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Journal of Bioscience and Bioengineering

VOL. 125 No. 1, 52e58, 2018


www.elsevier.com/locate/jbiosc

Inhibition of Saccharomyces cerevisiae growth by simultaneous uptake


of glucose and maltose

Haruyo Hatanaka,1, 2, * Hitoshi Mitsunaga,2, x and Eiichiro Fukusaki2

Research Institute, Suntoy Global Innovation Center Limited, 8-1-1 Seikadai, Seika-cho, Soraku-gun, 619-0284 Kyoto, Japan1 and Department of Biotechnology, Graduate School of
Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871 Osaka, Japan2

Received 6 April 2017; accepted 21 July 2017


Available online 15 September 2017
Saccharomyces cerevisiae expresses a-glucoside transporters, such as MalX1p (X [ 1(Agt1p), 2, 3, 4, and 6), which are
proton symporters. These transporters are regulated at transcriptional and posttranslational levels in the presence of
glucose. Malt wort contains glucose, maltose, and maltotriose, and the assimilation of maltose is delayed as a function of
glucose concentration. With the objective of increasing beer fermentation rates, we characterized a-glucoside trans-
porters and bred laboratory yeasts that expressed various a-glucoside transporters for the simultaneous uptake of
different sugars. Mal21p was found to be the most resistant transporter to glucose-induced degradation, and strain
(HD17) expressing MAL21 grew on a medium containing glucose or maltose, but not on a medium containing both
sugars (YPDM). This unexpected growth defect was observed on a medium containing glucose and >0.1% maltose but was
not exhibited by a strain that constitutively expressed maltase. The defect depended on intracellular maltose concen-
tration. Although maltose accumulation caused a surge in turgor pressure, addition of sorbitol to YPDM did not increase
growth. When strain HD17 was cultivated in a medium containing only maltose, protein synthesis was inhibited at early
times but subsequently resumed with reduction in accumulated maltose, but not if the medium was exchanged for
YPDM. We conclude that protein synthesis was terminated under the accumulation of maltose, regardless of extracel-
lular osmolarity, and HD17 could not resume growth, because the intracellular concentration of maltose did not
decrease due to insufficient synthesis of maltase. Yeast should incorporate maltose after expressing adequate maltase in
beer brewing.
Ó 2017, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Saccharomyces cerevisiae; Growth inhibition; a-Glucoside transporters; Maltose; Glucose]

The first step of sugar assimilation by Saccharomyces cerevisiae is Mal31p, Mal41p, and Mal61p, which are also called maltose
the transport of sugar into cells. S. cerevisiae produces sugar trans- transporters, can incorporate maltose and turanose. In contrast, the
porters that incorporate sugars from the medium, and HXT1eHXT17 substrate specificity of Agt1p is broad. For example, Agt1p can
encode transporters for hexose such as glucose, fructose, and transport maltose, sucrose, turanose, trehalose, and maltotriose as
mannose. Each hexose transporter possesses a different kinetic well as hexoses such as fructose and glucose (6,7).
property (1). S. cerevisiae regulates the expression of hexose trans- Besides MalX1p and Agt1p, the genome of a lager-brewing
porters to respond to the environment with priority for glucose yeast, which is a hybrid between S. cerevisiae and Saccharomyces
uptake (2,3). Hexose transporters transfer glucose through facili- eubaynus (8), encodes the Agt1p ortholog SeAgt1p as well as Mtt1p
tated diffusion and therefore do not require energy (1). that is 90% identical to Mal61p (9e11). These a-glucoside trans-
To assimilate a-glucosides such as maltose and maltotriose, porters are encoded by the MAL locus, which comprises transporter
S. cerevisiae produces the a-glucoside transporters MalX1p (MALX1), maltase (MALX2), and activator (MALX3) genes. The
(X ¼ 1(Agt1p), 2, 3, 4, and 6) that are encoded on chromosomes VII, transcription of genes encoding transporters and maltase are
III, II, XI, and VIII, respectively (4). The allele type and copy number driven by the bidirectional promoter between them, and their
of a-glucoside transporter genes vary depending on the strain. transcription is repressed by glucose and induced by maltose
Generally, the genomes of yeasts used to brew beer encode through the binding of the transcriptional activator MalX3p at the
numerous numbers of a-glucoside transporter genes (5). Mal21p, upstream-activating promoter sequence (12,13).
The results of a binding experiment using strains carrying wild-
type Mal63p, the constitutive mutant Mal63p, and the non-
* Corresponding author at: Research Institute, Suntoy Global Innovation Center inducible mutant Mal63p revealed that native, inducible Mal63p
Limited, 8-1-1 Seikadai, Seika-cho, Soraku-gun, 619-0284 Kyoto, Japan. Tel.: þ81 50 binds strongly to Ssa1p, Hsp82p, and Sti1p in the presence of
3182 0524; fax: þ81 774 98 6262.
glucose but is released in response to the inducer, maltose, after the
E-mail address: Haruyo_Hatanaka@suntory.co.jp (H. Hatanaka).
The study represents a portion of the dissertation submitted by Haruyo Hata- depletion of glucose (14).
naka to Osaka University in partial fulfillment of the requirement for her PhD. When glucose is added to the medium, Mal61p and Mal31p are
x
Present address: Research and Development Division, Kikkoman Corporation, immediately phosphorylated and are subsequently ubiquitinated
399 Noda, Noda-Shi, Chiba, Japan.

