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1.
Food Microbiol ; 94: 103650, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33279075

RESUMO

The positive impact of certain non-Saccharomyces yeasts on the aromatic profile of wines has been well documented in literature and their industrial use in association with S. cerevisiae is now recommended. Competition between non-Saccharomyces species and Saccharomyces cerevisiae for various nutrients, especially nitrogen sources, greatly impacts the production of aroma compounds. In this study, we further explored the impact of different nitrogen nutrition strategies on the production of carbon and sulphur volatile compounds of three non-Saccharomyces strains, namely Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae sequentially inoculated with S. cerevisiae in Sauvignon blanc and Shiraz grape musts. Nitrogen additions were implemented according the specific requirement of each species. At the end of fermentation, we observed specific metabolic signatures for each strain in response to the nature of the nitrogen source suggesting strain-specific metabolic fluxes present. Overall, these results confirmed and further explored the interconnection between nitrogen sources and aroma metabolism (including that of higher alcohols, fatty acids, esters and volatile sulphur compounds), and their variations according to species and the nature of the nitrogen source. The knowledge generated provides new insights to modulate the aroma profile of wines produced with non-Saccharomyces species.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33355112

RESUMO

Nitrogen is among the essential nutriments that govern interactions between yeast species in the wine environment. A thorough knowledge of how these yeasts assimilate the nitrogen compounds of grape juice is an important prerequisite for a successful co- or sequential fermentation. In the present study, we investigated the efficiency of 18 nitrogen sources for sustaining the growth and fermentation of two Starm. bacillaris strains displaying metabolic properties, compared to the reference yeast S. cerevisiae The analysis of growth and fermentation parameters provided a comprehensive picture of Starm. bacillaris preferences with respect to nitrogen sources for sustained growth and fermentation. Important differences were observed in S. cerevisiae regarding rates, final population and CO2 production. In particular, Lys and His supported substantial Starm. bacillaris growth and fermentation contrary to S. cerevisiae, while only 3 nitrogen sources, Arg, NH4+ and Ser, promoted S. cerevisiae growth more efficiently than that of Starm. bacillaris strains. Furthermore, Starm. bacillaris strains displayed a higher fermentative activity than S. cerevisiae during the first phase of culture with Gly or Thr, when the former species consumed solely fructose. Finally, no correlation has been shown between the ability of nitrogen sources to support growth and their fermentation efficiency. The specificities of Starm. bacillaris regarding nitrogen sources preferences are related to its genetic background, but further investigations are needed to elucidate the molecular mechanisms involved. These data are essential elements to be taken into account in order to make the best use of the potential of the two species.IMPORTANCE Mixed fermentations combining non-Saccharomyces and S. cerevisiae strains are increasingly implemented in the wine sector as they offer promising opportunities to diversify the flavour profile of end-products. However, competition for nutrients between species can cause fermentation problems, which is a severe hindrance to the development of these approaches. With the knowledge provided in this study on the nitrogen preferences of Starm. bacillaris, winemakers will be able to set up a nitrogen nutrition scheme adapted to the requirement of each species during mixed fermentation, through must supplementation with relevant nitrogen compounds. This will prevent nitrogen depletion or competition between yeasts for nitrogen sources, and consequently potential issues during fermentation. The data of this study highlight the importance of an appropriate nitrogen resource management during co- or sequential fermentation for fully exploiting the phenotypic potential of non-Saccharomyces yeasts.

3.
Microorganisms ; 8(6)2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32560056

RESUMO

Non-Saccharomyces yeast strains have become increasingly prevalent in the food industry, particularly in winemaking, because of their properties of interest both in biological control and in complexifying flavour profiles in end-products. However, unleashing the full potential of these species would require solid knowledge of their physiology and metabolism, which is, however, very limited to date. In this study, a quantitative analysis using 15N-labelled NH4Cl, arginine, and glutamine, and 13C-labelled leucine and valine revealed the specificities of the nitrogen metabolism pattern of two non-Saccharomyces species, Torulaspora delbrueckii and Metschnikowia pulcherrima. In T. delbrueckii, consumed nitrogen sources were mainly directed towards the de novo synthesis of proteinogenic amino acids, at the expense of volatile compounds production. This redistribution pattern was in line with the high biomass-producer phenotype of this species. Conversely, in M. pulcherrima, which displayed weaker growth capacities, a larger proportion of consumed amino acids was catabolised for the production of higher alcohols through the Ehrlich pathway. Overall, this comprehensive overview of nitrogen redistribution in T. delbrueckii and M. pulcherrima provides valuable information for a better management of co- or sequential fermentation combining these species with Saccharomyces cerevisiae.

