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Applied and Environmental Microbiology, February 2000, p. 744-753, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effect of Increased Yeast Alcohol Acetyltransferase
Activity on Flavor Profiles of Wine and Distillates
M.
Lilly,
M. G.
Lambrechts, and
I. S.
Pretorius*
Institute for Wine Biotechnology and
Department of Viticulture and Oenology, University of Stellenbosch,
ZA-7600 Stellenbosch, South Africa
Received 16 August 1999/Accepted 12 November 1999
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ABSTRACT |
The distinctive flavor of wine, brandy, and other grape-derived
alcoholic beverages is affected by many compounds, including esters
produced during alcoholic fermentation. The characteristic fruity odors
of the fermentation bouquet are primarily due to a mixture of hexyl
acetate, ethyl caproate (apple-like aroma), iso-amyl acetate
(banana-like aroma), ethyl caprylate (apple-like aroma), and
2-phenylethyl acetate (fruity, flowery flavor with a honey note). The
objective of this study was to investigate the feasibility of improving
the aroma of wine and distillates by overexpressing one of the
endogenous yeast genes that controls acetate ester production during
fermentation. The synthesis of acetate esters by the wine yeast
Saccharomyces cerevisiae during fermentation is ascribed to
at least three acetyltransferase activities, namely, alcohol
acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT.
To investigate the effect of increased AAT activity on the sensory
quality of Chenin blanc wines and distillates from Colombar base wines,
we have overexpressed the alcohol acetyltransferase gene
(ATF1) of S. cerevisiae. The ATF1
gene, located on chromosome XV, was cloned from a widely used
commercial wine yeast strain of S. cerevisiae, VIN13, and
placed under the control of the constitutive yeast phosphoglycerate
kinase gene (PGK1) promoter and terminator. Chromoblot
analysis confirmed the integration of the modified copy of
ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast
transformants. The levels of ethyl acetate, iso-amyl 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 strain 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. These changes in the wine and distillate composition had a pronounced effect
on the solvent or chemical aroma (associated with ethyl acetate and
iso-amyl acetate) and the herbaceous and heads-associated aromas of the
final distillate and the solvent or chemical and fruity or flowery
characters of the Chenin blanc wines. This study establishes the
concept that the overexpression of acetyltransferase genes such as
ATF1 could profoundly affect the flavor profiles of wines
and distillates deficient in aroma, thereby paving the way for the
production of products maintaining a fruitier character for longer
periods after bottling.
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INTRODUCTION |
The distinctive flavor of wine,
brandy, and other grape-derived alcoholic beverages is affected by many
variables. More specifically, grape variety, viticultural practices,
and soil affect vine development and berry composition and exert major
influences on the distinctiveness of wine and brandy flavor, as
evaluated by sensory descriptive analyses (5, 30). In
addition, enological practices, including yeast and fermentation
conditions, have a prominent effect on the primary flavors of
Vitis vinifera wines. In this regard, it is well documented
that the volatile profile of wines is dominated by those components
that are formed during fermentation, since these volatile compounds are
present in the highest concentrations (22, 32, 33).
Moreover, the character of brandy is further changed as the absolute
and relative amounts of volatiles are altered by distillation.
Furthermore, the flavor of both wine and brandy immediately after
fermentation or distillation only approximates that of the finished
product (5). After the sudden and dramatic changes in
composition during fermentation and distillation, chemical constituents
generally react slowly during aging to move to their equilibria,
resulting in gradual changes in flavor (2, 5, 27). The
harmonious complexity of wine and brandy can subsequently be further
increased by volatile extraction during oak barrel aging.
Despite the extensive information published on flavor chemistry, odor
thresholds, and aroma descriptions, the flavor of complex products such
as wine and brandy cannot be predicted. With a few exceptions
(e.g., terpenes in the aromatic varieties and alkoxypyrazines in the
vegetative or herbaceous cultivars), perceived flavor is the result of
specific ratios of many compounds rather than being attributable to a
single impact compound (5). In wines and brandies, the major
products of yeast fermentation, esters and alcohols, contribute to a
generic background flavor (22, 32, 33), whereas subtle
combinations of trace components derived from the grapes usually elicit
the characteristic aroma notes of these complex beverages (5,
30).
During the primary or alcoholic fermentation of grape sugars, the wine
yeast, Saccharomyces cerevisiae, produces ethanol, carbon
dioxide, and a number of by-products, including esters, of which
alcohol acetates and C4 to C10 fatty acid ethyl
esters are found in the highest concentrations in wine and brandy
(33, 38). Although these compounds are ubiquitous to all
wines and brandies, the level of esters formed varies significantly.
