Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e8, 2015

www.elsevier.com/locate/jbiosc

REVIEW

Advances in mechanisms and modifications for rendering yeast thermotolerance

Liman Gao,1 Yueqin Liu,1 Hun Sun,1 Chun Li,1 Zhiping Zhao,2 and Guiyan Liu1, *

School of Life Science, Beijing Institute of Technology, 5th Building, Zhongguancun South Street, Beijing 100081, China1 and School of Chemical Engineering and the Environment,
Beijing Institute of Technology, 5th Building, Zhongguancun South Street, Beijing 100081, China2

Received 30 June 2015; accepted 8 November 2015

Available online xxx
Thermotolerant Saccharomyces cerevisiae is widely regarded as an attractive strain with which to accomplish the
coupling of enzyme saccharification, ethanol fermentation and ethanol distillation in non-grain fuel bioethanol
fermentation systems, and it has many advantages for increasing the ethanol yield and reducing production costs. This
review provided an overview of the yeast heat-resistant mechanisms from six aspects, including gene expression re-
sponses, heat shock proteins, trehalose, ATPase, the ubiquitin-proteasome pathway and heat-induced antioxidant de-
fenses. Innovative methods, such as random and rational strategies for improving yeast thermotolerance, were further
discussed, and several special cases were provided. To rationally engineer thermotolerance in yeast, the advances in
employing heat-resistant mechanisms of thermophiles were particularly discussed. By designing and constructing heat-
resistant devices consists of heat-resistant parts from thermophiles to yeast, a superior thermotolerance of S. cerevisiae
has been achieved, providing a new system with important applications for research regarding the improvement of the
robustness of microbes.
Ó 2015, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Non-gain fuel bioethanol; Saccharomyces cerevisiae; Thermotolerant mechanism; Thermophiles;
Heat-resistant modification; Heat-resistant devices]

Non-grain fuel bioethanol has received considerable attention in
recent years as an economic, environmental friendly, practical and
renewable alternative to traditional energy sources. Simultaneous
saccharification and fermentation (SSF) is an attractive option
among all of the bioethanol production processes. Enzyme-induced
saccharification of polysaccharides and the subsequent fermenta-
tion of sugars to ethanol by yeast take place in the same vessel in
SSF, which enhances the enzyme hydrolysis rate of the poly-
saccharides due to the removal of end product inhibition (1).
Saccharomyces cerevisiae, which possesses high intracellular
enzyme activity, has an outstanding capacity to produce ethanol
and is fairly resistant to the inhibitors present in biomass hydro-
lysates. S. cerevisiae is currently the most widely used microbial cell
factory for producing non-grain fuel bioethanol. In addition,
S. cerevisiae is relatively tolerant to low pH values and high sugar
and high ethanol concentrations (2,3). Despite all of these advan-
tages, there are still several unsolved problems regarding the use of
S. cerevisiae as an ethanol producer in the SSF process. On the one
hand, thermal energy emission during the fermentation process
exposes yeast strains to elevated temperatures, which results in
significantly reduced cell viability and decreased ethanol produc-
tion. To address this problem, large quantities of cooling water and
cooling power are consumed, leading to high production costs. On
the other hand, most of the enzyme saccharifications have optimal
temperatures ranging from 45
C to 50
C, which are much higher
compared to the optimal temperature for yeast cell growth and
* Corresponding author. Tel.: þ86 10 68912140; fax: þ86 10 68913171.
E-mail address: gyliu@bit.edu.cn (G. Liu).
fermentation, of approximately 25e30
C, making it difficult to
synchronize the SSF process (4). Thus, breeding thermotolerant
S. cerevisiae has become a necessity in non-grain fuel bioethanol
fermentation systems. Performing fermentation at higher temper-
atures using thermotolerant yeast could not only achieve a higher
ethanol production with faster polysaccharide hydrolysis rates and
shorter SSF times but could also reduce the cost of cooling and the
rate of contamination (5). Moreover, high-temperature fermenta-
tion helps with simultaneous ethanol production and distillation
(45
C) (6). Hence, significant cost reductions and increased pro-
duction would be achieved if SSF is conducted using the thermo-
tolerant yeast.
An in-depth understanding of the mechanism of yeast ther-
motolerance provides a theoretical basis for breeding more robust
strains. In this review, we highlight and discuss, in detail, the heat-
resistant mechanisms of yeast, and review the recent advances in
breeding methods aimed at improving yeast thermotolerance,
including random and rational strategies. Rational strategies are
introduced from two aspects, namely, breeding thermotolerant
yeast based on its own heat-resistant mechanisms and based on the
heat resistance mechanisms of thermophiles.
