Introduction
Saccharomyces
cerevisiae is used wildly in fermentation fields, as in wine-making or production
of various high additional value biochemicals such as
ethanol, glycerol. Therefore, fermentation of S.
cerevisiae has always received a huge
amount of attention to academic and industrial groups. Currently, it is popular to
use various technologies to enhance reproduction stability and metabolic
activity of fermenting microorganisms, which almost determine production efficiency and quality of
fermentation. For example, ultrasonic or
magnetic fields can be used to assist the fermenting process by enhancing metabolite yield and growth (Santos et al.
2010; Ojha et al. 2017). Metabolic engineering has been
presented as promising solution, it is necessary to ensure growth rate, titer and yield (Semkiv et al. 2017; Lalwani et al. 2018). Various potential approaches were explored to enhance
the fermentation performance with considering factors such as usability,
stability and environment-friendly.
Pulsed electric field (PEF) has been normally used as a
non-thermal sterilization technique using low energy requirements to inactivate
microorganisms without destroying quality and flavor of the food and medicine (Zhao et al. 2019). PEF
is usually applied for short duration (μs)
by high intensity electric fields (1 to 50 kV·cm-1). The mechanism of PEF was commonly known as
bacterial inactivation caused by irreversible membrane rupture (Garner 2019). PEF also could modify the functional groups on
proteins located on cell membrane directly or indirectly (Hristov et al.
2018). Application values of PEF have received growing attention in recent
years, not just limited to food safety.
For instance, PEF was found to be a viable elicitor for stimulating secondary
metabolite biosynthesis in plant cell cultures (Cai et al. 2011). It was also
reported that PEF could enhance activity of enzymes and
improve the growth of Lactobacillus acidophilus and
Lactobacillus bulgaricus LB-12 (Najim and Aryana 2013; Martens et al. 2020). Proposed that PEF showed
a higher efficiency on enhancing the activity of anammox bacteria compared with
ultrasound and magnetic field (Yin et al. 2015). The fermentative
process of S. cerevisiae is also performed by a series of enzymatic
catalytic reactions against yeast cells. It is speculated that the PEF might play an active role on fermentation
of S. cerevisiae under appropriate
conditions (Mattar et al. 2015). There are
still few studies about direct effects of moderate PEF on
physiological behavior of S. cerevisiae. The underlying molecular mechanism of
PEF on fermentation cells is also poorly reported.
The aim of this study is to
investigate the effects of moderate PEF (1 to 3 kV·cm-1) on the growth and fermentation kinetics to S.
cerevisiae. We studied the stress responses mechanism by
analyzing changes of the expression
level of ten genes
involved in glycolysis pathway using the reverse transcription-quantitative
PCR (RT-qPCR) method. The
results revealed that moderate PEF can be applied to promote the fermentation
of S. cerevisiae.
Materials and
Methods
Strains, media and
inoculum culture
S. cerevisiae strain RV100, widely used
commercial yeast, was purchased from
the Angel company (Angel, Yichang, China). The active
dry yeast was rehydrated in 5%
glucose solution at 30°C for 30 min and spread on YPD
medium plates, then incubated at
30°C. Single
colonies from the agar plate were incubated in 5 mL YPD broth and culturing at
30°C with orbital shaking (200 rpm). The cells were then collected and washed
with a phosphate buffered saline (PBS) solution. The initial yeast suspension
(OD600=0.8) for the fermentation trials was prepared by inoculating
cells into synthetic medium (8% glucose, 1% tryptone, 0.5% w/v yeast extract, 0.1%
(NH4)2SO4, 0.1% MgSO4·7H2O,
0.1% KH2PO4).
PEF treatment and
fermentation
In this work,
a constant treatment protocol was designed to
investigate the effects of PEF on S. cerevisiae (Fig. 1). The experiments were operated on a cylinder glass bioreactor,
which exposed to the PEF treatment chamber. The treatment system contains a
circulation pump and temperature controller. The initial yeast suspension (5%
v/v) was inoculated to 300 mL fermentation media in the bioreactor under PEF processing
conditions, while pulse width is 5μs, frequency is 10 Hz. The treatment chamber and
bioreactor was also disinfected by 75% alcohol and washed by sterile
double distilled water. All experiments were performed in
triplicate.
