BLBS102-c33 BLBS102-Simpson March 21, 2012 14:5 Trim: 276mm X 219mm Printer Name: Yet to Come
636 Part 5: Fruits, Vegetables, and Cerealsester formation on a metabolic basis, it has further been shown
that ester synthesizing activity of AAT is dependent on its po-
sitioning within the yeast cell. An interesting feature of this
distribution pattern is that specific rates of acetate ester for-
mation varied directly with the level of cytosolic AAT activity
(Masschelein 1997).
TheATF1gene, which encodes AAT, has been cloned fromS.
cerevisiaeand brewery lager yeast (S. cerevisiae uvarum) (Fujii
et al. 1994). An hydrophobicity analysis suggested that AAT
does not have a membrane-spanning region that is significantly
hydrophobic, which contradicts the membrane-bound assump-
tion. A Southern analysis of the yeast genomes in which the
ATF1gene was used as a probe revealed thatS. cerevisiaehas
oneATF1gene, while brewery lager yeast has oneATF1gene
and another homologous gene (Lg-ATF1). The AAT activities
have been compared in vivo and in vitro under different fermen-
tation conditions (Malcorps et al. 1991). This study suggested
that ester synthesis is modulated by a repression–induction of
enzyme synthesis or processing the regulation of which is pre-
sumably linked to lipid metabolism. Other enzymes are Eht1
(ethanolhexanoyl transferase) and Eeb1, which are responsible
for the formation of ethyl esters (Verstrepen et al. 2003, Saerens
et al. 2006).
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 theLEU4
gene in theS. cerevisiaegenome. A comparableS. cerevisiae
uvarummutant 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 alcohol and
acetyl coenzyme A by AAT and is hydrolyzed by esterases at
the same time inS. cerevisiae. To study the effect of balancing
both enzyme activities, yeast strains with different numbers of
copies ofATF1gene and isoamyl acetate-hydrolyzing esterase
gene (IAH1) have been constructed and used in small-scale sake
brewing (Fukuda et al. 1998). Fermentation profiles as well as
components of the resulting sake were largely alike. However,
the amount of isoamyl acetate in the sake increased with in-
creasing ratio of AAT/Iah1p esterase activity. Therefore, it was
concluded that the balance of these two enzyme activities is
important for isoamyl acetate accumulation in sake mash.
The synthesis of acetate esters byS. cerevisiaeduring fer-
mentation is ascribed to at least three acetyltransferase activi-
ties, namely, AAT, ethanol acetyltransferase, and isoamyl AAT
(Lilly et al. 2000). To investigate the effect of increased AAT
activity on the sensory quality of Chenin blanc wines and distil-
lates from Colombar base wines, theATF1gene ofS. cerevisiae
was overexpressed. Northern blot analysis indicated constitu-
tive expression ofATF1at 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, respectively, depending on the fermentation temperature,
cultivar, and yeast used. The concentrations of ethyl caprate,
ethyl caprylate, and hexyl acetate only showed minor changes,
whereas the acetic acid concentration decreased by more than
half. This study established the concept that the overexpressionof acetyltransferase genes such asATF1could profoundly affect
the flavor profiles of wines and distillates deficient in aroma.
In order to investigate and compare the roles of the knownS.
cerevisiaeAATs, Atf1p, Atf2p, and Lg-Atf1p, in volatile ester
production, the respective genes were either deleted or overex-
pressed in a laboratory strain and a commercial 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. 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 re-
spect to the esters analyzed in this study, Atf2p seemed to play
only a minor role compared to Atf1p. Theatf1atf2double
deletion strain did not form any isoamyl acetate, showing that
together, Atf1p and Atf2p are responsible for the total cellu-
lar isoamyl AAT 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 al-
leles ofATF1andATF2led to different ester production rates,
indicating that differences in the aroma profiles of yeast strains
may be partially due to mutations in theirATFgenes.
Recently, it has been discovered that the Atf1 enzyme is lo-
calized inside lipid vesicles in the cytoplasm of the yeast cell
(Verstrepen 2003). Lipid vesicles are small organelles in which
certain neutral lipids are metabolized or stored. This indicates
that fruity esters are possibly by-products of these processes.
Ester formation is highly dependent on the yeast strain used
(Nyk ̈anen and Nyk ̈anen 1977, Peddie 1990, Verstrepen et al.
2003b) and on certain fermentation parameters such as tem-
perature (Engan and Aubert 1977, Gee and Ramirez 1994,
Sablayrolles and Ball 1995), specific growth rate (Gee and
Ramirez 1994), pitching rate (Maule 1967, D’Amore et al. 1991,
Gee and Ramirez 1994), and top pressure (NN 2000, Verstrepen
et al. 2003b). Additionally, the concentrations of assimilable ni-
trogen compounds (Hammond 1993, Calderbank and Hammond
1994, Sablayrolles and Ball 1995), carbon sources (Pfisterer and
Stewart 1975, White and Portno 1979, Younis and Stewart 1998,
2000), dissolved oxygen (Anderson and Kirsop 1975a, 1975b,
Avhenainen and M ̈akinen 1989, Sablayrolles and Ball 1995),
and fatty acids (Thurston et al. 1981, 1982) can influence the
ester production rate.
Acetate ester formation in brewer’s yeast is controlled mainly
by the expression level of the AATase-encoding genes (Ver-
strepen et al. 2003b). Additionally, changes in the availability
of the two substrates for ester production, higher alcohols and
acyl-CoA, also influences ester synthesis rates. Any factor that
influences the expression of the ester synthase genes and/or
the concentrations of substrates will affect ester production ac-
cordingly. Perhaps the most convenient and selective way to
reduce ester production is applying tank overpressure, if neces-
sary in combination with (slightly) lower fermentation temper-
atures, low wort free amino nitrogen (FAN) and glucose levels