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Yeast 2000; 16: 12871298.


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, This paper reviews our current knowledge of yeast alcohol acyltransferases. Much of this
PO Box 56, Dunedin, information has been gathered over the past 10 years through the application of powerful
New Zealand. yeast molecular biology techniques. Evidence from gene disruption and expression analysis
E-mail: jean-pierre.dufour@ 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

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

Table 1. Molecular and enzymatic characteristics of
(b) Biochemical synthesis (alcohol acyltransferase reaction): yeast alcohol acetyltransferase enzymes
(c) Activation of the acetyl/acyl moiety:
Pyruvate dehydrogenase reaction Gene ATF1 LgATF1 ATF2
Fatty acyl-CoA ligases reaction
Source S. cerevisiae S. pastorianus S. cerevisiae
Fatty acid synthase reaction ORF designation YOR377w Yscatf1ba YGR177c
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 57t3 kDa. The enzyme shows maximal activity at
products around pH 8.0 and has apparent Km values (acetyl-
CoA) of 25 mM and 45 mM for isoamyl acetate and
Purication of the enzyme(s) responsible for alcohol ethyl acetate synthesis, respectively; the apparent
acetyltransferase (AATase) activity in yeast pro- Km for isoamyl alcohol is 25 mM [30]. Oligonucleo-
vides compelling evidence for the existence of tide probes designed on the basis of peptide
multiple proteins with quite different physicochem- sequences established from puried isoamyl alcohol
ical properties [30]. To date, three distinct AATase acetyltransferase were used to identify the ATF1
genes (ATF1, LgATF1 and ATF2) have been gene in a l-EMBL3 library of genomic DNA from
cloned from several different yeast backgrounds the sake yeast Saccharomyces cerevisiae Kyokai
[13,32,40,44]. Selected properties of yeast alcohol No. 7 [13,31]. Given its particularly low codon bias
acetyltransferase enzymes characterized so far are index of 0.07 [2], Aft1p is predicted to be expressed
summarized in Table 1. The ATF1 gene and/or the poorly, a prediction consistent with the consider-
closely related LgATF1 gene have been found in all able increase in specic enzyme activity measured
the examined Saccharomyces strains [13,14,40].
during purication [30].
While products of the ATF1, LgATF1 and ATF2 A Southern analysis of S. cerevisiae genomic
gene may still not account for the full complement
DNA detected only one copy of ATF1 per haploid
of yeast alcohol acyltansferase activities, the follow-
S. cerevisiae genome [13]. By contrast, a S.
ing discussion covers what is currently known about
cerevisiae ATF1-derived DNA probe hybridized to
the structural organization and expression of these
two non-identical genes in genomic DNA of the
genes, together with insights as to their natural
bottom-fermenting lager yeast S. pastorianus
KBY001 [13]. The S. pastorianus ATF1 gene differs
from its S. cerevisiae ATF1 homologue by only
ATF1 (and LgATF1) three amino acid substitutions, giving a 99.4%
Acetyl-CoA : isoamyl alcohol acetyltransferase identity at the deduced amino acid sequence level.
(AATase I) catalyses the production of short-chain The second S. pastorianus ATF1-like gene (desig-
and medium-chain aliphatic esters from isoamyl nated LgATF1) encodes a protein that is 81%
alcohol or ethanol and acetyl-CoA [30]. Malcorps identical to S. cerevisiae Atf1p at the deduced
and Dufour [30] achieved a 19 000-fold purication amino acid sequence level and includes a 20 amino
of AATase I, as measured by the increase in specic acid N-terminal extension. The copy of LgATF1 in
activity, and estimated its molecular mass at 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 1289

