 |
INTRODUCTION |
It is well known that Saccharomyces cerevisiae converts
sugars to ethanol using the glycolytic pathway. In addition, it also produces a number of long chain and complex alcohols including isoamyl
alcohol, isobutanol, active amyl alcohol, 2-phenylethanol, and
tryptophol (1). These are all important flavor and aroma compounds in
yeast-fermented products and have interesting organoleptic properties
in their own right, many of which are both concentration- and
context-dependent. These long chain and complex alcohols
are the end products of amino acid catabolism. In this respect the pathways used by S. cerevisiae are very different from those
in other eukaryotes that have been well understood for many years (2).
We have sought to discover the genes and enzymes used by S. cerevisiae in the catabolism of leucine to isoamyl alcohol (3), valine to isobutanol (4), and isoleucine to active amyl alcohol (5). In
all cases, the general sequence of biochemical reactions is similar,
but the details for the formation of the individual alcohols are
surprisingly different. The branched-chain amino acids are first
deaminated to the corresponding
-ketoacids (
-ketoisocaproic acid
from leucine,
-ketoisovaleric acid from valine, and
-keto-
-methylvaleric acid from isoleucine). There are significant
differences in the way each
-ketoacid is subsequently
decarboxylated. In the leucine degradation pathway the major
decarboxylase is encoded by YDL080c. Consequently, we refer to this
open reading frame as KID1 for keto
isocaproate decarboxylase 1 (3). In
valine degradation any one of the three isozymes of pyruvate
decarboxylase encoded by PDC1, PDC5, and
PDC6 will decarboxylate
-ketoisovalerate (4), and in
isoleucine catabolism any one of the family of decarboxylases encoded
by PDC1, PDC5, PDC6, YDL080c, or
YDR380w is sufficient for the conversion of isoleucine to active amyl
alcohol (5).
There are three important questions that remain unanswered. First, our
earlier work had shown that the YDL080c-encoded decarboxylase was the
major route of decarboxylation of
-ketoisocaproate but that there
was at least one other unidentified decarboxylase involved in leucine
catabolism. This enzyme should be identified, and its metabolic
contribution should be quantified. Second, other than the fact that
tryptophan is the precursor of tryptophol, and phenylalanine is the
precursor of 2-phenylethanol (1), little else is known about the genes
and enzyme-catalyzed steps involved in the catabolism of these two
aromatic amino acids in S. cerevisiae except that Ydr380wp
has been suggested as a potential indolepyruvate decarboxylase (the
enzyme that would be required for the conversion of indolepyruvate to
indole acetaldehyde if tryptophan is catabolized in an analogous way to
the branched-chain amino acids) (6). Third, the final stage of long
chain and complex alcohol formation has always been assumed to be an
alcohol dehydrogenase-catalyzed step (1), but this has never been
rigorously examined. Because yeast has 20 genes that could potentially
encode this activity, seven putative aryl alcohol dehydrogenase genes
(7, 8) and 13 other alcohol dehydrogenase genes, ADH1,
ADH2, ADH3, ADH4, ADH5,
SFA1, BDH1, putative BDH2 (YAL061w),
SOR1, putative SOR2 (YDL246c), XDH1, the cinnamyl alcohol dehydrogenase CDH1 (YCR105w), and the
recently proposed ADH6 (YMR318c) (9), it is also necessary
to discover which of these gene products are required for long chain
and complex alcohol formation. This paper addresses all three questions.
 |
EXPERIMENTAL PROCEDURES |
Strains, Media, and Cultural Conditions--
The strains
used are shown in Table I. IWD72
is the wild-type strain that we have used for all studies of
amino acid catabolism. Strain YPH499 is the wild-type parent of strain
AAD7x. Standard genetic techniques were used in all cases (11, 12).