Apart from being dependent on factors such as grape cultivar and
rootstocks (34), as well as grape maturity (17,
18), the concentration of esters produced during fermentation is
dependent on the yeast strain (28), fermentation temperature
(8, 36), insoluble material in the grape must
(7), vinification methods (15), skin contact
(9, 26), must pH (25), the amount of sulfur dioxide (6), amino acids present in the must
(16), and malolactic fermentation (20).
Furthermore, the ester content of distilled beverages is greatly
dependent on whether the yeast lees are present at the time of
distillation (33).
The characteristic fruity odors of wine, brandy, and other
grape-derived alcoholic beverages are primarily due to a mixture of
hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate
(banana-like aroma), ethyl caprylate (apple-like aroma) and
2-phenylethyl acetate (fruity, flowery flavor with a honey note)
(11). The synthesis of acetate esters such as iso-amyl acetate and ethyl acetate in S. cerevisiae is ascribed to at
least three acetyltransferase activities, namely alcohol
acetyltransferase (AAT), ethanol acetyltransferase, and
iso-amyl AAT (24, 29). These acetyltransferases are
sulfhydryl enzymes which react with acetyl coenzyme A (acetyl-CoA) and,
depending on the degree of affinity, with various higher alcohols to
produce esters (31, 35, 42, 43). It has also been shown that
these enzymatic activities are strongly repressed under aerobic
conditions and by the addition of unsaturated fatty acids to a culture
(10, 23).
The ATF1-encoded AAT activity is the best-studied
acetyltransferase activity in S. cerevisiae (10, 11,
12, 23, 24). It has been reported that the 61-kDa ATF1
gene product (Atf1p) is located within the yeast's cellular vacuomes
(24) and plays a major role in the production of iso-amyl
acetate and to a lesser extent ethyl acetate during beer fermentation
(12). To investigate the role of AAT in wine and brandy
composition, we have cloned, characterized, and mapped the
ATF1 gene from a widely used commercial wine yeast strain,
VIN13. The aim of this study was to overexpress the ATF1
gene during fermentation to determine its effect on the yeast
metabolism, acetate ester formation, and flavor profiles of Chenin
blanc wines and distillates from Colombar base wines. This study could
ultimately lead to the development of a variety of wine yeast strains
for the improvement of the flavor profiles of different types and
styles of wines and distillates, especially of those products deficient
in aroma and lacking a long, fruity shelf life.
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MATERIALS AND METHODS |
Microbial strains, media, and genetic methods.
All yeast and
bacterial strains used in this study and their relevant genotypes are
listed in Table 1. Escherichia
coli cells were grown in Luria-Bertani broth at 37°C
(37). S. cerevisiae cells were grown at 30°C in
synthetic media SCD and SCDSM (containing 0.67% yeast nitrogen base
without amino acids [Difco], supplemented with either the required
amino acids and 2% glucose [for SCD] or 0.5% glucose and 60 µg of
sulfometuron methyl [Dupont] per ml dissolved in
N-,N-,dimethylformamide [for SCDSM]) as well as in a rich medium, YPD (containing 1% yeast extract, 2% peptone and
2% glucose). Laboratory strains were also grown in synthetic medium
SCDD (containing 0.67% yeast nitrogen base without amino acids,
supplemented with 10% glucose and the appropriate amino acids).
Synthetic media containing all the required amino acids except leucin
were designated SCD
Leu and SCDDLeu. Solid
media contained 2% agar (Difco). All bacterial transformations and
isolation of DNA were carried out according to standard protocols (37). Laboratory yeast strains were also transformed
according to standard protocols (1). Industrial wine yeast
strains were transformed by means of electroporation. YPD (10 ml) was
inoculated with yeast cells, and the cells were incubated at 30°C
until stationary phase. A prewarmed 100-ml volume of YPD was then
inoculated with 10 ml of the preculture and incubated until the
mid-logarithmic growth phase was reached (absorbance at 600 nm
[A600] of 1). The cells were then harvested,
washed with 50 ml of sterile water, resuspended in 50 ml of a 0.025 M
1,4-dithiothreitol solution, and incubated at room temperature for 10 min. Thereafter, the cells were harvested again and washed in 50 ml
Tris-EDTA disodium salt buffer (pH 7.5). Finally, the cells were
resuspended in 10 ml of Tris-EDTA disodium salt buffer. Linear DNA (10 to 15 µg) in a maximum volume of 20 µl was added to a 400-µl cell
suspension in a microcentrifuge tube and incubated on ice for 10 min.
Thereafter, 400 µl of a 70% polyethylene glycol solution was added
and mixed in thoroughly but carefully. The mixture was transferred to
electroporation cuvettes and incubated on ice for 5 to 10 min. The
EasyjecT + 450 V Twin pulse apparatus (EquiBio) was used for
electroporation. The pulse program was as follows: voltage, 1,300 V;
capacity, 25 µF; shunt, 329 
; and pulse, 8.2 ms. The yeast cells
were then immediately plated on SCDSM and incubated at 30°C for at
least 7 days.
