MAL62 overexpression and NTH1 deletion enhance the freezing tolerance and fermentation capacity of the baker’s yeast in lean dough
Sun et al. Microb Cell Fact
MAL62 overexpression and NTH1 deletion enhance the freezing tolerance and fermentation capacity of the baker's yeast in lean dough
Xi Sun 2 3
CuiY‑ing Zhang 0 1 5
MingY‑ue Wu 4
Zhi‑Hua Fan 2 3
Shan‑Na Liu 2 3
Wen‑Bi Zhu 3
Dong‑Guang Xiao 0 1 5
0 College of Biotechnology, Tianjin University of Science and Technology , Tianjin 300457 , People's Republic of China
1 Key Laboratory of Industrial Fermentation Microbiology, Ministry of Educa‐ tion, Tianjin Industrial Microbiology Key Laboratory, Tianjin University of Sci‐ ence and Technology , Tianjin 300457 , People's Republic of China
2 Tianjin Engineering Research Center of Agricultural Products Processing , Tianjin 300384 , People's Republic of China
3 College of Biological Engineering, Tianjin Agricultural University , Tian‐ jin 300384 , People's Republic of China
4 Diagreat Biotechnologies., Ltd , Beijing 101111 , People's Republic of China
5 College of Biotechnology, Tianjin University of Science and Technology , Tianjin 300457 , People's Republic of China
Background: Trehalose is related to several types of stress responses, especially freezing response in baker's yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose‑ related genes to create yeast strains that better tolerate freezing‑ thaw stress with improved fermentation capacity, which are in high demand in the baking industry. Results: The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention‑ freezing and long‑ term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing‑ thaw stress. Conclusions: The mutants of the industrial baker's yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.
Baker's yeast; MAL62; NTH1; Freezing tolerance; Cell viability; Leavening ability
Frozen dough technology has been used in bakery
industry to provide consumers with high-quality fresh bakery
and convenience. However, cellular macromolecules,
including proteins, nucleic acids and lipids of the yeast
used in frozen dough, could be seriously damaged under
the freezing and the subsequent thawing treatments,
leading to inhibition of cell growth, cell viability and the
leavening ability [
A great body of knowledge is already available
regarding the molecular responses of the baker’s yeast
(Saccharomyces cerevisiae) to frozen dough-associated stresses
]. Among other molecules, trehalose has been
highlighted due to its main function as a protective molecule
in stress response [
]. This effect is achieved either by
protecting membrane integrity through the union with
], or by preserving the native
conformation of proteins and preventing aggregation of partially
denatured proteins [
When yeast cells suffer from freezing stress, they
accumulate large amounts of trehalose [
]. The accumulation
is mainly induced by the classical the UDPG-dependent
trehalose synthesis pathway, or referred as system I. It
contains a trehalose-6-phosphate synthase encoded by
], a trehalose-6-phosphate phosphatase encoded
by TPS2 [
] and a trehalose-synthesis protein complex
encoded by TSL1 [
]. In addition, an alternative trehalose
synthesis pathway, called ADPG-dependent trehalose
synthesis pathway or the system II, has been proposed
]. It is specifically linked to maltose utilization.
Maltose metabolism in yeast depends on at least one
of the five unlinked MAL loci (MAL1 through MAL4
and MAL6). A typical MAL locus consists of a MALx1
(MALxT) gene (where x is the locus), encoding
maltose permease, a MALx2 (MALxS) gene, coding for
alpha-glucosidase (maltase), and a MALx3 (MALxR)
gene, encoding a positive regulatory protein [
]. It is
reported that the expression of any one of the MAL
loci in MAL-constitutive strains could elicit a
maltose-induced trehalose accumulation [
]. Studies have
shown that maltose and trehalose seem to share a
common regulating mechanism [
]. The maltose
permease has been considered the rate-limiting enzyme in
the MAL genes induction and maltose metabolism [
]. Hence, attempts to increase the trehalose content
by system II had been concentrated on the
modification of maltose permease or the entire MAL gene cluster
]. However, recent studies showed that the
alphaglucosidase (maltase) is more important than maltose
permease in maltose metabolism and leavening ability of
baker’s yeast in lean dough [
]. In addition, the
system II might be dependent of the system I, due to the fact
that the system II is completely prevented when TPS1, a
key gene in system I, is deleted .