Strain 59.2.1 was produced by crossing strain Y04623
(MATa his3
1 leu2
0 met15
0 ura3
0
adh4::kanMX4 from EUROSCARF) with strain 58.3.4 (MAT
ura3-52 lys2-801 ade2-101 trp1
63 his3
200
leu2
1). Strain 58.3.4 had been produced in an earlier cross
between IWD72 and YPH499. Strain 60.3.3 was produced by mating strain
900-17-101a to strain 59.2.1 and sporulation of the resultant diploid.
Strain 61.1.1 was produced by sporulating strain Y26236
(MATa/MAT
his3
1/his3
1 leu2
0/leu2
0
lys2
0/LYS2 MET15/met15
0 ura3
0/ura3
0 adh1::kanMX4/ADH1 from EUROSCARF) and then
dissection of the ascospores. Strain ESH380w was produced by a cross
between strain Y14216 (MAT
his3
1 leu2
0 lys2
0 ura3
0
ydr380w::kanMX4 from EUROSCARF) and
YSH5.127.17C. In all cases haploid progeny carrying a single gene
disrupted with the kanMX4 cassette were identified by their ability to grow in the presence of G418 (Geneticin) at 200 µg/ml. Strain 62.3.17 was produced by crossing strain 60.3.3 and strain Y13284
(MAT
his3
1 leu2
0 lys2
0 ura3
0
adh5::kanMX4 from EUROSCARF). Strain 66.1.3 was produced by crossing strain 60.3.3 and strain 63.2.1 (MAT
his3
1
sfa1::kanMX4), the latter having been
produced earlier by crossing IWD72 and Y03866. In progeny containing
two alleles that were both disrupted with kanMX4, the presence of the adh4::kanMX4 and
adh5::kanMX4 alleles or the
adh4::kanMX4 and
sfa1::kanMX4 alleles were confirmed by
diagnostic polymerase chain reactions using specific primers that
yielded products that were unique to each disruption allele.
Starter cultures were grown in a medium comprising 1% yeast extract,
2% Bactopeptone (Difco), and 2% carbon source. For studies of
amino acid catabolism cells were grown in minimal medium containing (per liter) 1.67 g of yeast nitrogen base (Difco), 20 g of a
single L-amino acid, and either 20 ml of ethanol or 20 g of glucose for the carbon source. Experiments involving
13C labeling used [2-13C]phenylalanine (99 atom %) or
[U-13C11-U-15N2]tryptophan
(98 atom % 13C, 96-99 atom % 15N, 95%
pure). Both labeled amino acids were obtained from Cambridge Isotope
Laboratories (Cambridge, MA). Auxotrophic requirements were supplied as
required at 20 µg/ml. Liquid cultures were grown in conical flasks
filled to 40% nominal capacity in a gyrorotatary shaker. Agar (2%)
was used to solidify media. All cell cultures were at 30 °C.
NMR and GC-MS1
Analyses--
13C NMR spectra were recorded, and signals
were identified exactly as described previously (3). Chemical shifts
(
, ppm) are reported relative to external tetramethylsilane in
C2HCl3; addition of sodium
trimethylsilylpropanesulfonate gave a methyl signal at
2.6 ppm under
the conditions used here. The concentrations of the various alcohol end
products of yeast amino acid catabolism were determined in culture
filtrates using GC-MS as described before (5). Standard solutions gave
linear calibrations over the range from 0 to 1000 µg/ml.
 |
RESULTS |
The YDR380w-encoded Decarboxylase Makes a Minor Contribution in
Leucine Catabolism--
Strain 56.9.2 (pdc1 pdc5 pdc6 ydl080c
ydr380w) was precultured in yeast extract-peptone-ethanol
medium for 48 h when its A600 nm had
reached 8.0. An aliquot (1 ml) of this culture was inoculated into
ethanol minimal medium in which leucine (2%) (w/v) was the sole source
of nitrogen, and the cells were then cultured for 407 h by which
time the A600 nm was 12.2, corresponding to
stationary phase for this strain in this medium. The concentration of
isoamyl alcohol remained zero throughout the experiment. When strain
JRD815-1.2 (pdc1 pdc5 pdc6 ydl080c) was cultured in the same medium for a similar time the concentration of isoamyl alcohol fell to ~6% of that produced by its parent YSH5.127.-17C (pdc1 pdc5 pdc6) (3). Thus, the ydl080c mutation had caused a
94% reduction in isoamyl alcohol levels (3). This indicates that the
YDL080c gene product is responsible for 94% of the decarboxylation of
-ketoisocaproic acid and that YDR380wp is the minor decarboxylase being responsible for only 6% of the flux in leucine degradation.
