Institute for Fundamental Research, Suntory
Research Center, Mishima-gun, Osaka 618-8503, Japan
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INTRODUCTION |
Lager brewing yeasts
(bottom-fermenting yeasts) were originally classified as
Saccharomyces carlsbergensis (8) but have been
recently reclassified as Saccharomyces pastorianus, which is
thought to be a natural hybrid between Saccharomyces
cerevisiae and Saccharomyces bayanus (25).
Tamai et al. (22) and Yamagishi and Ogata
(27) reported that the genome of lager brewing yeasts consists of both types of chromosomes, those originating from S. cerevisiae and those from S. bayanus, indicating that
the lager brewing yeasts have two types of allele, i.e., one that has
its origin from S. cerevisiae and the other from S. bayanus. Fujii et al. (5) and Tamai et al.
(23) reported that lager brewing yeasts contain two types
of ATF1 gene and HO gene, respectively, one
similar to that of S. cerevisiae and the other identical to that of S. bayanus. On the other hand, it has been reported
that lager brewing yeasts contain an S. cerevisiae type gene
and a Saccharomyces monacensis type gene (1, 9,
10). The amino acid sequence homology between the S. cerevisiae type gene and the non-S. cerevisiae type
gene in lager brewing yeasts is high (75 to 94%) (1, 5, 9, 10,
23). It is as yet unclear whether all the non-S.
cerevisiae type genes found in lager brewing yeasts originated
from the same species (e.g., S. bayanus or S. monacensis).
In brewing, transport of branched-chain amino acids (i.e., leucine,
valine, and isoleucine) is very important, specifically because the
metabolites of these compounds are converted to higher alcohols, which
are some of the most important flavors in alcoholic beverages. The
branched-chain amino acids are transported by at least four permeases,
which are the general amino acid permease (Gap1p) (7), the
branched-chain amino acid permeases (Bap2p and Bap3p) (2,
6), and the high-affinity tyrosine permease (Tat1p)
(4). Gap1p can transport all naturally occurring amino acids, including citrulline and D-amino acids (7,
12), and is active during growth on poor nitrogen sources, such
as proline. Gap1p activity is downregulated transcriptionally and
posttranslationally in response to preferred nitrogen sources, such as
glutamine, aspargine, and ammonia (21). Under these
conditions, most of the branched-chain amino acids are transported by
Bap2p, Bap3p, and Tat1p. The transcription of the branched-chain amino
acid permease genes (BAP2 and BAP3) is induced by
some amino acids, such as leucine and phenylalanine, in the medium
(2, 3), and this induction requires Ssy1p as a sensor for
external amino acids (4).
We found that the constitutive expression of BAP2 in a
brewing yeast strain accelerated the rates of assimilation for
branched-chain amino acids, while the disruption of BAP2 did
not affect assimilation rates for these amino acids during the brewing
process (13). This suggests that there are possibly other
functional permeases present during the brewing process. These could be
Bap3p, Tat1p, and/or other branched-chain amino acid permease
homologues, which exist in lager brewing yeasts.
In this paper, we report on the isolation and characterization of the
non-S. cerevisiae type BAP2 gene found in lager
brewing yeast.
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MATERIALS AND METHODS |
Strains and media.