genetic hybridization between S. cerevisiae and S. possible that the observed differences in relative
bayanus, the latter species having only LgATF1 and enzyme activities and ester synthesis may have been
no ATF1 equivalent [40,41,44]. ATF1 and LgATF1 affected, at least in part, by differences in transcrip-
may have diverged from a common ancestral gene tional efciency between plasmid constructs.
and therefore their cellular function(s) are likely to An atf1D::URA3 null mutant constructed in a
have been conserved. The LgATF1 gene is func- haploid S. cerevisiae strain displayed no visible
tional when expressed in S. cerevisiae [40] but it is phenotype, although IATase and ethanol acetyl-
not kown if it complements fully for an atf1D null transferase (EATase) activities were approximately
mutation. This question awaits determination of the 80% and 20% lower, respectively, than in wild-type
physiological function of the Atf1p enzyme. cells [15]. This resulted in an 80% decrease in
Hydrophobicity analysis of the deduced amino isoamyl acetate production and a 30% decrease in
acid sequence of Atf1p (mean hydrophobicity index ethyl acetate production by the atf1D mutant [15].
of x0.34) (Table 1) reveals short runs of hydro- Residual IATase and EATase activities in the atf1D
phobic residues, none of which is of sufcient length mutant could be further separated by chromatofo-
to span a membrane, suggesting that the enzyme cusing and also had contrasting thermotolerance
may be membrane/lipid-associated rather than an characteristics. These results provided evidence
integral membrane component [13,32]. Indeed, that, in addition to Aft1p, S. cerevisiae must possess
AATase I co-puries initially with high molecular at least two other polypeptides with AATase
weight lipoprotein components of the yeast cell and activity.
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 The ATF2 gene was cloned from a l-EMBL3 phage
vacuome [28] (vacuome: `general term including all library of S. cerevisiae Kyokai No. 7 (sake yeast)
the cellular compartments separated from the genomic DNA by plaque hybridization, using two
cytosol by the endomembrane system' [42]). oligonucleotide probes designed on the basis of
In addition to the presence of the specic internal peptide sequences obtained following pur-
polypeptide sequences used to identify ATF1 in the ication of a second isoamyl alcohol acetyltransfer-
deduced amino acid sequence of the cloned gene, ase activity, the AATaseII enzyme [32]. At the
early evidence that ATF1 did indeed code for an deduced amino acid level, Atf2p is 36.9% identical
isoamyl alcohol acetyltransferase (IATase) came to Atf1p (36.6% identical to LgAtf1) and its
from experiments in which ATF1 was over- hydropathy prole (mean hydrophobicity index of
expressed in S. cerevisiae [13]. Multicopy plasmid x0.36) is similar to that of Atf1p proteins
(Yep13)-based expression of S. pastorianus Aft1p (Table 1). Data from subcellular fractionation sug-
resulted in a 15-fold increase in specic activity of gest that Atf2p is mainly a soluble enzyme [5,32].
the AATase for isoamyl alcohol. Similar over- A variety of Saccharomyces strains were screened
expression of Atf1p from the S. cerevisiae sake for the presence of ATF2 by Southern analysis [32].
yeast and LgAtf1p from brewing yeast produced In all but one of the strains tested, a single copy of
increases in AATase activity of 11-fold and six-fold, ATF2 per haploid genome was detected. The
respectively, over the control strain. When acetate exception to this rule was S. bayanus, which did
ester synthesis was measured, overexpression of the not give an hybridization signal when probed using
lager yeast ATF1 gene caused a 27-fold increase in an internal DNA fragment from S. cerevisiae K7
isoamyl acetate (IA) levels and a nine-fold increase ATF2 [32]. An ATF2 homologue cloned from S.
in ethyl acetate (EA), compared to a strain pastorianus was 100% identical to that of S.
transformed with the vector only; the increases in cerevisiae at the deduced amino acid level [44].
acetate production due to LgATF1 were 17-fold A feature common to Atf1p, LgAtf1p and Atf2p
and two-fold for IA and EA, respectively [13]. In is an heptapeptide (164WRLICLP), which is fully
these expression experiments, Southern analysis conserved in each instance and which appears to be
suggested that between-strain differences in plasmid found only in these three Atf proteins in the entire
copy numbers varied by less than 10%. Nonetheless, yeast proteome. If this peptide constitutes part of
in the absence of RNA transcript analyses, it is the active site, it is possible that the cysteine residue