Analysis methods of fermentation kinetics
Growth dynamic of yeast was
monitored by measuring OD600. The dry
weight biomass was determined by weighing the cells from suspensions, which
washed twice with sterilized water, and then kept at 70°C until reaching a
constant weight in tubes. Fermentation performance was estimated by measuring
the residual glucose, ethanol and glycerol in medium
substrate. The concentrations
of glucose, ethanol and glycerol were determined by HPLC according to the (Seong et al. 2017). The HPLC column system
(Shimadzu, Japan) was set at a flow rate of 0.8 mL·min-1 with 65°C
on column.
RNA extraction and
cDNA synthesis
Cells were collected by centrifuged (5000 g, 4°C, 5 mins) and washed twice
with PBS buffer (pH7.2). After grinding with liquid nitrogen, total RNA was extracted from 2×108
cells using TransZol Up RNA kit
(TransGen). cDNA was
synthesized on a Thermal Cycler (Bio-Rad S1000) with
PrimeScriptTM RT kit (Takara). All operations were in accordance
with the protocol specified by the kit supplier. Qualitative and quantitative analysis of RNA and cDNA
were measured by NanoDrop (Thermo
Scientific). For each PEF condition, equal RNA amounts
were pooled from three independent tests.
Primers and
quantitative PCR
All primers were
designed by Primer Premier 5.0 based on the sequences
achieved from GenBank (Table 1). Reactions were performed in 96-well plates
on a Real-Time QPCR System (Agilent Mx3005P). Baseline and
threshold values were automatically determined by the MxPro3005 software. The 2-∆∆CT method was
used to calculate the relative expression levels.
Statistical
analysis
Statistical analysis was performed using StatSoft STATISTICA
(Statistica, Inc.). Results are reported as mean ± standard deviation.
Results
Effects of PEF on fermentation and growth of S. cerevisiae
To investigate the effects of moderate PEF on
physiological behavior of S. cerevisiae, the
constant PEF treatment was applied to initial yeast suspensions. The effects of PEF on the growth
dynamics and fermentation kinetics of S. cerevisiae were investigated.
The selection of PEF parameters was based on extensive
preliminary trials for the purpose of avoiding noticeable
Table 1: Genes and
Primers used in the RT-qPCR analyses
|
Genes |
ID |
Forward and reverse primer |
Description |
|
ACT1 |
850504 |
5'-CCA AGA CAC CAA GGT ATC ATG GTC G-3' |
actin, structural protein |
|
|
|
5'-CGG AAG AGT ACA AGG ACA AAA CGG C-3' |
|
|
GPD2 |
854095 |
5'-AAG ATC GGA CTC TGC CGT GTC AAT T-3' |
NAD-dependent glycerol 3-phosphate dehydrogenase |
|
|
|
5'-ACG TGG CCT TGC AAT TGT TTG ACT A-3' |
|
|
PDC1 |
850733 |
5'-AAG GTA TGA GAT GGG CTG GTA ACG C-3' |
pyruvate decarboxylase isoenzyme
(major one) |
|
|
|
5'-TGA AGT CAC CGT TAC CCA AGG TGT G-3' |
|
|
PDC6 |
852978 |
5'-GCT ACC AGG CGA CTT CAA CTT GT-3' |
pyruvate decarboxylase (Minor isoform) |
|
|
|
5'-TGA GAT ATT GGC GGA CAT TCT GTG A-3' |
|
|
ALD4 |
854556 |
5'-CGT GTT GAA GAC TGC CGA ATC CA-3' |
Mitochondrial aldehyde dehydrogenase |
|
|
|
5'-CGC ACA ACA GAC CTC ACC
AGA ATT-3' |
|
|
ALD6 |
856044 |
5'-AAC TTC ACC ACC TTA GAG
CCA ATC G-3' |
cytosolic aldehyde dehydrogenase |
|
|
|
5'-CGA CAG CAA CAC TCT TAC CGA CTT-3' |
|
|
ADH1 |
854068 |
5'-TGC TGC TGG TGG TCT AGG
TTC TT-3' |
alcohol dehydrogenase |
|
|
|
5'-GAG ATG GAC TTG ACG ACT TGG TTG A-3' |