Yeast strains used in this work are
listed in Table 1. The Escherichia
coli strain JM109 (recA1 
[lac-proAB]
endA1 gyrA96 thi-1 hsdR17 supE44 relA1 F'traD36 proAB
lacIqZ M15) (28) (TOYOBO
Co., Ltd.) served as the plasmid host. Growth and handling of E. coli bacteria, plasmids, and yeast strains followed standard
procedures (18, 19). Yeast cells were grown at 30°C in
yeast extract-peptone-dextrose (YPD) medium (18), YPM
medium (1% yeast extract, 2% bacto-peptone, 2% maltose), or SD
medium (2% glucose, 0.67% Yeast Nitrogen Base without amino acids;
Difco). Yeast transformation was performed using the lithium acetate
method (11). The selections for positive clones were carried out on either YPD plates supplemented with 300 µg of G418/ml, YPD plates supplemented with 10 mM formaldehyde, or YPD plates supplemented with 1 µg of aureobasidin A (Takara Shuzo Co., Ltd.)/ml. For the suppression of the growth defect of the gap1
ssy1 strain, SLD agar plates (0.17% Yeast Nitrogen Base
without amino acids and ammonium sulfate [Difco], 2% glucose, 0.1%
leucine, 2% agar) were used. For the transcription analysis in poor
nitrogen source, SPM medium (0.17% Yeast Nitrogen Base without amino
acids and ammonium sulfate [Difco], 2% maltose, 0.1% proline) was
employed.
Southern analysis.
Yeast genomic DNA was prepared according
to the standard method (18). The genomic DNA was digested
with appropriate restriction enzymes, fractionationed in a 1% agarose
gel, and transferred to a nylon membrane (Hybond-N+; Amersham
Pharmacia, Buckinghamshire, United Kingdom). Labeling probe DNA,
hybridization, membrane washing, and detection of hybridizing probe DNA
were carried out with a Gene Images random-prime labeling and detection
system (Amersham Pharmacia). The hybridization and washing temperature
was 60°C, except in the case of low-stringency conditions (50°C).
Cloning and DNA sequencing of Lg-BAP2.
Lg-BAP2 was cloned by colony hybridization from
SpeI libraries of the lager brewing yeast S. pastorianus BH-225 and S. bayanus IFO1127 constructed
in pBlueScript SK(
) (20). An about 2.7-kb SpeI fragment was cloned from each strain and sequenced
using an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems).
Construction of plasmids.
The construction of plasmids for
the disruption of the general amino acid permease gene
(GAP1) (12) and amino acid sensor gene
(SSY1) (4) was carried out using PCR
techniques. The plasmids and oligonucleotides used are listed in Table
2. Amplified PCR products were subcloned
using the TOPO TA Cloning kit (Invitrogen) according to the supplier's
instructions. The integrity of PCR fragments was verified by
sequencing. The GAP1 coding region was prepared by PCR with
genomic DNA of X2180-1A as template using the oligonucleotides 001 and
002 as primers. After digestion with SacI and
BamHI, the 1.9-kb GAP1 open reading frame (ORF)
was inserted into the SacI-BamHI gap of pUC19
(28) to yield pGAP1. The yeast glyceraldehyde- 3-phosphate
dehydrogenase (TDH3) promoter was amplified from pIGZ2
(14) by PCR using the oligonucleotides 003 and 004 as
primers. After digestion with SacI and HindIII, the TDH3 promoter was ligated with the 2.0-kb
HindIII-SalI fragment encoding
AUR1-C (aureobasidin A resistance gene) prepared from pAUR112 (Takara Shuzo Co., Ltd.), and inserted into the
SacI-SalI gap of pUC19 to yield pTDH3-AUR1C. The
plasmid pTDH3-AUR1C was digested with XbaI, repaired, and
resealed with phosphorylated BglII linkers. The 3.1-kb
KpnI-BglII TDH3-AUR1-C fragment was excised and inserted into the KpnI-BglII gap of
pGAP1 to give pGAP1. Plasmid pGAP1 was linearized at
SacI and XbaI sites before transformation of
yeast cells.