4.
Front Microbiol ; 11: 305, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184771

RESUMO

Non-Saccharomyces yeasts are currently widely used in winemaking to enhance aroma profile diversity among wines. The use of Metschnikowia pulcherrima in sequential inoculation with S. cerevisiae was compared to the inoculation of a pure culture of S. cerevisiae. Moreover, various concentrations of sugar, nitrogen and lipids were tested in synthetic must to assess their impact on fermentation and its outcomes using a Box-Behnken design. Due to its phenotypic specificities, early inoculation with M. pulcherrima led to important modifications, first altering the fermentation kinetics. This may relate, at least in part, to the depletion of some nitrogen sources by M. pulcherrima during the first part of fermentation. Beyond these negative interactions on fermentation performance, comparisons between pure cultures and sequentially inoculated cultures revealed changes in the distribution of carbon fluxes during fermentation in presence of M. pulcherrima, resulting in a positive impact on the production of central carbon metabolites and aromas. Furthermore, the expression of varietal thiols was strongly increased as a consequence of positive interactions between the two species. The mechanism of this release still needs to be investigated. Significant differences in the final concentrations of fermentative and varietal aromas depending on the initial must composition were obtained under both inoculation strategies. Interestingly, the response to changes in nutrient availability varied according to the inoculation modality. In particular, a greater incidence of lipids on the production of fatty acids and their ethyl esters derivatives was found during sequential fermentation compared with pure culture, to be viewed in combination with the metabolic characteristics of M. pulcherrima regarding the production of volatile compounds from acetyl-CoA. Overall, the importance of managing nutrient availability under M. pulcherrima/S. cerevisiae sequential inoculation in order to derive the maximum benefit from the potentialities of the non-Saccharomyces species while carrying out fermentation to dryness was highlighted.

5.
Sci Rep ; 10(1): 2162, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034164

RESUMO

The yeast Saccharomyces cerevisiae is an attractive industrial microorganism for the production of foods and beverages as well as for various bulk and fine chemicals, such as biofuels or fragrances. Building blocks for these biosyntheses are intermediates of yeast central carbon metabolism (CCM), whose intracellular availability depends on balanced single reactions that form metabolic fluxes. Therefore, efficient product biosynthesis is influenced by the distribution of these fluxes. We recently demonstrated great variations in CCM fluxes between yeast strains of different origins. However, we have limited understanding of flux modulation and the genetic basis of flux variations. In this study, we investigated the potential of quantitative trait locus (QTL) mapping to elucidate genetic variations responsible for differences in metabolic flux distributions (fQTL). Intracellular metabolic fluxes were estimated by constraint-based modelling and used as quantitative phenotypes, and differences in fluxes were linked to genomic variations. Using this approach, we detected four fQTLs that influence metabolic pathways. The molecular dissection of these QTLs revealed two allelic gene variants, PDB1 and VID30, contributing to flux distribution. The elucidation of genetic determinants influencing metabolic fluxes, as reported here for the first time, creates new opportunities for the development of strains with optimized metabolite profiles for various applications.