|
|
ADH2 |
855349 |
5'-ATT AGT TGG TGG TCA CGA
AGG TGC C-3' |
glucose-repressible alcohol dehydrogenase II |
|
|
|
5'-CGG TGA TAC CAG CAC ACA AGA TTG G-3' |
|
|
ADH3 |
855107 |
5'-CAT TGT TCA CCA GGC GTG TCC AA-3' |
mitochondrial alcohol dehydrogenase isoenzyme
III |
|
|
|
5'-AAA TCA CCG ACT TTC CAG CCC TTG-3' |
|
|
ADH4 |
852636 |
5'-TGG TTC TGC TCA CGA CAA TGC TAA G-3' |
alcohol dehydrogenase isoenzyme
type IV |
|
|
|
5'-GGG TTA GAG GCG GTG GAA ACA TAA G-3' |
|
|
ADH5 |
852442 |
5'-ACG AAG GTG CTG GTG TTG TTG
TT-3' |
alcohol dehydrogenase isoenzyme
V |
|
|
|
5'-GGC GTA TTG GAT TGC CAG AGA ACC-3' |
%20160-164_files/image002.png)
Fig. 1: Schematic diagram
of the experimental setup of constant PEF treatment
%20160-164_files/image004.png)
Fig. 2: The effects on growth of S. cerevisiae.
(a) Growth dynamics
of yeast cells. (b) Dry biomass
weight. Values represent the average means ± standard deviations (n ≥
3).
damage of yeast population caused by PEF. The growth
dynamics of S. cerevisiae was investigated by examining dry cell biomass
and medium absorbance of OD600, which showed in Fig. 2. The
weight of dry cell biomass was enhanced 20.6% (1 kV·cm-1) and 32.4% (3 kV·cm-1) respectively than those of control samples.
Glucose is the one
of the most important nutrient sources for yeast fermentation. The metabolic
activity of yeast cells was evaluated by monitoring the uptake of glucose. The residual glucose concentration is presented in Fig. 3a. At the initial stage of fermentation (0~3 h), the treated
sample (1
kV·cm-1) showed around 1.79 times of glucose consumption than control sample,
which it showed around 1.78 times under 3 kV·cm-1 PEF treatment. It showed
that the glucose consumption reached
%20160-164_files/image006.png)
Fig. 3: Effects of PEF on fermentation of S.
cerevisiae. (a) Residual glucose
in culture, (b)
Ethanol concentrations in culture, (c)
Glycerol concentrations in culture. Values represent the
average means ± standard deviations (n ≥ 3)
%20160-164_files/image008.png)
Fig. 4: Schematic
representation of the gene expression of the sugar metabolism pathway affected
by PEF. The relative fold change shown next to the gene, left (1 kV·cm-1),
and right (3 kV·cm-1)
the fastest rate at 9 h after PEF treatment (1 kV·cm-1), while the control samples
reached at 12 h after treatment. After 12 h, the residual glucose in treated samples were 4.23% (1
kV·cm-1)
and 11.19% (3 kV·cm-1) lower than those in control samples. During fermentation progress, the residual glucose content was lower in
PEF-treated samples than control ones.
Metabolite analysis of ethanol and glycerol was measured
among the fermentation progress, showed in Fig. 3b and c. PEF-treated samples increased
ethanol production significantly, which increased respectively 12.91% (1
kV·cm-1) and 18.26% (3 kV·cm-1) in ethanol production after 12h
fermentation (Fig. 3b). A
significant increase in ethanol accumulation was observed after 3h of
fermentation. The ethanol yield exceeded by 1.18 times (1
kV·cm-1) and 1.43 times (3
kV·cm-1) respectively in treated samples than the control ones after 9 h fermentation. The trends of glycerol production were similar
to ethanol (Fig. 3c). The final glycerol productions were 1.28 g·L-1(1 kV·cm-1) and 1.36 g·L-1(3 kV·cm-1),
which were
2.1 and 7.95% higher than control samples.
