Yeast

Yeast 2000; 16: 12871298.

Review

Alcohol acetyltransferases and the signicance of

ester synthesis in yeast
A. Brett Mason and Jean-Pierre Dufour*
Food Science Department, University of Otago, PO Box 56, Dunedin, New Zealand

* Correspondence to: Abstract

J.-P. Dufour, Food Science
Department, University of Otago,
PO Box 56, Dunedin,
New Zealand.
E-mail: jean-pierre.dufour@
stonebow.otago.ac.nz
This paper reviews our current knowledge of yeast alcohol acyltransferases. Much of this
information has been gathered over the past 10 years through the application of powerful
yeast molecular biology techniques. Evidence from gene disruption and expression analysis
of members of the alcohol acyltransferase (ATF) gene family indicates that different ester
synthases are involved in the synthesis of esters during alcoholic fermentation. The natural
physiological rationale behind these enzyme activities remains unclear. However, it is
believed that these enzymes may be involved in very different functions, including cellular
fatty acid homeostasis and detoxication mechanisms. Insights into the regulation of yeast
ester synthesis by oxygen and unsaturated fatty acids have contributed to our
understanding of the general mechanisms of gene regulation. In particular, control
mechanisms that underpin the oxygen-mediated regulation of ATF1 gene transcription
appear to be closely linked to those involved in the regulation of fatty acid metabolism.
Data pertaining to the regulation of ATF1 gene transcription have been integrated into a
working model for future research. Copyright # 2000 John Wiley & Sons, Ltd.
Keywords: yeast; esters; alcohol acetyltransferases; ATF1; ATF2; OLE1

Contents only isoamyl acetate concentrations are above

Formation of esters . . . . . . . . . . . . . . . . .
The ATF genes and their enzyme products .
Regulation of alcohol acetyltransferase gene
expression. . . . . . . . . . . . . . . . . . . . . . .
Physiological roles for ATF1 and ATF2 . . .
Additional alcohol ester synthesizing
activities? . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . .
Formation of esters
The synthesis of volatile aliphatic esters by yeast is
of major industrial interest because the presence of
these compounds determines the fruity aroma of
fermented beverages. Esters represent the largest
group of avour compounds in alcoholic beverages.
In beer, the major esters are ethyl acetate, isoamyl
acetate, phenylethyl acetate and the C6C10 short
chain fatty acid ethyl esters. Among these esters,
threshold level in most lager beers [9].
1287 The need to understand and control ester synth-
1288 esis is driven by problems encountered in brewing
procedures, such as high-gravity brewing (produc-
1290 tion of disproportionate amounts of ethyl acetate
1295 and isoamyl acetate), the use of large scale
cylindroconical fermenters (reduction of ester
1296 levels) or the production of reduced-alcohol beers
1296 (lack of avour compounds). An understanding of
1296 the molecular basis of ester synthesis should allow
for better control of ester production in these
processes.
Synthesis of esters requires two substrates: alco-
hol and carboxylic acid. Esters can be formed via a
chemical reaction but the reaction rate is too slow
to account for the amount of esters present in beer.
In 1962, Nordstrom [33] demonstrated that esters
are formed via an intracellular process catalysed by
an acyltransferase (EC 2.3.1) or `ester synthase'.
The reaction (Figure 1) requires energy provided by
the thioester linkage of the acyl-coenzyme A (CoA)
co-substrate. The most abundant acyl-CoA is

Copyright # 2000 John Wiley & Sons, Ltd.

1288 A. B. Mason and J.-P. Dufour

(a) Chemical synthesis:

Rf-OH+R-COOHaR-COO-Rf+H2O
Table 1. Molecular and enzymatic characteristics of
(b) Biochemical synthesis (alcohol acyltransferase reaction): yeast alcohol acetyltransferase enzymes
Rf-OH+R-CO~SCoAaR-COO-Rf+CoASH
(c) Activation of the acetyl/acyl moiety:
Pyruvate dehydrogenase reaction Gene ATF1 LgATF1 ATF2
CH3COCOOH+NAD++CoASHaCH3CO~SCoA+NADH+H++CO2
Fatty acyl-CoA ligases reaction
Source S. cerevisiae S. pastorianus S. cerevisiae
R-COOH+ATP+CoASHaR-CO~SCoA+AMP+PPi
Fatty acid synthase reaction ORF designation YOR377w Yscatf1ba YGR177c
R-CO~SCoA+HOOCCH2CO~SCoA+2NADPH+H+a
Rt-CO~SCoA+CoASH+2NADP++CO2+H2O Chromosome XV 850 kbb VII
ORF size (bp) 1575 1635 1605
Amino acid identity (%) 100 81 36.9
Figure 1. Chemical and biochemical synthesis of esters
Predicted Mr (kDa) 61.1 63.2 61.9
Mean hydrophobicity indexc x0.34 x0.34 x0.36
acetyl-CoA, which can be formed either by oxida- Predicted pId 6.94 7.78 5.77
tive decarboxylation of pyruvate or by direct Null mutant Viable ND Viable
activation of acetate with ATP. The majority of Km (isoamyl alcohol) 25 mM ND 25 mM
(pregnenolone) nda ND 0.5 mM
acetyl-CoA is formed by the oxidative decarboxyla-
tion of pyruvate, while most of the other acyl-CoAs a
Genbank locus designation; bsee Yoshimoto et al. [43]; csee Nagasawa
come from the acylation of free coenzyme A et al. [32]; dCalculated using ISOELECTRIC program from Program
(CoASH) catalysed by the acyl-CoA synthase Manual for the Wisconsin Package Version 8, August 1994, Genetics
(fatty acid metabolism). Computer Group, 575 Science Drive, Madison WI 53711, USA; ND,
not determined; nda, no detectable activity.