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2017.07.013
VOL. 125, 2018 GROWTH INHIBITION OF S. CEREVISIAE 53

by Npi1p/Rsp5p. The ubiquitinated transporters are endocytosed, concentration of sugar was 0.1 mM for HD17 and HD49, and 0.1, 1.0, or 2.0 mM for
transferred to vacuoles, and rapidly degraded (15e17). These reg- strains with designations starting with “HH.”

ulatory systems enable S. cerevisiae to primarily utilize glucose and Transporter degradation studies Degradation rates of transporters tagged
with HA in the presence of glucose were measured by Western blot analysis as
then other sugars. described in reference (19) with mouse anti-HA antibody (MMS-101P, Berkeley
Malt wort contains maltose, maltotriose, and glucose compa- Antibody Company, Richmond, CA, USA). The assays were repeated three times.
rable to approximately 20e25% of maltose, and beer-brewing yeast Measurement of maltase activity Maltase activity of cell extract was
start to assimilate maltose and maltotriose after depletion of measured using p-nitrophenyl-a-D-glucopyranoside as described in reference (23),
glucose. Fermentation with malt wort of a high specific gravity and the relative maltase activity per mg protein was calculated.
improves efficiency of beer production, although occasional prob- Measurement of b-galactosidase activity The Yeast b-Galactosidase Assay
Kit (Thermo Fisher Scientific) was used to determine b-galactosidase activity ac-
lems are caused by the delay in assimilating maltose and malto-
cording to the manufacturer’s manual. Briefly, cells (1.0 OD unit) were collected and
triose that is associated with the prolonged time required to extracted in a mildly alkaline solution. Hydrolysis of o-nitrophenyl-b-D-galactopyr-
consume glucose (18). Therefore, we characterized several a- anoside to o-nitrophenol and galactose was measured at 420 nm.
glucoside transporters and attempted to breed yeast that can Quantitative PCR RNA samples were prepared using the RNeasy Mini Kit
assimilate glucose and maltose simultaneously to achieve rapid (Qiagen, Hilden, Germany). Complementary DNA was synthesized using PrimeScript
fermentation. We report here that a strain that simultaneously RT Master Mix (Takara Bio, Shiga, Japan). Quantitative PCR was performed using the
SYBR premix Ex Taq kit (Takara Bio) with primer sets for MAL32 (25 þ 26), lacZ (27 þ
incorporated glucose and maltose from the medium did not grow
28), and PDA1 (29 þ 30) with a StepOnePlus System (Thermo Fisher Scientific).
unexpectedly, and we therefore investigated the reasons for this Expression levels of MAL32 and lacZ were normalized to those of PDA1.
growth defect. This would give a clue as to whether to select or Intracellular sugar analysis Cells (5 OD units ¼ OD  mL) were collected
breed an adequate brewing strain for rapid fermentation with malt from the culture, washed with ice-cold water and lyophilized, and then 1 mL of
wort of high specific gravity. chloroform:methanol:water (2:5:2), with 8.9 mg/mL ribitol as the internal
standard for GC/MS analysis, was added to the cells, and the mixture was
vortexed. The sample was centrifuged at 16,000 g and 4 C for 3 min, and 900 mL
of the supernatant was collected and mixed with 400 mL of water. The sample was
MATERIALS AND METHODS centrifuged, and 500 mL of the supernatant was centrifugally dried and then