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
1290 A. B. Mason and J.-P. Dufour

may be critical to enzyme activity, as Atf1p and to suppress AATase activity [9]. Addition of linoleic
Atf2p are both strongly inhibited by sulphydryl acid (50 mg/l) to the medium, prior to yeast
reagents [5,30]. inoculation, nullied induction of AATase activity
The relative contribution made by Atf2p to total and only a very low basal level of AATase activity
cellular AATase activity has not been determined. was observed. If added at the mid-exponential
Nagasawa and colleagues [32] refer to a S. phase of growth, linoleic acid caused a steady
cerevisiae atf1D atf2D double mutant which pro- decrease in AATase activity, although the total
duced no isoamyl acetate, suggesting that Atf2p specic activity of the pre-formed enzyme was
would be responsible for the entire 20% of isoamyl unaffected. Since incorporation of 14C-linoleic acid
acetate alcohol acetyltransferase activity remaining into yeast membranes occurred within 20 min,
in an atf1D mutant [15]. direct enzyme inhibition could not account for the
In a recent surprising discovery, S. cerevisiae gradual decrease in AATase activity, suggesting
Atf2p was identied as the key enzyme in a steroid that inhibition of enzyme synthesis was the most
detoxication mechanism in yeast [5]. In this report, likely mechanism [9]. This theory was further
Atf2p/acetyl-CoA:pregnenolone acetyltransferase strengthened when it was demonstrated that addi-
(APAT) activity was solely responsible for the esteri- tion of the protein synthesis inhibitor cycloheximide
cation of pregnenolone and related 3b-hydroxy- to yeast at the mid-exponential phase of growth
steroids by transferring an acetyl group from acetyl prevented induction of AATase activity. This result
Co-A to the 3b-hydroxyl group, converting them suggested that either the regulation was at the level
into substrates suitable for export by the ABC-type of AATase synthesis, or that synthesis of a
transporters Pdr5p and Snq2p. The enzyme showed regulatory co-factor was required in order to
a clear structural specicity for D5- or D4- 3b- activate the AATase.
hydroxysteroids, such as pregnenolone, 17a- Using the atf1D null mutant, Fujii et al. [15]
hydroxy pregnenolone and dehydroepiandrosterone demonstrated that the remaining AATase activity in
(DHEA). Deletion/disruption of ATF2 eliminated this strain was not affected by aeration or by UFAs,
all detectable in vitro and in vivo pregnenolone suggesting that Atf1p was responsible for most, if
acetylation activity but had no other apparent effect not all, oxygen- and UFA-repressible AATase
on cell phenotype; activity was restored when the activity. But were these factors acting indepen-
atf2 null mutant was transformed with ATF2 on a dently, or was oxygen contributing to UFA synth-
multicopy (2m) plasmid [5]. No residual APAT esis, which then repressed AATase activity? As a
activity could be demonstrated in an atf2D null partial answer to this question, Fujii et al. [15]
mutant, even under semi-anaerobic conditions, showed that, while both aeration and certain UFAs
suggesting that pregnenolone cannot be acetylated caused transcriptional repression of ATF1 within
by Atf1p. Pregnenolone inhibition of the growth 1 h of exposure, aeration did not lead to a
rate of the atf2D mutant was exacerbated when signicant change in the fatty acid composition of
atf2D was introduced into a pdr5D snq2D back- yeast membranes within that time interval. This
ground. suggested that O2-mediated repression occurred via
a mechanism that was independent of changes in
membrane UFA levels. Complementing this result,
Regulation of alcohol acetyltransferase
the ability of UFAs to independently regulate
gene expression
transcription of ATF1 was demonstrated recently
by the UFA-mediated inhibition of ATF1lacZ
Effect of aeration and unsaturated fatty acids
expression under anaerobic conditions in rox1,
on expression of ATF1
tup1 and ssn6 null mutants ( [18], and see below).
Inhibition of yeast AATase activity by trace Analysis of the promoter region upstream of the
amounts of oxygen and/or unsaturated fatty acids ATF1 ORF has established that this regulation of
(UFAs) was reported many years prior to the expression was mediated primarily at the transcrip-
cloning of the ATF1 gene [45]. Repression of tional level. Replacement of the natural promoter
enzyme synthesis as a means of regulating ester of the ATF1 gene with the constitutive promoter
synthesis was rst proposed by Malcorps et al. [29] (pGAPDH) from the yeast glyceraldehyde-3-phos-
and supported by studies using linoleic acid (18 : 2) phate dehydrogenase gene released expression of