Plasmid construction and recombinant DNA methods.
Standard
procedures for isolation and manipulation of DNA were used throughout
this study (1). Restriction enzymes, T4 DNA ligase, and
Expand Hi-Fidelity DNA polymerase (Boehringer Mannheim) were used in
the enzymatic manipulation of DNA according to the specifications of
the supplier.
The restriction maps of the gene constructs and plasmids are shown in
Fig.
1. The following two primers were
synthesized to
amplify the coding region of
ATF1 by means of
the PCR technique:
ATF'F
(5'-GATC
CTCGAGATGAATGAAATCGATGAGAA-3')
and ATF'R
(5'-GATC
CTCGAGGTAAGGGCCTAAAAGGAGAG-3').
Both the forward
(ATF'F) and reverse (ATF'R) primers contain an
XhoI site (boldface).
Twenty of the bases in each primer
are homologous to
ATF1 (underlined).
Genomic DNA from
the commercial wine yeast strain VIN13 was used
as a template to
amplify the coding sequence of the
ATF1 gene.
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FIG. 1.
(A) Restriction map of episomal plasmid pATF1-m. (B)
Restriction map of integrating plasmid pATF1-s. In both plasmids the
ATF1 gene is inserted between the yeast phosphoglycerate
kinase gene (PGK1) promoter (PGK1P)
and terminator (PGK1T).
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A multicopy, episomal
S. cerevisiae-E. coli shuttle plasmid
containing the
ATF1 gene under the control of the regulatory
sequences
of the yeast phosphoglycerate kinase gene (
PGK1),
pATF1-m ("m"
refers to multicopy) (Fig.
1A), was constructed as
follows: the
PCR-generated 1,580-bp fragment was digested with
XhoI, subcloned
into plasmid pHVX2 (
40), and
digested with
XhoI, thereby generating
plasmid pATF1-m.
Plasmid pHVX2 contains the
PGK1 gene promoter
(
PGK1P) and terminator
(
PGK1T), with the unique restriction sites
EcoRI,
XhoI, and
BglII located in
between them. Plasmid pATF1-m
was transformed into laboratory strains
of
S. cerevisiae, ISP15
and FY10, and maintained as an
episomal plasmid in SCD
Leu and SCDD
Leu
selection
media.
Similarly, a single-copy integrating
S. cerevisiae-E. coli
shuttle plasmid containing the
PGK1P-ATF1-PGK1T gene cassette,
pATF1-s ("s" refers to single copy) (Fig.
1B), was constructed
as
follows: the
HindIII-
HindIII fragment
containing the
PGK1P and
PGK1T sequences was obtained from plasmid pHVX2
(
40) and
inserted into the unique
HindIII
site of plasmid YIpLac128 (
14),
generating plasmid pPGK1.
The 1,580-bp
XhoI-
XhoI PCR fragment,
containing
the coding region of the
ATF1 gene, was then inserted
into
the unique
XhoI site of plasmid pPGK1, thereby generating
plasmid pPATX2. A 3,200-bp
SacI-
SalI fragment
containing the dominant
selectable
SMR1-410 marker gene (a
mutant allele of an endogenous
gene of
S. cerevisiae
conferring resistance to the herbicide sulfometuron
methyl, i.e.,
Sm
r) was obtained from plasmid pWX509 (
4) and
subcloned into the
unique
SacI and
SalI sites of
plasmid pPATX2, thereby generating
plasmid pATF1-s. Plasmid pATF1-s was
linearized with
EcoRV in
the
LEU2 gene for
integration into the genomes of laboratory strains
ISP15 and FY10 as
well as wine yeast strains VIN7, VIN13, and
WE228.
Hybridization probes.
All blots were performed using a
1,580-bp XhoI-XhoI DNA fragment from plasmid
pATF1-m as a probe for the ATF1 gene. ATF1, ACT1, and lambda DNA probes were labeled with
[32P]dATP, using the Prime-It II randomly primed labeling
kit (Stratagene). The actin-encoding gene (ACT1) was used as
the internal control for Northern blotting and a 563-bp ClaI
fragment isolated from pBR322-ACT1 (13) was used
to probe for ACT1 transcripts.
Southern blot analysis.
Genomic DNA was isolated from the
control yeast strains (VIN7, VIN13, and WE228) as well as the
corresponding transformed S. cerevisiae strains
[VIN7(pATF1-s), VIN13(pATF1-s), and WE228(pATF1-s)], using the
standard mechanical method (1), and digested with EcoRV. The DNA fragments were separated by agarose gel
electrophoresis and transferred to a Hybond-N nylon membrane
(Amersham), and Southern blot hybridization was performed.