Assuntos
Redes e Vias Metabólicas , Locos de Características Quantitativas , Saccharomyces cerevisiae/genética , Carbono/metabolismo , Glucose/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética
6.
Food Microbiol ; 85: 103287, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31500707

RESUMO

Wine-related non-Saccharomyces yeasts are becoming more widely used in oenological practice for their ability to confer wine a more complex satisfying aroma, but their metabolism remains unknown. Our study explored the nitrogen utilisation profile of three popular non-Saccharomyces species, Torulaspora delbrueckii, Metschnikowia pulcherrima and Metschnikowia fructicola. The nitrogen source preferences to support growth and fermentation as well as the uptake order of different nitrogen sources during wine fermentation were investigated. While T. delbrueckii and S. cerevisiae strains shared the same nitrogen source preferences, Metschnikowia sp. Displayed a lower capacity to efficiently use the preferred nitrogen compounds, but were able to assimilate a wider range of amino acids. During alcoholic fermentation, the non-Saccharomyces strains consumed different nitrogen sources in a similar order as S. cerevisiae, but not as quickly. Furthermore, when all the nitrogen sources were supplied in the same amount, their assimilation order was similarly affected for both S. cerevisiae and non-Saccharomyces strains. Under this condition, the rate of nitrogen source consumption of non-Saccharomyces strains and S. cerevisiae was comparable. Overall, this study expands our understanding about the preferences and consumption rates of individual nitrogen sources by the investigated non-Saccharomyces yeasts in a wine environment. This knowledge provides useful information for a more efficient exploitation of non-Saccharomyces strains that improves the management of the wine fermentation.


Assuntos
Fermentação , Nitrogênio/metabolismo , Vinho/microbiologia , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismo , Aminoácidos/metabolismo , Metschnikowia/crescimento & desenvolvimento , Odorantes , Saccharomyces cerevisiae , Torulaspora/crescimento & desenvolvimento
7.
Int J Food Microbiol ; 316: 108441, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31778839

RESUMO

Even though non-Saccharomyces yeasts were regarded as spoilage microorganisms for a long time, their abilities to improve and diversify the aromatic profile of wines are now well recognized. Consequently, their use in combination with S. cerevisiae strains during winemaking has attracted substantial attention over the last decade. However, our limited understanding of the metabolism and physiology of these species remains a barrier to promoting efficient exploitation of their full potential. In this study, we further explored the metabolism involved in the production of fermentative volatile compounds of two commercial non-Saccharomyces strains, T. delbrueckii Biodiva™ and M. pulcherrima Flavia®, in comparison with the reference wine yeast S. cerevisiae Lalvin EC1118®. After growing these strains in the presence of 24 different N-compounds, particular attention was paid to the influence of the nitrogen source on the profile of aroma compounds synthesized by these yeasts (higher alcohols and acids, medium-chain fatty acids and their acetate or ethyl esters derivatives). A comprehensive analysis of the dataset showed that these three species were able to produce all the fermentative aromas, regardless of the nitrogen source, demonstrating the key contribution of the central carbon metabolism to the formation of volatile molecules. Nevertheless, we also observed some specific phenotypic traits for each of the strains in their assimilation capacities for the various nitrogen nutrients as well as in their response to the nature of the nitrogen source in terms of the production of volatile molecules. These observations revealed the intricacy and interconnection between the networks involved in nitrogen consumption and aroma production. These differences are likely related to the genetic backgrounds of the strains. Overall, this study expands our understanding of the metabolic processes responsible for the formation of volatile compounds during wine fermentation and their variations according to species and the nature of the nitrogen source. This knowledge provides a new platform for the more efficient exploitation of non-Saccharomyces strains during winemaking, improving the management of the fermentation.


Assuntos
Nitrogênio/metabolismo , Odorantes , Saccharomycetales/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Fermentação , Odorantes/análise , Fenótipo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/classificação , Especificidade da Espécie , Compostos Orgânicos Voláteis/análise , Vinho/análise , Vinho/microbiologia
8.
Environ Microbiol ; 21(11): 4076-4091, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31336027

RESUMO

In grape must, nitrogen is available as a complex mixture of various compounds (ammonium and amino acids). Wine yeasts assimilate these multiple sources in order to suitably fulfil their anabolic requirements during alcoholic fermentation. Nevertheless, the order of uptake and the intracellular fate of these sources are likely to differ between strains and species. Using a two-pronged strategy of isotopic filiation and RNA sequencing, the metabolic network of nitrogen utilization and its regulation in Kluyveromyces marxianus were described, in comparison with those of Saccharomyces cerevisiae. The data highlighted differences in the assimilation of ammonium and arginine between the two species. The data also revealed that the metabolic fate of certain nitrogen sources differed, thereby resulting in the production of various amounts of key wine aroma compounds. These observations were corroborated by the gene expression analysis.