freeze-dried. Samples were derivatized as follows: (i) Oximation, 50 mL of
methoxyamine hydrochloride in pyridine (20 mg/mL) was added before
Yeast strains, media, and culture conditions The S. cerevisiae strains used
incubation at 30 C for 90 min. (ii) Trimethyl silylation, 50 mL of N-methyl-N-
in this study are listed in Table S1. Strain JH1032 is the ura3-derivative of strain
trimethylsilyltrifluoroacetamide was added before incubation at 37 C for 30 min.
X2180-1A, created by integration of a constitutive expression unit of MAL32 (19).
GC/MS analysis was performed using a GC-2010 Plus gas chromatograph
The strains indicated by “HH” are derivatives of JH1032, in which an expression
(Shimadzu, Kyoto, Japan) with an AOC-20 in-series injector/autosampler (Shimadzu)
unit of a transporter gene is integrated into the ura3 site. D152 was generated by
and a GCMS-QP2010 Ultra mass spectrometer (Shimadzu). We used a fused silica
disruption of MAL61 with TRP1 in strain ATCC96955 (19). D152U and D152MS are
capillary column (30 m long  0.25 mm inner diameter) coated with 0.25 mm CP-SIL
strains which were inserted URA3 or URA3::TDH3p::MAL62 (encoding maltase) to
8-CB low bleed/MS (Agilent Technologies, Tokyo, Japan). The front inlet temperature
ura3 in D152, respectively. HD93 and HD94 are strains with inserted b-
was 230 C. The flow of helium through the column was 1.12 mL/min. The column
galactosidase (lacZ) expressed under the control of the TPI1 promoter in D152U or
temperature was held at 80 C for 2 min and then ramped from 80 C to 330 C at
D152MS, respectively, that harbors MAL21 on the plasmid pYCGPY (20). The
15 C/min and held isothermally for 6 min. The transfer line and ion-source tem-
strains with designations starting with “HD” are transformants of D152U or
peratures were 250 C and 200 C, respectively. Scans (20/s) were recorded from 85
D152MS harboring a transporter gene (MAL61, MAL21, AGT1-2HA, SeAGT1, or MTT1)
to 500 m/z. Sugar concentration was quantified using an external standard, and the
on the plasmid pYCGPY. YP5D, YP0.5M, YP5M, YP5S, YP5F, YP4D0.01M,
intracellular sugar concentration was calculated using a cell size that was measured
YP4D0.05M, YP4D0.1M, YP4D0.25M, YP4D0.5M, YP4D0.75M, YP4D1M, and
using a CDA-500 particle counter (Sysmex Corporation), according to the electrical
YP4D1S are designations of media based on 1% yeast extract and 2% yeast
sensing zone method (24), and the cell number per OD unit was counted using a
peptone. D, M, S, and F indicate glucose, maltose, sucrose, and fructose,
hemocytometer.
respectively, and the numbers preceding D, M, S, and F indicate the percentage
concentration of each sugar. YPD and YPM contain 2% glucose or 2% maltose, Analysis of nucleotide species and metabolites produced by
respectively. Glucose or maltose-based synthetic complete medium (SCD or SCM) glycolysis Cultured cells (10 OD units of cells per sample) were collected by
was prepared as previously described (19). SCM medium with or without filtration using 0.2 mm isopore membrane filters (Millipore Corporation, Billerica,
antimycin A (3 mg/L) was used to measure maltose uptake. To test the resistance MA, USA) and washed twice with ice-cold water. The cells were sonicated in 1.6 mL
of transporters to glucose-induced degradation, we used YNBMH medium (19) of methanol for 30 s to inactivate enzymes. Next, the cell extract was treated with