YEAST THERMOTOLERANT MECHANISM
The comparison and analysis of the differences between ther-
mophilic and mesophilic yeast in terms of the enzymes, nucleic
acids, structure and function of the cell membrane and biosynthetic
systems have been the focus of early research on heat-resistant
mechanisms. Since the discovery of the heat shock phenomenon
in prokaryotes and eukaryotes, heat shock proteins (Hsps) and heat

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2015.11.002

Please cite this article in press as: Gao, L., et al., Advances in mechanisms and modifications for rendering yeast thermotolerance, J. Biosci.
Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.11.002
2 GAO ET AL.
shock response (HSR) have become the focal point of research, and
a new field has begun to expand in different directions (7). In
addition, heat shock can accelerate the production of large amounts
of reactive oxygen species (ROS), including superoxide anion (O 2 ),
hydrogen peroxide (H2O2) and highly reactive hydroxyl radical,
resulting in oxidative damage. Subsequently, S. cerevisiae produces
corresponding antioxidants against heat-induced oxidative stress
(8). So far, several molecules have been shown to be involved in
heat resistance, including Hsps, trehalose, ATPase, ubiquitin and
antioxidant enzymes, all of which play important roles in yeast
heat-resistant mechanisms.
Gene expression responses in heat-shocked yeast
S. cerevisiae, the expression of Hsps is increased through the
mediation of the so-called heat shock factor (HSF) after heat shock,
and the phenomenon was described as HSR. Four distinct HSF
members have been described in mammals and plants, termed
HSF1 to HSF4. However, yeast expresses only a single and
indispensable HSF, which is functionally equivalent to HSF1. In
most eukaryotes, HSF1 exists as an inactive monomer or dimer
with hidden acidic groups in the cytoplasm. Under heat stress,
HSF1 forms a homotrimer and binds to the heat shock element
(HSE) with repeating units of the sequence nGAAn to upregulate
the expression of Hsps. However, unlike in other eukaryotes, Hsf1
in S. cerevisiae is constitutively bound to HSE as a trimer under
normal temperature. Phosphorylation and other posttranslational
modifications stimulate Hsf1 activity and regulate the
transcription of Hsps after heat shock (8e10).
All aerobic microorganisms, including yeast, continue to
generate ROS during normal aerobic metabolism. ROS are toxic
agents that cause membrane lipid peroxidation, nucleic acid
damage and protein oxidation toxicity. Heat shock can accelerate
the production of ROS, leading to severe oxidative damage to cells,
and can induce the oxidative stress response. Thermotolerance in
yeast cells is tightly linked to intracellular ROS levels. Yeast cells
that are cultured anaerobically display a 102- to 103-fold increase in
thermotolerance than those grown under aerobic conditions (11).
The main regulators of the antioxidant response under oxidative
stress conditions are the transcription factors Yap1 and Skn7 (12).
In the absence of oxidants, Yap1 is primarily found in the cyto-
plasm. Upon H2O2 stress, Yap1 is activated by covalently interacting
with a sensor protein, Gpx3, causing it to move to the nucleus and
upregulate antioxidant gene expression (13). Contrary to Yap1,
Skn7 acts as a transcription regulator that remains in the nucleus.
Skn7 has a similar DNA binding domain to Hsf1 and plays a sup-
porting role in combining Hsf1 with HSE (8). In addition to the
Hsf1-mediated HSR and Yap1- or Skn7-mediated oxidative stress
response, another heat-induced stress response pathway, defined
as the environmental stress response (ESR), exists in S. cerevisiae
and is regulated by the transcription factors Msn2p and Msn4p (14).
Both Msn2p and Msn4p have been reported to bind to stress
response elements (STREs), which are composed of a conserved
pentameric core of CCCCT. ESR responds to a remarkable variety of
stressors and induces the expression of w200 genes with different
functions to resist stress (15e17).
Heat shock proteins and yeast thermotolerance Hsps are
needed by almost all organisms to resist high temperatures. Hsps
protect thermally damaged proteins from aggregation, refold
damaged proteins, clear irreversibly aggregated proteins and
improve the thermal stability of soluble proteins, SOD and proton
pumps in stressed cells. The thermotolerance of most Hsp-deficient
strains is recovered by the corresponding gene transfection and
expression (18). Moreover, Hsps function in cross protection, that
is, Hsps induced by a mild dose of one type of stress can
subsequently provide protection against a lethal dose of stress
(19). In yeast, the predominant Hsps, as molecular chaperones,
Please cite this article in press as: Gao, L., et al., Advances in mechanisms and modifications for rendering yeast thermotolerance, J. Biosci.
Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.11.002
J. BIOSCI. BIOENG.,
mainly include Hsp12, small Hsps (sHsps), Hsp70s, Hsp90s and
Hsp100, which are named and classified according to their
apparent molecular masses and functions (20,21). The Hsp70
chaperone system is the largest and highly conserved family of
Hsps. Most Hsp70s are constitutively expressed, and their
expression is further increased in stressed cells. Under non-stress
conditions, Hsp70s assist with the de novo folding of proteins,
whereas under stressed conditions, they participate in the
resolubilization of proteins that have aggregated and maintain
unfolding proteins in a soluble state (22). Moreover, Hsp70 acts
as a sensor for Hsf1-mediated cytoprotection (23). The protection
mechanisms of yeast Hsp90s are relatively complex and require a
In
large number of cochaperones to regulate the Hsp90 cycle (24).
Hsp90s participate in the maturation and stability of a special
class of client proteins, including kinases and transcription
factors. In addition, Hsp82 has been reported to be involved in
the DNA repair pathway under normal, unstressed conditions
(25). The expression of Hsp82 of S. cerevisiae increases 20 to 30
times when cells are cultured at 39
C than 25
C; however, at
lethal temperatures, the growth of the Hsp82 deficient strain is
similar to the growth of the wild strain, illustrating the
dispensability of Hsp82 in coping with extreme temperature
stress (26). In contrast to Hsp70s and Hsp90s, the yeast protein
Hsp104 functions as an oligomeric complex in extreme
temperatures. In S. cerevisiae, Hsp104 is essential for
thermotolerance. Hsp104D cells grow at the same rate as wild-
type cells when cultured at 25
C and 39
C; nevertheless, a 100-
to 1000-fold difference was found in the viability of the mutant
and wild-type cells after a temperature change to 44
C (27).
Hsp104 solubilizes protein aggregates in cooperation with Hsp70
and Hsp40. Recent study has shown that Hsp70 and Hsp40
prevent small aggregates from forming larger aggregates and that
the binding of these proteins to Hsp104 stimulates the prolonged
association of Hsp104 with aggregates, leading to efficient
disaggregation (28,29). Similarly, small heat shock protein Hsp26
interacts with damaged substrates in a mixed oligomeric
agglomeration to prevent the irreversible aggregation of its client
proteins (30). Hsp12 is a special molecular chaperone that is
related with the cell membrane and functions unlike almost all
other Hsps, including sHsp. Under physiological conditions,
Hsp12 dissolves in the cytoplasm and has an entirely amorphous
structure; however, under heat shock conditions, it associates
with yeast membranes in a helical structure. The interaction of
Hsp12 with the cell membrane stabilizes membrane fluidity and
enhances thermotolerance in yeast (21).
Trehalose and yeast thermotolerance When exposed to
high temperatures, S. cerevisiae can protect itself through the
regulation of trehalose biosynthesis. There have been numerous
studies on the correlation between thermotolerance and trehalose
(31e33). Trehalose is a well-known storage carbohydrate for yeast
and contributes to the stabilization of biological membranes,
proteins and nucleic acids under stress conditions (34,35).
Trehalose biosynthesis involves two steps. First, trehalose-6-
phosphate synthase (Tps1) catalyzes the conversion of glucose-6-
phosphate and UDP-glucose to trehalose-6-phosphate (T6P).
Second, trehalose-6-phosphatase (Tps2) dephosphorylates T6P to
trehalose (36). Moreover, the trehalose degradation pathway,
catalyzed by neutral trehalases (Nth) and acidic trehalases (Ath),
also exists in S. cerevisiae, forming the trehalose metabolism
cycle. Nth, encoded by the genes NTH1 and NTH2, and Ath,
encoded by the gene ATH1, can convert trehalose into glucose,
which accelerates the return of cells to normal growth after being
released from thermal stress (37). The trehalose metabolism in
S. cerevisiae is an intricate process. The TPS complex in
S. cerevisiae is composed of two enzyme subunits (Tps1, Tps2)
VOL. xx, 2015
and two regulatory subunits (Tsl1, Tps3). Under heat shock, TSL1
encoding Tsl1 is strongly induced, activating the TPS complex
largely for the synthesis of trehalose. When yeast recovers from
heat stress, Tps3 is phosphorylated and inhibits the activity of
Tps2, leading to the accumulation of T6P (38). The increased T6P
levels can, in turn, inhibit Tps1 activity, pausing the synthesis of
trehalose (39). Meanwhile, phosphorylation and activation of
Nth1 accelerate the degradation of trehalose (40). Different views
on the thermoprotection mechanism of trehalose have been
proposed. Hottiger et al. proposed that trehalose acts as a protein
stabilizer and an inhibitor of nonspecific protein aggregation
against thermal denaturation (41). Simola et al. have postulated
that trehalose, together with Hsp104, facilitates the
conformational repair of heat-damaged proteins in the cytosol
and the ER lumen and concluded that it has no role in the
thermoprotection of membranes engaged in vesicular traffic (42).
