Rosana Chiva

Nitrogen requirements of commercial wine yeast strains during fermentation of a synthetic grape must

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen (usually ammonium phosphate). However, it is important to know the specific nitrogen requirement of each strain, to avoid excessive addition that can lead to microbial instability and ethyl carbamate accumulation. In this study, we aimed to determine the effect of increasing nitrogen concentrations of three different nitrogen sources on growth and fermentation performance in four industrial wine yeast strains. This task was carried out using statistical modeling techniques. The strains PDM and RVA showed higher growth-rate and maximum population size and consumed nitrogen much more quickly than strains ARM and TTA. Likewise, the strains PDM and RVA were also the greatest nitrogen demanders. Thus, we can conclude that these differences in nitrogen demand positively correlated with higher growth rate and higher nitrogen uptake rate. The most direct effect of employing an adequate nitrogen concentration is the increase in biomass, which involves a higher fermentation rate. However, the impact of nitrogen on fermentation rate is not exclusively due to the increase in biomass because the strain TTA, which showed the worst growth behavior, had the best fermentation activity. Some strains may adapt a strategy whereby fewer cells with higher metabolic activity are produced. Regarding the nitrogen source used, all the strains showed the better and worse fermentation performance with arginine and ammonium, respectively.

Proteomic evolution of a wine yeast during the first hours of fermentation

The inoculation of active dry wine yeast (ADWY) is one of the most common practices in winemaking. This inoculation exposes the yeast cells to strong osmotic, acidic and thermal stresses, and adaptation to the new medium is crucial for successful fermentation. We have analysed the changes that occur in the ADWY protein profile in the first hours after inoculation under enological-like conditions at a low temperature. Protein changes mainly included enzymes of the nitrogen and carbon metabolism and proteins related to the cellular stress response. Most of the enzymes of the lower part of the glycolysis showed an increase in their concentration 4 and 24 h after inoculation, indicating an increase in glycolytic flux and in ATP production. However, the shift from respiration to fermentation was not immediate in the inoculation because some mitochondrial proteins involved in oxidative metabolism were induced in the first hours after inoculation. Inoculation in this fresh medium also reduced the cellular concentration of stress proteins produced during industrial production of the ADWY. The only exception was Cys3p, which might be involved in glutathione synthesis as a response to oxidative stress. A better understanding of the yeast stress response to rehydration and inoculation will lead to improvements in the handling efficiency of ADWY in winemaking and presumably to better control of fermentation startup.

The role of GAP1 gene in the nitrogen metabolism of Saccharomyces cerevisiae during wine fermentation

Aim:  The aim of this study was to analyse the relevance of the general amino acid permease gene (GAP1) of the wine yeast Saccharomyces cerevisiae on nitrogen metabolism and fermentation performance.

Functional analysis to identify genes in wine yeast adaptation to low‐temperature fermentation

Aims:  To identify genes and proteins involved in adaptation to low‐temperature fermentations in a commercial wine yeast.

Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation

Fermentations carried out at low temperatures, that is, 10-15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set-up close to industrial conditions.

Comparative genomic analysis of Saccharomyces cerevisiae yeasts isolated from fermentations of traditional beverages unveils different adaptive strategies.

Saccharomyces cerevisiae strains are the main responsible of most traditional alcohol fermentation processes performed around the world. The characteristics of the diverse traditional fermentations are very different according to their sugar composition, temperature, pH or nitrogen sources. During the adaptation of yeasts to these new environments provided by human activity, their different compositions likely imposed selective pressures that shaped the S. cerevisiae genome. In the present work we performed a comparative genomic hybridization analysis to explore the genome constitution of six S. cerevisiae strains isolated from different traditional fermentations (masato, mescal, cachaça, sake, wine, and sherry wine) and one natural strain. Our results indicate that gene copy numbers (GCN) are very variable among strains, and most of them were observed in subtelomeric and intrachromosomal gene families involved in metabolic functions related to cellular homeostasis, cell-to-cell interactions, and transport of solutes such as ions, sugars and metals. In many cases, these genes are not essential but they can play an important role in the adaptation to new environmental conditions. However, the most interesting result is the association observed between GCN changes in genes involved in the nitrogen metabolism and the availability of nitrogen sources in the different traditional fermentation processes. This is clearly illustrated by the differences in copy numbers not only in gene PUT1, the main player in the assimilation of proline as a nitrogen source, but also in CAR2, involved in arginine catabolism. Strains isolated from fermentations where proline is more abundant contain a higher number of PUT1 copies and are more efficient in assimilating this amino acid as a nitrogen source. A strain isolated from sugarcane juice fermentations, in which arginine is a rare amino acid, contains less copies of CAR2 and showed low efficiency in arginine assimilation. These results suggest that nitrogen metabolism has played an important role in the adaptive evolution of S. cerevisiae strains.

Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: implication in growth at low temperatures.

The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10-15°C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition.

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast

Low‐temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low‐temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature.

Biodiversity of non-Saccharomyces yeasts in distilleries of the La Mancha region (Spain)

The aim of this pioneering study was to determine the biodiversity of non-Saccharomyces yeasts in ancient distilleries located in the La Mancha region, which is the principal area for the production of bioethanol and grape-based distillates in Spain. In this study, the yeast populations that were present during the process of extraction of alcohol and residual sugars from the byproducts of vinification, such as piquettes, pomace and grape skins, were studied. Non-Saccharomyces yeasts were identified by PCR-RFLP analysis of the 5.8S rRNA genes and, when necessary, by sequencing the D1/D2 domain of the 26S and/or 5.8S rRNA genes. Further, fermentation and the assimilation of carbon compounds were studied, to identify potential industrial applications. Phylogenetic trees and heat-maps were constructed for the genetic and phenotypic traits, respectively. Twenty yeast species belonging to eight genera were identified (Torulaspora, Candida, Zygosaccharomyces, Pichia, Hanseniaspora, Kluyveromyces, Ogataea and Saccharomycodes). Pichia galeiformis, Candida lactis-condensi, Hanseniaspora osmophila and Torulaspora delbrueckii were the most abundant species and were found principally in sweet and fermented piquettes.

Biomarkers for detecting nitrogen deficiency during alcoholic fermentation in different commercial wine yeast strains

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Several putative biomarkers were tested in order to analyze their appropriateness to detect nitrogen stress in the yeast. To this aim, four commercial wine strains (PDM, ARM, RVA and TTA) were grown in a synthetic grape must with different nitrogen concentrations. Trehalose accumulation, arginase activity and the expression of eleven genes were tested in these wine strains, known to have different nitrogen requirements. The overall response of the four strains was similar, with differences in response intensity (PDM and RVA with higher intensity) and response time (which was also related with nitrogen consumption time). Trehalose response was mostly related to entry into the stationary phase, whereas arginase activity was responsive to nitrogen depletion, although its measurement is too complicated to be used for routine monitoring during winemaking. The expression of the genes DAL4, DAL5, DUR3 and GAP1 was clearly related to nitrogen depletion and thus, GAP1 and DAL4 were selected as markers of nitrogen deficiency. In order to adapt expression analysis to winemaking conditions, the original strains were transformed into reporter strains based on the expression of green fluorescent protein (GFP) under control of the promoters for GAP1 and DAL4. The transformants had a similar fermentative capacity to the parental strains and were able to detect alterations in yeast physiological status due to nitrogen limitations.