The ATF genes and their enzyme
products
Purication of the enzyme(s) responsible for alcohol
acetyltransferase (AATase) activity in yeast pro-
vides compelling evidence for the existence of
multiple proteins with quite different physicochem-
ical properties [30]. To date, three distinct AATase
genes (ATF1, LgATF1 and ATF2) have been
cloned from several different yeast backgrounds
[13,32,40,44]. Selected properties of yeast alcohol
acetyltransferase enzymes characterized so far are
summarized in Table 1. The ATF1 gene and/or the
closely related LgATF1 gene have been found in all
the examined Saccharomyces strains [13,14,40].
While products of the ATF1, LgATF1 and ATF2
gene may still not account for the full complement
of yeast alcohol acyltansferase activities, the follow-
ing discussion covers what is currently known about
the structural organization and expression of these
genes, together with insights as to their natural
function.
ATF1 (and LgATF1)
Acetyl-CoA : isoamyl alcohol acetyltransferase
(AATase I) catalyses the production of short-chain
and medium-chain aliphatic esters from isoamyl
alcohol or ethanol and acetyl-CoA [30]. Malcorps
and Dufour [30] achieved a 19 000-fold purication
of AATase I, as measured by the increase in specic
activity, and estimated its molecular mass at
57t3 kDa. The enzyme shows maximal activity at
around pH 8.0 and has apparent Km values (acetyl-
CoA) of 25 mM and 45 mM for isoamyl acetate and
ethyl acetate synthesis, respectively; the apparent
Km for isoamyl alcohol is 25 mM [30]. Oligonucleo-
tide probes designed on the basis of peptide
sequences established from puried isoamyl alcohol
acetyltransferase were used to identify the ATF1
gene in a l-EMBL3 library of genomic DNA from
the sake yeast Saccharomyces cerevisiae Kyokai
No. 7 [13,31]. Given its particularly low codon bias
index of 0.07 [2], Aft1p is predicted to be expressed
poorly, a prediction consistent with the consider-
able increase in specic enzyme activity measured
during purication [30].
A Southern analysis of S. cerevisiae genomic
DNA detected only one copy of ATF1 per haploid
S. cerevisiae genome [13]. By contrast, a S.
cerevisiae ATF1-derived DNA probe hybridized to
two non-identical genes in genomic DNA of the
bottom-fermenting lager yeast S. pastorianus
KBY001 [13]. The S. pastorianus ATF1 gene differs
from its S. cerevisiae ATF1 homologue by only
three amino acid substitutions, giving a 99.4%
identity at the deduced amino acid sequence level.
The second S. pastorianus ATF1-like gene (desig-
nated LgATF1) encodes a protein that is 81%
identical to S. cerevisiae Atf1p at the deduced
amino acid sequence level and includes a 20 amino
acid N-terminal extension. The copy of LgATF1 in
S. pastorianus was probably acquired as a result of