It is surprising to find that Tps1, rather than trehalose, is essential
for the maintenance of energy homeostasis and is a key player in
yeast survival during heat shock (43).
ATPase and yeast thermotolerance Heat shock can result in
a substantial decrease in cytoplasmic pH due to membrane per-
meabilization (44), which perturbs redox regulation in the
cytoplasm and reduces the cellular capacity to maintain pH
homeostasis. Proton-pumping ATPase can transport hydrogen
against the concentration gradient through membrane-integrated
glycoproteins, driven by the energy of ATP hydrolysis, thereby
generating a pH gradient and potential gradient across the
plasmalemma, activating membrane transport and keeping the
intracellular pH stable (45). Therefore, ATPase is essential for
thermotolerance in yeast. Pma1 Hþ-ATPase (P-ATPase), as a major
ATPase in yeast, has been reported to be responsible for
maintaining the structural integrity of cells (46), and for
thermotolerance in yeast as evidenced by the significantly
decreased resistance of mutant yeast with reduced PMA1
expression to lethal temperature (50
C). Prior exposure to sub-
lethal temperatures resulted in an increase in thermotolerance to
more severe and otherwise fatal stress, which is referred to as
induced thermotolerance. However, the results showed that the
induced thermotolerance of the mutant yeast was similar to that
of the parent strain, suggesting that Pma1 Hþ-ATPase is not
involved in the development of induced thermotolerance and
that the mechanisms of induced thermotolerance and basal
resistance to lethal temperatures are not identical (47). Moreover,
vacuolar Hþ-ATPases (V-ATPase) are critical for the maintenance
of pH homeostasis under heat shock in yeast. Mutants that are
defective in vacuolar Hþ-ATPases (vma2D, vma3D, and vma21D)
are among the most heat-shock sensitive strains (48).
Ubiquitin-proteasome pathway Proteins function optimally
only within a relatively narrow temperature range. Heat stress
leads to multiple changes, including the accumulation of delete-
rious proteins that result in changes in the microbial morphology
and phenotype and even in death. Heat-stressed cells initiate
various measures to protect themselves from proteotoxicity,
including the activation of the ubiquitin-proteasome pathway
(49), a highly effective protein degradation pathway in eukaryotic
cells that participates in many eukaryotic cellular processes,
including cell cycle regulation, receptor endocytosis, regulation of
transcription, signal transduction, gene silencing, DNA repair and
the stress response (50). The ubiquitin-proteasome pathway is a
cascade of reactions that involves ubiquitin (Ub), ubiquitin-
activating enzyme (E1), ubiquitin-conjugating enzymes (E2s),
ubiquitin-protein ligases (E3s), 26S proteasomes and
deubiquitinating enzymes. Under heat shock conditions, ubiquitin
covalently links to the lysine residues of thermally damaged
proteins in a reaction catalyzed by E1, E2s and E3s. The
Please cite this article in press as: Gao, L., et al., Advances in mechanisms and modifications for rendering yeast thermotolerance, J. Biosci.
Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.11.002
YEAST THERMOTOLERANT MECHANISM AND MODIFICATION 3
ubiquitinated protein is then recognized and degraded by 26s
proteasomes (51). Ubiquitin ligases play a large role in protecting
cells against acute proteotoxic stress (52). A number of ubiquitin
ligases acting in distinct compartments have been identified in
yeast. The Rsp5 ubiquitin ligase, acting in the cytosol, is
responsible for the thermotolerance phenotype and is identified
as the major constituent involved in the disposal of misfolded
proteins after exposure to heat (53,54). The Hul5 ubiquitin ligase,
also found in the yeast cytoplasm, is reported to maintain cell
fitness by degrading short-lived misfolded proteins (55). In
addition, some ubiquitin ligases, such as Hrd1, Doa10 and Rma1,
are found at the endoplasmic reticulum membrane and clear the
misfolded membrane proteins or proteins destined for secretion
(56). Moreover, yeast nuclear protein quality control is
accomplished mainly by ubiquitin ligase San1, which directly
binds to misfolded nuclear substrates and clears them by
recognizing hydrophobic patches exposed on the surface of
misfolded proteins (57).
Heat-induced antioxidant defenses In addition to thermal
insult, oxidative damage is one of the major secondary conse-
quences of heat shock. S. cerevisiae, like all organisms, contains
multiple antioxidant defense mechanisms to address oxidative
stress. Enzymatic antioxidant defenses, including a number of
protective enzymes, such as superoxide dismutase (SOD), catalase
and peroxiredoxin (Prx), are the predominant protective mecha-
nism against oxidative damage. SOD is one of the most important
antioxidants because it provides a protective role in vivo by con-
verting the superoxide anion to hydrogen peroxide. Two SODs that
exist in yeast are cytoplasmic Sod1 (Cu/Zn-SOD) and mitochondrial
Sod2 (MnSOD). ROS are mainly produced in eukaryotic mitochon-
dria, which means that mitochondria are the primary organelles
damaged by ROS. Sod2, which functions as a mitochondrial anti-
oxidase, protects the mitochondrial electron transport chain from
disruption and thereby maintains the intracellular redox environ-
ment (58). Yeast contains peroxisomal catalase A, encoded by CTA1
and a cytosolic catalase T encoded by CTT1. Catalase T appears to
play a broader role as an antioxidant because its expression can
be induced by multiple stress conditions (59).