Assuntos
Compostos de Amônio/metabolismo , Kluyveromyces/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Fermentação , Perfilação da Expressão Gênica , Kluyveromyces/genética , Redes e Vias Metabólicas/fisiologia , Saccharomyces cerevisiae/genética , Vitis/microbiologia , Vinho/microbiologia
9.
Food Microbiol ; 76: 29-39, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30166153

RESUMO

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas.


Assuntos
Nitrogênio/farmacologia , Odorantes/análise , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia , Acetiltransferases/efeitos dos fármacos , Acetiltransferases/genética , Álcoois/metabolismo , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Compostos de Amônio/metabolismo , Compostos de Amônio/farmacologia , Meios de Cultura/síntese química , Ésteres/metabolismo , Fermentação , Cinética , Nitrogênio/metabolismo , Proteínas/efeitos dos fármacos , Proteínas/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo , Vinho/análise
10.
Appl Environ Microbiol ; 84(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29858207

RESUMO

Over the last few years, the potential of non-Saccharomyces yeasts to improve the sensory quality of wine has been well recognized. In particular, the use of Starmerella bacillaris in mixed fermentations with Saccharomyces cerevisiae was reported as an appropriate way to enhance glycerol formation and reduce ethanol production. However, during sequential fermentation, many factors, such as the inoculation timing, strain combination, and physical and biochemical interactions, can affect yeast growth, the fermentation process, and/or metabolite synthesis. Among them, the availability of yeast-assimilable nitrogen (YAN), due to its role in the control of growth and fermentation, has been identified as a key parameter. Consequently, a comprehensive understanding of the metabolic specificities and the nitrogen requirements would be valuable to better exploit the potential of Starm. bacillaris during wine fermentation. In this study, marked differences in the consumption of the total and individual nitrogen sources were registered between the two species, while the two Starm. bacillaris strains generally behaved uniformly. Starm. bacillaris strains are differentiated by their preferential uptake of ammonium compared with amino acids that are poorly assimilated or even produced (alanine). Otherwise, the non-Saccharomyces yeast exhibits low activity through the acetaldehyde pathway, which triggers an important redistribution of fluxes through the central carbon metabolic network. In particular, the formation of metabolites deriving from the two glycolytic intermediates glyceraldehyde-3-phosphate and pyruvate is substantially increased during fermentations by Starm. bacillaris This knowledge will be useful to better control the fermentation process in mixed fermentation with Starm. bacillaris and S. cerevisiaeIMPORTANCE Mixed fermentations using a controlled inoculation of Starmerella bacillaris and Saccharomyces cerevisiae starter cultures represent a feasible way to modulate wine composition that takes advantage of both the phenotypic specificities of the non-Saccharomyces strain and the ability of S. cerevisiae to complete wine fermentation. However, according to the composition of grape juices, the consumption by Starm. bacillaris of nutrients, in particular of nitrogen sources, during the first stages of the process may result in depletions that further limit the growth of S. cerevisiae and lead to stuck or sluggish fermentations. Consequently, understanding the preferences of non-Saccharomyces yeasts for the nitrogen sources available in grape must together with their phenotypic specificities is essential for an efficient implementation of sequential wine fermentations with Starm. bacillaris and S. cerevisiae species. The results of our study demonstrate a clear preference for ammonium compared to amino acids for the non-Saccharomyces species. This finding underlines the importance of nitrogen sources, which modulate the functional characteristics of inoculated yeast strains to better control the fermentation process and product quality.