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
Alcohol acetyltransferases and ester synthesis in yeast
genetic hybridization between S. cerevisiae and S.
bayanus, the latter species having only LgATF1 and
no ATF1 equivalent [40,41,44]. ATF1 and LgATF1
may have diverged from a common ancestral gene
and therefore their cellular function(s) are likely to
have been conserved. The LgATF1 gene is func-
tional when expressed in S. cerevisiae [40] but it is
not kown if it complements fully for an atf1D null
mutation. This question awaits determination of the
physiological function of the Atf1p enzyme.
Hydrophobicity analysis of the deduced amino
acid sequence of Atf1p (mean hydrophobicity index
of x0.34) (Table 1) reveals short runs of hydro-
phobic residues, none of which is of sufcient length
to span a membrane, suggesting that the enzyme
may be membrane/lipid-associated rather than an
integral membrane component [13,32]. Indeed,
AATase I co-puries initially with high molecular
weight lipoprotein components of the yeast cell and
further separation requires detergent solubilization
[30]. While the exact cellular location of Aft1p has
not been determined, during differential fractiona-
tion the enzyme segregates with components of the
vacuome [28] (vacuome: `general term including all
the cellular compartments separated from the
cytosol by the endomembrane system' [42]).
In addition to the presence of the specic
polypeptide sequences used to identify ATF1 in the
deduced amino acid sequence of the cloned gene,
early evidence that ATF1 did indeed code for an
isoamyl alcohol acetyltransferase (IATase) came
from experiments in which ATF1 was over-
expressed in S. cerevisiae [13]. Multicopy plasmid
(Yep13)-based expression of S. pastorianus Aft1p
resulted in a 15-fold increase in specic activity of
the AATase for isoamyl alcohol. Similar over-
expression of Atf1p from the S. cerevisiae sake
yeast and LgAtf1p from brewing yeast produced
increases in AATase activity of 11-fold and six-fold,
respectively, over the control strain. When acetate
ester synthesis was measured, overexpression of the
lager yeast ATF1 gene caused a 27-fold increase in
isoamyl acetate (IA) levels and a nine-fold increase
in ethyl acetate (EA), compared to a strain
transformed with the vector only; the increases in
acetate production due to LgATF1 were 17-fold
and two-fold for IA and EA, respectively [13]. In
these expression experiments, Southern analysis
suggested that between-strain differences in plasmid
copy numbers varied by less than 10%. Nonetheless,
in the absence of RNA transcript analyses, it is
Copyright # 2000 John Wiley & Sons, Ltd.
1289
possible that the observed differences in relative
enzyme activities and ester synthesis may have been
affected, at least in part, by differences in transcrip-
tional efciency between plasmid constructs.
An atf1D::URA3 null mutant constructed in a
haploid S. cerevisiae strain displayed no visible
phenotype, although IATase and ethanol acetyl-
transferase (EATase) activities were approximately
80% and 20% lower, respectively, than in wild-type
cells [15]. This resulted in an 80% decrease in
isoamyl acetate production and a 30% decrease in
ethyl acetate production by the atf1D mutant [15].
Residual IATase and EATase activities in the atf1D
mutant could be further separated by chromatofo-
cusing and also had contrasting thermotolerance
characteristics. These results provided evidence
that, in addition to Aft1p, S. cerevisiae must possess
at least two other polypeptides with AATase
activity.
ATF2
The ATF2 gene was cloned from a l-EMBL3 phage
library of S. cerevisiae Kyokai No. 7 (sake yeast)
genomic DNA by plaque hybridization, using two
oligonucleotide probes designed on the basis of
internal peptide sequences obtained following pur-
ication of a second isoamyl alcohol acetyltransfer-
ase activity, the AATaseII enzyme [32]. At the
deduced amino acid level, Atf2p is 36.9% identical
to Atf1p (36.6% identical to LgAtf1) and its
hydropathy prole (mean hydrophobicity index of
x0.36) is similar to that of Atf1p proteins
(Table 1). Data from subcellular fractionation sug-
gest that Atf2p is mainly a soluble enzyme [5,32].
A variety of Saccharomyces strains were screened
for the presence of ATF2 by Southern analysis [32].
In all but one of the strains tested, a single copy of
ATF2 per haploid genome was detected. The
exception to this rule was S. bayanus, which did
not give an hybridization signal when probed using
an internal DNA fragment from S. cerevisiae K7
ATF2 [32]. An ATF2 homologue cloned from S.
pastorianus was 100% identical to that of S.
cerevisiae at the deduced amino acid level [44].
A feature common to Atf1p, LgAtf1p and Atf2p
is an heptapeptide (164WRLICLP), which is fully
conserved in each instance and which appears to be
found only in these three Atf proteins in the entire