Phenylalanine Catabolism in a Wild-type Strain--
Fig.
1 shows the 13C NMR spectrum
of a culture supernatant of wild-type strain IWD72 that had been
cultured for 24 h in a minimal medium in which glucose was the
carbon source and [2-13C]phenylalanine was the sole
nitrogen source. The largest signal was C-2 of phenylalanine (the
13C-labeled substrate). A number of resonances were
observed because of natural abundance in the phenylalanine substrate:
C-1 (at 174 ppm appearing as a doublet J = 54 Hz because of the
adjacent labeled C-2), C-1 of the phenyl ring (135.1 ppm), C-2, C-3,
C-4, C-5, and C-6 of the phenyl ring (129.3 ppm, 129.1 ppm, 128.6 ppm,
127.6 ppm and 126.4 ppm), and C-3 (at 36.4 ppm appearing as a doublet J = 34 Hz because of the adjacent labeled C-2). There were no natural abundance signals because of glucose indicating that it had all
been consumed, but the resonances at 57.5 ppm and 16.8 ppm were because
of C-1 and C-2 (respectively) of ethanol, which had been produced from
the glucose. Another resonance that was observed because of natural
abundance was C-1 of the phenyl ring of 2-phenylethanol at 139.1 ppm,
which, along with the large resonance at 62.6 ppm, because of C-1 of
2-phenylethanol, and the smaller doublet at 37.8 ppm (J = 37 Hz),
the C-2 of 2-phenyl ethanol, indicates that large quantities of this
compound were produced from the [2-13C]phenylalanine
substrate. Other important resonances identified were C-2 of
3-phenylpyruvate (204.2 ppm) and C-2 of 3-phenyllactate (73.4 ppm). A
very small resonance at 214.7 ppm and a larger one at 64.1 ppm remain
unidentified. The former is very likely a ketone. The latter is
characteristic of a CH2 group adjacent to oxygen in
an ester. Likely candidates were thought to include ethyl acetate, the
ethyl ester of phenylalanine, and 2-phenylethyl acetate, but this was
found not to be the case.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
The 13C NMR spectrum of the
wild-type strain in [2-13C] phenylalanine. Wild-type
strain IWD72 was cultured for 24 h in glucose minimal medium with
[2-13C]phenylalanine as the sole nitrogen source. The
resonances marked 1, 2, and 3 are C-1
to C-3 (respectively) of phenylalanine. The other resonances identified
are PP2, C-2 of 3-phenylpyruvate; A, C-1 of the
phenyl ring of 2-phenylethanol; B, C-1 of the phenyl ring of
phenylalanine; V, C-3, C-3, C-4, C-5, and C-6 of the phenyl
ring of phenylalanine; PL2, C-2 of 3-phenyllactate;
PE1, PE2, C-1, and C-2 (respectively) of 2-phenylethanol;
E1, E2, C-1, and C-2 (respectively) of ethanol.
|
|
The metabolites identified and the positions that were labeled with
13C indicate that in yeast the pathway of phenylalanine
catabolism is as shown in Fig. 2 in which
phenylalanine is first deaminated to 3-phenylpyruvate. The
3-phenylpyruvate is then decarboxylated to 2-phenylacetaldehyde, which
is subsequently reduced to 2-phenylethanol. The presence of
13C label in C-2 of 3-phenyllactate can be explained by the
reduction of 3-phenylpyruvate to 3-phenyllactate in an analogous
fashion to the conversion of
-ketoisocaproate to
-hydroxyisocaproate and
-ketoisovalerate to
-hydroxyisovalerate, which were observed during the catabolism of
leucine and valine (respectively) (3, 4).
