Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine

Abstract Control of volatile acidity (VA) is a major issue for wine quality. In this study, we investigated the production of VA by a deletion mutant of the fermentation stress response gene AAF1 in the budding yeast Saccharomyces cerevisiae. Fermentations were carried out in commercial Chardonnay grape must to mimic industrial wine-making conditions. We demonstrated that a wine yeast strain deleted for AAF1 reduced acetic acid levels in wine by up to 39.2% without increasing the acetaldehyde levels, revealing a potential for industrial application. Deletion of the cytosolic aldehyde dehydrogenase gene ALD6 also reduced acetic acid levels dramatically, but increased the acetaldehyde levels by 41.4%, which is not desired by the wine industry. By comparison, ALD4 and the AAF1 paralog RSF2 had no effects on acetic acid production in wine. Deletion of AAF1 was detrimental to the growth of ald6D and ald4Dald6D mutants, but had no effect on acetic acid production. Overexpression of AAF1 dramatically increased acetic acid levels in wine in an Ald6p-dependent manner, indicating that Aaf1p regulates acetic acid production mainly via Ald6p. Overexpression of AAF1 in an ald4Dald6D strain produced significantly more acetic acid in wine than the ald4Dald6D mutant, suggesting that Aaf1p may also regulate acetic acid synthesis independently of Ald4p and Ald6p

Functional Characterization of Individual- and Mixed-Burgundian Saccharomyces cerevisiae Isolates for Fermentation of Pinot Noir

Pinot noir has traditionally been fermented by native flora of multiple yeasts producing a complex combination of aromas and flavors. With the use of industrial dry yeasts, winemakers gained enological reliability and consistency in their wines, but lost diversity and complexity. This research evaluated the use of co-culturing yeasts to fulfill this dual role. Fermentations of Burgundian Saccharomyces cerevisiae isolates and their mixtures were evaluated for their enological characteristics and production of volatile compounds, at 22 °C and 27 °C. The novel isolates were genetically unique and enologically equivalent to the industrial strains. Analysis of variance and principal component analysis of 25 headspace volatiles revealed differences among the yeasts and between the fermentation temperatures. Wines from the mixed-Burgundian isolates were most similar to one another and could be differentiated from the industrial strains at both 22 °C and 27 °C. Mixed-Burgundian wines at both temperatures had higher concentrations of ethyl esters and acetate esters, compared to the industrial strains which had higher concentrations of higher alcohols at 27 °C and higher concentration of other ethyl esters at 22 °C. Given the unique profiles of the co-cultured wines, this research offers winemakers a strategy for producing wines with unique and more complex characters without the risk of spontaneous fermentations.Land and Food Systems, Faculty ofNon UBCReviewedFacult

Transcriptomic Response of Saccharomyces cerevisiae during Fermentation under Oleic Acid and Ergosterol Depletion

Under anaerobic/hypoxic conditions, Saccharomyces cerevisiae relies on external lipid supplements to modulate membrane lipid fraction in response to different stresses. Here, transcriptomic responses of two S. cerevisiae wine strains were evaluated during hypoxic fermentation of a synthetic must with/without ergosterol and oleic acid supplementation. In the absence of lipids, the two strains, namely EC1118 and M25, showed different behaviour, with M25 significantly decreasing its fermentation rate from the 72 h after inoculum. At this time point, the whole genome transcriptomic analysis revealed common and strain-specific responses to the lack of lipid supplementation. Common responses included the upregulation of the genes involved in ergosterol biosynthesis, as well as the seripauperin and the heat shock protein multigene families. In addition, the upregulation of the aerobic isoforms of genes involved in mitochondrial electron transport is compatible with the previously observed accumulation of reactive oxygen species in the two strains during growth in absence of lipids. Considering the strain-specific responses, M25 downregulated the transcription of genes involved in glucose transport, methionine biosynthesis and of those encoding mannoproteins required for adaptation to low temperatures and hypoxia. The identification of these pathways, which are presumably involved in yeast resistance to stresses, will assist industrial strain selection.Other UBCNon UBCReviewedFacult

Functional analyses of NSF1 in wine yeast using interconnected correlation clustering and molecular analyses.

Analyzing time-course expression data captured in microarray datasets is a complex undertaking as the vast and complex data space is represented by a relatively low number of samples as compared to thousands of available genes. Here, we developed the Interdependent Correlation Clustering (ICC) method to analyze relationships that exist among genes conditioned on the expression of a specific target gene in microarray data. Based on Correlation Clustering, the ICC method analyzes a large set of correlation values related to gene expression profiles extracted from given microarray datasets. ICC can be applied to any microarray dataset and any target gene. We applied this method to microarray data generated from wine fermentations and selected NSF1, which encodes a C2H2 zinc finger-type transcription factor, as the target gene. The validity of the method was verified by accurate identifications of the previously known functional roles of NSF1. In addition, we identified and verified potential new functions for this gene; specifically, NSF1 is a negative regulator for the expression of sulfur metabolism genes, the nuclear localization of Nsf1 protein (Nsf1p) is controlled in a sulfur-dependent manner, and the transcription of NSF1 is regulated by Met4p, an important transcriptional activator of sulfur metabolism genes. The inter-disciplinary approach adopted here highlighted the accuracy and relevancy of the ICC method in mining for novel gene functions using complex microarray datasets with a limited number of samples