yeast proteome. If this peptide constitutes part of
the active site, it is possible that the cysteine residue
Yeast 2000; 16: 12871298.
1290
may be critical to enzyme activity, as Atf1p and
Atf2p are both strongly inhibited by sulphydryl
reagents [5,30].
The relative contribution made by Atf2p to total
cellular AATase activity has not been determined.
Nagasawa and colleagues [32] refer to a S.
cerevisiae atf1D atf2D double mutant which pro-
duced no isoamyl acetate, suggesting that Atf2p
would be responsible for the entire 20% of isoamyl
acetate alcohol acetyltransferase activity remaining
in an atf1D mutant [15].
In a recent surprising discovery, S. cerevisiae
Atf2p was identied as the key enzyme in a steroid
detoxication mechanism in yeast [5]. In this report,
Atf2p/acetyl-CoA:pregnenolone acetyltransferase
(APAT) activity was solely responsible for the esteri-
cation of pregnenolone and related 3b-hydroxy-
steroids by transferring an acetyl group from acetyl
Co-A to the 3b-hydroxyl group, converting them
into substrates suitable for export by the ABC-type
transporters Pdr5p and Snq2p. The enzyme showed
a clear structural specicity for D5- or D4- 3b-
hydroxysteroids, such as pregnenolone, 17a-
hydroxy pregnenolone and dehydroepiandrosterone
(DHEA). Deletion/disruption of ATF2 eliminated
all detectable in vitro and in vivo pregnenolone
acetylation activity but had no other apparent effect
on cell phenotype; activity was restored when the
atf2 null mutant was transformed with ATF2 on a
multicopy (2m) plasmid [5]. No residual APAT
activity could be demonstrated in an atf2D null
mutant, even under semi-anaerobic conditions,
suggesting that pregnenolone cannot be acetylated
by Atf1p. Pregnenolone inhibition of the growth
rate of the atf2D mutant was exacerbated when
atf2D was introduced into a pdr5D snq2D back-
ground.
Regulation of alcohol acetyltransferase
gene expression
Effect of aeration and unsaturated fatty acids
on expression of ATF1
Inhibition of yeast AATase activity by trace
amounts of oxygen and/or unsaturated fatty acids
(UFAs) was reported many years prior to the
cloning of the ATF1 gene [45]. Repression of
enzyme synthesis as a means of regulating ester
synthesis was rst proposed by Malcorps et al. [29]
and supported by studies using linoleic acid (18 : 2)
Copyright # 2000 John Wiley & Sons, Ltd.
A. B. Mason and J.-P. Dufour
to suppress AATase activity [9]. Addition of linoleic
acid (50 mg/l) to the medium, prior to yeast
inoculation, nullied induction of AATase activity
and only a very low basal level of AATase activity
was observed. If added at the mid-exponential
phase of growth, linoleic acid caused a steady
decrease in AATase activity, although the total
specic activity of the pre-formed enzyme was
unaffected. Since incorporation of 14C-linoleic acid
into yeast membranes occurred within 20 min,
direct enzyme inhibition could not account for the
gradual decrease in AATase activity, suggesting
that inhibition of enzyme synthesis was the most
likely mechanism [9]. This theory was further
strengthened when it was demonstrated that addi-
tion of the protein synthesis inhibitor cycloheximide
to yeast at the mid-exponential phase of growth
prevented induction of AATase activity. This result
suggested that either the regulation was at the level
of AATase synthesis, or that synthesis of a
regulatory co-factor was required in order to
activate the AATase.
Using the atf1D null mutant, Fujii et al. [15]
demonstrated that the remaining AATase activity in
this strain was not affected by aeration or by UFAs,
suggesting that Atf1p was responsible for most, if
not all, oxygen- and UFA-repressible AATase
activity. But were these factors acting indepen-
dently, or was oxygen contributing to UFA synth-
esis, which then repressed AATase activity? As a
partial answer to this question, Fujii et al. [15]
showed that, while both aeration and certain UFAs
caused transcriptional repression of ATF1 within
1 h of exposure, aeration did not lead to a
signicant change in the fatty acid composition of
yeast membranes within that time interval. This
suggested that O2-mediated repression occurred via
a mechanism that was independent of changes in
membrane UFA levels. Complementing this result,
the ability of UFAs to independently regulate
transcription of ATF1 was demonstrated recently
by the UFA-mediated inhibition of ATF1lacZ
expression under anaerobic conditions in rox1,
tup1 and ssn6 null mutants ( [18], and see below).
Analysis of the promoter region upstream of the
ATF1 ORF has established that this regulation of
expression was mediated primarily at the transcrip-
tional level. Replacement of the natural promoter
of the ATF1 gene with the constitutive promoter
(pGAPDH) from the yeast glyceraldehyde-3-phos-
phate dehydrogenase gene released expression of