A formaldehyde resistance gene, SFA1 (24) of
S. cerevisiae, was used as a dominant selective marker in
the construction of the ssy1 strain. The SFA1
gene fragment (nucleotide positions 1 to 1401) was prepared by PCR
with genomic DNA of X2180-1A as a template using the oligonucleotides
005 and 006 as primers. The oligonucleotide primers 007 and 008 were
used for amplification of the TDH3 promoter as described
above. The resultant 1.4-kb HindIII-SalI
fragment for SFA1 and 1.1-kb
KpnI-HindIII fragment for the promoter
sequence were ligated together into the KpnI and
SalI sites of the pUC119 vector (26) to give
pUC-SFA1. The 0.7-kb SSY1 5'-flanking region (nucleotide
positions 715 to 4) was obtained by PCR with genomic DNA of
X2180-1A as a template by using the oligonucleotide primers 009 and
010. The 0.7-kb SSY1 3'-flanking region (nucleotide
positions 1817 to 2550) was obtained in the same way, using the
oligonucleotide primers 011 and 012. The 5'-flanking fragment was
digested with EcoRI and KpnI, and the 3'-flanking
fragment was digested with KpnI and BamHI. The resultant fragments were ligated together into the BamHI and
EcoRI sites of the pUC18 vector (28), in which
the original SalI site had been eliminated. The resulting
plasmid was designated pUC-SSY1. To construct pSSY1, the 2.5-kb
KpnI-SalI fragment was excised from pUC-SFA1 and
inserted into the KpnI-SalI gap of pUC-SSY1. pSSY1 was digested with EcoRI and BamHI prior
to the transformation of YK006 (gap1) to obtain a
gap1 ssy1 double disruptant (YK007).
The plasmid pYCGPY (Fig. 1A) is a
centromeric vector that allows expression of genes placed downstream of
the yeast pyruvate kinase (PYK1) promoter and contains the
kanamycin resistance (G418r) gene (15) for use
as a selective marker. The G418r gene embraced by the
TDH3 promoter and terminator sequences was prepared as a
2.5-kb BamHI fragment from pIGZ2 (14) and
cloned into the BamHI site of YCp50 (17) to
give YCpG418r. YCpG418r was partially digested
with BamHI and self-ligated with a phosphorylated oligonucleotide, 013, to eliminate one of the two BamHI
sites present in YCpG418r. The resulting plasmid
(YCpG418rSma) was then digested with AatII and
BamHI to release a 2.9-kb AatII-BamHI
fragment. To obtain the PYK1 promoter region (nucleotide positions 798 to 1), PCR was carried out with the oligonucleotides 014 and 015 as primers and genomic DNA of X2180-1A as the PCR template.
The TDH3 terminator region was amplified from plasmid pIGZ2
by PCR, with the oligonucleotides 016 and 017 as primers. The 0.8-kb
HindIII-XbaI PYK1 promoter
fragment and the 170-bp XbaI-EcoRI
TDH3 terminator fragment were coligated into the
HindIII-EcoRI gap of pUC19 to create pPTPYK1.
The 1.0-kb BglII-SpeI fragment was then excised
from pPTPYK1 and ligated with the 2.9-kb
AatII-BamHI fragment from YCpG418rSma
and the 4.6-kb SpeI-AatII fragment from YCp50 to
obtain pYCGPY.
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FIG. 1.
The structures of the plasmids. (A) PYCGPY is
a centromeric vector containing yeast centromere sequence
(CEN4), yeast autonomously replicating sequence
(ARS1), yeast glyceraldehyde-3-phosphate dehydrogenase
promoter (TDH3p), the G418 resistance gene (G418r), the
yeast pyruvate kinase promoter (PYK1p), the yeast
glyceraldehyde-3-phosphate dehydrogenase terminator (TDH3t),
and the ampicillin resistance gene (Ampr). (B and C)
BAP2 and Lg-BAP2 were inserted in the
SacI-BamHI site of PYCGPY and named
PYCGPYBP2 and PYCGPYLgBP, respectively.