Ydl080cp Has No Role in Phenylalanine Catabolism--
To confirm
the metabolic pathway of phenylalanine catabolism indicated by the
13C NMR study and to ascertain which of the
-ketoacid
decarboxylases are involved, a number of mutants were analyzed for
their ability to grow and produce 2-phenylethanol in minimal medium in
which phenylalanine was the sole nitrogen source. All of the strains were capable of using phenylalanine as the sole source of nitrogen (Fig. 3), and all had reached stationary
phase by 100 h. Strain 53.1.4 (lpd1) grew conspicuously
less well than all of the other strains. As noted previously, strains
carrying lpd1 mutations have reduced energy production and,
consequently, reduced growth on glucose minimal media compared with the
wild-type (4, 15). Because the various strains grew to different
extents, comparisons of the amount of 2-phenylethanol produced are
better on a per cell basis rather than on the basis of absolute
concentration in the growth medium. Table
II shows that a significant reduction in
2-phenylethanol occurred in the pdc1 pdc5 pdc6 ydr380w
strain (ESH380w). The pdc1 pdc5 pdc6 triple mutant
(YSH5.127.17C) and the ydr380w single mutant (55.2.1) did
not show similar reductions in the amount of 2-phenylethanol
synthesized. This indicates that the decarboxylation of
3-phenylpyruvate can proceed if any one of the four genes,
PDC1, PDC5, PDC6, or YDR380w, is
functional. A mutation in YDL080c either singly or in combination with
any of the other mutations did not result in a lowering of
2-phenylethanol levels indicating that this open reading frame has no
role in the decarboxylation of 3-phenylpyruvate. The lpd1
mutant 53.1.4 that is defective in lipoamide dehydrogenase produced
wild-type levels of 2-phenylethanol. Lipoamide dehydrogenase is an
essential component of branched-chain
-ketoacid dehydrogenase,
pyruvate dehydrogenase,
-ketoglutarate dehydrogenase, and glycine
decarboxylase (13-16). Hence, because strain 53.1.4 lacks
branched-chain
-ketoacid dehydrogenase, it can be concluded that
this activity is not involved in the formation of 2-phenylethanol from
phenylalanine.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
The growth of various strains in minimal
medium with phenylalanine as the sole nitrogen source. The carbon
source was 2% ethanol except where indicated. with solid
line, IWD72 (wild-type); with solid line, IWD72
glucose as carbon source; with solid line,
ESH380w; with solid line, 55.2.1; with solid
line, 56.9.2; with dashed line, 57.2.13; X with
solid line, YSH5.127.-17C; with dashed line,
51.1.3; * with solid line, JRD815-1.2; with dotted line, 53.1.4, glucose as
carbon source.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
The amount of 2-phenylethanol produced by various yeast strains growing
in a minimal medium with phenylalanine as sole source of nitrogen
All pdc1 pdc5 pdc6 triple mutants cannot utilize glucose;
these had ethanol (2%) as carbon source. A lpd1 mutant
cannot use ethanol; this strain had glucose (2%) as carbon source.
2-Phenylethanol was determined by GC-MS when each strain had reached
stationary phase. The values are the means of two separate experiments.
Differences between samples were <5%.
|
|
Tryptophan Catabolism in a Wild-type Strain--
Fig.
