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

Insect-Induced Conifer Defense. White Pine Weevil and Methyl Jasmonate Induce Traumatic Resinosis, de Novo Formed Volatile Emissions, and Accumulation of Terpenoid Synthase and Putative Octadecanoid Pathway Transcripts in Sitka Spruce

Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system

Modelling Changes in Volatile Compounds in British Columbian Varietal Wines That Were Bottle Aged for Up to 120 Months

This research quantified 46 volatile compounds in vintage wines (1998–2005) from British Columbia (BC), which had been bottle-aged for up to 120 months. Wines were analyzed up to five times, between December 2003 and October 2008. Compounds were identified using gas chromatography mass spectrometry (GC-MS) and their concentrations were related to “wine age” using single linear regression (SLR). SLR models were developed for each wine compound (eight alcohol, 12 ester/acetate, one acid, one aldehyde, one sulfur) in eight varietal wines: six red (Cabernet franc, Cabernet Sauvignon, Meritage, Merlot, Pinot noir, Syrah) and two white (Chardonnay, Pinot gris). Parameter estimates (b₀, intercept; b₁, slope) and R² values for models were reported for each compound and each variety. Most of the significant SLR models (109/123) had negative slopes (−b₁ coefficients), indicating a decrease in the compounds’ concentration with “wine age”. The b₁ coefficients were very small for isobutyl acetate, ethyl isovalerate and ethyl decanoate (−0.00013 to −0.0006 mg/L/mon) and largest (most negative) for 3-methyl-1-butanol, ethyl lactate and isobutyl alcohol (−2.26 to −6.26 mg/L/mon). A few SLR models (14/123) had positive slopes (+b₁ coefficients), indicating an increase in the compounds’ concentration with “wine age”, particularly for acetaldehyde, diethyl succinate, ethyl formate and dimethyl sulfide. The +b₁ coefficients were smallest for ethyl decanoate (0.0001 mg/L/mon) and dimethyl sulfide (0.00024 mg/L/mon) and largest for dimethyl succinate and acetaldehyde (0.06 mg/L/mon). These values varied by four orders of magnitude (104), reflecting the large concentration range observed for the different volatile compounds. The work provided, for the first time, an empirical (non-theoretical) approach to documenting the evolution of volatile compounds in BC wines. It equipped the industry with an easy-to-use new tool for predicting the concentration of desirable or undesirable compounds in their wines and assisted the industry with decision making regarding the release of their wines into the marketplace.Land and Food Systems, Faculty ofOther UBCNon UBCReviewedFacult

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

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

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

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

Yml081Wp regulates <i>ALD</i> gene expression and protein levels.

<p>(<b>A</b>) Acetate biosynthesis pathway, adapted from Saint-Prix <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051551#pone.0051551-SaintPrix1" target="_blank">[6]</a>. (<b>B</b>) <i>ALD</i> gene expression was measured in mid-log phase wild-type and YML081W-null cells by quantitative PCR. Removal of YML081W resulted in a significant reduction in <i>ALD4</i> and <i>ALD6</i> mRNA levels. (<b>C</b>) <i>ALD</i> gene expression was measured in mid-log phase yeast cells overexpressing YML081W under the control of the <i>PGK1</i> promoter, and compared to wild-type cells. YML081W-overexpressing cells produced significantly higher levels of ALD4 and ALD6 mRNA compared to wild-type cells. (<b>D</b>) <i>ALD4</i> and <i>ALD6</i> genes were tagged with the FLAG epitope at the C-terminus in wild-type, YML081W-null and YML081Woverexpressing strains. The cells were grown to mid-log phase, then harvested and lysed. The lysates were immunoblotted with an α-FLAG antibody to detect the levels of Ald4p-FLAG and Ald6p-FLAG protein. Cells lacking YML081W produced lower levels of FLAG-tagged Ald4p and Ald6p, compared to wild-type cells. Cells overexpressing YML081W produced significantly higher levels of Ald4p-FLAG protein compared to wild-type cells. ALD6p-FLAG levels did not appear to change significantly. Membrane staining shown below the immunoblots indicates the equivalance of total protein loading between lanes.</p