29 Biochemistry and Fermentation of Beer 669ture of this distribution pattern is that specific rates of
acetate ester formation varied directly with the level
of cytosolic AAT activity (Masschelein 1997).
The ATF1gene, which encodes alcohol acetyl-
transferase, has been cloned from S. cerevisiaeand
brewery lager yeast (S. cerevisiae uvarum) (Fujii et
al. 1994). A hydrophobicity analysis suggested that
alcohol acetyltransferase does not have a membrane-
spanning region that is significantly hydrophobic,
which contradicts the membrane-bound assumption.
A Southern analysis of the yeast genomes in which
the ATF1gene was used as a probe, revealed that S.
cerevisiaehas one ATF1gene, while brewery lager
yeast has one ATF1gene and another, homologous
gene (Lg-ATF1). The AAT activity of S. cerevisiae
has been compared in vivo and in vitro under differ-
ent fermentation conditions (Malcorps et al. 1991).
This study suggested that ester synthesis is modulat-
ed by a repression-induction of enzyme synthesis or
processing, the regulator of which is presumably
linked to yeast metabolism.
The ester production can be altered by changing
the synthesis rate of certain fusel alcohols. Hirata et
al. (1992) increased the isoamyl acetate levels by
introducing extra copies of the LEU4gene in the S.
cerevisiaegenome. A comparable S. cerevisiae uva-
rummutant has been isolated (Lee et al. 1995). The
mutants have an altered regulation pattern of amino
acid metabolism and produce more isoamyl acetate
and phenylethyl acetate.
Isoamyl acetate is synthesized from isoamyl alco-
hol and acetyl-CoA by AAT and is hydrolyzed by
esterases at the same time in S. cerevisiae. To study
the effect of balancing both enzyme activities, yeast
strains with different numbers of copies of the ATF1
gene and isoamyl acetate–hydrolyzing esterase gene
(IAH1) have been constructed and used in small-
scale sake brewing (Fukuda et al. 1998). Fermen-
tation profiles as well as components of the resulting
sake were largely alike. However, the amount of
isoamyl acetate in the sake increased with increas-
ing ratio of AAT:Iah1p esterase activity. Therefore,
it was concluded that the balance of these two en-
zyme activities are important for isoamyl acetate
accumulation in sake mash.
The synthesis of acetate esters by S. cerevisiae
during fermentation is ascribed to at least three
acetyltransferase activities, namely alcohol acetyl-
transferase (AAT), ethanol acetyltransferase, and
isoamyl AAT (Lilly et al. 2000). To investigate the
effect of increased AAT activity on the sensory qual-
ity of Chenin blanc wines and distillates from
Colombar base wines, the ATF1gene of S. cerevisi-
aewas overexpressed. Northern blot analysis indi-
cated constitutive expression of ATF1at high levels
in these transformants. The levels of ethyl acetate,
isoamyl acetate, and 2-phenylethyl acetate increased
3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, re-
spectively, depending on the fermentation tempera-
ture, cultivar, and yeast used. The concentrations of
ethyl caprate, ethyl caprylate, and hexyl acetate only
showed minor changes, whereas the acetic acid con-
centration decreased by more than half. This study
established the concept that the overexpression of
acetyltransferase genes such as ATF1could pro-
foundly affect the flavor profiles of wines and distil-
lates deficient in aroma.
In order to investigate and compare the roles of
the knownS. cerevisiaealcohol acetyltransferases
Atf1p, Atf2p, and Lg-Atf1p in volatile ester produc-
tion, the respective genes were either deleted or
overexpressed in a laboratory strain and a commer-
cial brewing strain (Verstrepen et al. 2003). Analysis
of the fermentation products confirmed that the
expression levels ofATF1andATF2greatly affect
the production of ethyl acetate and isoamyl acetate.
Gas chromatography/mass spectrometry (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 phenylethyl acetate. With respect
to the esters analyzed in this study, Atf2p seemed to
play only a minor role compared to Atf1p. Theatf1
atf2double deletion strain did not form any iso-
amyl acetate, showing that together, Atf1p and
Atf2p are responsible for the total cellular isoamyl
alcohol acetyltransferase activity. However, the dou-
ble 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, over-
expression of different alleles ofATF1andATF2led
to different ester production rates, indicating that
differences in the aroma profiles of yeast strains
may be partially due to mutations in theirATF
genes.
Recently, it has been discovered that the Atf1 en-
zyme is localized inside lipid vesicles in the cy-
toplasm of the yeast cell (Verstrepen 2003). Lipid