Prxs serve various defensive functions, ranging from playing
roles as antioxidants or molecular chaperones to regulators of signal
transduction (60). Redox-active Cys residues detoxify peroxide in
peroxiredoxins and can be divided into 1-Cys Prx and 2-Cys Prx
based on the number of Cys residues involved in catalysis. Four
classes of 2-Cys Prxs (Tsa1, Tsa2, Ahp1 and Dot5) and one class of 1-
Cys Prx (Prx1) have been described in yeast. Tsa1, Tsa2 and Ahp1 are
cytoplasmic 2-Cys Prxs, all of which have thioredoxin peroxidase
activity, but possess diverse physiological roles. Tsa1 and Tsa2 are
characterized by having similar functions as antioxidants in the
reduction of hydroperoxides and as chaperones that can boost heat
resistance, but they differ in gene expression. Non-analogous to
Tsa1, Ahp1 mainly acts as an antioxidant in the detoxification of alkyl
hydroperoxides. Dot5 is located in the nucleus and has a weak
antioxidant activity, and its function is predominantly related to
telomeric silencing. Yeast Prx1 is a mitochondrial 1-Cys Prx and
displays a number of similar functions as the thioredoxin and the
glutathione systems in the mitochondria (8).
HEAT-RESISTANT MODIFICATION IN YEAST
Microbial strains form the basis of the fermentation industry.
Improving the thermotolerance of microbial strains can benefit the
fermentation industry by both significantly reducing energy con-
sumption and the use of water resources and by simplifying and
optimizing the fermentation process. Recent advances in breeding
methods to improve yeast thermotolerance have mainly focused on
4 GAO ET AL. J. BIOSCI. BIOENG.,

random and rational strategies. Two aspects of rational strategies elucidated. Accordingly, the understanding of yeast stress
are introduced, namely, breeding thermotolerant yeast based on its tolerance provided solutions for breeding yeast using various
own heat-resistant mechanism and the mechanisms of thermo- methods ranging from random strategies to rational methods.
philes heat resistance. Therefore, breeding yeast strains with improved thermotolerance
by regulating the heat-resistant genes of S. cerevisiae itself has
Random strategies for breeding thermotolerant
been widely used recently.
yeast Breeding yeast strains with improved thermotolerance is
Protein ubiquitination or poly-ubiquitination contributes to the
a hot topic in the fermentation industry. At present, random stra-
degradation of abnormal intracellular proteins after thermal insult,
tegies to improve thermotolerance in yeast mainly include classical
which has been reported to be a key process for improving heat
strain improvement methods, such as natural breeding, mutagen-
resistance in yeast. The overexpression of ubiquitin ligase Rsp5 and
esis, adaptation and modern biotechnology breeding, including
ubiquitin-conjugating enzymes results in improved multiple stress
genome shuffling.
tolerance, including thermotolerance, at a high growth tempera-
Classical strain improvement methods have effectively pro-
ture (39
C) (71). Shahsavarani has suggested that the over-
moted the development of the microbial industry because of their
expression of ubiquitin ligase Rsp5 contributes to the rise in the
simple operation and low technology threshold. The three ther-
upper limit of yeast thermotolerance (53).
motolerant S. cerevisiae strains, which can grow at 41
C and pro-
As a large leap in evolution, the emergence of biomembranes,
duce approximately 38 g/L ethanol with 10% glucose after 24 h at
which enable cells to exist independent of the environment, was
41
C, were isolated from Thai fruits. These strains may have ac-
vital for the maintenance of the biological processes of cells. Welker
quired thermotolerance by natural breeding under high tempera-
et al. have shown that Hsp12 is able to render thermotolerance in
ture in tropical area (61). By random respiration-deficient
yeast to resist lethal temperatures for a short time by stabilizing
mutagenesis using ethidium bromide, four S. cerevisiae strains
membrane fluidity (21). Caspeta et al. (63) improved the heat
named mbc 1e4 with a significantly higher growth and fermen-
resistance of S. cerevisiae by increasing the robustness of the cell
tation ability at 42
C were screened. SSF was performed with
membrane by changing the sterol composition of the cell mem-
thermotolerant mutant mbc 2 at 42
C. The results showed that the
brane through a nonsense mutation of C-5 sterol desaturase.