Relative
expression levels of glycolysis genes
In order to reveal the molecule physiological mechanism caused by PEF
treatment, relative expression levels of 10 genes (ADH1, ADH2, ADH3, ADH4,
ADH5, GPD2, PDC1, PDC6, ALD4, ALD6) were analyzed by RT-qPCR method which
involved in glycolysis pathway (Fig. 4).
The expression
level of ADH1 was up-regulated by around 4.2-folds (1 kV·cm-1) and 8.9-folds (3 kV·cm-1), while ADH2 showed 3.4-folds (1 kV·cm-1) and 5.6-folds (3 kV·cm-1) than the control samples. The
up-regulation of GPD2 has also observed 2.9-folds (1 kV·cm-1) and 3.9-folds (3 kV·cm-1) than the control ones (Fig. 4).
The expression changes in metabolism and fermentation-related genes
indicated that PEF stimulated cells by regulating the expression levels of
those genes. Cells could respond quickly to PEF stress by enhancing their
metabolic bio-synthesis rate. The up-regulation of these genes indicates that
the glycolysis reaction and metabolism rate are also enhanced. These results
confirm the positive effect of PEF in fermentation capacity.
In this study, variations of physiological behaviors were characterized of S. cerevisiae treated by moderate PEF treatment (1 kV·cm-1, 3 kV·cm-1). The
results showed that fermentation capacity was promoted which manifested in
increased consumption of glucose, increased production of metabolites (ethanol
and glycerol) and accelerated growth of yeast biomass. The changes in sugar metabolism
and fermentation-related gene expression levels also indicated that PEF
accelerates yeast fermentation capacity by inducing higher efficiency sugar
metabolism. In addition, we also attempted to clarify the
correlation between the fermentation phenotype and gene regulation in response
to PEF stress.
The electro-poration or electro-permeabilization,
which caused by PEF, is a well-recognized physical process in biological cells
that influences permeability and diffusion across membranes of cells (Garner
2019). It was reported that the higher uptake of
molecules and the rise of ions accumulation in cells under PEF stress (Pankiewicz et
al. 2017). In our work, the increasing fermentation capacity
after PEF treatment may be due to the increased permeability of the cell
membrane. PEF could also directly induce an increase in enzyme activity (Mannozzi et al.
2019; Benede and Molina 2020). We
speculate that the dynamic enzymes induced by PEF may also be one of the
reasons for the promoted fermentation capacity.
The changes in gene expression are consistent with PEF
fermentation phenomena. The glycolysis
pathway is the key one in the growth and metabolism of yeast. GPD2 plays a crucial role in
osmoregulation and redox balancing, by encoding the enzyme which controls the glycerol formation in the
absence of oxygen (Jagtap et al. 2019). Up-regulation
of gene GPD2 and higher glycerol yield were observed after PEF
stimulation or inducing in this work. Approved that oxidation stress response of genes (SOD1,
SOD2 and GSH1) were induced by PEF stimulation, and stress
responding caused by PEF was different from heat stress (Tanino et al.
2012). Reported nsPEFs (Nanosecond pulsed electric fields)
induced a transient activation of signaling pathways involving
mitogen-activated protein kinases (MAPKs) which
regulating diverse cellular programs including embryogenesis, proliferation,
differentiation and apoptosis (Morotomi-Yano and
Akiyama 2011). We speculated that PEF treatment induces higher glucose
uptake, and could also directly affect the proliferation of S. cerevisiae
by regulation of associated-genes.
Conclusion
In this study we
report the positive effects of moderate PEF on both metabolic activity and cell
growth of S. cerevisiae. Meanwhile, analysis of the transcriptional level
of genes involved in glucose metabolism also indicates that PEF contribute to S.
cerevisiae fermentation. It provides a promising method for fermentation in
industrial production. In order to obtain higher production efficiency, it is necessary to determine the optimum conditions in
actual production applications. It will be helpful to clarify the complexity
responses and mechanism to PEF stress on S.
cerevisiae. We believe that the combined use of PEF and other
techniques such as genetic modification is also feasible to meet the practical
production requirements.
Acknowledgement
This study was
funded by the National Natural Science Foundation of China (31401667).
Author
Contributions
Methods were devised by NJY, WJL and CKF. Experiments
were performed by WJL, QSM, SSL and the data analysis by SSL and LZ. The
manuscript was written by all authors.
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