Assuntos
Compostos de Amônio/metabolismo , Fermentação , Nitrogênio/metabolismo , Fenótipo , Saccharomycetales/metabolismo , Vinho/microbiologia , Aminoácidos/metabolismo , Carbono/metabolismo , Glicerol/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/análise
11.
FEMS Yeast Res ; 18(5)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29741618

RESUMO

Saccharomyces cerevisiae is currently the most important yeast involved in food fermentations, particularly in oenology. However, several other yeast species occur naturally in grape must that are highly promising for diversifying and improving the aromatic profile of wines. If the nitrogen requirement of S. cerevisiae has been described in detail, those of non-Saccharomyces yeasts remain poorly studied despite their increasingly widespread use in winemaking. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we explored the fermentation performances, the utilisation of nitrogen sources and the volatile compound production of 10 strains of non-conventional yeasts in pure culture. Two different conditions were tested: one mimicking the grape juice's nitrogen composition and one with all the nitrogen sources at the same level. We highlighted the diversity in terms of nitrogen preference and amount consumed among the yeast strains. Some nitrogen sources (arginine, glutamate, glycine, tryptophan and γ-aminobutyric acid) displayed the largest variations between strains throughout the fermentation. Several non-Saccharomyces strains produced important aroma compounds such as higher alcohols, acetate and ethyl esters in significantly higher quantities than S. cerevisiae.


Assuntos
Fermentação , Nitrogênio/metabolismo , Odorantes/análise , Vinho/microbiologia , Leveduras/metabolismo , Acetatos/análise , Álcoois/análise , Ésteres/análise , Saccharomyces cerevisiae
12.
Mol Biol Evol ; 35(7): 1712-1727, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29746697

RESUMO

The budding yeast Saccharomyces cerevisiae can be found in the wild and is also frequently associated with human activities. Despite recent insights into the phylogeny of this species, much is still unknown about how evolutionary processes related to anthropogenic niches have shaped the genomes and phenotypes of S. cerevisiae. To address this question, we performed population-level sequencing of 82 S. cerevisiae strains from wine, flor, rum, dairy products, bakeries, and the natural environment (oak trees). These genomic data enabled us to delineate specific genetic groups corresponding to the different ecological niches and revealed high genome content variation across the groups. Most of these strains, compared with the reference genome, possessed additional genetic elements acquired by introgression or horizontal transfer, several of which were population-specific. In addition, several genomic regions in each population showed evidence of nonneutral evolution, as shown by high differentiation, or of selective sweeps including genes with key functions in these environments (e.g., amino acid transport for wine yeast). Linking genetics to lifestyle differences and metabolite traits has enabled us to elucidate the genetic basis of several niche-specific population traits, such as growth on galactose for cheese strains. These data indicate that yeast has been subjected to various divergent selective pressures depending on its niche, requiring the development of customized genomes for better survival in these environments. These striking genome dynamics associated with local adaptation and domestication reveal the remarkable plasticity of the S. cerevisiae genome, revealing this species to be an amazing complex of specialized populations.


Assuntos
Adaptação Biológica , Evolução Biológica , Domesticação , Alimentos e Bebidas Fermentados/microbiologia , Saccharomyces cerevisiae/genética , Variações do Número de Cópias de DNA , Fermentação , Transferência Genética Horizontal , Genoma Fúngico , Seleção Genética
13.
BMC Genomics ; 19(1): 166, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29490607

RESUMO

BACKGROUND: The volatile metabolites produced by Saccharomyces cerevisiae during alcoholic fermentation, which are mainly esters, higher alcohols and organic acids, play a vital role in the quality and perception of fermented beverages, such as wine. Although the metabolic pathways and genes behind yeast fermentative aroma formation are well described, little is known about the genetic mechanisms underlying variations between strains in the production of these aroma compounds. To increase our knowledge about the links between genetic variation and volatile production, we performed quantitative trait locus (QTL) mapping using 130 F2-meiotic segregants from two S. cerevisiae wine strains. The segregants were individually genotyped by next-generation sequencing and separately phenotyped during wine fermentation. RESULTS: Using different QTL mapping strategies, we were able to identify 65 QTLs in the genome, including 55 that influence the formation of 30 volatile secondary metabolites, 14 with an effect on sugar consumption and central carbon metabolite production, and 7 influencing fermentation parameters. For ethyl lactate, ethyl octanoate and propanol formation, we discovered 2 interacting QTLs each. Within 9 of the detected regions, we validated the contribution of 13 genes in the observed phenotypic variation by reciprocal hemizygosity analysis. These genes are involved in nitrogen uptake and metabolism (AGP1, ALP1, ILV6, LEU9), central carbon metabolism (HXT3, MAE1), fatty acid synthesis (FAS1) and regulation (AGP2, IXR1, NRG1, RGS2, RGT1, SIR2) and explain variations in the production of characteristic sensorial esters (e.g., 2-phenylethyl acetate, 2-metyhlpropyl acetate and ethyl hexanoate), higher alcohols and fatty acids. CONCLUSIONS: The detection of QTLs and their interactions emphasizes the complexity of yeast fermentative aroma formation. The validation of underlying allelic variants increases knowledge about genetic variation impacting metabolic pathways that lead to the synthesis of sensorial important compounds. As a result, this work lays the foundation for tailoring S. cerevisiae strains with optimized volatile metabolite production for fermented beverages and other biotechnological applications.