4 shows the 13C NMR spectrum
of a culture supernatant of wild-type strain IWD72 that had been
cultured for 24 h in a minimal medium in which glucose was the
carbon source and
[U-13C11-U-15N2]tryptophan
was the sole nitrogen source. The two 15N atoms showed
negligible coupling to 13C atoms, and the NMR spectrum was
effectively the same as if the tryptophan was labeled only with
13C atoms. All of the C atoms of tryptophan were observed:
C-1 (at 174.5 ppm appearing as a doublet J = 54 Hz), C-7' of the
indole ring (a triplet at 136 ppm), the C-6', C-5', C-4', C-3
',
C-2
', C-3', and C-2' atoms in the indole ring (multiplets at 126.6, 125.2, 122.0, 119.4, 118.4, 112.0, and 107.5 ppm), C-2 (a double doublet at 55.2 ppm), and C-3 (a double doublet at 26.4 ppm). As in the
phenylalanine experiment, there were no natural abundance signals
because of glucose indicating that it had all been consumed, but the
resonances at 57.5 and 16.8 ppm (C-1 and C-2, respectively, of ethanol,
produced from the glucose) were evident. Also evident were the C-1 (a
doublet J = 37 Hz at 61.8 ppm) and C-2 (a double doublet at 27.3 ppm) of tryptophol, which had been produced from the tryptophan. The
small resonance at 22.5 ppm could not be identified. The most likely
candidate was thought to be the ethyl ester of tryptophan. S. cerevisiae forms a wide variety of esters, the quantities formed
being largely determined by the concentrations of acids and alcohols
present (17). With the high concentrations of tryptophan and ethanol
observed in this experiment, the formation of tryptophan ethyl ester
would not have been a surprise. However, examination of the spectrum of
the bona fide compound showed that it was not tryptophan
ethyl ester. The resonance at 62.6 ppm is attributed to C-1 of
2-phenylethanol and is a singlet indicating that it was observed
because of natural abundance and thus was not derived directly from
tryptophan. The significance of this is further considered below.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 4.
The 13C NMR spectrum of the
wild-type strain in
[U-13C11-U-15N2]tryptophan.
The wild-type strain IWD72 was cultured for 24 h in glucose
minimal medium containing
[U-13C11-U-15N2]tryptophan
as the sole nitrogen source. The resonances marked 1,
2, and 3 are C-1 to C-3 (respectively) of
tryptophan. The other resonances identified are 7', C-7' of
the indole ring of tryptophan; IR, all of the other C atoms
in the indole ring of tryptophan; PE1, C-1 of
2-phenylethanol; T1, T2, C-1, and C-2 (respectively) of
tryptophol; E1, E2, C-1, and C-2 (respectively) of
ethanol.
|
|
From the metabolites identified it is clear that tryptophan catabolism
to tryptophol must proceed as shown in Fig.
5. The fact that no signals were observed
because of 3-indolepyruvate indicates that the pool size of this
compound must be extremely low, and/or its subsequent decarboxylation
is very rapid. This is consistent with the fact that no 3-indolelactate
was observed although the absence of this compound may be because there
is no enzyme in yeast that is capable of reducing 3-indolepyruvate to
3-indolelactate.
Ydl080cp also Has No Role in Tryptophan Catabolism--
The same
set of mutants used to study phenylalanine catabolism were also
analyzed for their ability to grow and produce tryptophol in minimal
medium in which tryptophan was the sole nitrogen source. All of the
strains were capable of using tryptophan as the sole source of nitrogen
(Fig. 6), and all except ESH380w had
reached stationary phase by 144 h. Strain ESH380w (pdc1 pdc5
pdc6 ydr380w) had a very prolonged deceleration phase and did not
reach stationary phase until 550 h. Upon inspecting the final cell
density, it can be seen that there were two groups of strains. The
majority, including the wild-type, reached
A600 nm in the range from 6 to 7, whereas four
strains (ESH380w, 55.2.1, 57.2.13, and 53.1.4) only reached half that
density. As before, to allow for the fact that the various strains grew
to different extents, comparisons of the amount of tryptophol produced
are better made on a per cell basis rather than simply on the
concentration in the growth medium. Table
III shows that most of the strains
produced roughly similar amounts of tryptophol to the wild-type, but
strains ESH380w (pdc1 pdc5 pdc6 ydr380w) and 56.9.2 (pdc1 pdc5 pdc6 ydl080c ydr380w) did not produce this
compound. Because the pdc1 pdc5 pdc6 mutant YSH5.127.17C and
the ydr380w single mutant 55.2.1 did not produce lowered
levels of tryptophol, and ydl080c mutations (singly or in
combination with other mutations) did not result in a lowering of
tryptophol levels, it is apparent that tryptophan can be catabolized to
tryptophol if any one of the four genes, PDC1,
PDC5, PDC6, or YDR380w, is functional and that,
as with phenylalanine catabolism in this yeast, YDL080c has no
role.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
The growth of various strains in minimal
medium with tryptophan as the sole nitrogen source. The carbon
source was 2% ethanol except where indicated. with solid
line, IWD72 (wild-type); with solid line, IWD72
glucose as carbon source; with solid line,
ESH380w; with solid line, 55.2.1; with solid
line, 56.9.2; with dashed line, 57.2.13; X with
dashed line, YSH5.127.-17C; with dashed line,
51.1.3; * with solid line, JRD815-1.2; with
dotted line, 53.1.4, glucose as carbon source.