Yeast 2000; 16: 12871298.
Alcohol acetyltransferases and ester synthesis in yeast
IATase activity from repression by O2 and/or UFA.
The specic non-coding DNA sequences implicated
in this regulation were delimited to a 150 bp stretch
of DNA between x50 and x200 bp upstream of
the initiation codon of the S. pastorianus LgATF1
gene [12,14]. An homologous control region is
found in the equivalent position upstream from
the S. cerevisiae Kyokai No. 7 ATF1 gene [12].
When this 150 bp stretch of the S. cerevisiae ATF1
promoter region was fused to the Escherichia coli
lacZ gene, b-galactosidase activity was rendered
subject to repression by both O2 and UFA [12].
Transcriptional co-regulation of ATF1 and
OLE1 by oxygen and unsaturated fatty acid
Transcription of ATF1 is inhibited by a variety of
UFAs, including palmitoleic (16 : 1), oleic (18 : 1),
linoleic (18 : 2) and linolenic (18 : 3) acids [12,18].
Only palmitoleic and oleic acids are found naturally
in yeast membranes and these are produced
exclusively by the activity of the D9-fatty acid
desaturase. This enzyme is encoded by the OLE1
gene, which is present as a single copy per haploid
S. cerevisiae genome. The desaturase introduces a
double bond between carbons 9 and 10 of palmi-
toyl- or steary1-CoA substrates to produce palmi-
toleic and oleic acids, respectively [38,39]. A
discussion of the OLE1 gene and its role in yeast
fatty acid metabolism is beyond the scope of this
review; however, given the common responses of
both OLE1 and ATF1 gene to O2 and UFAs, some
examination of the mechanisms of transcriptional
regulation of these two genes is warranted. Figure 2
presents a current view of regulatory elements, both
demonstrated and putative, that are located
upstream of the OLE1 and ATF1 ORFs.
ATF1 and OLE1: two hypoxic genes
In their response to oxygen, both OLE1 and ATF1
are examples of hypoxic genes whose transcription
is induced to enable S. cerevisiae to grow under
conditions of severe oxygen limitation, but which
are otherwise repressed by means of a heme-
dependent mechanism (see Figure 2; for review see
[46]). The biosynthesis of heme is oxygen-depen-
dent. As heme accumulates, it combines with the
transcriptional activator Hap1p to form a complex
which transcriptionally activates the ROX1 gene.
The ROX1 gene product, Rox1p, in association
with the Tup1Ssn6 general repressor complex [25],
Copyright # 2000 John Wiley & Sons, Ltd.
1291
represses a subset of hypoxic genes by binding to its
cognate site (i.e. Rox1p consensus recognition
sequence, YYYATTGTTCTC, where Y represents
pyrimidine) located within the promoter regions of
those genes. This subset of genes includes the ROX1
gene itself. The Rox1p autoregulatory binding site
may serve as a means of restricting cellular levels of
Rox1p in order to avoid over-repression of genes
such as OLE1, whose functional product is also
required for aerobic growth.
As heme levels fall during hypoxia, Hap1p still
binds to its DNA recognition site but in combina-
tion with other non-heme proteins. This large
complex represses ROX1 transcription which, in
turn, has the effect of de-repressing transcription of
the subset of hypoxic genes. There are two Rox1p
binding sites located upstream of the OLE1 initia-
tion codon (Figure 2). The rst runs from x272 to
x261 bp (x272CCTATTGTTACG; nucleotides
numbered such that the rst base 5k of the ATG
start codon is designated x1) and the second
lies between bases x130 and x119
(x130TTTATTGTTCTA). Of the two sequences,
the latter conforms more closely to the Rox1p
consensus sequence, but transcriptional analyses
have not been performed to see if there is
preferential usage of one site over the other
or if, in fact, both sites are involved in trans-
criptional control. A Rox1p binding site
(x120CCTATTGTTTTTT) is also found between
x120 and x108 bp upstream of the ATF1 initia-
tion codon. Specic binding of Rox1p to this
promoter element in vitro has been demonstrated
recently [17]. Furthermore, analysis of an ATF1
promoterlacZ fusion construct expressed in rox1,
tup1 and ssn6 null mutants under aerobic growth
conditions showed approximately three- to four-
fold increases in b-galactosidase activity in each
case [17]. No signicant changes in b-galactosidase
activity were seen in the same mutants grown
anaerobically in the presence of oleic acid. Taken
together, these results indicate that the Rox1p
Tup1pSsn6p complex is responsible for transcrip-
tional repression of the ATF1 gene by oxygen but
plays no signicant role in UFA-mediated repres-
sion.
A series of nested deletions of the ATF1
promoter fused to the S. cerevisiae PHO5 gene
was also used by Fujiwara and colleagues to analyse
ATF1 expression as a function of acid phosphatase
(rAPase) activity in a pho5-1 mutant lacking
Yeast 2000; 16: 12871298.
Copyright # 2000 John Wiley & Sons, Ltd.