Trehalose degradation could also be induced under
certain stress [
]. The best characterized trehalase is
the neutral trehalase encoded by the NTH1 gene, which
is induced by stress, such as heat. Nth1p is involved in
thermos-tolerance and hydrolyzes intracellular trehalose
into glucose [
]. Deletion of NTH1 results in
accumulation of trehalose, and heat sensitivity.
To better understand the role of trehalose in freezing
tolerance of baker’s yeast in lean dough, and its possible
mechanism, we investigated the effects of overexpression
of MAL62, the gene encoding an alpha glucosidase, and
deletion of NTH1 gene, on trehalose accumulation and
on the freezing tolerance and leavening ability of baker’s
yeast in lean dough.
Strains, plasmids and growth conditions
The genetic properties of all S. cerevisiae strains and
plasmids used in the present study are summarized
in Table 1. The BY14a was selected as a high leavening
capacity haploid from 32 clones derived from the diploid
BY14 strain, which has been maintained at the Tianjin
Key Laboratory of Industrial Microbiology, Tianjin
University of Science and Technology.
Recombinant DNA was amplified in Escherichia
coli DH5a. Transformants were grown in
Luria–Bertani medium (10 g/L tryptone, 5 g/L yeast extract, and
10 g/L NaCl) with 100 mg/L ampicillin. The plasmid was
obtained using Plasmid Mini Kit II (D6945, Omega, USA).
The yeast strain was grown at 30 °C in yeast extract
peptone dextrose (YEPD) medium (10 g/L yeast extract,
20 g/L peptone, and 20 g/L dextrose). Approximately
800 mg/L of G418 was added to the YEPD plates for
selecting Geneticin (G418)-resistant transformants. After
cultivation in YEPD for 24 h, 20 mL of the cell culture
was inoculated into 200 mL of cane molasses medium
(5 g/L yeast extract, 0.5 g/L (NH4)2SO4, and 12° Brix
cane molasses) at the initial OD600 = 0.4 and cultivated
for 24 h at 30 °C with 180 rpm rotary shaking to the final
OD600 = 1.8. Cells were harvested through centrifugation
(4 °C, 1500×g, 5 min) and were washed twice with
sterile water at 4 °C for the succeeding fermentation
experiments. To investigate the degradation of trehalose during
prefermentation and the freezing tolerance, a modified
the low sugar model liquid dough (LSMLD) medium
was used [
]. The modified medium contains 2.5 g/L
(NH4)2SO4, 5 g/L urea, 16 g/L KH2PO6, 5 g/L Na2HPO4,
0.6 g/L MgSO4, 22.5 mg/L nicotinic acid, 5 mg/L
Capantothenate, 2.5 mg/L thiamine, 1.25 g/L pyridoxine,
1 mg/L riboflavin, and 0.5 mg/L folic acid and carbon
sources (33.25 g/L maltose with 5 g/L glucose).
Plasmid construction and yeast transformation
Genomic yeast DNA was prepared from the industrial
baker’s yeast BY14a using a yeast DNA kit (D3370-01,
Omega, Norcross, GA, USA). Table 2 shows the PCR
primers used in this study.
Plasmid Yep-PMK (Yep-PGK1-MAL62-KanMX), an
episomal plasmid with MAL62 under the control of the
constitutive yeast phosphoglycerate Kinase gene (PGK1)
promoter (PGK1P) and terminator (PGK1T), was
constructed as follows: a KpnI/BamHIKanMX fragment,
which was the dominant selection marker during yeast
conversion, was amplified through PCR using pUG6
as template with Kan-U and Kan-D primers, and was
cloned to the Yep352 vector to construct the empty
plasmid Yep-K (Yep-KanMX). A XhoI fragment of MAL62
amplified with MAL62-U and MAL62-D primers from
the genomes of the parental strain BY14a was inserted
into the PGK1 fragment of pPGK1 vector and resulted in
plasmid pPGKM. Then, the BamHI fragment of PGKM
(the entire PGK1 and the inserted MAL62) amplified
with PGK-U and PGK-D from pPGKM was cloned to
Yep-K to produce the final plasmid Yep-PMK.