Assuntos
Álcoois/metabolismo , Mapeamento Cromossômico , Fermentação , Locos de Características Quantitativas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Substituição de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Estudos de Associação Genética , Genoma Fúngico , Genômica/métodos , Escore Lod , Redes e Vias Metabólicas , Modelos Biológicos , Fenótipo , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável , Metabolismo Secundário , Açúcares/metabolismo
14.
Front Microbiol ; 9: 196, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29487584

RESUMO

The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance, fermentations performed with K. marxianus were characterized by the formation of phenylethanol and phenylethyl acetate, while those performed with P. burtonii or Z. meyerae displayed higher production of isoamyl alcohol and ethyl esters. When considering sequential inoculation of yeasts, the nutritional requirements of the yeasts used should be carefully considered and adjusted accordingly. Finally, our chemical data suggests that the organoleptic properties of the wine are altered in a species specific manner.

15.
PLoS One ; 13(2): e0192383, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29432462

RESUMO

Saccharomyces cerevisiae strains are genetically diverse, largely as a result of human efforts to develop strains specifically adapted to various fermentation processes. These adaptive pressures from various ecological niches have generated behavioral differences among these strains, particularly in terms of their nitrogen consumption capacities. In this work, we characterize this phenotype by the specific quantity of nitrogen consumed under oenological fermentation conditions using a new approach. Indeed, unlike previous studies, our experiments were conducted in an environment containing excess nitrogen, eliminating the nitrogen limitation/starvation factor that is generally observed in fermentation processes. Using these conditions, we evaluated differences in the nitrogen consumption capacities for a set of five strains from diverse origins. The strains presented extremely different phenotypes and variations in their capacities to take up nitrogen from a wine fermentation environment. These variations reflect the differences in the nitrogen uptake capacities between wine and non-wine strains. Finally, the strains differed in their ability to adapt to the nitrogen composition of the environment, leading to variations in the cellular stress states, fermentation performances and the activity of the nitrogen sensing signaling pathway.


Assuntos
Fermentação , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho , Genes Fúngicos , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Transcriptoma
16.
J Vis Exp ; (131)2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29443074

RESUMO

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations. We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples-facilitated by a robot-assisted system for online monitoring of fermentations-and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.


Assuntos
Isótopos de Carbono/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Marcação por Isótopo/métodos , Isótopos de Carbono/análise , Fluxo de Trabalho
17.
Microb Biotechnol ; 10(6): 1649-1662, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28695583

RESUMO

Nitrogen and lipids are key nutrients of grape must that influence the production of fermentative aromas by wine yeast, and we have previously shown that a strong interaction exists between these two nutrients. However, more than 90% of the acids and higher alcohols (and their acetate ester derivatives) were derived from intermediates produced by the carbon central metabolism (CCM). The objective of this study was to determine how variations in nitrogen and lipid resources can modulate the contribution of nitrogen and carbon metabolisms for the production of fermentative aromas. A quantitative analysis of metabolism using 13 C-labelled leucine and valine showed that nitrogen availability affected the part of the catabolism of N-containing compounds, the formation of α-ketoacids from CCM and the redistribution of fluxes around these precursors, explaining the optimum production of higher alcohols occurring at an intermediate nitrogen content. Moreover, nitrogen content modulated the total production of acids and higher alcohols differently, through variations in the redox state of cells. We also demonstrated that the phytosterol content, modifying the intracellular availability of acetyl-CoA, can influence the flux distribution, especially the formation of higher alcohols and the conversion of α-ketoisovalerate to α-ketoisocaproate.