Northern blot analysis.
Total RNA was isolated from cells by
using the FastRNA Kit-RED product (BIO 101). Cells were precultured
in YPD and then inoculated into Chenin blanc grape juice. Total RNA was
isolated on days 3, 5, 7, 9, and 11 after inoculation of the juice. The
RNA (10 µg) from each culture was subjected to formamide gel
electrophoresis. The RNA was then transferred to Hybond-N nylon
membranes, and Northern blotting was performed according to standard
procedures (1).
Preparation of intact chromosomal DNA for pulsed-field gel
electrophoresis and chromoblotting.
Chromosomal DNA samples were
prepared according to the embedded-agarose procedure (3).
Intact chromosomal DNAs were separated using contour-clamped
homogeneous electric field (CHEF) electrophoresis. The apparatus used
was the CHEF-MAPPER (Bio-Rad Laboratories, Richmond, Va.). All CHEF
separations were carried out according to the method of Van der
Westhuizen and Pretorius (39). The chromosomes were
transferred to Hybond-N nylon membranes, and Southern blotting was
performed according to standard procedures (1).
Fermentation trials with laboratory yeasts.
Laboratory
strains of S. cerevisiae, ISP15, ISP15(pATF1-m),
ISP15(pATF1-s), FY10, FY10(pATF1-m), and FY10(pATF1-s) (Table 1),
were each inoculated into 100 ml of SCDDLeu medium and
incubated at 30°C. The yeast cells were then harvested and
resuspended in 10 ml of grape juice and incubated for 30 min at 30°C;
samples of the suspension were then inoculated (2 × 106 cells/ml) into 400 ml of Colombar juice and left to
ferment for 18 days at 15°C. Routine analysis, sugar and volatile
acid determination, and gas chromatographic (GC) analysis were done on
the wines (results not shown).
White wine production.
The wine yeast strains VIN7,
VIN7(pATF1-s), VIN13, and VIN13(pATF1-s) were each inoculated (2 × 106 cells/ml) into 4.5 liters of Chenin blanc grape
juice and fermented at 15°C until dry. The wines were then cold
stabilized, filtered, and bottled according to standard practices for
white wine production. All fermentations were done in triplicate.
Base wine production and small-scale distillation.
Wine
yeast strains VIN13, VIN13(pATF1-s), WE228, and WE228(pATF1-s) were
each inoculated into 10 liters of Colombar grape juice, to which no
sulfur dioxide was added, and fermented at 15°C until dry. These
fermentations were also done in triplicate. Two 5-liter round-bottom
flasks were each filled with 4.5 liters of base wine and yeast lees
derived from the original 10-liter base wine fermentation volume. Two
copper plates and 3 g of copper-sulfate were added to the base
wine and heated in heating mantles. The distillation flow rate was
maintained at 5 ml/min, and the distillate was collected until 30%
(vol/vol) alcohol was reached. The same procedure was followed with the
second distillation, except that the first 40 ml of distillate
collected at a flow rate of 2 ml/min was discarded. The flow rate was
then adjusted to 5 ml/min, and the heart was collected until 70%
(vol/vol) alcohol was reached.
GC analysis.
To a 50-ml volume of each Chenin blanc wine or
Colombar base wine were added 4 ml of a solution (2.2 mg/liter) of
4-methyl-2-pentanol (internal standard) and 30 ml of diethyl ether. The
flask was then mechanically rotated at 60 rpm for 30 min. The top ether layer was separated and the extracts were then analyzed. The internal standard was added directly to the distilled samples and injected into
the gas chromatograph.
Analyses were done on a Hewlett-Packard model 5890 series II gas
chromatograph with a Lab Alliance capillary column (length,
60 m;
inside diameter, 0.32 µm; film width, 0.5 µm). The injection
block
and detector temperatures were kept constant at 200 and
250°C,
respectively. Hydrogen was used as the carrier gas, with
an injection
volume of 3 µl (split, 20 ml/min). A Hewlett-Packard
3396A integrator
was used to quantify the peaks by using standard
solutions. The oven
temperature was programmed as follows: 35°C
(10 min) to 230°C at
3°C/min. For the distillate analysis (4 µl
was injected), the
conditions were as described above, except
that a different oven
program was used, as follows: 30°C (5 min)
to 80°C at 2°C/min,
and 80 to 230°C at 3°C/min.
Chenin blanc extractions were done after alcoholic fermentation and 6 months after bottling. Extractions from the Colombar
base wine were
made after alcoholic fermentation. Samples from
the distillate were
taken after the second
distillation.
Sensory evaluations.