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A 9.0-kb DNA fragment containing the BAP2 locus was isolated
from a genomic library derived from S. cerevisiae (strain
X2180-1A). The 4.6-kb SphI fragment containing the
BAP2 ORF was excised and subcloned into the SphI
gap of pUC18 to give pBAP2Sph. pBAP2Sph was digested with
SmaI and Eco47III and resealed to give pBAP2ES. The 5'-flanking region was trimmed with exonuclease III, and a 500-bp
SacI-PstI fragment encompassing the 60-bp
5'-flanking region and part of the BAP2 ORF was prepared and
ligated together with a 1.5-kb PstI-KpnI fragment
from pBAP2ES into the SacI-KpnI gap of pUC18 to
give pBAP2ORF. Eventually the SacI-BamHI fragment from pBAP2ORF, containing the BAP2 ORF, was inserted into
the SacI-BamHI gap of pYCGPY, thus creating
pYCGPYBP2 (Fig. 1B), in which the BAP2 gene is controlled by
the constitutive PYK1 promoter. The Lg-BAP2 ORF
was prepared as a SacI-BamHI fragment by PCR with genomic DNA of the brewing yeast BH-225 as a template using the oligonucleotides 018 and 019 as primers. The fragment of
Lg-BAP2 ORF was inserted into the SacI and
BamHI sites of pYCGPY and placed under control of the
constitutive PYK1 promoter, to yield pYCGPYLgBP (Fig. 1C).
Northern analysis.
Total RNA was prepared according to the
standard method (18). Agarose gel electrophoresis was
carried out with 45 µg of RNA per lane, and subsequent Northern
analysis was performed with 32P-labeled DNA fragments as
probes. After hybridization, the blot was washed twice with 0.1× SSC
(1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) as described
previously (18). This condition is sufficiently stringent
to distinguish the transcripts of cer-BAP2 and
Lg-BAP2 in lager brewing yeast BH-225 using a
BAP2 fragment (nucleotide positions +180 to +891) isolated
from S. cerevisiae X2180-1A and an Lg-BAP2
fragment (+1 to +1300) from BH-225 as probes. For the detection of
other genes, approximately 700-bp PCR products from genomic DNA of
X2180-1A were used as probes.
Fermentation conditions.
The fermentation was performed in a
2-liter fermentation tube. The initial wort gravity was 12% (wt/vol)
prepared with 100% malt. The fermentation was carried out at 12°C
with a pitching rate of 15 × 106 cells/ml of wort and
a dissolved oxygen content of 9 ppm.
Nucleotide sequence accession numbers.
The nucleotide
sequence data of Lg-BAP2 from lager brewing yeast BH-225 and
by-BAP2-1 from S. bayanus IFO1127 have been
deposited with DDBJ under the accession numbers AB049008 and AB049009, respectively.
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RESULTS |
Southern analysis of BAP2 in lager brewing yeast,
S. cerevisiae, and S. bayanus.
To
investigate the existence of the non-S. cerevisiae-type of
BAP2 in a lager brewing yeast, low-stringency (50°C)
Southern analysis of yeast genomic DNAs from S. cerevisiae
X2180-1A, lager brewing yeast strain BH-225, and S. bayanus
IFO1127 was carried out using a fragment of the BAP2 gene
(nucleotide positions +180 to +891) from S. cerevisiae
X2180-1A as a probe. A 5.5-kb DNA fragment from S. cerevisiae X2180-1A hybridized to this probe (Fig. 2, lane 1),
while two fragments from S. bayanus IFO1127 hybridized to
this probe (Fig. 2, lane 3). Lager brewing yeast strain BH-225 showed
two fragments, one identical in size to that of S. cerevisiae and the other identical to one of the two bands of
S. bayanus IFO1127 (Fig. 2, lane 2). This result suggests
that the lager brewing yeast BH-225 possesses two BAP2
genes, one similar to that of S. cerevisiae and the other
similar to that of S. bayanus. As shown in Fig.