|
|
View this table:
[in this window]
[in a new window]
|
Table III
The amount of tryptophol and 2-phenylethanol produced by various yeast
strains growing in a minimal medium with tryptophan as sole source
of nitrogen
All pdc1 pdc5 pdc6 triple mutants cannot utilize glucose;
these had ethanol (2%) as carbon source. A lpd1 mutant
cannot use ethanol; this strain had glucose (2%) as carbon source.
Tryptophol and 2-phenylethanol were determined by GC-MS when each
strain had reached stationary phase. The values are the means of two
separate experiments. Differences between samples were <4%.
|
|
Tryptophol levels were also reduced in the lpd1 mutant
53.1.4. This could be interpreted to mean that lipoamide dehydrogenase is required for tryptophan catabolism to tryptophol and that there is
an alternative metabolic route involving one or more of branched-chain
-ketoacid dehydrogenase, pyruvate dehydrogenase,
-ketoglutarate dehydrogenase, or glycine decarboxylase. However, we do not believe this to be the case for three reasons. First, the NMR and GC-MS analyses of the wild-type strain provide no evidence of alternative intermediates or end products. Second, the lpd1 mutant does
not accumulate any intermediates that could be considered to be derived from tryptophan. Third, the tryptophol level produced by strain 53.1.4 is not reduced as drastically as in strains ESH380w and 56.9.2. On a
per cell basis it is still 31% of the level produced by the wild-type
in the same (glucose minimal) medium. We suspect that the reduced
tryptophol formation in strain 53.1.4 is an indirect consequence of
both its impaired energy metabolism and the fact that the absence of
pyruvate dehydrogenase and
-ketogluratarate dehydrogenase activities
will result in shortages of
-ketogluratarate, which is required in
the initial deamination of tryptophan. The lpd1 mutant
uniquely did not consume all of the tryptophan by the end of the experiment.
The NMR analysis of the catabolism of 13C-labeled
tryptophan indicated the presence of unlabeled 2-phenylethanol. The
results in Table III that were obtained using GC-MS confirm the
presence of 2-phenylethanol at concentrations that were between
one-third and one-half of the concentrations of tryptophol. The levels
of 2-phenylethanol produced by cells grown on tryptophan as the sole nitrogen source were about one-tenth of the levels produced when the
cells were grown on phenylalanine as the sole nitrogen source. This
indicates wasteful catabolism of phenylalanine when yeast is growing on
tryptophan as the sole nitrogen source.
Any Ethanol Dehydrogenase or Sfa1p Is Sufficient for Fusel Alcohol
Formation--
S. cerevisiae has 20 enzymes that
potentially could be involved in the formation of long chain and
complex alcohols. This includes the putative aryl alcohol
dehydrogenases encoded by the seven-member family of AAD
genes, all of which are of unknown function (7, 8), and 13 other
alcohol dehydrogenases. Seven of these can be eliminated after a
consideration of their properties. For example, BDH1 (YAL060w) has been
shown recently (18) to be involved in the formation of 2,3-butanediol.
