cerevisiae would eliminate the cultural and cellar operations undertaken to adjust the musts acidity, therefore improving wine quality and reducing production costs.
Assuntos
Malatos/metabolismo , Pichia/metabolismo , Vinho/microbiologia , Fermentação , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/metabolismo , Leveduras/isolamento & purificação , Leveduras/metabolismoRESUMO
During alcoholic fermentation, Saccharomyces cerevisiae is exposed to continuously changing environmental conditions, such as decreasing sugar and increasing ethanol concentrations. Oxygen, a critical nutrient to avoid stuck and sluggish fermentations, is only discretely available throughout the process after pump-over operation. In this work, we studied the physiological response of the wine yeast S. cerevisiae strain EC1118 to a sudden increase in dissolved oxygen, simulating pump-over operation. With this aim, an impulse of dissolved oxygen was added to carbon-sufficient, nitrogen-limited anaerobic continuous cultures. Results showed that genes related to mitochondrial respiration, ergosterol biosynthesis, and oxidative stress, among other metabolic pathways, were induced after the oxygen impulse. On the other hand, mannoprotein coding genes were repressed. The changes in the expression of these genes are coordinated responses that share common elements at the level of transcriptional regulation. Beneficial and detrimental effects of these physiological processes on wine quality highlight the dual role of oxygen in 'making or breaking wines'. These findings will facilitate the development of oxygen addition strategies to optimize yeast performance in industrial fermentations.