Fatty acid ethyl esters are secondary metabolites produced bySaccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of mediumchain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a ␣/␤-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in mediumchain fatty acid ethyl ester formation.The synthesis of fatty acid ethyl esters (FAEEs) 3 is widely distributed in microorganisms, higher plants, and mammals. In mammals, FAEEs are the result of the nonoxidative pathway for the metabolism of ethanol, after ethanol intake (1, 2). In higher plants and microorganisms, FAEEs are formed as secondary metabolites. Because of their strong fruit flavor, ethyl esters of short-and medium-chain fatty acids (MCFAs) constitute a large group of flavor compounds particularly important in the food, beverage, cosmetic, and pharmaceutical industries. The biosynthesis of FAEEs proceeds by two different enzymatic mechanisms, esterification or alcoholysis (3). Esterification is the formation of esters from alcohols and carboxylic acids and is catalyzed by FAEE synthases/carboxylesterases. Alcoholysis is the production of esters from alcohols and acylglycerols or from alcohols and fatty acylCoAs derived from metabolism of fatty acids. Alcoholysis is essentially a transferase reaction in which fatty acyl groups from acylglycerols or acyl-CoA derivatives are directly transferred to alcohols. The formation of FAEEs by alcoholysis is catalyzed by acyl-CoA:ethanol O-acyltransferases (AEATases) (4).Ester biosynthesis is very common in microorganisms, especially in bacteria and yeasts that are used in the fermentation of alcoholic beverages and food products. Information about the metabolic pathways and enzymology of ester biosynthesis in these microorganisms, however, is still v...

SummaryThe need to understand and control ester synthesis is driven by the fact that esters play a key role in the sensorial quality of fermented alcoholic beverages like beer, wine and sake. As esters are synthesized in yeast via several complex metabolic pathways, there is a need to gain a clear understanding of ester metabolism and its regulation. The individual genes involved, their functions and regulatory mechanisms have to be identified. In alcoholic beverages, there are two important groups of esters: the acetate esters and the medium‐chain fatty acid (MCFA) ethyl esters. For acetate ester synthesis, the genes involved have already been cloned and characterized. Also the biochemical pathways and the regulation of acetate ester synthesis are well defined. With respect to the molecular basis of MCFA ethyl ester synthesis, however, significant progress has only recently been made. Next to the characterization of the biochemical pathways and regulation of ester synthesis, a new and more important question arises: what is the advantage for yeast to produce these esters? Several hypotheses have been proposed in the past, but none was satisfactorily. This paper reviews the current hypotheses of ester synthesis in yeast in relation to the complex regulation of the alcohol acetyl transferases and the different factors that allow ester formation to be controlled during fermentation.

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 ...

The Saccharomyces cerevisiae genome encodes a Flo (flocculin) adhesin family responsible for cell-cell and cell-surface adherence. In commonly used laboratory strains, these FLO genes are transcriptionally silent, because of a nonsense mutation in the transcriptional activator FLO8, concealing the potential phenotypic diversity of fungal adhesion. Here, we analyse the distinct adhesion characteristics conferred by each of the five FLO genes in the S288C strain and compare these phenotypes with a strain containing a functional copy of FLO8. Our results show that four FLO genes confer flocculation, but with divergent characteristics such as binding strength, carbohydrate recognition and floc size. Adhesion to agar surfaces, on the other hand, largely depended on two adhesins, Flo10 and Flo11. Expression of any FLO gene caused a significant increase in cell wall hydrophobicity. Nevertheless, the capacity to adhere to plastic surfaces, which is believed to depend on hydrophobic interactions, differed strongly between the adhesins. Restoring Flo8 yielded both flocculation and cell-surface adherence, such as invasive growth, a phenotype not observed when any of the single FLO genes was overexpressed. Taken together, this study reveals how S. cerevisiae carries a small reservoir of FLO genes that allows cells to display a wide variety of adhesive properties.