2, we named the S. cerevisiae
type BAP2 gene and the non-S. cerevisiae type
BAP2 homologue in BH-225 cer-BAP2 and
Lg-BAP2, respectively, and we named the two BAP2
homologues in S. bayanus IFO1127 by-BAP2-1 and
by-BAP2-2, respectively. A DNA fragment encompassing the
5'-flanking region and the open reading frame of cer-BAP2
(nucleotide positions 886 to +1827) in BH-225 was isolated by PCR
using chromosomal DNA of BH-225 as a template. The sequence similarity
between the isolated fragment and the BAP2 sequence in the
Saccharomyces Genome Database (SGD) (http://genome-www.stanford.edu/Saccharomyces) was proven to be 99.3%
(data not shown).
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FIG. 2.
Genomic Southern hybridization with S. cerevisiae
BAP2 probe (nucleotides 180 to 891). Genomic DNA was digested with
XbaI. Lane 1, S. cerevisiae X2180-1A; lane 2, lager brewing yeast BH-225; lane 3, S. bayanus IFO1127.
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Cloning of the BAP2 homologue from lager brewing yeast
and S. bayanus.
For the cloning of the non-S.
cerevisiae type BAP2 homologue (Lg-BAP2)
from lager brewing yeast, we obtained a 1.3-kb PCR fragment of
Lg-BAP2 from lager brewing yeast BH-225 by using primers designed on the BAP2 sequence. The DNA homology between the
BAP2 sequence in SGD and Lg-BAP2 in this region
was about 80% (data not shown). Southern analysis was carried out
using this PCR fragment of Lg-BAP2 as a probe. As shown in
Fig. 3A, this fragment did not hybridize
to X2180-1A (lane 1 and lane 4). Lager brewing yeast BH-225 showed one
fragment that hybridized with this probe, which was similar in size to
one of the two fragments of S. bayanus IFO1127 (lanes 2 and
lane 5). S. bayanus IFO1127 showed two fragments that
hybridized with this probe, one identical in size to that of
Lg-BAP2 of BH-225 (by-BAP2-1) and another one
(by-BAP2-2) (lane 3 and lane 6). The hybridization intensity
of by-BAP2-1 with the probe (Lg-BAP2) was higher
than that of by-BAP2-2, suggesting that the DNA homology
between by-BAP2-1 and Lg-BAP2 is higher than that
between by-BAP2-2 and Lg-BAP2.
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FIG. 3.
Genomic Southern hybridization with the
Lg-BAP2 probe. (A) Genomic DNA was digested with
EcoRV (lanes 1 to 3) and with XbaI (lanes 4 to
6). Lane 1 and lane 4, S. cerevisiae X2180-1A; lane 2 and
lane 5, lager brewing yeast BH-225; lane 3 and lane 6, S. bayanus IFO1127. (B) Genomic DNA was digested with
SpeI. Lane 1, lager brewing yeast BH-225; lane 2, S. bayanus IFO1127.
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Further Southern blot analysis indicated that Lg-BAP2 from
BH-225 and by-BAP2-1 from IFO1127 could be cloned as
approximately 2.7-kb SpeI fragments (Fig. 3B). Thus,
SpeI-libraries of genomic DNA of BH-225 and IFO1127 were
constructed in pBlueScript SK() and the clones containing the
Lg-BAP2 or by-BAP2-1 were isolated by colony
hybridization, using the PCR fragment of Lg-BAP2 as a probe.
The SpeI fragment of approximately 2.7 kb in size which contains the whole ORF and 5'-flanking region (about 800 bp) was obtained from each strain. From the results of DNA sequencing, it was
revealed that the sequence of Lg-BAP2 from BH-225 was 100% identical to that of by-BAP2-1 from S. bayanus
IFO1127. DNA homology of the 5'-flanking region (about 800 bp) and the
open reading frame between the BAP2 sequence in SGD and
Lg-BAP2 was 60 and 80%, respectively. The amino acid
sequence comparison between Bap2p from SGD and Lg-Bap2p from BH-225 is
shown in Fig. 4. Deduced amino acid
sequence homology between Bap2p and Lg-Bap2p was 88%. This homology
seems to be comparable with previously reported homology between lager
brewing yeast-specific proteins and their S. cerevisiae
counterparts: Met2p (94%), Ilv1p (95.7%), Ilv2p (92.3%), partial
Ura3p (93%) (9) and Atf1p (75.7%) (5).