ethanol concentration and theoretical ethanol yields by mbc 2 in
In addition, thermal stress induces ROS insult, including lipid
48 h were 15.3 g/L and 90.1%, respectively, far higher than the 8.3 g/
peroxidation, nucleic acid damage and protein oxidation. Therefore,
L and 49.3% of the control (62). Evolutionary engineering approach
the fast removal of large amounts of ROS generated under heat stress
is also useful for rendering thermotolerance in yeast. Caspeta et al.
is the key to improving thermotolerance in yeast (72). The intra-
selected yeast strains with improved growth and ethanol produc-
cellular GSH content has a positive effect on the robustness of yeast.
tion at 40
C by using laboratory evolution (63). Based on this
It has been shown that the engineered yeast strain BY-G, with
method, Cakar et al. successfully obtained multiple stress-resistant
threefold higher GSH content, have a highly increased thermotol-
yeast strains, with the best yeast exhibiting a 89-fold increase in
erance and twofold higher ethanol concentration in SSF than control
thermal tolerance, a 62-fold increase in ethanol tolerance, a 1429-
(73). Nasution et al. (74) have also shown that thermotolerance in
fold increase in oxidative stress tolerance and a 102-fold increase
yeast can be improved by knocking out the
in freeze-thaw tolerance (64). In addition, continuous cultivation
glycosylphosphatidylinositol-anchored membrane protein coding
followed by haploidization, and mating also proved to be a
gene DFG5. dfg5D exhibited lower levels of ROS and decreased
powerful approach for enhancing the tolerance in yeast strains,
membrane permeability compared with the parent strain.
with a significant improvement in acid and heat tolerance (65).
Recent research in the acquisition of thermotolerant yeast has
Genome shuffling, which takes advantage of the genome di-
mainly focused on the activation and regulation of specific genes
versity derived from mutation and selection, can provide an effi-
that mediate heat resistance in yeast. Although this idea provides a
cient way to accumulate beneficial mutations by recursive
rapid and convenient method of developing thermotolerance in
protoplasts (66). The robustness of yeast under heat, osmotic and
yeast strains, it is limited by the heat-resistant capacity of the yeast
acid conditions as well as the bioethanol fermentation performance
itself and hardly generates desired thermotolerant yeast (75).
were improved after genome shuffling. Compared with parent
strains, the ethanol yields of the shuffled yeast increased by 10.31%
Microbial thermotolerance modification based on the
at 42
C (67). Genome shuffling was also applied to an ethanolo-
mechanisms of thermophiles heat resistance Recent studies
genic S. cerevisiae. After three rounds of genome shuffling, the re-
have shown great interest in utilizing thermophiles. Thermophiles
combinant strain R32 was obtained, with greatly improved heat,
can be divided into three groups based on the temperatures that
acetic acid and furfural tolerance; high cell viability at 45
C; and the
they are able to resist. Thermophiles can resist temperatures be-
ability to produce 81.4  2.7 g/L ethanol from 194.4  1.4 g/L
tween 50
C and 75
C, extreme thermophiles can resist tempera-
glucose after 42 h at 42
C (68).
tures between 75
C and 90
C and hyperthermophiles can resist
Classical methods for strain improvement and genome shuffling
temperatures greater than 90
C (76). Those thermophiles, which
allow for the modification of strains with poorly understood
can thrive even at extreme temperatures employ a number of
genome sequences and heat-resistant mechanisms and have been
distinct mechanisms. First, compared to mesophiles, the
successfully used to improve the thermotolerance of strains as well
intracellular components of thermophiles have a high intrinsic
as tolerance to multiple stressors in harsh surroundings. However,
stability and exhibit unique features that include special
classical methods for strain improvement and genome shuffling
membrane composition, high GþC% content, nearly perfect DNA
were time consuming, resource intensive and had poor stability. In
repair and replication mechanisms, and high protein thermal
addition, these methods often come at the cost of reduced fitness
stability due to their high content of specific amino acids, such as
and/or diminished biofuel production after random modification
Ile, Pro, Glu, Arg, and lower content of amino acids such as Cys,
(69,70), leading to the potential of being unsuitable for use for SSF
Ser, Thr, Asn and Asp (77,78). Second, thermophiles have a
process.
powerful protein synthesis machinery that ensures that proteins
Breeding thermotolerant yeast based on its own heat- can be synthesized at sufficient amounts for functional
resistant mechanism In the post-genome era, the functions of performance after heat shock (79). However, the translation-
a large number of genes have been demystified and the yeast related functions of mesophilic yeast are suppressed at high
response mechanisms and defense mechanisms have been temperatures to prevent unfolded proteins from tangling and

Please cite this article in press as: Gao, L., et al., Advances in mechanisms and modifications for rendering yeast thermotolerance, J. Biosci.
Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.11.002
VOL. xx, 2015
aggregating by reducing protein synthesis (80). Third, the
regulation of HSR of thermophiles was found to be drastically
different from that of yeast. In yeast, the regulators Hsf1, Msn2/4,
Yap1 and Skn7 are mainly responsible for the strong upregulation
of genes induced by heat shock. However, in thermophiles, the
critical regulator is a global s factor, which can constitutively
regulate gene expression to cope with heat shock (81). Finally,
the Hsp composition differs between thermophiles and yeast.
Two main classes of Hsp, Hsp100s and Hsp90s, are absent in
hyperthermophiles, leaving only Hsp60s, Hsp70s, sHsps, prefoldin
(PFD) and AAA (ATPase associated with various cellular activities)
proteins. This simplified protein renaturation and folding system
is expected to function as a powerful model for the study of more
complex chaperone systems (82).
Although the strategies of responding to thermal stress are quite
different between thermophiles and yeast, there are several com-
mon features. HSR and Hsps exist in thermophiles as they do in
yeast. Hsps in thermophiles have a greater ability to protect cells
from thermal killing. After heat shock, thermophiles can regulate
the synthesis of multiple chaperones, including the ATP-dependent
chaperonin and sHsp. These chaperones are involved in a
comprehensive range of protein-folding processes and enable
thermotolerance in thermophiles (82). In addition, heat shock
could lead to oxidative stress in both thermophiles and yeast. Some
antioxidant proteins, such as thioredoxin peroxidase, rubredoxin,
and SOD, are upregulated after heat shock in thermophiles (83).
Furthermore, metabolic pathways are changed in response to heat
shock to recognize and enhance the energy supply of the cell. For
example, proteomics evidence has demonstrated that the glycolysis
pathway-related proteins of thermophiles and yeast are all upre-
gulated to cope with heat stress by providing energy (79,84).
Mushroom development in synthetic biology paves the way for
designing more systematic, predictable, scalable and efficient bio-
logical systems (85). Applications of synthetic biology span across a
wide variety of fields, including industrial manufacturing, envi-
ronment, food, health and medicine (86). The biological parts are
the simplest and most basic building blocks that exhibit a basic
function in synthetic biology. Massive biological parts can be
characterized using bioinformatics tools, such as promoters, ter-
minators, operons, transcription factors and transcriptional factor
binding sites, ribosome binding sites and protein encoding regions
(87). Thermophiles, as microorganisms grown at extreme temper-
atures, can provide valuable biological parts including thermo-
zymes encoded genes, heat-inducible genes, and so on, which have
the potential to enable the generation of novel or more robust cell
factories (88e90).
Heat-inducible proteins are essential for thermotolerance in
both yeast and thermophiles. However, heat-inducible proteins
from thermophiles have proven to be superior to the native pro-
teins in terms of their thermostability and thermal protection
abilities (79,91). Therefore, heat-inducible genes from thermo-
philes are valuable biological parts that can be used to enhance the
heat-resistant defense system and improve thermotolerance in
yeast. The application of thermophiles for breeding thermotolerant
yeast strains is focused on the mining and construction of heat-
resistant devices using synthetic biology (89,90,92). Heat-
resistant devices with heat-inducible genes from thermophiles as
the functional parts and with regulator parts from S. cerevisiae are
designed and assembled using synthetic biology. Heat-resistant
devices are then introduced into S. cerevisiae to improve its ther-
motolerance by protecting critical intracellular components from
denaturation with an enhanced thermotolerant defense mecha-
nism (89,90).
Hsps and ubiquitins are important for cells to survive at high
temperatures. Liu et al. (89) constructed 10 engineered thermoto-
lerant yeast strains by introducing heat-resistant devices
Please cite this article in press as: Gao, L., et al., Advances in mechanisms and modifications for rendering yeast thermotolerance, J. Biosci.
Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.11.002
YEAST THERMOTOLERANT MECHANISM AND MODIFICATION 5
containing Hsp encoding genes and ubiquitin encoding genes from
Thermoanaerobacter tengcongensis, which were expressed with a
strong constitutive FBA1 (the gene coding fructose-bisphosphate
aldolase of S. cerevisiae) promoter. From the engineered strains,
T.te-Tte2469, T.te-Gros2 and T.te-IbpA were selected because these
strains displayed an increase in cell density of over 25% and a
significantly higher cell viability than control at high temperatures.
