Acetate ester production by Saccharomyces cerevisiae lacking the ATF1 gene encoding the alcohol acetyltransferase

414

260

Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of mediumchain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.During fermentation the yeast Saccharomyces cerevisiae produces a broad range of aroma-active substances, which are vital for the complex flavor of fermented beverages, such as beer and wine. In particular, volatile esters are of major industrial interest because the presence of these compounds determines the fruity aroma of beer and wine (6,7,9,20,24,26,27,30,31,36). Even small changes in the concentrations of these secondary metabolites can have large effects on the final sensorial quality of fermented beverages. There are two main groups of flavor-active esters in fermented beverages. The first group contains the acetate esters (in which the acid group is acetate and the alcohol group is ethanol or a complex alcohol derived from amino acid metabolism), such as ethyl acetate (solventlike aroma), isoamyl acetate (banana aroma), and phenyl ethyl acetate (roses, honey). The second group is the ethyl esters (in which the alcohol group is ethanol and the acid group is a medium-chain fatty acid [MCFA]) and includes ethyl hexanoate (anise seed, applelike aroma), ethyl octanoate (sour apple aroma), and ethyl decanoate (floral odor). Of these two groups, the acetate esters have received the most attention, not because they are more important but because ...

Section: Resultssupporting

confidence: 81%

Parameters Affecting Ethyl Ester Production bySaccharomyces cerevisiaeduring Fermentation

Saerens

Verstrepen

Dijck

et al. 2008

414

260

“…In haploid S. cerevisiae atf1⌬ strains, isoamyl acetate transferase and ethanol acetate transferase activities were 80 and 20% lower, respectively, than in the wild-type strain. This resulted in an 80% decrease in isoamyl acetate production and a 30% decrease in ethyl acetate production compared to rates in wild-type cells (24). In another study, overexpression of ATF1 derived from an industrial lager brewer's yeast strain resulted in a 27-fold increase in isoamyl acetate production and a 9-fold increase in ethyl acetate production compared to rates in empty-vector transformants (22).…”

mentioning

confidence: 95%

Expression Levels of the Yeast Alcohol Acetyltransferase GenesATF1,Lg-ATF1, andATF2Control the Formation of a Broad Range of Volatile Esters

Verstrepen

Laere

Vanderhaegen

et al. 2003

345

311

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1⌬ atf2⌬ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.During fermentation processes, yeast cells produce a broad range of aroma-active substances which greatly affect the complex flavor of fermented alcoholic beverages. While these secondary metabolites are often formed only in trace amounts, their concentrations determine the distinct aroma of these beverages. Flavor-active substances produced by fermenting yeast cells can be divided into five main groups: sulfur-containing molecules, organic acids, higher alcohols, carbonyl compounds, and volatile esters (32,39,56,57,66). Of these categories, volatile esters represent the largest and most important group. They are responsible for the highly desired fruity character of beer and, to a lesser extent, other alcoholic beverages, such as wine.The major flavor-active esters in beer are acetate esters such as ethyl acetate (solvent-like aroma), isoamyl acetate (banana flavor), and phenylethyl acetate (flowery, rose aroma). In addition, C 6 -C 10 medium-chain fatty acid ethyl esters such as ethyl hexanoate (ethyl caproate) and ethyl octanoate (ethyl caprylate), which have "sour apple" aromas, are also important for the overall bouqu...

“…It has been reported that the 61-kDa ATF1 gene product (Atf1p) is located within the yeast's cellular vacuomes (24) and plays a major role in the production of iso-amyl acetate and to a lesser extent ethyl acetate during beer fermentation (12). To investigate the role of AAT in wine and brandy composition, we have cloned, characterized, and mapped the ATF1 gene from a widely used commercial wine yeast strain, VIN13.…”

mentioning

confidence: 99%

“…The ATF1-encoded AAT activity is the best-studied acetyltransferase activity in S. cerevisiae (10,11,12,23,24). It has been reported that the 61-kDa ATF1 gene product (Atf1p) is located within the yeast's cellular vacuomes (24) and plays a major role in the production of iso-amyl acetate and to a lesser extent ethyl acetate during beer fermentation (12).…”

mentioning

confidence: 99%

Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates

Lilly

Lambrechts

Pretorius

2000

313

165

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3-to 10-fold, 3.8-to 12-fold, and 2-to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.