Baker’s yeast transformation was achieved through
lithium acetate/PEG method [
]. The deletion
cassette of NA-loxP-KanMX-loxP-NB was amplified with
N-S and N-X and transformed into the industrial
baker’s yeast BY14a. The fragment was integrated into the
chromosome at the NTH1 locus of BY14a by
homologous recombination to construct the NTH1 deletion
strain. The selection of NTH1 deletion strain was
performed using the YEPD medium supplemented with
800 mg/L geneticin (G418). After selection, recombinant
strains were verified with the primers N-S, K-S and N-X,
K-X. Cre recombinase was expressed and KanMX was
excised after introducing the plasmid pSH-Zeocin into
a BY14a was selected as high leavening capacity haploid from 32 clones derived from BY14 strain (data not shown)
the NTH1 deletion strain, thus resulting in B-NTH1.
The respective transformation plasmids Yep-K,
YepPMK were then transformed to select the
G418-resistant strains BY14a + K, B-NTH1 + K, B + MAL62 and
B-NTH1 + MAL62. BY14a + K and B-NTH1 + K were
BY14a and B-NTH1 carrying the vector Yep-K,
respectively, used as a blank control to demonstrate any possible
effect of the empty vector. The transformants were then
verified by PCR using the primers Kan-U and Kan-D.
Assay of the intracellular trehalose content
Fresh yeast cells were dried overnight at 85 °C to
calculate the cell dry weight (CDW). Trehalose was extracted
from 0.1 g of fresh yeast cells (previously washed with
distilled water twice) with 4 mL of 0.5 mol/L cold
trichloroacetic acid and the extract was employed for
measuring the trehalose content as described previously [
Experiments were conducted three times.
Determination of neutral trehalase activity
The activities of neutral trehalase in crude extracts were
measured as described previously [
]. The liberated
glucose was analyzed by HPLC employing an Aminex
HPX-87H column with 5 mmol/L H2SO4 as the mobile
phase at a flow rate of 0.6 mL/min at 65 °C. One unit of
trehalase activity was defined as the amount of trehalase
producing 1.0 μm glucose per min under assay
conditions. The specific trehalase activity was expressed as the
units per gram CDW. Experiments were conducted three
Determination of Tps1 (trehalose‑6‑phosphate synthase) activity
Tps1 activity was measured as described previously [
The trehalose-6-phosphate formed during the reaction
was quantitatively determined using the Anthrone
]. One unit of Tps1 activity was defined as the
amount of Tps1 producing 1.0 μm 6-phosphate-trehalose
per min under assay conditions. The specific Tps1 activity
was expressed as the units per gram CDW. Experiments
were conducted three times.
Determination of alpha‑glucosidase activity
Crude extracts were prepared using the Salema-Oom
method to determine enzyme activities [
Alphaglucosidase were determined following the
HoughtonLarsen method [
]. Standard errors were less than
Determination of the cell viability of baker’s yeast after freezing and thaw
For the freeze–thaw stress, yeast cells were harvested
from the cane molasses medium and inoculated into the
LSMLD medium at 30 °C for 25 min. One milliliter of
cell culture was shifted to −20 °C and at 5 min intervals
for different prefermentation time periods. After
freezing for 1–3 week, the frozen suspensions were thawed at
30 °C for 30 min then diluted and plated on YEPD plates
for 2 days. Cell viability was determined by the
percentage of the number of colonies after stressing relative to
the number of colonies before stress. Three independent
experiments were performed.