Particularly, strain T.te-Gros2 had a 4-fold increase in cell viability
compared to control after culture at 42
C for 72 h. Heat-inducible
genes TTE2496, GROS2 and IBPA encode ubiquitin, Hsp10 and
Hsp20, respectively. Overexpression of these heat-inducible genes
may endow yeast with increased thermotolerance by protecting
proteins from damage. In addition, it can indirectly affect and
upregulate heat-inducible genes in yeast, such as TPS1 and CDC19
(the gene coding pyruvate kinase of S. cerevisiae), resulting in
increased trehalose accumulation and energy supply, helping
strains withstand adverse heat stress. Furthermore, the engineered
strains showed greater resistance to Congo Red, a reagent that in-
terferes with cell wall integrity, indicating that these exogenous
heat-inducible proteins may contribute to the maintenance of cell
wall integrity under heat stress. Therefore, heterogenous expres-
sion of heat-inducible genes from thermophiles can efficiently
improve thermotolerance in yeast via multiple protective roles (89).
In another study, five HSP genes, GROES, HSP20, HSP33, DNAK and
CLPP, encoding Hsp10, Hsp20, Hsp33, Hsp70 and Hsp100, respec-
tively, from Thermus thermophiles were cloned. Corresponding
heat-resistant devices were constructed using synthetic biology
with the HSP genes as the functional parts and with a strong
constitutive FBA1 promoter and the SLM5 (the gene coding
asparagine-tRNA ligase of S. cerevisiae) terminator as the regulatory
part, and were implanted into S. cerevisiae. The thermotolerance of
S. cerevisiae was significantly enhanced by heterologous expression
of the GROES gene with a 29.2% higher increase in cell density after
72 h of incubation with a gradually increasing temperature and 3-
fold increase in cell viability after 48 h at 42
C compared to control.
In addition, the engineered strain is protected against oxidative
damage with a ROS level reduction of 36.7% at 42
C and cell
viability increase of 1.62-fold after treatment with 2 mM H2O2
compared to control. These results indicate that heat-resistant not
only improve thermotolerance but also contribute to the reduction
in heat-induced oxidative damage. Furthermore, the introduction
of heat-resistant device can improve the metabolic rate of ethanol.
The engineered S. cerevisiae displayed a 25% and 13.8% increase in
ethanol yield over control when cultured at 30
C and 40
C,
respectively (90). Thermophiles show superior robustness under
harsh conditions due to the adaptation of their thermal response
genes over the course of evolution. The use of heat-resistant
mechanisms found in thermophiles allows for quick modifications
for rendering yeast thermotolerance and has opened a new field of
technology. In addition, it provides a good reference for engineering
robust strains with other resistant properties. However, this
method is still in the early stages of development and no literature
exists describing the applications of the engineered thermotolerant
yeast in the SSF process. However, as a very interesting and more
robust method, it potentially has broad applications in ethanol
production by the SSF process and is likely to lead to new de-
velopments in the future.
CONCLUSION AND FUTURE PROSPECTS
S. cerevisiae is a potential bioconversion platform for non-grain
fuel bioethanol fermentation, and its ability to resist high temper-
atures is an important criterion for efficient bioethanol fermenta-
tion. Understanding the molecular and biochemical basis for yeast
thermotolerance enables the breeding of stress resistant strains
6 GAO ET AL.
based on the mechanisms of yeast heat resistance. This method, as
a rapid and convenient way to develop thermotolerance in yeast, is
limited, however, by the yeast’s own heat-resistant capacity, mak-
ing it difficult to obtain desired thermotolerant yeast. Extreme
thermophiles represent the limits of life with a complete set of
heat-resistant mechanisms, which provide a valuable library of
functional parts for improving the thermotolerance of microor-
ganisms. Different properties of heat-resistant devices are con-
structed through artificial design and optimization and are
assembled into host cells to render thermotolerance in yeast by
strengthening the heat defense mechanisms. Using the thermo-
philes’ mechanisms of heat resistance may become the most effi-
cient way of improving thermotolerance in yeast in the future and
therefore has a broad prospect for development.
However, the application of thermophiles to the breeding of
thermotolerant yeast still needs to be improved. The genetic basis
of yeast and thermophilic thermotolerance is a complex network
that is influenced by multiple genes and still needs to be fully
elucidated. Furthermore, the manipulation of yeast by the heter-
ogenous expression simply of one or a very few genes from ther-
mophiles is still difficult and not an efficient method for improving
the thermotolerant properties of yeast. Therefore, the objective and
focus of future research should be to further elucidate the ther-
motolerant mechanisms in yeast and thermophilic microorgan-
isms, which would provide a more detailed theoretical foundation
for the successful construction of thermotolerant yeast with effi-
cient rational strategies and greatly contribute to the improvement
of the production efficiency in microbial fermentations systems.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation of
China (grant number 21276024). The authors declare that they have
no conflict of interest.
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Please cite this article in press as: Gao, L., et al., Advances in mechanisms and modifications for rendering yeast thermotolerance, J. Biosci.
Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.11.002