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FIG. 4.
Amino acid sequence homology between the Lg-Bap2 protein
of the lager brewing yeast BH-225 and the Bap2 protein of S. cerevisiae. The numbers indicate the amino acid positions. Amino
acid sequence identities between two proteins are shaded.
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There are some reports regarding the chromosomal structure of lager
brewing yeasts (22, 27). In these reports, it is shown that lager brewing yeasts have chromosomes that are a mix of those from
S. cerevisiae and from S. bayanus. From the
results of pulsed-field electrophoresis and subsequent Southern
analysis, the sizes of chromosomes carrying Lg-BAP2 and
by-BAP2-1 in the lager brewing yeast BH-225 and S. bayanus IFO1127, respectively, were identical (S. bayanus chromosome 12). Additionally, the sizes of chromosomes harboring cer-BAP2 and BAP2 in BH-225 and
S. cerevisiae X2180-1A, respectively, were identical
(S. cerevisiae chromosome II) (data not shown).
Function of Lg-Bap2p as a branched-chain amino acid permease.
Branched-chain amino acids are transported by at least four
transporters, which are the general amino acid permease (Gap1p) and
high- and low-affinity transporters specific for branched-chain amino
acids (Bap2p, Bap3p, and Tat1p). Didion et al. (4) showed that deletion of the gene for amino acid sensor SSY1 in a
gap1 strain abolishes branched-chain amino acid uptake to
an extent similar to that of the gap1 bap2
bap3 tat1 strain. To confirm the function
of Lg-Bap2p as a branched-chain amino acid permease, we attempted the
constitutive expression of Lg-BAP2 in YK007
(gap1 ssy1) and investigated the
complementation of the growth defect of YK007 on SLD agar plates, which
contained leucine as the sole nitrogen source. Lg-BAP2 could
complement the growth defect of YK007 on SLD agar plates as well as
BAP2 (Fig. 5). These results indicate that Lg-Bap2p, as well as Bap2p, is functional as a leucine transporter.
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FIG. 5.
Growth phenotypes of strains X2180-1A (wild type), YK006
(gap1), YK007 (gap1 ssy1),
YK008 (gap1 ssy1, pYCGPY
[vector]), YK009 (gap1 ssy1,
pYCGPYBP2 [PYK1p-BAP2]), and YK010
(gap1 ssy1, pYCGPYLgBP
[BPPYK1p-Lg-BAP2]) after 3 days of growth
on an SLD agar plate which contained leucine as the sole nitrogen
source.
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The expression profile of branched-chain amino acid permease genes
in lager brewing yeast BH-225.
Since DNA homology of the promoter
region between BAP2 in SGD and Lg-BAP2 from lager
brewing yeast BH-225 was rather low, we anticipated that
cer-BAP2 and Lg-BAP2 in BH-225 are differently regulated. As it is reported that transcription of the branched-chain amino acid permease genes (BAP2 and BAP3) is
induced by several amino acids, especially by leucine (2,
3), we investigated the transcriptional induction of
cer-BAP2 and Lg-BAP2 in BH-225 in response to
leucine addition. The transcription of cer-BAP2 (detected
with a BAP2 probe) was induced by leucine 30 min after addition, while the transcription of Lg-BAP2 was not induced
(Fig. 6).
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FIG. 6.
The transcription of BAP2 homologues in the
lager brewing yeast BH-225 after addition of leucine was analyzed by
Northern blotting. Cells were pregrown overnight on SD medium at
30°C. From these precultures, main cultures were inoculated at an
optical density at 600 nm of 0.5 in fresh SD medium and grown
subsequently to an optical density at 600 nm of 0.65 (for 4 h) at
30°C. Then, leucine was added to a final concentration of 2 mM. Total
RNA was isolated at different time points after leucine addition and
hybridized with BAP2, Lg-BAP2, and
ACT1 as probes. ACT1 was used as a loading
control.
