Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted

Fukuda, Katsuhiko; Yamamoto, N.; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

doi:10.1016/s0922-338x(97)80362-5

Cited by 43 publications

(21 citation statements)

References 14 publications

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“…As esterases are responsible for the breakdown of esters, it is clear that their activity may affect the concentration of certain volatile esters. In S. cerevisiae, for example, it has been shown that the balance between ester-synthesizing enzymes and esterases, such as Iah1p (also known as Est2p), is important for the net rate of ester accumulation (27)(28)(29)(30) (59,60,61,62,70). This indicates that esterases may also play a role in the formation of certain esters (42,62,70).…”

Section: Discussionmentioning

confidence: 99%

“…However, firm evidence for the ester-synthesizing capacity of yeast esterases is lacking. Deletion of the esterase-encoding gene IAH1 results in an increase in the production of ethyl acetate and isoamyl acetate, showing that Iah1p mainly hydrolyzes these esters and is not responsible for a major part of their production (29,30). However, the effect of Iah1p on other esters, such as the group of ethyl esters, has not yet been thoroughly studied.…”

Section: Discussionmentioning

confidence: 99%

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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

Appl Environ Microbiol

336

322

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Appl Environ Microbiol

336

322

View full text Add to dashboard Cite

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“…While the amount of isoamyl acetate produced is low in quantitative terms, it is one hundred times higher than the taste threshold for isoamyl acetate (1.6 ppm) and a concentrated product could be prepared by counter-current solvent extraction or other extraction techniques, such as freeze-trapping the off-gases of fermentation 4 . The ability of saké yeast strains to produce isoamyl acetate in increased quantities has been greatly enhanced by recombinant DNA technologies, such as gene dosing of the ATF1 gene 6 (encoding the AATase enzyme), also, by the disruption of the isoamyl acetate hydrolysing esterase gene 5 (EST2) and by various genetic manipulations of the leucine biosynthetic and catabolic pathways 8 . It is believed that a combination of these genetic manipulations, allied to the adjustments in fermentation conditions outlined in this paper, could ultimately lead to a situation where brewing strains of S. cerevisiae could be used to produce commercially viable levels of natural isoamyl acetate.…”

Section: (-7'977-32mentioning

confidence: 99%

The Production of Isoamyl Acetate from Amyl Alcohol bySaccharomyces cerevisiae

Quilter

Hurley²,

Lynch³

et al. 2003

Journal of the Institute of Brewing

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Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as "lees oil" or "fusel oil", of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L-leucine and amyl alcohol in fermentations carried out by a high ester-producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro-DL-leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.

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“…Their sensory threshold is very low, and thus they have a strong influence on the aroma of alcoholic beverages, giving characteristic flavours to, for example, wine 25 , beer 23 and sake 7 . Therefore, the study of enzyme activities, affecting the ester content and spectrum, is of primary importance in elucidating the mechanisms leading to the formation of the aroma profile of a fermented beverage such as wine.…”

Section: -2863(9'8-32mentioning

confidence: 99%

“…By regulating the equilibrium between esters and free acids, esterases might determine ester synthesis 3,12,20,23,25 , but seem to be mainly involved in their breakdown 6,8,12 . For example, isoamyl acetate, which is thought to be synthesized from isoamyl alcohol and acetyl CoA by yeast alcohol acetyltransferase, is hydrolyzed by yeast esterases in sake mash 7,8 thus affecting the flavour quality of this beverage.…”

Section: -2863(9'8-32mentioning

confidence: 99%

Solubilization and Activity Detection in Polyacrylamide Gels of a Membrane-Bound Esterase from an Oenological Strain of Saccharomyces cerevisiae

Rizzi

Lomolino

Lante

et al. 2003

Journal of the Institute of Brewing

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Due to the importance of esters in determining wine aroma, the presence of different esterases in soluble and insoluble cell fractions of an oenological strain of Saccharomyces cerevisiae was studied. Cells of an oenological yeast strain were separated into three fractions corresponding to the cytoplasm, periplasm and plasma membrane, all showing esterase activity. The conditions for optimal solubilization of the membrane-bound esterase were found, as well as those allowing its electrophoretic analysis, using the detergent disodium-n-lauryl-β-iminodipropionate (Deriphat) in the cathodic buffer. Comparison of the activity bands detected on gels revealed the presence of different esterase isoforms in the three sub-cellular fractions. The method can also be used as the first electrophoretic step in two-dimensional PAGE. Therefore, the procedures described are also a promising tool for the study of the yeast enzymes in relation to their effects during winemaking.

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Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted

Cited by 43 publications

References 14 publications

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

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

The Production of Isoamyl Acetate from Amyl Alcohol bySaccharomyces cerevisiae

Solubilization and Activity Detection in Polyacrylamide Gels of a Membrane-Bound Esterase from an Oenological Strain of Saccharomyces cerevisiae

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