with 0e2 mM 2-deoxyglucose (2-DOG). YNBDS medium (19) was used for 1.1 mL of Milli-Q water containing internal standards (H3304-1002, Human
transporter degradation studies (21). When the strains designated HD were Metabolome Technologies, Inc., Tsuruoka, Japan) and incubated for another 30 s.
cultivated, geneticin (300 mg/mL) was added to all media to maintain the plasmids To remove proteins, the extract was centrifuged at 2300 g at 4 C for 5 min, and
constructed using pYCGPY. then 1.6 mL of the upper aqueous layer was centrifugally filtered through a
Millipore 5 kDa cutoff filter at 9100 g at 4 C for 120 min. For CE/MS analysis, the
DNA manipulations and mutagenesis All PCR primers are listed in Table S2.
filtrate was centrifugally concentrated and resuspended in 50 mL of Milli-Q water.
The construction of plasmids pJHIMAL61 and pJHIMAL21 was previously described
Human Metabolome Technologies, Inc. performed the metabolome analyses.
(19). AGT1 was obtained from S288C, SeAGT1 (AGT1 ortholog of Saccharomyces
Analysis was conducted using independent triplicate samples.
eubayanus-type gene of Saccharomyces pastrianus) and MTT1 from Weihenstephan
German Lager Dry Yeast, SafLager W-34/70 (22) by PCR, and they were inserted Determination of intracellular pH Intracellular pH was determined using
downstream of TPI1 promoter of pJHIXSB (19). Two tandem hemagglutinin tags flow cytometry with the fluorescent pH-indicator carboxy SNARF-4F AM acetate
(2HA) were inserted at XhoI site created immediately upstream of the stop codon from Thermo Fisher Scientific, according to the reference method (25).
of AGT1 to generate pJHIAGT1-2HA. Mutant alleles of MAL61[E161A], MAL21
[E161A], AGT1-2HA[E51P], AGT1-2HA[L55P], and AGT1-2HA[E51G, L55H] were
created from pJHIMAL61, pJHIMAL21, and pJHIAGT1-2HA using a GeneArt
mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The pJHIXSB-based RESULTS AND DISCUSSION
plasmids were integrated into the ura3 site of JH1032. MAL61, MAL21, MAL61
[E161A], MAL21[E161A], AGT1-2HA, AGT1-2HA[L55P], Se-AGT1, and MTT1 were
Characterization of a-glucoside transporters Each a-
transferred from each pJHIXSB plasmid into the centromeric expression vector
pYCGPY under the control of the PYK1 promoter. The pYCGPY-based plasmids glucoside transporter expression unit driven from TPI1 promoter
were used to transform D152U and D152MS. The GenBank accession numbers of was integrated into JH1032, which overexpresses Mal32p maltase.
nucleotide sequences used in this study are as follows: MAL21, GenBank: Maltose uptake activities of a-glucoside transporters were
AB453253.1; MAL61, GenBank: X17391.1; MTT1, GenBank: EF650853.1; and AGT1, measured using these strains. The activities of Agt1p, SeAgt1p, and
GenBank: Z73074.1. SeAGT1 is LBYG13187 (22) obtained from the WS34/70
genome sequence, GenBank: ABPO00000000.1.
Mtt1p, which have broad substrate specificities, were much lower
Measurement of maltose and glucose uptake activities Using cells (10
than those of Mal61p and Mal21p (Table 1). To compare the
OD660 unit ¼ OD  mL) grown in each condition, maltose or glucose uptake rate was degradation of transporters in the presence of glucose, each
measured with [14C]-labeled substrates as described in reference (19). The final strain was spotted on maltose-based SCM containing 2-DOG
54 HATANAKA ET AL. J. BIOSCI. BIOENG.,