The Chenin blanc wines were sensorially
evaluated for fruity or flowery and solvent or chemical intensity and
the distillates were evaluated for solvent or chemical,
heads-associated, and herbaceous intensity by a panel of five
experienced judges. The wines and distillates were evaluated on a
scale from 1 to 5, where 1 represented the absence or very low
intensity of a specific flavor and 5 represented a very high intensity
of the flavor.
Statistical analysis.
Statistical differences between the
results for wines produced by the control yeasts and those for wines
produced by the modified yeasts were determined by applying standard
analysis of variance methods to the data. The significant differences
between values were determined by a two-tailed test.
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RESULTS |
Cloning and constitutive expression of ATF1 in
laboratory and industrial yeast strains.
With the aim to produce
increased levels of AAT throughout fermentation, the ATF1
gene was cloned from a widely used commercial wine yeast strain,
VIN13, and placed under the constitutive regulatory sequences of the
phosphoglycerate kinase gene (PGK1) of S. cerevisiae to generate plasmids pATF1-m (Fig. 1A) and
pATF1-s (Fig. 1B).
Both plasmids pATF1-m and pATF1-s were transformed into two laboratory
strains of
S. cerevisiae, ISP15 and FY10, and
Leu
+ transformants were screened on SCD
Leu
agar plates. Plasmid pATF1-m was maintained as an episomal plasmid
in
selection media, and transformants ISP15(pATF1-m) and FY10(pATF1-m)
yielded a strong fruity or pineapple- or banana-like aroma on
the
selective SCD
Leu agar plates. Colombar juice was
fermented with these transformed
laboratory strains. Although these
ethanol-sensitive transformants
did not ferment the juice until
dry, the results indicated increased
levels of ethyl- and iso-amyl
acetate production (data not
shown).
To allow for the stable maintenance of the
PGK1P-ATF1-PGK1T gene construct in
nonselective grape juice medium, the commercial
wine yeast strains
VIN7, VIN13, and WE228 were transformed with
linearized (in the
LEU2 gene) plasmid pATF1-s to facilitate direct
integration
into the
LEU2 gene located on chromosome III. Integration
of
pATF1-s into the genomes of Sm
r transformants
VIN7(pATF1-s), VIN13(pATF1-s), and WE228(pATF1-s)
was
confirmed by Southern blot and chromoblot analysis (Fig.
2).
A single
ATF1
hybridization band of 4,361 bp was obtained with
the control host yeast
strains (VIN7, VIN13, and WE228), whereas
two hybridization bands of
4,361 and 9,416 bp, corresponding to
the wild-type
ATF1 gene
and the integrated
PGK1P-ATF1-PGK1T
gene
cassette, respectively, were obtained with the recombinant wine
yeast strains [VIN7(pATF1-s), VIN13(pATF1-s), and
WE228(pATF1-s)]
(Fig.
2A). To avoid any chromosomal positional
effect on the regulation
of the
PGK1P-ATF1-PGK1T gene cassette, a
chromoblot was performed
to determine whether integration occurred at
the targeted
LEU2-site
on chromosome III. Figure
2B clearly
shows that all untransformed
strains (VIN7, VIN13, and WE228) contain a
single hybridization
band corresponding to chromosome XV, on which the
wild-type
ATF1 gene is located. Furthermore, only the
transformed wine yeast
strains [VIN7(pATF1-s), VIN13(pATF1-s),
and WE228(pATF1-s)] contain
an additional hybridization band
corresponding to chromosome III,
thereby confirming an integration
event (presumably by homologous
recombination) at the
LEU2
target site (Fig.
2B).
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FIG. 2.
(A) Genomic DNA analysis of ATF1. Lanes were
loaded with EcoRI-HindIII-digested lambda
DNA (lane 1) or EcoRV-digested genomic DNA of the yeast
strains WE228, WE228(pATF1-s), VIN13, VIN13(pATF1-s), VIN7, and
VIN7(pATF1-s) (lanes 2 to 7, respectively). (B) Chromoblot analysis
of ATF1. The chromosomes of wine yeast strains WE228
(lane 1), WE228(pATF1-s) (lane 2), VIN13 (lane 3),
VIN13(pATF1-s) (lane 4), VIN7 (lane 5), and VIN7(pATF1-s)
(lane 6) were transferred to nylon membranes and probed.
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Northern blotting was performed with the control and transformed
industrial yeast strains to study the expression levels of
the
ATF1 gene during fermentation. Intense
ATF1
mRNA hybridization
bands on the Northern blots were observed for
transformants VIN7(pATF1-s),
VIN13(pATF1-s), and
WE228(pATF1-s) at all the sampling time points,
whereas intense
ATF1 transcript bands in the wild types were only
detectable
from day 7 of the fermentation (Fig.
3).