1292
A. B. Mason and J.-P. Dufour
Yeast 2000; 16: 12871298.

Figure 2. Oxygen- and UFA-dependent regulatory pathways controlling transcriptional expression of ATF1 and OLE1 genes
Alcohol acetyltransferases and ester synthesis in yeast
endogenous rAPase activity [17]. An 18 bp DNA
sequence (x85 to x68 bp upstream of ATF1 start
codon) was identied which appears to be essential
for the activation of ATF1 transcription and which
contains a consensus binding sequence for the
ubiquitous transcription factor Rap1p [37]. As
binding of Rap1p to the 18 bp element was
demonstrated in vitro, it is possible that Rap1p
acts as a positive factor in transcription of the
ATF1 gene.
ATF1: a stress response gene?
Another interesting DNA motif found upstream of
ATF1 but not OLE1 is the sequence x650CCCCT
(see C4T, Figure 2). The `C4T' sequence (STRE:
stress response element) is associated with global
stress response genes induced as a result of stress
conditions such as heat shock, osmotic stress,
oxidative stress, acid pH, glucose or nitrogen
starvation, and DNA-damaging agents [27]. The
signal transduction pathways that detect and com-
municate these various stress conditions ultimately
converge, activating the Rox3p effector, which then
activates transcription of the global stress response
genes. Rox3p, an essential component of the RNA
polymerase II complex, can act as either an
activator or repressor of gene transcription but has
no inherent DNA-binding activity of its own [21].
The transcription factors that mediate this interac-
tion may be a pair of functionally redundant
proteins, Msn2p and Msn4p, which have been
shown to activate genes in response to various
stresses [10]. During growth on glucose, the C4T
element mediates repression by the cyclic AMP
(cAMP)-signalling pathway, thus many genes with
this stress-responsive element (STRE) are induced
at the diauxic shift, when glucose is depleted and
cAMP levels fall.
Other than hypoxia, the effects of stress condi-
tions on ATF1 transcriptional expression have not
been measured directly. A two- to three-fold
increase in AATase activity on the second day of a
barley shochu yeast fermentation was observed as a
consequence of heat shock treatment [23]. After this
peak of activity, the AATase activity level waned to
that of untreated wild-type cells. Although this
response was not characterized at the mRNA level,
the pattern of expression could reect transcrip-
tional induction of ATF1. In another study, heat
shock-resistant (HSR) yeast mutants, obtained after
two rounds of heat shock selection (45uC) of the S.
Copyright # 2000 John Wiley & Sons, Ltd.
1293
cerevisiae shochu yeast BAW-6, not only produced
higher levels of glycerol than the parental strain but
also showed potentially signicant (approximately
two-fold) increases in isoamyl acetate, ethyl
caproate and ethyl caprate production [34]. These
characteristics were apparently stable through sub-
sequent generations. The selection, by heat shock,
of yeast mutants with elevated AATase activity
suggests either that this trait specically contributes
to enhanced thermotolerance, or that increased
AATase production is a serendipitous consequence
of a generalized stress response.
It should be noted that in the regulation of the
ROX3 gene, the upstream region of ROX3 does not
include the C4T motif. Instead, heme-independent
transcriptional induction of ROX3 in response to
heat shock, osmotic stress and particularly hypoxia
apparently depends upon a DNA sequence motif
(5k-GA10GGAA-3k) that is repeated three times in
the non-coding sequences upstream of ROX3 [10].
Even a single copy of this motif conferred increased
expression when placed upstream of an heterolo-
gous gene [11]; thus, it may be signicant that a
copy of this motif is also present upstream of the
OLE1gene (x803GA10GGAA) (Figure 2). The fac-
tors responsible for this mode of heme-independent
oxygen sensing have not been elucidated. No
similar motif is present upstream of either the
ATF1 or ATF2 genes (see below).
Unsaturated fatty acid-mediated regulation of ATF1
and OLE1 expression
Unsaturated fatty acids repress transcription of
ATF1 and OLE1 but it is not clear if this repression
is effected via the same signal transduction path-
ways. Analysis of the OLE1 promoter region
identied a fatty acid response (FAR) element of
111 bp running from x576 to x466 bp upstream of
the ATG start codon [7]. This stretch of DNA
includes sites for both UFA-mediated repression
and activation of OLE1 transcription. The tran-
scription factor Hap1p appears to be responsible
for over 50% of the activation of OLE1 transcrip-
tion in wild-type cells. This was demonstrated by
the signicant decreases in transcription of OLE1
promoterlacZ reporter constructs expressed in a
hap1D::LEU2 background [7]. However, disruption
of HAP1 made no difference to either the SFA/
UFA ratio or absolute fatty acid levels, suggesting
that transcription factor(s) other than Hap1p must
Yeast 2000; 16: 12871298.
1294
be involved in the regulation of the FAR element
and maintenance of normal fatty acyl composition.
While no specic transcription factor has been
implicated in the UFA-mediated regulation of
ATF1, the regulatory DNA sequence(s) appears to
be included in the 150 bp (x50 to x200) of ATF1
promoter sequence which incorporates the Rox1p
binding site [12], and may be contained entirely
within an 18 bp DNA sequence identied by
Fujiwara et al. [17]. Another sequence within the
x50 to x200 region of the ATF1 promoter was
identied in a search of the S. cerevisiae genome
database for oleate response element (ORE)
sequences targeted by the transcription factors
Oaf1p and Pip2p (Oaf2p) [24]. Pip2p was rst
described by Rottensteiner et al. [36]. These oleate-
activated transcription factors are instrumental in
driving the increased transcription of peroxisomal
protein genes which are induced when S. cerevisiae
is grown on a fatty acid as sole carbon source,
resulting in a proliferation in the number and size of
peroxisomes in the cell. ATF1 was one of 40 genes
identied as having an ORE within 500 bp of the
ATG start codon [24]. Like ATF1, a number of
these genes did not encode peroxisomal compo-
nents. Transcriptional expression of all 40 genes