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
Alcohol acetyltransferases and ester synthesis in yeast 1291

IATase activity from repression by O2 and/or UFA. represses a subset of hypoxic genes by binding to its
The specic non-coding DNA sequences implicated cognate site (i.e. Rox1p consensus recognition
in this regulation were delimited to a 150 bp stretch sequence, YYYATTGTTCTC, where Y represents
of DNA between x50 and x200 bp upstream of pyrimidine) located within the promoter regions of
the initiation codon of the S. pastorianus LgATF1 those genes. This subset of genes includes the ROX1
gene [12,14]. An homologous control region is gene itself. The Rox1p autoregulatory binding site
found in the equivalent position upstream from may serve as a means of restricting cellular levels of
the S. cerevisiae Kyokai No. 7 ATF1 gene [12]. Rox1p in order to avoid over-repression of genes
When this 150 bp stretch of the S. cerevisiae ATF1 such as OLE1, whose functional product is also
promoter region was fused to the Escherichia coli required for aerobic growth.
lacZ gene, b-galactosidase activity was rendered As heme levels fall during hypoxia, Hap1p still
subject to repression by both O2 and UFA [12]. binds to its DNA recognition site but in combina-
tion with other non-heme proteins. This large
Transcriptional co-regulation of ATF1 and complex represses ROX1 transcription which, in
OLE1 by oxygen and unsaturated fatty acid turn, has the effect of de-repressing transcription of
the subset of hypoxic genes. There are two Rox1p
Transcription of ATF1 is inhibited by a variety of binding sites located upstream of the OLE1 initia-
UFAs, including palmitoleic (16 : 1), oleic (18 : 1), tion codon (Figure 2). The rst runs from x272 to
linoleic (18 : 2) and linolenic (18 : 3) acids [12,18]. x261 bp (x272CCTATTGTTACG; nucleotides
Only palmitoleic and oleic acids are found naturally numbered such that the rst base 5k of the ATG
in yeast membranes and these are produced start codon is designated x1) and the second
exclusively by the activity of the D9-fatty acid lies between bases x130 and x119
desaturase. This enzyme is encoded by the OLE1 (x130TTTATTGTTCTA). Of the two sequences,
gene, which is present as a single copy per haploid the latter conforms more closely to the Rox1p
S. cerevisiae genome. The desaturase introduces a consensus sequence, but transcriptional analyses
double bond between carbons 9 and 10 of palmi- have not been performed to see if there is
toyl- or steary1-CoA substrates to produce palmi- preferential usage of one site over the other
toleic and oleic acids, respectively [38,39]. A or if, in fact, both sites are involved in trans-
discussion of the OLE1 gene and its role in yeast criptional control. A Rox1p binding site
fatty acid metabolism is beyond the scope of this (x120CCTATTGTTTTTT) is also found between
review; however, given the common responses of x120 and x108 bp upstream of the ATF1 initia-
both OLE1 and ATF1 gene to O2 and UFAs, some tion codon. Specic binding of Rox1p to this
examination of the mechanisms of transcriptional promoter element in vitro has been demonstrated
regulation of these two genes is warranted. Figure 2 recently [17]. Furthermore, analysis of an ATF1
presents a current view of regulatory elements, both promoterlacZ fusion construct expressed in rox1,
demonstrated and putative, that are located tup1 and ssn6 null mutants under aerobic growth
upstream of the OLE1 and ATF1 ORFs. conditions showed approximately three- to four-
fold increases in b-galactosidase activity in each
ATF1 and OLE1: two hypoxic genes case [17]. No signicant changes in b-galactosidase
In their response to oxygen, both OLE1 and ATF1 activity were seen in the same mutants grown
are examples of hypoxic genes whose transcription anaerobically in the presence of oleic acid. Taken
is induced to enable S. cerevisiae to grow under together, these results indicate that the Rox1p
conditions of severe oxygen limitation, but which Tup1pSsn6p complex is responsible for transcrip-
are otherwise repressed by means of a heme- tional repression of the ATF1 gene by oxygen but
dependent mechanism (see Figure 2; for review see plays no signicant role in UFA-mediated repres-
[46]). The biosynthesis of heme is oxygen-depen- sion.
dent. As heme accumulates, it combines with the A series of nested deletions of the ATF1
transcriptional activator Hap1p to form a complex promoter fused to the S. cerevisiae PHO5 gene
which transcriptionally activates the ROX1 gene. was also used by Fujiwara and colleagues to analyse
The ROX1 gene product, Rox1p, in association ATF1 expression as a function of acid phosphatase
with the Tup1Ssn6 general repressor complex [25], (rAPase) activity in a pho5-1 mutant lacking