Assuntos
Isótopos de Carbono/química , Aromatizantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/análise , Isótopos de Carbono/metabolismo , Fermentação , Aromatizantes/análise , Marcação por Isótopo , Leucina/química , Leucina/metabolismo , Saccharomyces cerevisiae/química , Valina/química , Valina/metabolismo , Vitis/metabolismo , Vitis/microbiologia , Vinho/microbiologia
18.
BMC Genomics ; 18(1): 455, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28595605

RESUMO

BACKGROUND: During must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to floral and fruity aromas. The action of yeast, in particular Saccharomyces cerevisiae, on the must components will build the architecture of the wine flavour and its fermentation bouquet. The objective of the present work was to better understand the molecular and metabolic bases of aroma production during a fermentation process. For such, comparative transcriptomic and metabolic analysis was performed at two time points (5 and 50 g/L of CO2 released) in fermentations conducted by four yeast strains from different origins and/or technological applications (cachaça, sake, wine, and laboratory), and multivariate factorial analyses were used to rationally identify new targets for improving aroma production. RESULTS: Results showed that strains from cachaça, sake and wine produced higher amounts of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain. At fermentation time T1 (5 g/L CO2 released), comparative transcriptomics of the three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain also showed upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50 g/L CO2 released), again sake strain, but also VL1 strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed. CONCLUSIONS: Our approach revealed successful to integrate data from several technologies (HPLC, GC-MS, microarrays) and using different data analysis methods (PCA, MFA). The results obtained increased our knowledge on the production of wine aroma and flavour, identifying new gene in association to the formation of flavour active compounds, mainly in the production of fatty acids, and ethyl and acetate esters.


Assuntos
Perfilação da Expressão Gênica , Metabolômica , Odorantes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fermentação , Fenótipo
19.
Appl Environ Microbiol ; 83(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28115380

RESUMO

During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the α-keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources.IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines.


Assuntos
Fermentação , Nitrogênio/química , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia , Aminoácidos/metabolismo , Biomassa , Carbono/metabolismo , Meios de Cultura/análise , Indústria Alimentícia , Microbiologia de Alimentos , Sucos de Frutas e Vegetais , Marcação por Isótopo , Redes e Vias Metabólicas , Metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Açúcares/metabolismo , Vitis/química , Vinho/análise
20.
Microb Cell Fact ; 15: 58, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27044358

RESUMO

BACKGROUND: S. cerevisiae has attracted considerable interest in recent years as a model for ecology and evolutionary biology, revealing a substantial genetic and phenotypic diversity. However, there is a lack of knowledge on the diversity of metabolic networks within this species. RESULTS: To identify the metabolic and evolutionary constraints that shape metabolic fluxes in S. cerevisiae, we used a dedicated constraint-based model to predict the central carbon metabolism flux distribution of 43 strains from different ecological origins, grown in wine fermentation conditions. In analyzing these distributions, we observed a highly contrasted situation in flux variability, with quasi-constancy of the glycolysis and ethanol synthesis yield yet high flexibility of other fluxes, such as the pentose phosphate pathway and acetaldehyde production. Furthermore, these fluxes with large variability showed multimodal distributions that could be linked to strain origin, indicating a convergence between genetic origin and flux phenotype. CONCLUSIONS: Flux variability is pathway-dependent and, for some flux, a strain origin effect can be found. These data highlight the constraints shaping the yeast operative central carbon network and provide clues for the design of strategies for strain improvement.


Assuntos
Carbono/metabolismo , Ecossistema , Variação Genética , Redes e Vias Metabólicas , Fenótipo , Saccharomyces cerevisiae/metabolismo , Adaptação Biológica/genética , Microambiente Celular/genética , Evolução Molecular , Genótipo , Glicólise/genética , Redes e Vias Metabólicas/genética , Técnicas Microbiológicas/métodos , Via de Pentose Fosfato/genética , Saccharomyces cerevisiae/genética
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