was measured in wild-type, oaf1D, pip2D or oaf1D/
pip2D mutants grown on different carbon sources.
By comparison with wild-type expression levels, no
changes in ATF1 mRNA transcription were
observed, although it should be noted that these
tests were made under aerobic growth conditions,
and it is possible that the ORE upstream of ATF1
may assume some signicance in the regulation of
ATF1 expression when the same mutants are grown
under hypoxic or anaerobic conditions. Given the
repressive effect of UFAs on ATF1 expression, it
seems more likely that this ORE would be utilized
in a negative regulatory context in ATF1 transcrip-
tion. The OLE1 gene has no ORE consensus
sequence within its promoter region.
Fatty acids supplied exogenously to yeast must be
converted into their acy1-CoA derivatives prior to
incorporation into membrane and/or storage lipids.
This function is fullled by the fatty acy1-CoA
synthetases encoded by the FAA genes [22]. For
some time it had been suspected that at least part of
the fatty acid signalling pathways that control
OLE1 and ATF1 transcription may be shared.
This hypothesis relied heavily on the observation
that UFA-mediated transcriptional repression of
Copyright # 2000 John Wiley & Sons, Ltd.
A. B. Mason and J.-P. Dufour
both OLE1 and ATF1 genes was relieved by
deletion of the FAA1 and/or FAA4 genes [7,18]. In
a recent study, Choi and Martin [6] established that
Faa1p and Faa4p are long-chain (C14C18) fatty
acyl-CoA synthetases that are, in fact, essential for
the import of long-chain saturated and unsaturated
fatty acids (Figure 2). Thus, the similarity in
transcriptional behaviour of the OLE1 and ATF1
genes was attributable to the fact that UFAs were
not getting into the mutant cells. Further research is
needed to establish the extent of commonality
between the fatty acid signal transduction pathways
that regulate OLE1 and ATF1 expression.
Recently, Fujiwara et al. considered the inhibi-
tory effects of a variety of fatty acids on transcrip-
tional expression of ATF1 and OLE1 [18] by
measuring the b-galactosidase activity expressed
from ATF1lacZ or OLE1lacZ reporter con-
structs. Their results indicated an inverse relation-
ship between the degree of transcriptional
repression of ATF1 or OLE1 and UFA melting
point. The OLE1lacZ strains were grown under
anaerobic conditions in order to avoid the repres-
sive effect of Rox1p during aerobic growth.
Increased repression due to UFAs with lower
melting points might indicate that membrane
uidity is a key parameter in regulating desaturase
synthesis in order to maintain or modify the ratio of
SFA : UFA. Such a relationship has been previously
postulated to account for the effects of changes in
membrane UFA content on the induction of
transcription of heat shock genes in yeast [4].
In the control of D9-desaturase activity, addi-
tional regulation is provided by post-transcriptional
modulation of OLE1 mRNA half-life, and is
mediated, at least in part, by the activity of the
Xrn1 ribonuclease [20]. Post-transcriptional regula-
tion of ATF1 mRNA half-life has not been studied.
As illustrated in Figure 2, the transcriptional
regulation of ATF1 and OLE1 expression involves
numerous controlling elements. The oxygen-
mediated regulation of both genes appears to
depend upon the same factors, although some
additional mechanism(s) of differential regulation
must exist to allow for expression of the D9-
desaturase under aerobic conditions, where Atf1p
is almost completely repressed. The effectors of
UFA-induced repression of each of the two genes
have not been well dened and there appear to be
clear differences in the nature of the interactive sites
involved in transcriptional control. It is interesting
Yeast 2000; 16: 12871298.
Alcohol acetyltransferases and ester synthesis in yeast
to note that both OLE1 and ATF1 have upstream
sequences that may constitute STREs (Figure 2).
The signicance of these characteristic sequences
has not been investigated. One can understand the
yeast cell's even greater need to maintain tight
control over membrane fatty acid composition
under stress conditions. On the other hand, the
potential signicance of Atf1p function in response
to a stress condition(s) is perhaps less obvious. The
C4T motif upstream of ATF1 is present as a single
copy, some distance from the initiation codon. In
most cases where this STRE has been identied
upstream of a stress-induced gene, multiple copies
have been found [3]. Since ATF1 is poorly
expressed, the magnitude of stress induction would
need to be only small in order to have a signicant
effect on Atf1p levels.
Regulation of ATF2 expression
When compared with a vector-only control strain,
multicopy plasmid-based expression of ATF1 or
ATF2 in S. cerevisiae produced 4.7- and 2.4-fold
increases, respectively, in total AATase activity
levels measured in cell-free extracts [32]. Signi-
cantly, the AATase activity from cells over-expres-
sing Atf2p was much less susceptible to inhibition
by linoleic acid in vitro than the AATase from cells
overexpressing Atf1p. Yoshimoto et al. [44] found
that the S. pastorianus ATF2 gene produced func-
tional AATase activity when expressed in an S.
cerevisiae atf1 null mutant, resulting in a 2.5-fold
increase in total AATase activity. In the same
report, b-galactosidase activity in a S. pastorianus
strain expressing an ATF2lacZ construct was
repressed by aeration but activated by the addition
of 1 mM oleic acid [44].
The promoter region of the ATF2 gene has a
`poor-t' Rox1p consensus sequence which runs
from x139 to x128 upstream of both S. cerevisiae
and S. pastorianus ATF2 genes, and Yoshimoto
et al. [44] reported induction of an ATF2lacZ
expression construct under anaerobic conditions.
While this nding appears contradictory to an
earlier report that, in an atf1D null mutant,
AATase activity was no longer subject to repression
by either UFAs or oxygen [15], the disparity may be
due to the relatively low levels of activity being
measured in each case. Collectively, these prelimin-
ary ndings as to the regulation of Atf2p expression
suggest that the ATF2 gene may be subject to
Copyright # 2000 John Wiley & Sons, Ltd.
1295