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Furthermore, we investigated the change in the transcription levels of
cer-BAP2 and Lg-BAP2 in response to nitrogen
starvation, because brewing yeast cells undergo the nitrogen starvation
during the latter period of fermentation. The cells were transferred from amino acid-rich medium (YPM) to nitrogen-poor medium (SPM), and
the mRNA level was analyzed (Fig. 7A).
The transcription levels of both cer-BAP2 (detected with the
BAP2 probe) and Lg-BAP2 did not change after
transfer to nitrogen-poor medium, while the transcription of
DUR1 (urea amidolyase gene, inducible in response to
nitrogen starvation) was induced after transfer to nitrogen-poor
medium.
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FIG. 7.
(A) The transcription of BAP2 homologues in
the lager brewing yeast BH-225 in response to nitrogen starvation was
analyzed by Northern blotting. Total RNA was isolated after cultivation
for 4 h in YPM medium (lane 1) and after transfer to SPM medium
and was cultivated for 2 h (lane 2) and hybridized with
BAP2, Lg-BAP2, DUR1, and
ACT1 as probes. The blot that was hybridized with
Lg-BAP2 probe was overexposed for comparison of the
transcriptional level in these conditions. ACT1 was used as
a loading control, and DUR1 was used as a control nitrogen
starvation-induced gene. (B) The transcription of BAP2
homologues in the lager brewing yeast BH-225 in response to various
stresses was analyzed by Northern blotting. Total RNA was isolated
after incubation in YPM medium at 30°C (lane 1); in YPM medium
containing 8% ethanol at 30°C (lane 2); in YPM medium containing 1 mM sorbate (pH 4.5) at 30°C (lane 3); in YPM medium containing 27%
maltose at 30°C (lane 4); and in YPM medium at 37°C (lane 5) and
hybridized with BAP2, Lg-BAP2, HSP30,
and ACT1 as probes. ACT1 was used as a loading
control, and HSP30 was used as a control stress-induced
gene.
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We also investigated the transcription of cer-BAP2 and
Lg-BAP2 in response to various stresses, because brewing
yeast cells are put under such stresses as a high concentration of
alcohol, low pH, and osmotic stress due to a high concentration of
sugars during beer fermentation. Some stress-inducible genes, such as a
heat-shock-protein-encoding gene, HSP30, are induced in the latter period of fermentation (data not shown). As shown in Fig. 7B,
the transcription of Lg-BAP2 was repressed when cells were treated with ethanol and weak organic acid (1 mM sorbate), while other
treatment, such as osmotic stress (27% maltose) and heat shock
(37°C), did not affect its transcription. Conversely, the transcription of cer-BAP2 (detected with the BAP2
probe) was not affected by any of these treatments. The control gene
for stress induction, HSP30, was induced when cells were
treated with ethanol, weak organic acid, and heat shock.
Finally, we investigated the transcription of cer-BAP2 and
Lg-BAP2 during beer fermentation by Northern analysis. The
transcription level of Lg-BAP2 was rather low at the
beginning of the fermentation period, while cer-BAP2
(detected with the BAP2 probe) was highly expressed
throughout the fermentation (Fig. 8).
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FIG. 8.
The transcription of BAP2 homologues in the
lager brewing yeast BH-225 during wort fermentation was analyzed by
Northern blotting. Total RNA was isolated after 1, 2, 3, 4, 5, 6, and 7 days of the fermentation period and hybridized with BAP2,
Lg-BAP2, and ACT1 as probes. ACT1 was
used as a loading control.
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DISCUSSION |
We have been investigating the transport of branched-chain amino
acids during the brewing process, specifically because metabolic regulation of these compounds is important in the flavor control of
alcohol beverages.