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
Copyright # 2000 John Wiley & Sons, Ltd.

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 1293

endogenous rAPase activity [17]. An 18 bp DNA cerevisiae shochu yeast BAW-6, not only produced
sequence (x85 to x68 bp upstream of ATF1 start higher levels of glycerol than the parental strain but
codon) was identied which appears to be essential also showed potentially signicant (approximately
for the activation of ATF1 transcription and which two-fold) increases in isoamyl acetate, ethyl
contains a consensus binding sequence for the caproate and ethyl caprate production [34]. These
ubiquitous transcription factor Rap1p [37]. As characteristics were apparently stable through sub-
binding of Rap1p to the 18 bp element was sequent generations. The selection, by heat shock,
demonstrated in vitro, it is possible that Rap1p of yeast mutants with elevated AATase activity
acts as a positive factor in transcription of the suggests either that this trait specically contributes
ATF1 gene. to enhanced thermotolerance, or that increased
AATase production is a serendipitous consequence
ATF1: a stress response gene? of a generalized stress response.
Another interesting DNA motif found upstream of It should be noted that in the regulation of the
ATF1 but not OLE1 is the sequence x650CCCCT ROX3 gene, the upstream region of ROX3 does not
(see C4T, Figure 2). The `C4T' sequence (STRE: include the C4T motif. Instead, heme-independent
stress response element) is associated with global transcriptional induction of ROX3 in response to
stress response genes induced as a result of stress heat shock, osmotic stress and particularly hypoxia
conditions such as heat shock, osmotic stress, apparently depends upon a DNA sequence motif
oxidative stress, acid pH, glucose or nitrogen (5k-GA10GGAA-3k) that is repeated three times in
starvation, and DNA-damaging agents [27]. The the non-coding sequences upstream of ROX3 [10].
signal transduction pathways that detect and com- Even a single copy of this motif conferred increased
municate these various stress conditions ultimately expression when placed upstream of an heterolo-
converge, activating the Rox3p effector, which then gous gene [11]; thus, it may be signicant that a
activates transcription of the global stress response copy of this motif is also present upstream of the
genes. Rox3p, an essential component of the RNA OLE1gene (x803GA10GGAA) (Figure 2). The fac-
polymerase II complex, can act as either an tors responsible for this mode of heme-independent
activator or repressor of gene transcription but has oxygen sensing have not been elucidated. No
no inherent DNA-binding activity of its own [21]. similar motif is present upstream of either the
The transcription factors that mediate this interac- ATF1 or ATF2 genes (see below).
tion may be a pair of functionally redundant
proteins, Msn2p and Msn4p, which have been
shown to activate genes in response to various Unsaturated fatty acid-mediated regulation of ATF1
stresses [10]. During growth on glucose, the C4T and OLE1 expression
element mediates repression by the cyclic AMP Unsaturated fatty acids repress transcription of
(cAMP)-signalling pathway, thus many genes with ATF1 and OLE1 but it is not clear if this repression
this stress-responsive element (STRE) are induced is effected via the same signal transduction path-
at the diauxic shift, when glucose is depleted and ways. Analysis of the OLE1 promoter region
cAMP levels fall. identied a fatty acid response (FAR) element of
Other than hypoxia, the effects of stress condi- 111 bp running from x576 to x466 bp upstream of
tions on ATF1 transcriptional expression have not the ATG start codon [7]. This stretch of DNA
been measured directly. A two- to three-fold includes sites for both UFA-mediated repression
increase in AATase activity on the second day of a and activation of OLE1 transcription. The tran-
barley shochu yeast fermentation was observed as a scription factor Hap1p appears to be responsible
consequence of heat shock treatment [23]. After this for over 50% of the activation of OLE1 transcrip-
peak of activity, the AATase activity level waned to tion in wild-type cells. This was demonstrated by
that of untreated wild-type cells. Although this the signicant decreases in transcription of OLE1
response was not characterized at the mRNA level, promoterlacZ reporter constructs expressed in a
the pattern of expression could reect transcrip- hap1D::LEU2 background [7]. However, disruption
tional induction of ATF1. In another study, heat of HAP1 made no difference to either the SFA/
shock-resistant (HSR) yeast mutants, obtained after UFA ratio or absolute fatty acid levels, suggesting
two rounds of heat shock selection (45uC) of the S. that transcription factor(s) other than Hap1p must