TABLE 1. Maltose uptake rates of a-glucoside transporters. analysis of the Agt1-2HAp revealed that its half-life was
a-Glucoside Maltose uptake rate (nmol/min/10 OD) 13.9  4 min (Fig. S2), which was significantly shorter than those of
transporters Mal61p (25  6 min) and Mal21p (118  5 min) (19). Although
0.1 mM maltose 1.0 mM maltose
Agt1p and SeAgt1p can transport oligosaccharides such as tur-
Mal61p 3.74  0.09 33.3  3.5
anose, isomaltose, sucrose, trehalose, and maltotriose as well as
Mal21p 7.51  0.40 66.0  7.0
Agt1p 0.66  0.01 6.30  0.1 hexoses, such as fructose and glucose (6,7), Mtt1p inefficiently
SeAgt1p 0.33  0.05 2.62  0.19 transported glucose or fructose (data not shown). Therefore, we
Mtt1p 0.84  0.04 7.51  0.50 anticipated that broader substrate specificity is associated with
relatively increased degradation rate.

Cells expressing Mal21p fail to grow on medium containing


glucose and maltose Mal21p was not significantly degraded in
the presence of glucose, and the strain expressing Mal21p was
expected to simultaneously transport glucose and maltose. Each a-
glucoside transporter gene on pYCGPY was transformed to D152U.
D152U has the maltase genes MAL12 and MAL62, which are driven
by the binding of the maltose activator protein encoded by MAL63
to their native promoters. When we measured the growth of cells
expressing Mal61p, Mal21p, Agt1-2HAp, Mtt1p, or SeAgt1p on
plates containing various sugars, we found that, except for cells
expressing Mal21p (HD17), all strains grew on each medium.
Thus, HD17 did not grow on YP4D1M, YP4F1M, or YP4S1M
(Fig. 1B). Further, HD17 did not grow on YP medium containing
2% maltose and 3% glucose, 3% maltose and 2% glucose, or 4%
maltose and 1% glucose (data not shown).
We speculated that Mal21p overexpression may inhibit the
localization of other membrane proteins to cause membrane
FIG. 1. Growth of cells expressing a-glucoside transporters. (A) Growth on maltose- integrity loss or trigger an inappropriate signal for growth on
based minimum medium containing 2-deoxyglucose (2-DOG). Host strain is JH1032
YP4D1M. To determine if this growth defect was caused by maltose
(overexpressing maltase). (B) Growth on rich media containing different sugars as a
carbon source. Host strain is D152U (inducible maltase).
uptake, but not by localization of excess Mal21p on plasma mem-
brane, we isolated an inactive transporter mutant. The alignment of
the amino acid sequences of Mal61p and Mal21p showed 10 dif-
(Fig. 1A). Although 2-DOG is not metabolized further than 2-DOG- ferences (Fig. S1). Maltose transporters, which belong to the major
6-phosphate and is not assimilated as a carbon source, it induces facilitated sugar transporter (MFS) family, have 12 membrane-
the inactivation and degradation of maltose transporters to the spanning regions, and function as proton symporters (27,28). Pro-
same extent as glucose (26). tonated acidic amino acid residues in the membrane-spanning re-
Cells expressing Mal21p were significantly more resistant to 2- gions are likely required for uptake (29,30), and Glu113 resides at
DOG than the other strains. Although cells expressing Mal61p or the end of the first membrane-spanning region and Glu161 at the
Mtt1p grew on the medium containing 0.5 mM 2-DOG, cells middle of the second one.
expressing Agt1p or SeAgt1p did not (Fig. 1A). Agt1p and SeAgt1p Therefore, we reasoned that Glu161 is likely involved in proton
seemed to be readily degraded. To measure the half-life of Agt1p, relay. MAL61[E161A] or MAL21[E161A] expression unit was inte-
we used a strain that expresses HA-tagged Agt1p. Western blot grated to JH1032. The uptake rate of cells expressing Mal61p

FIG. 2. The influence of the uptake of maltose and constitutive maltase expression on growth on YP4D1M. (A) Maltose uptake rates of cells expressing parental or mutant maltose
transporters. Host strain is JH1032 (overexpressing maltase). Substrate concentration was 0.1 or 2.0 mM. (B) Growth of cells expressing parental or mutant maltose transporters on
glucose or maltose minimum medium with or without antimycin. Host strain is JH1032. (C) Growth of cells expressing parental or mutant maltose transporters on YP4D1M. Host
strain is D152U (inducible maltase) or D152MS (overexpressing maltase).
VOL. 125, 2018 GROWTH INHIBITION OF S. CEREVISIAE 55