Determination of leavening ability
The leavening ability of yeast cells was assayed by
measuring the CO2 production in lean dough. Lean dough was
composed of 280 g of standard flour, 150 mL of water, 4 g
of salt, and 9 g of fresh yeast. The dough was evenly and
rapidly stirred for 5 min at 30 ± 0.2 °C then divided into
pieces (50 g each) and placed in a fermentograph box 171
(Type JM451, Sweden). CO2 production was recorded
at 30 °C for 120 min. Experiments were conducted three
To assay the leavening ability after freeze–thaw, the
mixed dough was stored at −20 °C. After freezing for
1 week, the frozen dough was thawed at 30 °C for 30 min,
and the CO2 production was assayed for 120 min at
30 °C. Experiments were conducted at least thrice.
Data were expressed as mean ± SD and were
accompanied by the number of experiments independently
performed. Differences among all the strains were analyzed
using ANOVA. P < 0.05 were considered statistically
significant. The differences between the transformants
and the parental strain were confirmed by Student’s t
test. Differences at P < 0.05 were considered statistically
Overexpression of MAL62 enhances the Tps1 activity and intracellular trehalose content of baker’s yeast
Previous studies have reported that the MAL gene has
a positive effect on the activity of Tps1, a
trehalose6-phosphate synthase that synthesizes trehalose under
stress conditions [
]. We first tested if the Tps1
activity is affected by MAL62 overexpression. As shown in
Table 3, overexpression of MAL62 (in both B + MAL62
and B-NTH1 + MAL62) significantly increased the
Tps1 activity (P < 0.05). The alpha-glucosidase
activities of these two strains were also increased significantly
(Table 3). These results suggest that overexpression of
MAL62 induces trehalose production.
To further confirm this, we measured and
compared the trehalose levels in different strains. We found
that all six strains (BY14a, B-NTH1, B + MAL62,
B-NTH1 + MAL62, BY14a + K and B-NTH1 + K)
had similar growth curves. Cells entered exponential
phase 3 h after inoculation, and stationary phase 10 h
after inoculation (data not shown). Our results showed
in strains overexpressing MAL62 (B + MAL62 and
Values shown represent at least three independent experiments (data are mean ± SD). Significant difference of the transformants (BY14a + K, B-NTH1,B-NTH1 + K,
B + MAL62, B-NTH1 + MAL62) from the parental strain was confirmed by Student’s t-test (**P < 0.01,*P < 0.05, n = 3)
a Alpha-glucosidase activities and Tps1 activities were calculated from the cells grown in cane molasses medium
b Neutral trehalase activities were calculated from the cells prefermentation in LSMLD medium
B-NTH1 + MAL62), trehalose started to accumulate
in late exponential stage at a rate of 21.9 mg/h/g CDW.
In contrast, in strains having no MAL62 overexpression
(BY14a, B-NTH1, BY14a + K and B-NTH1 + K),
trehalose accumulation started only in stationary phase and at
a lower rate (19.1 mg/h/g CDW) (Fig. 1).
MAL62 overexpression does not affect the rate of trehalose degradation
To examine if MAL62 overexpression or NTH1 is
involved in trehalose degradation, we compared the
neutral trehalase activity and the degradation rate of
intracellular trehalose among the six strains. As shown in Table 3,
the B + MAL62 strain had a similar neutral trehalase
activity compared to its control (BY14a and BY14a + K),
suggesting that overexpression of MAL62 did not affect
the trehalose degradation. This is further confirmed by
direct measurement of the intracellular trehalose content
(Fig. 2), which showed a similar degradation rate among
B + MAL62, BY14a and BY14a + K. In addition, both the
neutral trehalase activity and the rate of trehalose
degradation were significantly lower in all NTH1 deletion
strains (B-NTH1, B-NTH1 + K and B-NTH1 + MAL62)
(Table 3, Fig. 2), regardless whether MAL62 was
overexpressed or not. These results suggest that NTH1, but not
MAL62, is important for trehalose degradation.