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
1294 A. B. Mason and J.-P. Dufour

be involved in the regulation of the FAR element both OLE1 and ATF1 genes was relieved by
and maintenance of normal fatty acyl composition. deletion of the FAA1 and/or FAA4 genes [7,18]. In
While no specic transcription factor has been a recent study, Choi and Martin [6] established that
implicated in the UFA-mediated regulation of Faa1p and Faa4p are long-chain (C14C18) fatty
ATF1, the regulatory DNA sequence(s) appears to acyl-CoA synthetases that are, in fact, essential for
be included in the 150 bp (x50 to x200) of ATF1 the import of long-chain saturated and unsaturated
promoter sequence which incorporates the Rox1p fatty acids (Figure 2). Thus, the similarity in
binding site [12], and may be contained entirely transcriptional behaviour of the OLE1 and ATF1
within an 18 bp DNA sequence identied by genes was attributable to the fact that UFAs were
Fujiwara et al. [17]. Another sequence within the not getting into the mutant cells. Further research is
x50 to x200 region of the ATF1 promoter was needed to establish the extent of commonality
identied in a search of the S. cerevisiae genome between the fatty acid signal transduction pathways
database for oleate response element (ORE) that regulate OLE1 and ATF1 expression.
sequences targeted by the transcription factors Recently, Fujiwara et al. considered the inhibi-
Oaf1p and Pip2p (Oaf2p) [24]. Pip2p was rst tory effects of a variety of fatty acids on transcrip-
described by Rottensteiner et al. [36]. These oleate- tional expression of ATF1 and OLE1 [18] by
activated transcription factors are instrumental in measuring the b-galactosidase activity expressed
driving the increased transcription of peroxisomal from ATF1lacZ or OLE1lacZ reporter con-
protein genes which are induced when S. cerevisiae structs. Their results indicated an inverse relation-
is grown on a fatty acid as sole carbon source, ship between the degree of transcriptional
resulting in a proliferation in the number and size of repression of ATF1 or OLE1 and UFA melting
peroxisomes in the cell. ATF1 was one of 40 genes point. The OLE1lacZ strains were grown under
identied as having an ORE within 500 bp of the anaerobic conditions in order to avoid the repres-
ATG start codon [24]. Like ATF1, a number of sive effect of Rox1p during aerobic growth.
these genes did not encode peroxisomal compo- Increased repression due to UFAs with lower
nents. Transcriptional expression of all 40 genes melting points might indicate that membrane
was measured in wild-type, oaf1D, pip2D or oaf1D/ uidity is a key parameter in regulating desaturase
pip2D mutants grown on different carbon sources. synthesis in order to maintain or modify the ratio of
By comparison with wild-type expression levels, no SFA : UFA. Such a relationship has been previously
changes in ATF1 mRNA transcription were postulated to account for the effects of changes in
observed, although it should be noted that these membrane UFA content on the induction of
tests were made under aerobic growth conditions, transcription of heat shock genes in yeast [4].
and it is possible that the ORE upstream of ATF1 In the control of D9-desaturase activity, addi-
may assume some signicance in the regulation of tional regulation is provided by post-transcriptional
ATF1 expression when the same mutants are grown modulation of OLE1 mRNA half-life, and is
under hypoxic or anaerobic conditions. Given the mediated, at least in part, by the activity of the
repressive effect of UFAs on ATF1 expression, it Xrn1 ribonuclease [20]. Post-transcriptional regula-
seems more likely that this ORE would be utilized tion of ATF1 mRNA half-life has not been studied.
in a negative regulatory context in ATF1 transcrip- As illustrated in Figure 2, the transcriptional
tion. The OLE1 gene has no ORE consensus regulation of ATF1 and OLE1 expression involves
sequence within its promoter region. numerous controlling elements. The oxygen-
Fatty acids supplied exogenously to yeast must be mediated regulation of both genes appears to
converted into their acy1-CoA derivatives prior to depend upon the same factors, although some
incorporation into membrane and/or storage lipids. additional mechanism(s) of differential regulation
This function is fullled by the fatty acy1-CoA must exist to allow for expression of the D9-
synthetases encoded by the FAA genes [22]. For desaturase under aerobic conditions, where Atf1p
some time it had been suspected that at least part of is almost completely repressed. The effectors of
the fatty acid signalling pathways that control UFA-induced repression of each of the two genes
OLE1 and ATF1 transcription may be shared. have not been well dened and there appear to be
This hypothesis relied heavily on the observation clear differences in the nature of the interactive sites
that UFA-mediated transcriptional repression of involved in transcriptional control. It is interesting

