Short‐chain and medium‐chain aliphatic‐ester synthesis in Saccharomyces cerevisiae

Abstract

In the yeast Saccharnmyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties.Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 3 kDa and its apparent pZ is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-~~-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA : 0-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.Short-chain and medium-chain aliphatic esters have been reported as by-products of oxidative metabolism in higher plants and fungi [I, 21. Although volatile aliphatic esters are important in producing the fruity aroma of foods and fermented beverages [3,4], the biochemical significancc of short-chain and medium-chain aliphatic-ester synthesis in cellular metabolism is still unknown.In most cases described in the literature, the synthesis of aliphatic esters involves alcoholysis of an acyl-CoA intermediate and is catalysed by acyltransferases or ester synthases. Such enzymes have been reported in various microorganisms[5 -91 and during the ripening of banana [lo]. On the other hand, enzyme-catalysed esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A has been reported in human and rabbit myocardium [l I].In the yeast Saccharomyces cerevisiae, esters are mainly synthesized and excreted under anaerobic fermentation conditions. It has been demonstrated that the synthesis of isoamyl acetate is catalysed by an acetyl-CoA : 0-isoamyl-alcohol acetyltransferase which is metabolically induced in the exponential phase of growth. The higher specific rate of ester production observed under anaerobiosis has been related to the induction of isoamyl-acetate synthase, the regulator of which is presumably linked to lipid metabolism [22].It would be interesting to elucidate the molecular basis of ester synthase regulation, as ester synthesis could be one of the functions specifically induced during anaerobic growth of yeast [13].Due to a lack of a rapid screening method for mutants affected in ester synthesis, characterization of ester synthases Correspondence to J.-P. Dufour, Unite de Brasserie et des Industries Alimentaires, UniversitP Catholique de Louvain, Place Croix du Sud 2jbte 7, B-1348 Louvain-La-Neuve, Belgium Fax: +3210472178.

Evaluation of Beer Fermentation with a Novel Yeast Williopsis saturnus

“…These fatt y acids are important for beer flavour because they themselves are odour-active and can lead to rancid off -flavour at high concentrations. Moreover, they are also one of the starting materials for the synthesis of volatile esters (32). In general, fatt y acid production by Safale US-05 was higher than by NCYC 500 strain.…”

Impact of Dark Specialty Malts on Extract Composition and Wort Fermentation

“…Evolution of isoamyl alcohol (A) and isobutanol (B) levels during fermentation of wort brewed with 100% Scarlett or 50% Scarlett and 50% of dark malt (Melanoidin, Cara-aroma and roasted malt). Properties of these wort samples are presented in Table VII. are the key enzymes involved in the biosynthesis of acetate esters 15,25,27 . Deletion and overexpression studies indicated that the ATF1 gene is responsible for the formation of both isoamyl acetate and ethyl acetate, whereas ATF2 mainly leads to isoamyl acetate 35 .…”

Acetate ester production by Chinese yellow rice wine yeast overexpressing the alcohol acetyltransferase-encoding gene ATF2

“…Similarly, Verstrepen et al (2003b) demonstrated that ATF2 deletion resulted in an 18% decrease in isoamyl acetate formation, while ATF2 overexpression resulted in a 6-to 8-fold increase. In consequence, the expression level of ATF2 in the current study was expected to be a limiting factor for ester synthesis in S. cerevisiae, confirming the hypothesis of Malcorps (Malcorps et al, 1991;Malcorps and Dufour, 1992). However, because different research groups have used different yeast strains and different fermentation conditions in ATF2 studies, it is difficult to compare results effectively.…”

“…The key enzymes involved in ester synthesis are alcohol acetyltransferases (AATases; EC 2.3.1.84) (Nordström, 1962(Nordström, , 1963(Nordström, , 1964. AATase II, which is encoded by ATF2, is 1 of the 2 main AATases in S. cerevisiae (Yoshioka and Hashimoto, 1981;Malcorps and Dufour, 1992;Fujii et al, 1994Fujii et al, , 1996aNagasawa et al, 1998;Yoshimoto et al, 1998Yoshimoto et al, , 1999. It catalyzes the formation of acetate esters from 2 substrates: an alcohol and an acetyl-CoA.…”