High trehalose content increases viability of yeast cells after freezing
Although a number of reports have shown that the
degradation of trehalose during prefermentation is necessary
], the residual intracellular trehalose is still considered
to be important to freezing tolerance of yeast [
Hereby, we assessed the cell viability of the six strains to
investigate the effect of MAL62 overexpression and/or
NTH1 deletion on the freezing tolerance of yeasts after
prefermentation and 7 d freezing.
As shown in Fig. 3, the cell viability of strains
with MAL62 overexpression (B + MAL62 and
B-NTH1 + MAL62) was significantly higher than the
other strains before prepermentation (time = 0 min).
Cell viability of all strains decreased as prefermentation
time increased. 25 min after prefermentation, the cell
viability of the strain with both MAL62 overexpression
Fig. 3 Cell viability of strains after prefermentation for different time
periods in LSMLD and frozen for 7 d at −20 °C. Data are average of
three independent experiments, and error bars represent ± SD
and NTH1 deletion (B-NTH1 + MAL62) was
significantly higher than other strains (ANOVA, P < 0.05). The
cell viability of strains with either MAL62
overexpression or NTH1 deletion remained in the middle, while the
control strains (BY14a and BY14a + K) had the lowest
viability, dropping from about 80 % to about 40 %. The
cell viability is in agreement with the trehalose content
(Fig. 2), which showed that 25 min after
prefermentation, the B-NTH1 + MAL62 had the highest trehalose
content (95 mg/g CDW) and the controls (BY14a and
BY14a + K) had the lowest (about 55 mg/g CDW). These
results suggest that the residual trehalose content has a
positive correlation with the viability of yeast cells after
prefermentation and freezing [
Overexpression of MAL62 or deletion of NTH1 confers long‑term freezing tolerance of baker’s yeast
In order to access the long-term freezing tolerance of the
NTH1-deletion and/or the MAL62-overexpression strains,
we examined the trehalose content before freezing and the
cell viability 21d after freezing (Fig. 4). As shown in Fig. 4,
both the trehalose content and the cell viability were
significantly higher in strains with MAL62 overexpression
(B + MAL62 and B-NTH1 + MAL62) (ANOVA, P < 0.05).
Compared with the control (BY14a and BY14a + K),
deletion of NTH1 alone (B-NTH1 and B-NTH1 + K) also
induced a higher trehalose content and higher cell viability,
which is in agreement with previous studies [
Overexpression of MAL62 and deletion of NTH1 enhance the fermentation characteristics of baker’s yeast exposed to freezing‑thaw stress
Leavening ability is an important fermentation
characteristic of baker’s yeast used in frozen dough. We
next explored the possible effects of MAL62
overexpression and NTH1 deletion on the leavening ability
after freezing and thaw by measuring the CO2
production. Our results showed that freezing-thaw caused a
reduction of CO2 production in all strains (comparing
Fig. 5a with 5b). However, either before or after
freezing-thaw, overexpression of MAL62 (B + MAL62 and
B-NTH1 + MAL62) significantly enhanced the CO2
production (ANOVA, P < 0.05). NTH1 deletion alone had no
effect on CO2 production before freezing-thaw (Fig. 5a)
but enhanced the CO2 production after freezing-thaw
(Fig. 5b). Interesting, MAL62 overexpression and NTH1
deletion (B-NTH1 + MAL62) had a lower CO2
production than MAL62 overexpression alone (B + MAL62)
before freezing-thaw, but the CO2 production was higher
after the freezing-thaw, suggesting that MAL62
overexpression and NTH1 deletion provide the best
enhancement on leavening ability upon freezing-thaw stress.
Biological macromolecules and membranes are liable to
denaturation under freezing conditions [
], Freezing also
causes the formation of intracellular ice crystals, which
are harmful to cells. It has been suggested that trehalose
could act as a stabilizer of cellular membranes and
proteins under freezing stress [
]. Previous studies have
reported that the modification of the whole MAL gene
cluster is necessary to elicit trehalose synthesis [
this study, we demonstrated that the
single-gene-overexpression of MAL62 in industrial baker’s yeast is capable of
increasing trehalose accumulation and cell viability under
freezing stress. Trehalose formation in MAL62
overexpressing strains (B + MAL62 and B-NTH1 + MAL62)
was earlier and faster than the controls (Fig. 1), suggesting
the positive effects on the intracellular trehalose content
and freezing tolerance (Figs. 2, 4). Moreover, although
MAL62 overexpression had little effect on protecting
trehalose against degradation during prefermentation, the
cell viability assay showed that the MAL62 overexpression
could protect cells against freezing stress after
prefermentation. This is in line with a previous report , showing
that the trehalose accumulation before the induction of
stress was more important for stress tolerance.