These results
indicate that expression of the integrated
ATF1 gene under the
control of the
PGK1 promoter
and terminator sequences in the transformed
strains was high throughout
the fermentation, whereas in the untransformed
strains it was only high
during the later stages of fermentation.
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FIG. 3.
Northern blot analysis of ATF1 transcripts
prepared from yeast cells during fermentation. RNA from commercial wine
yeast strains VIN7, VIN13, and WE228 together with their corresponding
transformants carrying the constitutively expressed
PGK1P-ATF1-PGK1T gene cassette,
VIN7(pATF1-s), VIN13(pATF1-s), and WE228(pATF1-s), was
isolated on days 3, 5, 7, 9, and 11, subjected to formamide gel
electrophoresis, and probed for ATF1 and ACT1.
The sizes of the ATF1 and ACT1 transcripts were
determined relative to a Bio-Rad Laboratories RNA molecular size
marker.
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GC analyses of wines and distillates.
Concentrations of
certain esters, higher alcohols, and acids were determined for the
wines and distillates (Table 2). The GC
analysis results confirmed high levels of AAT activity. Specifically, 3- to 10-fold and 3.5- to 12-fold increases in the production of ethyl
acetate and iso-amyl acetate, respectively, were observed for
both the Chenin blanc and Colombar base wines fermented with VIN7(pATF1-s), VIN13(pATF1-s), and WE228(pATF1-s) compared
to the wines fermented with VIN7, VIN13, and WE228. Hexyl acetate and 2-phenylethyl acetate concentrations increased from 1.4- to 2-fold
and from 2.4- to 10.8-fold, respectively, in the wines fermented with
VIN7(pATF1-s), VIN13(pATF1-s), and WE228(pATF1-s). The
concentrations of ethyl caproate were 1.4-fold greater in both of the
Chenin blanc wines fermented with VIN7(pATF1-s) and VIN13(pATF1-s) than in the wines fermented with VIN7 and
VIN13. The concentrations of ethyl caprate and ethyl caprylate were not significantly different for the Chenin blanc wines fermented with VIN7 and VIN7(pATF1-s), or for those fermented with VIN13 and VIN13(pATF1-s). The ethyl caprylate concentration was similar for
the Colombar wines fermented with VIN13, VIN13(pATF1-s), WE228, and
WE228(pATF1-s), but the ethyl caprate concentration showed a slight
decrease in the Colombar wines fermented with VIN13(pATF1-s) compared to that in the wine fermented with VIN13. The total ester concentrations showed 9.8- and 6.1-fold increases in the Chenin blanc wines fermented with VIN7(pATF1-s) and
VIN13(pATF1-s), respectively, compared to the wines fermented
with VIN7 and VIN13. The total ester concentrations of the Colombar
wines fermented with VIN13(pATF1-s) and WE228(pATF1-s) showed
3.9- and 3.2-fold increases, respectively.
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TABLE 2.
Yeast strain effect on the concentrations of major
volatiles in Chenin blanc wines as well as Colombar base wines and the
respective 70% distillates
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In all of the experimental wines the methanol concentration decreased
as a result of the use of the modified strains. This
is considered to
be a positive tendency, since excessive levels
of methanol could be
considered toxic. The propanol, hexanol,
iso-amyl alcohol, and
2-phenylethyl alcohol concentrations decreased
in the wines fermented
with the modified strains compared to those
fermented with VIN7, VIN13,
and WE228. These decreases in the
alcohol concentrations corresponded
with the increase in the ester
concentrations. AAT uses the different
alcohols and acetyl-CoA
as substrates for the production of the
corresponding esters.
The iso-butanol levels differed between the
different yeast strains.
The concentrations of iso-butanol in the
Chenin blanc and Colombar
wines fermented with VIN13(pATF1-s) and
WE228(pATF1-s) were decreased
compared to those in the control
wines, but they were increased
in the Chenin blanc wines fermented with
VIN7 and VIN7(pATF1-s).
The acetic acid concentrations in the wines produced by the transformed
strains were drastically decreased compared to those
in wines produced
by the control strains, which is a positive
result, since the volatile
acidity of the wine was therefore decreased.
Acid concentrations,
excluding acetic acid, appear to have remained
constant.