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
Alcohol acetyltransferases and ester synthesis in yeast 1295

to note that both OLE1 and ATF1 have upstream transcriptional repression by oxygen but is rela-
sequences that may constitute STREs (Figure 2). tively unaffected by the presence of UFAs at either
The signicance of these characteristic sequences the transcriptional or post-transcriptional level.
has not been investigated. One can understand the In batch culture, Aft2p (APAT) is present at very
yeast cell's even greater need to maintain tight low protein levels and there is a rapid decline in
control over membrane fatty acid composition abundance of Atf2p after glucose exhaustion [5].
under stress conditions. On the other hand, the The mechanistic basis for this decline is unknown.
potential signicance of Atf1p function in response In contrast to the promoter regions of the ATF1
to a stress condition(s) is perhaps less obvious. The and OLE1 genes, the ATF2 promoter region
C4T motif upstream of ATF1 is present as a single includes no putative STRE and there is no evidence
copy, some distance from the initiation codon. In of an ORE element.
most cases where this STRE has been identied
upstream of a stress-induced gene, multiple copies
have been found [3]. Since ATF1 is poorly Physiological roles for ATF1 and ATF2
expressed, the magnitude of stress induction would
need to be only small in order to have a signicant Just what physiological function Atf1p might full
effect on Atf1p levels. remains open to speculation but available evidence,
derived mostly from the remarkable parallels
between the regulation of expression of ATF1and
Regulation of ATF2 expression
OLE1 genes, suggests a specialized niche for Atf1p
When compared with a vector-only control strain, in fatty acid metabolism. The evidence includes: (a)
multicopy plasmid-based expression of ATF1 or the repressive effect of UFAs (particularly those
ATF2 in S. cerevisiae produced 4.7- and 2.4-fold with a double bond in the D9 position) on Atf1p
increases, respectively, in total AATase activity expression; (b) the apparent correlation between
levels measured in cell-free extracts [32]. Signi- fatty acid melting point and magnitude of the
cantly, the AATase activity from cells over-expres- repressive effect on ATF1 transcription, suggesting
sing Atf2p was much less susceptible to inhibition that membrane uidity may be of fundamental
by linoleic acid in vitro than the AATase from cells signicance to Atf1p function; (c) the association of
overexpressing Atf1p. Yoshimoto et al. [44] found Atf1p with (membrane) lipids, and nally; (d) the
that the S. pastorianus ATF2 gene produced func- observation that 12-DL-hydroxy stearate can be
tional AATase activity when expressed in an S. esteried by puried IATase [30] and potentially
cerevisiae atf1 null mutant, resulting in a 2.5-fold utilized as an unsaturated fatty acid analogue under
increase in total AATase activity. In the same anaerobic conditions [26]. The nal point is con-
report, b-galactosidase activity in a S. pastorianus sistent with the classication of ATF1 as an hypoxic
strain expressing an ATF2lacZ construct was gene whose expression may be regulated, along with
repressed by aeration but activated by the addition that of OLE1, by the heme-dependent transcription
of 1 mM oleic acid [44]. factor Rox1p. Given the multiplicity of regulatory
The promoter region of the ATF2 gene has a elements upstream of the OLE1 and ATF1 genes
`poor-t' Rox1p consensus sequence which runs and the apparent contrast in signicance of the two
from x139 to x128 upstream of both S. cerevisiae gene products to cell metabolism, it would be an
and S. pastorianus ATF2 genes, and Yoshimoto overstatement to claim that they are co-regulated or
et al. [44] reported induction of an ATF2lacZ coordinately expressed. Nonetheless, it is still
expression construct under anaerobic conditions. possible that the activity of Atf1p is signicant
While this nding appears contradictory to an under an as-yet-undened stress condition(s).
earlier report that, in an atf1D null mutant, An indication as to a possible biological function
AATase activity was no longer subject to repression of Atf2p is provided by the ndings from the recent
by either UFAs or oxygen [15], the disparity may be study by Cauet et al. [5]. The authors suggest that
due to the relatively low levels of activity being since progesterone, its structural relative pregneno-
measured in each case. Collectively, these prelimin- lone and various other steroids can bind with high
ary ndings as to the regulation of Atf2p expression afnity to the sterol C8C7 isomerase (the ERG2
suggest that the ATF2 gene may be subject to gene product involved in ergosterol biosynthesis),