One explanation is that MAL62 overexpression
enhances the activity of Tps1. This hypothesis relies
on the fact that maltose constitutive genes could
partially relieve Tps1 from the catabolite repression [
and the alpha-glucosidase (coded by gene MAL62) is
the rate-limiting factor in maltose metabolism [
Our result is consistent with this hypothesis, since
Tps1 activity could increase when the
alpha-glucosidase activity was enhanced by MAL62 overexpression
(Table 3). Another explanation is that the existence of
trehalose synthase, which requires ADPG instead of UDPG
as donor of glucose units for trehalose synthesis [
Since the expression of ADPG-pyrophosphorylase gene
and MAL genes shared the common regulation, any of
the MAL gene products either by means of control at the
transcription level, or by acting directly on enzyme
activity could regulate the activity of the
ADPG-pyrophosphorylase activity [
]. Hence, overexpression of MAL62
alone could increase intracellular trehalose content and
bring about further enhancements in freezing tolerance.
The fermentation characteristics of baker’s yeast as a
strong correlation with the tolerance in stress conditions
]. After exposure to freeze–thaw stress, response to the
environmental change involved in rapid accumulation of
relevant protectants and rapid production of enzymes related
to stress-protective effect [
]. In this work, we found that
the freezing tolerance and the fermentation characteristics
of the double mutant (B-NTH1 + MAL62) were
significantly enhanced than that of either single mutant (B-NTH1
or B + MAL62) after the freezing-thaw stress (Figs. 4, 5b).
In addition, we found that NHT1 deletion (B-NTH1,
B-NTH1 + K and B-NTH1 + MAL62) induced a low neutral
trehalase (Table 3), which caused a lower level of trehalose
degradation. High activity of trehalose synthase (+MAL62)
 and low activity of neutral trehalase (−NTH1) increase
the intracellular trehalose level [
], which explains why the
double mutant (B-NTH1 + MAL62) provides the best
freezing tolerance and fermentation characteristics [
In summary, our study showed that MAL62
overexpression and NTH1 deletion in baker’s yeast significantly
enhanced the freezing tolerance and fermentation
characteristics, which is in high demand in the frozen dough
baking industry [
]. On the basis of our findings, it is
also possible to lengthen the storage period of frozen
dough through overexpression of the MAL62 with NTH1
deletion. It provides valuable insights for breeding novel
stress-tolerant and fast-fermented baker’s yeast strains
that are useful for baking industry.
The results of this study show that overexpression of
MAL62 was an effective way of increasing trehalose
content and cell viability after prefermention-freezing and
long-term frozen. Deletion of NTH1 in combination of
MAL62 overexpression could further strengthen freezing
tolerance and improve the leavening ability after
freezingthaw stress. Furthermore, the single-gene-overexpression
of MAL62 in industrial baker’s yeast is capable of
increasing trehalose accumulation, therefore, promoting cell
viability and the leavening ability of baker’s yeast in lean
dough under freezing stress. Hence, such baker’s yeast
has excellent commercial and industrial applications.
XS carried out the experiments and drafted the manuscript. MYW and SNL
participated in the plasmid and strain construction. ZHF and WBZ assisted in
fermentation experiments. CYZ and DGX conceived the study and reviewed
the final manuscript. All authors read and approved the final manuscript.
The current study was financially supported by the National Natural Science
Foundation of China (31571809) and the Research Fund for the Doctoral
Program of Tianjin Agricultural University (2013N04).
The authors declare that they have no competing interests.
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