In this report, we investigated the branched-chain amino acid permease
genes in a lager brewing yeast and found that it has two divergent
BAP2 genes, one similar to that of S. cerevisiae and the other similar to that of S. bayanus. We have cloned
the non-S. cerevisiae type BAP2 homologue from a
brewing yeast, BH-225 (Lg-BAP2), and another BAP2
homologue from S. bayanus IFO1127 (by-BAP2-1),
and found that they were 100% identical to each other. This result
substantiates the notion that lager brewing yeast is a hybrid between
S. cerevisiae and S. bayanus.
The results of Southern blot analysis revealed that S. bayanus IFO1127 has another BAP2 homologue
(by-BAP2-2), which is different from Lg-BAP2 and
by-BAP2-1 (Fig. 2 and 3). We have found that Saccharomyces uvarum IFO 0615 (type strain) showed a
fragment that hybridized with the Lg-BAP2 probe, which is
similar in size to by-BAP2-2 (data not shown). It suggests
that IFO1127 could be a hybrid between S. bayanus and
S. uvarum. Rainieri et al. (16) have reported
that the type strain of S. bayanus and other strains that
have been classified as S. bayanus lack homogeneity and have
hypothesized that they are natural hybrids. Our results may support
this hypothesis.
The transcription analysis revealed that Lg-BAP2 is
regulated differently from cer-BAP2 in the brewing yeast.
The former is not induced by the addition of leucine, whereas the
latter is. The BAP2 promoter harbors putative binding sites
for Gen4p and Leu3p (6), which also exist in the
Lg-BAP2 promoter, but it has been shown that these sites are
not involved in the transcriptional induction of BAP2 by
leucine (3). De Boer et al. (2) showed that a
portion of the BAP3 promoter (from 418 to 392 relative to the ATG start codon [UASaa]) is necessary and
sufficient for the induction of BAP3 transcription by amino
acids. The element found in the BAP2 promoter (nucleotide
positions 417 to 400) is very similar to the UASaa of
the BAP3 promoter, while the corresponding region of the
Lg-BAP2 promoter is rather different (data not shown). This
result supports the hypothesis that the UASaa is necessary for the induction of transcription by leucine. Since the wort prepared
with 100% malt contains about 1 to 2 mM leucine, the difference of the
transcription level of cer-BAP2 and that of Lg-BAP2 in the beginning of the fermentation period may be
due to the difference of these genes in responsiveness to leucine.
The transcription of Lg-BAP2 seemed to be induced in the
latter period of fermentation. In this period, most amino acids are exhausted and yeast cells are exposed to nitrogen starvation. However,
the transcription of Lg-BAP2 was not induced in a poor nitrogen source, suggesting that the induction of Lg-BAP2
transcription in the latter period of fermentation is not due to
nitrogen starvation. Yeast cells are also put under a lot of stress
during fermentation. The concentration of alcohol increases and wort pH
decreases in the latter period of fermentation. However, since the
transcription of Lg-BAP2 was repressed in the presence of
alcohol and weak acid (Fig. 7B), the induction of Lg-BAP2
transcription in the latter period of fermentation is not due to these stresses.
The mechanisms that differentiate the transcription profiles of
cer-BAP2 and Lg-BAP2 during fermentation are
expected to be very complicated because there are likely a lot of
factors which could affect the transcription of these two genes. One of
them could be the leucine concentration in wort. Further investigations are under way to clarify the factors involved in the distinct regulation of cer-BAP2 and Lg-BAP2. It is our aim
to determine which phenotypes of lager brewing yeast are mainly
attributed to the genes derived from non-S. cerevisiae type
chromosomes. We hope that the identification and analysis of lager
brewing yeast-specific genes that are essential for beer fermentation will help us improve the quality of beer production.
We thank Y. Kaneko, G. G. Stewart, C. Slaughter, and O. Younis for critical reading of the manuscript. The expert technical assistance of Y. Itokui is gratefully acknowledged.
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Abstract
Introduction