On the basis of similarity it is safe to assume that YAL061W can be
called BDH2 and that it has a similar activity.
SOR1 (YJR159W), SOR2 (YDL246C), and
XDH1 (YLR070C) encode sugar alcohol dehydrogenases (19, 20), which are not the type of enzyme activities sought in this investigation. CDH1 (YCR105W) and ADH6 (YMR318C)
encode NADPH-dependent enzymes (9) and thus are also
unlikely to be the type of enzyme activities involved here. This leaves
six other alcohol dehydrogenases, of which perhaps the first four
(Adh1p-Adh4p) are the best known. Adh1p is the main cytosolic enzyme
involved in the formation of ethanol during glycolysis. Adh2p, which is
also cytosolic, is the glucose-repressed enzyme that is needed for
growth on ethanol. Adh3p is mitochondrial, it is induced on glucose,
and its role is not really understood. Recent work from Pronk and
co-workers (21) suggest it is involved in a redox shuttle. Adh4p is
present at only very low levels in most laboratory strains, but is
plentiful in brewing strains (22). Adh5p was discovered by genome
sequencing, and its role is unexplained. Sfa1p is part of a
bifunctional enzyme that has both glutathione-dependent
formaldehyde dehydrogenase activity (the gene name comes from
sensitive to formaldehyde, which is
the phenotype of mutants affected in this gene); it is also described
as being capable of catalyzing the breakdown of long chain alcohols
(23). Hence, each of Adh1p-Adhp5 and Sfa1p has at least one feature
suggesting that it could be responsible for the synthesis of long chain
alcohols. For example, Adh1p, Adh2p, and Adh3p all require zinc, and
brewers have known for years that the concentration of zinc in the wort
affects the concentrations of long chain alcohols that are formed.
Alternatively, Adh3p and Adh4p appear to be good candidates as no
certain role has yet been ascribed to them. Finally, because it has
been shown that Sfa1p can degrade long chain alcohols in
vitro, perhaps its true role in vivo is the formation
of these compounds.
To determine which alcohol dehydrogenase is/are required for the
formation of fusel alcohols we used a strain mutated in all seven of
its AAD genes and constructed other strains containing all
possible combinations of single and multiple mutations affecting the
six alcohol dehydrogenases. These strains were then grown in glucose
minimal medium containing a single amino acid as the sole nitrogen
source and analyzed for the ability to form the alcohol end product
corresponding to the amino acid supplied. Table
IV shows the amount of isoamyl alcohol
produced when a selection of these strains were grown on leucine as the
sole nitrogen source. All of the strains produced isoamyl alcohol.
Equivalent results were obtained using other combinations of mutations
and different single amino acid nitrogen sources. It appears that
the AAD genes have no role in long chain or complex alcohol
formation. Using both conventional crosses and by trying to transform
strains carrying five alcohol dehydrogenase mutations, it proved
impossible to construct a haploid that had mutations in all six alcohol
dehydrogenase genes. From this we conclude that as long as yeast has
one enzyme out of Adh1p-Adh5p or Sfa1p that is functional, it is viable
and able to form long chain and complex alcohols.
View this table:
[in this window]
[in a new window]
|
Table IV
The amount of isoamyl alcohol produced by various yeast strains growing
in glucose minimal medium with leucine as sole source of nitrogen
Isoamyl alcohol was determined by GC-MS when each strain had reached
stationary phase. The values are the means of two separate experiments.
Differences between samples were <5%.
|
|
 |
DISCUSSION |
Our previous study of leucine catabolism had identified Ydl080cp
as the major decarboxylase, because mutation of this open reading frame
alone led to an almost total (94%) abolition of the formation of
isoamyl alcohol (3). The current study has identified Ydr380wp as the
minor decarboxylase accounting for only 6% of the flux. When both
YDL080c and YDR380w are mutated a haploid strain of S. cerevisiae growing in minimal medium with leucine as the sole
nitrogen source makes no isoamyl alcohol indicating that no other
pathway exists for the synthesis of this compound.