To determine the effect of bottle-aging on the ester concentrations and
aroma profiles, the Chenin blanc wines were bottle
aged for 6 months at
8°C and then subjected to GC analysis (Table
2). Ethyl acetate,
iso-amyl acetate, and hexyl acetate levels
decreased drastically in all
the wines during this storage period,
but their concentrations still
remained 4- to 5.7-fold, 5.4- to
7.9-fold, and 1.5- to 2-fold higher in
the wines fermented with
VIN7(pATF1-s) and VIN13(pATF1-s) than
in the control wines. The
concentration of 2-phenylethyl acetate
also decreased during this
period, but the concentration in the
Chenin blanc and Colombar
wines fermented with VIN7(pATF1-s)
(9-fold) and VIN13(pATF1-s)
(4.3-fold) and WE228(pATF1-s)
(2.4-fold) and VIN13(pATF1-s) (2.8-fold),
respectively, remained
higher. The concentrations of ethyl caprate,
ethyl caproate, and ethyl
caprylate showed an overall increase
during this storage period, but
the difference in the concentrations
between the wines fermented with
the control yeast strains and
those fermented with the modified yeast
strains were not significant.
The acetic acid concentration increased
drastically due to ester
hydrolysis in both the VIN7(pATF1-s)- and
VIN7-fermented wines,
but still was lower in the wine fermented with
VIN7(pATF1-s) than
in the VIN7-fermented Chenin blanc.
Similar results were obtained
for the Chenin blanc wines fermented with
VIN13 and VIN13(pATF1-s)
(Table
2).
The analysis of the low wine (first distillate of base wine) indicated
that all of the measured compounds were concentrated
during the first
distillation (data not shown). After the second
distillation the
content of the distillate changed, since only
the aroma compounds
present in the heart were concentrated while
the rest of the
components, such as some acids, were discarded
in the heads and
tails. The spirit produced by the recombinant
strains
WE228(pATF1-s) and VIN13(pATF1-s) contained higher
concentrations
of total esters and lower concentrations of total higher
alcohols
than the respective control strains (Table
2). Concentrations
of ethyl acetate, iso-amyl acetate, hexyl acetate, and 2-phenylethyl
acetate were elevated in the distillates fermented with recombinant
yeast strains VIN13(pATF1-s) and WE228(pATF1-s). The
concentrations
of propanol, iso-butanol, iso-amyl alcohol, hexanol, and
acetic
acid were lower in the same
distillates.
The GC results were statistically evaluated by a two-tailed test (Table
3). The results indicated that the values
obtained
for all the wines and distillates for ethyl acetate, iso-amyl
acetate, iso-amyl alcohol and acetic acid differed significantly
(
P 
0.05). The values for 2-phenylethyl acetate,
2-phenylethyl
alcohol, propanol, hexyl acetate, hexanol, iso-butanol,
and
n-butanol
differed significantly for the 70% spirit
(
P 0.05), but only
in some cases for the Chenin
blanc wines.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Statistical differences between wines and distillates
produced by control and modified yeast strains with respect to
certain fermentation bouquet volatilesa
|
|
Sensory analyses.
The solvent or chemical aroma, associated
with ethyl- and iso-amyl acetate, was detected in all the distillates,
as were herbaceous and heads-associated aromas (Fig.
4). The aroma which differed the most
between the wines fermented with the industrial strains and those
fermented with the genetically modified strains was the solvent or
chemical aroma. The fruity or flowery character was stronger in
the Chenin blanc wines fermented with VIN7(pATF1-s) and
VIN13(pATF1-s) than in the control wines (Fig.
5). However, these wines were also
dominated by the solvent/chemical aroma. Nevertheless, the wines
fermented with the modified wine yeast strains had a much more intense
and complex aroma than the control wines.
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 4.
The aroma property intensities of distillate, distilled
from Colombar base wines fermented with VIN13 (control) and
VIN13(pATF1-s) (modified yeast strain).
|
|
View larger version (76K):
[in this window]
[in a new window]
|
FIG. 5.
The aroma property intensities of Chenin blanc
wine fermented with yeast strains VIN7 and VIN13 (controls) and
VIN7(pATF1-s) and VIN13(pATF1-s) (modified yeast
strains).
|
|
The sensory evaluation data were also statistically evaluated (Table
4). The results indicated that the
solvent or chemical
property differed significantly between the control
and modified
yeast strains for both the distillate and the Chenin blanc
wines.
The fruity or flowery property differed significantly between
the control and modified yeast strains for the Chenin blanc wines,
and
the herbaceous and heads-associated properties differed significantly
for the distillate. Only those properties with a significant difference
are shown in Fig.