[Ala161] was 13% compared with that of Mal61p in the presence of The growth defect depends on the maltose
2 mM maltose that is approximately a half of Km (3e4 mM) of the concentration Cells expressing Mal21p under inducible maltase
maltose transporter (31,32) (Fig. 2A). Cells expressing Mal61p background, HD17, only grew on media containing <0.05% maltose
[Ala161] or Mal21p[Ala161] were able to grow on synthetic mini- but not on media containing >0.1% maltose (Fig. 3A). We next
mal medium containing 2% (55 mM) maltose, but not when the determined the intracellular sugar concentrations of cells cultured
medium was supplemented antimycin A, indicating that they had in liquid media. Intracellular maltose detected in cells expressing
little activity compared with their parental constructs (Fig. 2B). Mal21p under Mal32p-overexpressing background, HD49, was
Slight growth of strains designated mal was often observed on much less than that in HD17, indicating rapid hydrolysis by
maltose-based media. Therefore maltose assimilation ability, maltase (Fig. 3B). In contrast, the concentration of intracellular
especially fermentation ability, has been measured on maltose- maltose of HD17 cultured in media containing maltose and glucose
based medium with antimycin A. increased as a function of maltose concentration and time in
MAL21[E161A] on pYCGPY was introduced into D152U, which culture. Although HD17 cultured in YP5M accumulated more
inducibly expresses maltase. In addition, MAL21 or MAL21[E161A] maltose than HD17 grown in YP4D0.25M at 0.5 h, the intracellular
on pYCGPY was also introduced into D152MS, which was identical maltose began decreasing after 1 h and was reduced to the same
to D152U, except for overexpressing Mal32p. Cells expressing level as that of HD17 grown on YP4D0.05M after 2 h, indicating
Mal21p[Ala161] under inducible maltase background and cells that the intracellular maltose concentration was involved in the
expressing Mal21p under Mal32p-overexpressing background growth defect. Intracellular glucose concentrations in HD17 and
(HD49) grew on YP4D1M as well as on YP5D and YP5M (Fig. 2C). HD49 grown on YP4D1M were high at 0.5 h and then rapidly
Therefore, we speculated that this growth defect was caused by the decreased. These findings support the conclusion that HD17
uptake of maltose and insufficient hydrolysis of maltose. We con- cultured in YP4D1M was unable to grow despite the presence of
structed Agt1-2HAp[Pro55], a mutant Agt1-2HAp that was resis- intracellular glucose to be metabolized through glycolysis.
tant to glucose-induced degradation to induce cells to
simultaneously transport sucrose and glucose (Fig. S2). Cells Maltose and glucose uptake rates and maltase activities of
expressing Agt1-2HAp[Pro55] under inducible maltase background HD17 and HD49 The uptake rates of maltose and glucose by
grew very slowly on YP4D1M, YP4D1S, and YP5S (Fig. S3). In HD17 grown in YP4D1M were lower than those of HD17 and HD49
contrast, cells expressing Agt1-2HAp[Pro55] under Mal32p- cultured in YP5M and YP4D1M, respectively (Fig. 4A). Nevertheless,
overexpressing background grew well on these media. Consid- HD17 grown in YP4D1M had substantial uptake activity of glucose
ering that maltase can also catalyze the hydrolysis of sucrose after 160 min. It indicated that maltose accumulated in HD17 did
(33,34), these findings indicated that the intracellular accumulation not inhibit the incorporation of glucose into cells. The level of
of sucrose caused a growth defect. It is known that S. cerevisiae maltase gene expression of HD17 cultured in YP5M at 2 h was
expresses two forms of invertase mRNAs with different 50 -termini 100% (as well as HD49 cultured on YP4D1M) and was 15% for
(35). The major species is induced in the presence of sucrose and HD17 cultured in YP4D1M, regardless of glucose repression
translated with a leader peptide, which extracellularly digests su- (Fig. 4C). Nevertheless, the maltase activities at 2 h of HD49 in
crose, and the minor species that lacks a signal peptide is intra- YP4D1M, HD17 in YP5M, and HD17 in YP4D1M were 100%, 9.8%,
cellular and is constitutively synthesized at low levels. If sucrose, and 0.6%, respectively (Fig. 4B). The maltase activity of HD17 in
which is much more abundant than maltose in nature, was added YP5M increased to 6 h but was unchanged in YP4D1M. Therefore,
to cells expressing Agt1p, it would be transported into cells to delay HD17 catabolized intracellular maltose in YP5M but did not
growth. To avoid such a risk, S. cerevisiae might express a minor synthesize maltase in YP4D1M, independent of maltase gene
form of invertase. expression.

FIG. 3. Influence of the maltose concentration of media and intracellular maltose on the growth of HD17. (A) Growth of cells carrying Mal61p or Ma21p on YP4D containing 0.01e1%
maltose. Host strain is D152U (inducible maltase). (B) Intracellular sugars of HD17 (Mal21p, inducible maltase) and HD49 (Mal21p, overexpressing maltase) grown in media with
different maltose concentrations. (C) Intracellular sugar concentrations of HD17 and HD49 grown in YP4D1M at 2 h.
56 HATANAKA ET AL. J. BIOSCI. BIOENG.,

FIG. 4. Uptake rates of maltose and glucose, maltase gene expression, and maltase activities of HD17 and HD49. HD17 (Mal21p, inducible maltase) was grown in YP5D, YP4D1M, and
YP5M and HD49 (Mal21p, overexpressing maltase) was grown in YP4D1M. (A) Maltose and glucose uptake rates. Substrate concentration was 10 mM. (B) Maltase activities: Relative
maltase activities per mg protein are indicated, and the activity of HD49 grown in YP4D1M for 2 h was defined as 100%. (C) Maltase gene expression: Expression levels of maltase
were normalized to those of PDA1 and indicated relative to that of HD49 grown on YP4D1M for 2 h (100%).

