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Applied and Environmental Microbiology, September 2003, p. 5228-5237, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5228-5237.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

Kevin J. Verstrepen,1* Stijn D. M. Van Laere,1 Bart M. P. Vanderhaegen,1 Guy Derdelinckx,1 Jean-Pierre Dufour,2 Isak S. Pretorius,3 Joris Winderickx,4 Johan M. Thevelein,5,6 and Freddy R. Delvaux1

Centre for Malting and Brewing Science, Department of Food and Microbial Technology,1 Functional Biology, Department of Biology,4 Laboratory of Molecular Cell Biology, Department of Biology, Katholieke Universiteit Leuven,5 VIB Department of Molecular Microbiology, B-3001 Louvain (Heverlee), Belgium,6 Department of Food Science, University of Otago, Dunedin, New Zealand,2 The Australian Wine Research Institute, Glen Osmond, Adelaide, South Australia 5064, Australia3

Received 31 March 2003/ Accepted 29 June 2003

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{Delta} atf2{Delta} 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.

* Corresponding author. Mailing address: Centre for Malting and Brewing Science, Department of Food and Microbial Technology, K.U.Leuven, Kasteelpark Arenberg 22, B-3001 Louvain (Heverlee), Belgium. Phone: 32-(0)16-329627. Fax: 32-(0)16-321576. E-mail: Kevin.Verstrepen{at}agr.kuleuven.ac.be.

Applied and Environmental Microbiology, September 2003, p. 5228-5237, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5228-5237.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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