transcriptional repression by oxygen but is rela-
tively unaffected by the presence of UFAs at either
the transcriptional or post-transcriptional level.
In batch culture, Aft2p (APAT) is present at very
low protein levels and there is a rapid decline in
abundance of Atf2p after glucose exhaustion [5].
The mechanistic basis for this decline is unknown.
In contrast to the promoter regions of the ATF1
and OLE1 genes, the ATF2 promoter region
includes no putative STRE and there is no evidence
of an ORE element.
Physiological roles for ATF1 and ATF2
Just what physiological function Atf1p might full
remains open to speculation but available evidence,
derived mostly from the remarkable parallels
between the regulation of expression of ATF1and
OLE1 genes, suggests a specialized niche for Atf1p
in fatty acid metabolism. The evidence includes: (a)
the repressive effect of UFAs (particularly those
with a double bond in the D9 position) on Atf1p
expression; (b) the apparent correlation between
fatty acid melting point and magnitude of the
repressive effect on ATF1 transcription, suggesting
that membrane uidity may be of fundamental
signicance to Atf1p function; (c) the association of
Atf1p with (membrane) lipids, and nally; (d) the
observation that 12-DL-hydroxy stearate can be
esteried by puried IATase [30] and potentially
utilized as an unsaturated fatty acid analogue under
anaerobic conditions [26]. The nal point is con-
sistent with the classication of ATF1 as an hypoxic
gene whose expression may be regulated, along with
that of OLE1, by the heme-dependent transcription
factor Rox1p. Given the multiplicity of regulatory
elements upstream of the OLE1 and ATF1 genes
and the apparent contrast in signicance of the two
gene products to cell metabolism, it would be an
overstatement to claim that they are co-regulated or
coordinately expressed. Nonetheless, it is still
possible that the activity of Atf1p is signicant
under an as-yet-undened stress condition(s).
An indication as to a possible biological function
of Atf2p is provided by the ndings from the recent
study by Cauet et al. [5]. The authors suggest that
since progesterone, its structural relative pregneno-
lone and various other steroids can bind with high
afnity to the sterol C8C7 isomerase (the ERG2
gene product involved in ergosterol biosynthesis),
Yeast 2000; 16: 12871298.
1296
Atf2p and the efux pumps may function coopera-
tively to rid the yeast cell of growth-inhibitory 3b-
hydroxysteroids generated as products of defective
ergosterol biosynthesis. Such a scenario might
provide a rational explanation for preferential
expression of Atf2p under anaerobic conditions,
where yeast cells are unable to biosynthesize
ergosterol [1]. The high afnity of Atf2p/APAT for
pregnenolone (Km, pregnenolone=0.5 mM) is a
strong indication that the enzyme has evolved to
interact with a steroidal substrate; Atf2p's Km for
isoamyl alcohol is 50 000-fold higher at 25 mM. In
their natural environment, it is possible that yeast
would come into contact with plant compounds
such as avonoids, which have some steroid-like
structural properties and may be substrates for such
a detoxication mechanism.
Additional alcohol ester synthesizing
activities?
Homology-based searches of the entire S. cerevisiae
genome database [19] using amino acid sequences
deduced from ATF1 and ATF2 have not identied
any other closely related genes. Biochemical frac-
tionation of a wild-type yeast cell extract separated
at least three distinct enzyme activities involved in
alcohol esterication [30]. The isoamyl alcohol
acetyltransferase Atf1p accounts for around 80%
of isoamyl acetate production and 30% of ethyl
acetate production. The remainder of the IATase
activity may be contributed by the Atf2p enzyme. If
this is so, an atf1D atf2D double mutant should be
devoid of isoamyl alcohol acetyltransferase activity.
However, this has yet to be established. Indeed,
ATF2 expression and the proportions of total
cellular AATase activity that can be attributed to
the Atf2p enzyme in wild-type and atf1D mutants
are parameters that require further investigation.
The relative contribution (if any) of Atf2p activity
to ethyl acetate production remains unclear. The
biochemical evidence originally presented by Mal-
corps and Dufour [30] suggests that, in addition to
Atf1p and Atf2p, there should be at least one more
yeast enzyme with ethyl alcohol acetyltransferase
activity. A fourth ester-synthesizing enzyme, etha-
nol hexanoyl transferase, is responsible for generat-
ing ethyl hexanoate from ethanol and hexanoyl-
CoA. This alcohol acyltransferase, encoded by the
EHT1 gene, has not been studied in any detail. At
Copyright # 2000 John Wiley & Sons, Ltd.
A. B. Mason and J.-P. Dufour
the deduced amino acid sequence level, Eht1p is
only 17% identical to the alcohol acetyltransferases
Atf1p and Atf2p and does not include the `con-
served' WRLICLP motif common to the three
AATase enzymes Atf1p, LgAtf1p and Atf2p. If
Eht1p has relatively broad substrate specicity, it
may also possess some ethanol esterication activ-
ity. An atf1D atf2D double mutant should facilitate
characterization of Eht1p and its relative contribu-
tion to the avour content of fermented products.
Concluding remarks
Our limited understanding of the physiological role
of yeast ester synthases detracts from our ability to
fully control ester production during fermentation.
The regulation of the alcohol acyltransferase gene
expression in yeast appears to be extremely compli-
cated. More and more, yeast genetic engineering
presents itself as the approach most likely to help
elucidate the mechanisms underlying ester synthesis.
To date, only four alcohol acyltransferase-encod-
ing genes have been cloned. Further studies are
needed to identify the remaining ester-synthesizing
enzymes, in particular the major enzyme involved in
ethyl acetate synthesis. With the completed S.
cerevisiae genome sequence database and the
powerful tools of molecular biology, major break-
throughs as to the role of ester synthesis in yeast
can be expected in the near future. Ultimately, these
insights should lead to better management of ester
levels in alcoholic beverages.
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