Addition of sorbitol to YP4D1M does not influence the except IMP, increased in HD17 and HD49 with time, but all were
growth of HD17 Cells maintain higher intracellular osmolarity higher in HD17 than those of HD49 at all times. Energy level,
to maintain their shape through turgor pressure caused by water ATP/(ADP þ AMP) ratio was speculated to be lower in HD17 than
entering the cells. Simultaneously, the yeast cell protects itself with in HD49.
a sturdy and elastic cell wall to avoid bursting, which can be caused The metabolites of glycolysis in HD17 and HD49 cultured in
by turgor pressure. When intracellular osmolarity increases during YP4D1M are shown in Fig. 5B. The levels of all metabolites gener-
sugar uptake, yeast cells transiently incorporate water to reduce the ated during the initial stages of glycolysis were low in HD17.
sugar concentration to counter the risk of damage to the plasma Phosphofructokinase, a key allosteric enzyme that is positively
membrane and cell wall (36,37). The intracellular maltose regulated by fructose-2,6P and AMP and inhibited by ATP, catalyzes
concentration of HD17 grown on YP4D1M reached 2.57% the rate-limiting step of glycolysis (42,43). The level of its product,
(71.4 mM) after 2 h, which was significantly higher than the fructose 1,6-bisphosphate, in HD17 was 35% compared with that of
maltose concentration in the medium (1%) (Fig. 3C). Considering HD49 at 0.5 h. The concentration of glucose-6P, the product of the
that 30% of a cell’s volume is filled with macromolecules (38), first reaction of glycolysis, was lower in HD17 than in HD49
maltose concentration would be 3.67% (102 mM). On the other (Fig. 5B). When grown on YP4D1M, HD17 seemed to decelerate
hand, maltose concentration of HD49 was 0.05%, and glucose glycolysis at a very early step despite the rich nutrient environment,
concentrations of HD17 and HD49 were 0.25% vs 4% (222 mM) in and then reduce energy level.
the medium, respectively. To examine whether differences Inhibition of protein synthesis in HD17 is associated with the
between intracellular and extracellular pressure had an effect on growth defect The maltase activities of HD17 grown in YP5M
this growth defect, sorbitol was added to the medium. The and YP4D1M were lower than its expression level, indicating the
growth of HD17 did not resume when sorbitol (25 mMe150 mM) possibility that protein synthesis was inhibited in HD17 in both
was added to YP4D1M (Fig. S4). Therefore, elevated turgor media. To investigate protein synthesis with strong promoter,
pressure did not explain the growth defect. As intracellular TPI1p::lacZ was introduced into HD17 and HD49. Both strains were
osmotic pressure is normally approximately 1 M, cells would deal cultured in YP5D, YP5M, and YP4D1M, and lacZ gene expression
with intracellular maltose of 71.4 mM from the point of view of was measured. Although lacZ gene expression of HD17 harboring
turgor pressure. TPI1p::lacZ decreased at 6 h in YP5M, the levels of all other samples
Changes in the intracellular pH of HD17 and HD49 As were similar (Fig. S6A). The b-galactosidase activities of cells
maltose transporters are proton symporters, simultaneous uptake cultured in YP5D, YP5M, and YP4D1M with and without
of maltose and protons occurs in cells (23). Cells must pump cycloheximide were measured (Fig. S6B).
protons out to avoid acidification by consuming one ATP It was verified that HD17 harboring TPI1p::lacZ could not syn-
molecule per proton through the function of Hþ-ATPase (23). thesize b-galactosidase when it was grown in YP4D1M. On the
Acidification possibly influences the reaction rates of numerous other hand, it could not synthesize b-galactosidase at first in YP5M,
enzyme reactions, including those that are required for glycolysis. but subsequently resumed after 4 h. The intracellular maltose
The pH optima of hexokinase, phosphofructokinase, and triose concentration started to decrease after 1 h in HD17 grown on YP5M
isomerase, which mediate glycolysis, are 7.5, 7.8, and 7.8, (Fig. 3B), and therefore, elimination of intracellular maltose was
respectively (39e41), leading us to hypothesize that acidification likely a key for reinitiating protein synthesis for cell growth.
reduces the rate of glycolysis. Intracellular pH of HD1(vector), A proliferating cell with surges of turgor pressure has a risk of
HD17, and HD49 cultured in YP4D1M was determined with bursting, and therefore, cells down-regulate gene expression,
carboxy SNARF4 AM ester, acetate. The intracellular pH of HD17 translation, or both to halt growth (44,45). However, we show here
was 5.79  0.06 at 0.5 h, vs 6.19  0.13 of HD49 at 0.5 h, although that the addition of sorbitol did not affect the growth of HD17 in
it returned to the same value as that of HD49 after 4 h (Fig. S5). A YP4D1M. Therefore, cells may stop protein synthesis by sensing an
rapid decrease in pH might represent one of a number of possible increase in intracellular osmolarity, regardless of extracellular os-
reasons for the transient growth defect, but it was not considered molarity. In HD17 grown in YP4D1M, stagnation of glycolysis and
to be the cause of the permanent growth defect. reduction of ATP and GTP levels, which are required for protein
synthesis, were observed. It was possibly one of the reasons that
Energy level and the rate of glycolysis in HD17 decreases in protein synthesis halted, but the reduction in ATP and GTP levels
YP4D1M To investigate energy levels, nucleotide species in should be restored if glycolysis, respiration, or both function nor-
HD17 and HD49 grown in YP4D1M were analyzed using CE/TOFMS mally. There is a possibility that accumulated maltose might inhibit
(Fig. 5A). All nucleotide triphosphate species decreased in HD17 crucial reactions in cells. In any case, the halting of protein synthesis
with time, although those in HD49 were increased with time. In under the condition of maltose accumulation affected numerous
contrast, all nucleotide diphosphate and monophosphate species, reactions, including glycolysis, leading to growth defect. HD17
VOL. 125, 2018 GROWTH INHIBITION OF S. CEREVISIAE 57