The Fermentation Stress Response Protein Aaf1p/Yml081Wp Regulates Acetate Production in <em>Saccharomyces cerevisiae</em>

<div><p>The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of <em>ALD4 and ALD6</em>, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of <em>ALD6</em>, YML081W had no effect on acetic acid levels, suggesting that this transcription factor’s effects are mediated primarily through this gene. <em>lacZ</em> reporter assays revealed that Yml081wp stimulates <em>ALD6</em> transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in <em>Saccharomyces cerevisiae.</em> We propose <em>AAF1</em> as a gene name for the YML081W ORF.</p> </div

Yml081Wp regulates acetic acid levels.

<p>(<b>A</b>) Wild-type, YML081W-null and YML081W-overexpressing M2 yeast cells were used in a wine fermentation with sterile Chardonnay grape juice. Once fermentation was complete, the wine was assayed for acetic acid by HPLC. YML081W-null cells produced significantly less acetic acid, while YML081W-overexpressing cells produced significantly more acetic acid. In this figure, and all subsequent figures, * indicates p<0.05 for a two-tailed Student t-test, compared to wild-type. (<b>B</b>) These same strains were grown in triplicate in YPD media. At 4 hour intervals, media samples were removed, and assayed for acetic acid as above. YML081-null cells (▪, dotted line) produced significantly less acetic acid in the media than wild-type cells (•, solid line), while YML081W-overexpressing cells (⧫, dashed line) produced significantly more acetic acid.</p

Yml081Wp regulates acetaldehyde dehydrogenase activity.

<p>M2 yeast cells were grown to mid-log phase, then harvested and lysed. ACDH specific activity was assayed as described in the Materials and Methods section. The numbers represent (nmol NAD(P)H formed/min) per mg protein. Cells without Yml081Wp contained lower ACDH activity, while cells overexpressing YML081W contained higher ACDH activity. These differences were statistically significant (p<0.05).</p

The acetic acid effect is much stronger with YML081W than with its ohnolog <i>RSF2</i>.

<p>Wild-type, YML081W-null and <i>RSF2</i>-null strains were grown in triplicate to mid-log phase in YPD media, and the resulting media was assayed for acetic acid. The dashed line indicates the initial acetic acid level of the media. Elimination of YML081W has a much more dramatic effect on acetic acid levels than elimination of <i>RSF2</i>.</p

Yml081Wp regulates the <i>ALD6</i> promoter.

<p>(<b>A</b>) A schematic diagram of the <i>ALD6</i> promoter sequence. Numbers on the left indicate the position of the first nucleotide in the promoter construct relative to the start codon. The positions and sequences of sites matching the previously published Yml081Wp binding sites are highlighted above the full-length promoter. (<b>B</b>) LacZ reporter activity from the <i>ALD6</i> promoter. Reporter plasmids carrying <i>ALD6</i> promoter fragments of the indicated lengths were transformed into wild-type M2 and YML081W-null strains. Three independent transformants for each reporter were grown to log phase, and assayed for β-galactosidase activity. Truncation of the reporter resulted in progressively lower activity levels. Importantly, cells lacking Yml081Wp produced significantly lower β-galactosidase activity than their wild-type counterparts (except for the shortest reporter fragment), suggesting that this transcription factor plays a positive role in stimulating <i>ALD6</i> transcription. (<b>C</b>) A consensus binding site at −590 plays an important role in mediating Yml081Wp transcriptional activity on the <i>ALD6</i> promoter. A reporter plasmid carrying a mutation of the YML081W consensus binding site was constructed for comparison to its wild-type counterpart. In cells producing normal levels of Yml081Wp, the mutation resulted in a 54% reduction in β-galactosidase activity (* indicates p<0.05 for a two-tailed Student t-test, compared to wild-type). However, in cells lacking Yml081Wp, the mutation had no significant effect on β-galactosidase activity. This result suggests that the consensus binding site at position −590 mediates Yml081Wp transcriptional activity on the <i>ALD6</i> promoter.</p

The Yml081Wp effect on acetate production requires <i>ALD6</i>.

<p><i>ALD4</i>-null and <i>ALD6</i>-null M2 yeast strains with and without YML081W were grown to mid-log phase, and then assayed for acetic acid production by HPLC. The dashed line indicates the initial acetic acid level of the media. As expected, with the full complement of <i>ALD</i> genes, eliminating YML081W produced a significant reduction in acetic acid levels (compare the two leftmost columns). Eliminating <i>ALD6</i> produced a lower acetic acid level compared to wild-type (compare the first and fifth columns). However, the further elimination of YML081W had no effect (compare the fifth and sixth columns). Therefore, YML081W requires <i>ALD6</i> to mediate its effects on acetic acid levels.</p