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 12871298.
1296 A. B. Mason and J.-P. Dufour

Atf2p and the efux pumps may function coopera- the deduced amino acid sequence level, Eht1p is
tively to rid the yeast cell of growth-inhibitory 3b- only 17% identical to the alcohol acetyltransferases
hydroxysteroids generated as products of defective Atf1p and Atf2p and does not include the `con-
ergosterol biosynthesis. Such a scenario might served' WRLICLP motif common to the three
provide a rational explanation for preferential AATase enzymes Atf1p, LgAtf1p and Atf2p. If
expression of Atf2p under anaerobic conditions, Eht1p has relatively broad substrate specicity, it
where yeast cells are unable to biosynthesize may also possess some ethanol esterication activ-
ergosterol [1]. The high afnity of Atf2p/APAT for ity. An atf1D atf2D double mutant should facilitate
pregnenolone (Km, pregnenolone=0.5 mM) is a characterization of Eht1p and its relative contribu-
strong indication that the enzyme has evolved to tion to the avour content of fermented products.
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 Concluding remarks
would come into contact with plant compounds
such as avonoids, which have some steroid-like Our limited understanding of the physiological role
structural properties and may be substrates for such of yeast ester synthases detracts from our ability to
a detoxication mechanism. fully control ester production during fermentation.
The regulation of the alcohol acyltransferase gene
expression in yeast appears to be extremely compli-
Additional alcohol ester synthesizing cated. More and more, yeast genetic engineering
activities? presents itself as the approach most likely to help
elucidate the mechanisms underlying ester synthesis.
Homology-based searches of the entire S. cerevisiae To date, only four alcohol acyltransferase-encod-
genome database [19] using amino acid sequences ing genes have been cloned. Further studies are
deduced from ATF1 and ATF2 have not identied needed to identify the remaining ester-synthesizing
any other closely related genes. Biochemical frac- enzymes, in particular the major enzyme involved in
tionation of a wild-type yeast cell extract separated ethyl acetate synthesis. With the completed S.
at least three distinct enzyme activities involved in cerevisiae genome sequence database and the
alcohol esterication [30]. The isoamyl alcohol powerful tools of molecular biology, major break-
acetyltransferase Atf1p accounts for around 80% throughs as to the role of ester synthesis in yeast
of isoamyl acetate production and 30% of ethyl can be expected in the near future. Ultimately, these
acetate production. The remainder of the IATase insights should lead to better management of ester
activity may be contributed by the Atf2p enzyme. If levels in alcoholic beverages.
this is so, an atf1D atf2D double mutant should be
devoid of isoamyl alcohol acetyltransferase activity.
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