FIG. 5. Comparison of metabolites in cells grown in YP4D1M between HD17 and HD49. (A) Nucleotides. (B) Metabolites of glycolysis: PEP, phosphoenolpyruvate; GA3P, glyceral-
dehyde 3-phosphate; DHAP, dihydroxyacetone phosphate.

seemed not to resume growth due to the impossibility of elimi- 9. Dietvorst, J., Londesborough, J., and Steensma, H. Y.: Maltotriose utilization
nating maltose with maltase on YP4D1M. Our data strongly suggest in lager yeast strains: MTT1 encodes a maltotriose transporter, Yeast, 22,
775e788 (2005).
that yeast, which expresses adequate maltase antecedent to uptake 10. Dietvorst, J., Walsh, M. C., van Heusden, P. H., and Steensma, H. Y.: Com-
maltose into the cells, should be selected or bred for brewing with a parison of the MTT1- and MAL31-like maltose transporter genes in lager yeast
high gravity wort. strains, FEMS Microbiol. Lett., 310, 152e157 (2010).
Supplementary data to this article can be found online at http:// 11. Cousseau, F. E., Alves, S. L., Jr., Trichez, D., and Stambuk, B. U.: Character-
ization of maltotriose transporters from the Saccharomyces eubayanus sub-
dx.doi.org/10.1016/j.jbiosc.2017.07.013.
genome of the hybrid Saccharomyces pastorianus lager brewing yeast strain
Weihenstephan 34/70, Lett. Appl. Microbiol., 56, 21e29 (2013).
12. Hu, Z., Yue, Y., Jiang, H., Zhang, B., Sherwood, P. W., and Michels, C. A.:
ACKNOWLEDGMENTS
Analysis of the mechanism by which glucose inhibits maltose induction of MAL
gene expression in Saccharomyces, Genetics, 154, 121e132 (2000).
We are very grateful to Prof. M. Ishiguro for the suggestions with 13. Wang, X. and Michels, C. A.: Mutations in SIN4 and RGR1 cause constitutive
regard to the active site of the maltose transporter and to Mrs. Y. expression of MAL structural genes in Saccharomyces cerevisiae, Genetics, 168,
Yasue for the technical support. 747e757 (2004).
14. Ran, F., Bali, M., and Michels, C. A.: Hsp90/Hsp70 chaperone machine regu-
lation of the Saccharomyces MAL-activator as determined in vivo using non-
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