This study has shown that in S. cerevisiae the catabolism of
phenylalanine to 2-phenylethanol and of tryptophan to tryptophol are
both accomplished in a similar way corresponding to what has been
called the Ehrlich pathway (1). Based on piecemeal evidence derived
from studies in different yeast species, it is often asserted (24) that
yeasts catabolize phenylalanine via trans-cinnamate to
benzoate and protocatechuate and that the benzene ring is subsequently opened to give maleylacetate, which is then supposedly reduced to
3-ketoadipate. The same authors state that tryptophan degradation involves ring opening by a dioxygenase to yield
N-formyl-L-kynurenine. Deformylation is said to
yield L-kynurenine, which then has an alanine moiety
removed to produce anthranilate. The pathway subsequently envisages
2,3-dihydroxybenzoate, catechol, cis,cis-muconate,
and finally 3-ketoadipate. Evidence for both pathways comes mainly from
Trichosporon cutaneum (25, 26). Our NMR study showed no
evidence of any of the intermediates of either of these pathways. Furthermore, 3-ketoadipate, the supposed end product of both
phenylalanine and tryptophan catabolism, would give a clear signal in
the region 200-210 ppm because of the C=O group. There was no such
resonance in either spectrum. The only conclusion possible is that in
S. cerevisiae phenylalanine and tryptophan are catabolized
according to the Ehrlich pathway.
In both phenylalanine and tryptophan catabolism the decarboxylation
step is carried out by any one of the gene products of PDC1,
PDC5, PDC6, or YDR380w. YDL080c has no part in
the catabolism of either of these amino acids. Thus, it is apparent
that in each of the amino acid catabolic pathways that we have studied,
the crucial decarboxylation step is accomplished in subtly different ways. In leucine catabolism Ydl080cp is the major decarboxylase, and
Ydr380wp is the minor decarboxylase. In valine catabolism any one of
the pyruvate decarboxylase isozymes (Pdc1p, Pdc5p, or Pdc6p) can
perform the decarboxylation of
-ketoisovaleric acid (4), whereas in
isoleucine catabolism any one of the five-membered family of
decarboxylases (Pdc1p, Pdc5p, Pdc6p, Ydl080cp, or Ydr380wp) is
sufficient. In complete contrast, for catabolism of the aromatic amino
acids phenylalanine and tryptophan Ydl080c is irrelevant; it is not
needed for these two pathways.
This study showed that any one of six alcohol dehydrogenases (encoded
by ADH1, ADH2, ADH3, ADH4,
ADH5, or SFA1) is sufficient for the final stage
of long chain or complex alcohol formation in S. cerevisiae.
Of course, this does not mean that every 5-fold alcohol dehydrogenase
mutant will proliferate and produce fusel alcohols under all
conditions. For example, an adh1 adh3 adh4 adh5 sfa1 mutant
will not grow in high concentrations of glucose, because the remaining
alcohol dehydrogenase (Adh2p) is repressed by glucose. More than 40 years ago, well before the genome and proteome of this yeast were fully
characterized, it had been concluded that the final step in fusel
alcohol formation was catalyzed by either of the enzymes that are now
known as Adh1p or Adh2p (27). This correct but incomplete conclusion
was made without a knowledge of all of the alcohol dehydrogenases that
yeast has. The functions of the seven AAD genes remain unknown.
In S. cerevisiae catabolism of the three branched-chain
amino acids (leucine, valine, and isoleucine) and the two aromatic amino acids (phenylalanine and tryptophan) proceeds essentially as
Ehrlich proposed nearly 100 years ago (28). However, it is much more
complex than Ehrlich ever imagined, because yeast uses at least three
aminotransferases, five decarboxylases, and six alcohol dehydrogenases.
The precise combination of enzymes used at a particular time depends
upon the amino acid, the carbon source, and the stage of growth of the
culture. In our experience, the major specificity resides with the
decarboxylases. It is the regulation of these decarboxylases that is
the current focus of our interest.