4 and
5.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Statistical differences between wines and distillates,
produced by control and modified yeast strains, with respect to
sensory evaluations
|
|
| |
DISCUSSION |
The absolute and relative amounts of specific fermentation-derived
alcohols and esters, such as alcohol acetates, contribute significantly
to the fruity odors and fermentation bouquet of wines and brandies
(11, 22, 32, 33). AAT, ethanol acetyltransferase, and
iso-amyl AAT represent the key AATs in yeast strains that play a
pivotal role in the production of alcohol acetates during fermentation
(24, 29). However, it was previously shown that the
AAT-encoding gene (ATF1) of brewer's yeast is not
transcribed in the presence of oxygen or unsaturated fatty acids and
that it is only expressed during the later stages of the fermentation (10). At the beginning of fermentation, ester synthesis is
very slow due to the high metabolic demand for acetyl-CoA for yeast growth (43). During this phase of the fermentation curve,
oxygen and acetyl-CoA are rapidly consumed to support the production of
unsaturated fatty acids and sterols. Immediately following this, an
equilibrium is established between acetyl-CoA consumption for fatty
acid and sterol synthesis and that for ester production. This
represents the first induction for ester synthesis and occurs after
about 8 h of beer wort fermentation. When fatty acid and sterol
synthesis finally stops, there is a peak in cellular acetyl-CoA levels,
and at this point the second induction of ester synthesis occurs. This
happens near the midpoint of the fermentation curve and is relatively
short-lived. However, this contributes significantly to the overall
ester level in beer (43).
In an attempt to increase the yeast's AAT production
throughout the fermentation of grape juice, and thereby enhance the
synthesis of important acetate esters and presumably the fruitiness of
Chenin blanc wine and Colombar base wine, the ATF1 gene was
constitutively expressed (PGK1 promoter and terminator) in
three commercial wine yeast strains VIN7, VIN13, and WE228. Northern
blot analyses showed that the
PGK1P-ATF1-PGK1T gene cassette was
transcribed at high levels throughout the fermentation, resulting in
higher concentrations of several esters in the wines and distillates.
The drastic increases in the levels of ethyl acetate and iso-amyl
acetate had a pronounced effect on the aroma of the fermented and
distilled products. Some of the other esters whose concentrations were
also increased as a result of the overexpression of ATF1
include hexyl acetate, ethyl caproate, and 2-phenylethyl acetate,
presenting a flowery and fruity aroma. By comparison, the
overexpression of ATF1 in brewing yeast strains resulted in
beer with increased levels of ethyl acetate and iso-amyl acetate and
decreased concentrations of the corresponding alcohols (21).
As can be expected, the sensorial evaluation of the wines and
distillates in this study indicated that excessively high
concentrations of ethyl acetate, however, did not improve
the fermentation bouquet and aroma of the young wines. A more
controlled increase of the level of this ester should have a more
positive effect on aroma. However, an important fact is that the ethyl
acetate/iso-amyl acetate ratio decreased from 11.1 just after the
alcoholic fermentation to 4.9 after 6 months of bottle aging.
Therefore, the hydrolysis of ethyl acetate occurs at a faster rate than
that of iso-amyl acetate. This is also true for 2-phenylethyl acetate
(twofold reduction). Therefore, further bottle aging might reduce the
negative effect of the initial high levels of ethyl acetate. These
products may also be used for blending purposes to improve the aroma of
a neutral wine or distillate. Distillates are normally matured in
wooden barrels for a minimum of 3 years, during which the
concentrations of the compounds are changed. New aroma compounds can be
extracted from the wood, and the concentrations of others can increase
or decrease. Therefore, when the spirit is matured, ethyl acetate and
iso-amyl acetate concentrations would likely decrease due to hydrolysis
and evaporation. Apart from cultivar-specific wines, white wine also
has a generic fruity character, which often disappears during bottle
aging. Therefore, higher initial levels of esters (including ethyl
acetate) could lead to a more complex brandy and to white wines with a
more fruity character. This is substantiated by the statistical
differences found between the wines and distillates fermented by
industrial and genetically modified yeast strains, with respect to both
composition (fermentation-produced volatiles) and quality (flavor properties).
In conclusion, this study has clearly demonstrated that the
manipulation of the expression of a single yeast gene such as ATF1 could alter the ester production significantly during
wine fermentation, thereby adjusting the aroma profiles of wine and distillates considerably. This paper indicates the enormous effect that
gene technology is likely to have on our understanding of flavor
chemistry, paving the way for the production of higher quality wines
and brandies with fruitier and even novel aromas.
| |
ACKNOWLEDGMENTS |
We thank Distillers Corporation for performing the GC analyses.
We also express our sincere gratitude to the ARC-Nietvoorbij Centre for
Vine and Wine for the use of their distillation equipment. We are also
grateful to Johan Marais (Nietvoorbij) and Enzo D'Aguanno for the
critical reading of the manuscript and to William Lilly for the drawing
of the graphics and tables.
We express our sincere gratitude to the South African wine industry
(Winetech) and the National Research Foundation (NRF) for financial support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Wine Biotechnology, University of Stellenbosch, Stellenbosch
7600, South Africa. Phone: (27) 21-8084730. Fax: (27) 21-8083771. E-mail: isp{at}maties.sun.ac.za.
| |
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Abstract
Introduction
