Review

Adaptive response and tolerance to sugar and salt stress in the food yeast
Zygosaccharomyces rouxii
Tikam Chand Dakal, Lisa Solieri, Paolo Giudici
Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42122, Reggio Emilia, Italy
a b s t r a c t a r t i c l e i n f o
Article history:
Received 14 November 2013
Received in revised form 18 April 2014
Accepted 4 May 2014
Available online 25 May 2014
Keywords:
Zygosaccharomyces rouxii
Spoilage yeast
Osmotolerance
Halotolerance
Glycerol accumulation and retention
Cation homeostasis
The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to growand survive
in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na
+
cations)
solutes. This ability determines the success of fermentationonhighosmolarity foodmatrices andleads to spoilage of
high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits
shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation con-
trol, optimizationof foodstarter culture, and preventionof foodspoilage. This reviewsummarizes recent insights on
the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses.
Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts inte-
grate general and osmoticum-specic adaptive responses under sugar and salts stresses, including regulation of
Na
+
and K
+
-uxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte produc-
tionandaccumulation, andstress signalling pathways. We suggest howanintegratedandsystem-basedknowledge
on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in
food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms.
2014 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2. A matter of nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3. Osmotolerant and halotolerant yeasts in food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4. Gene circuits and metabolic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
4.1. Cell wall and plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
4.2. Cation homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4.2.1. Na
+
inward and outward movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4.2.2. K
+
inward and outward movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4.3. Sugar transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4.4. Production and accumulation of osmolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.4.1. Glycerol metabolic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.4.2. Glycerol biosynthesis in non-stressed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.4.3. Glycerol biosynthesis in osmo-stressed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.4.4. Glycerol retention and active transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.4.5. Other compatible solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5. Signal transduction and cis/trans-acting regulatory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.1. High osmolarity glycerol (HOG) pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2. Calcineurin/Crz1 pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5.3. Ras-cAMP signalling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
6. Non genetic regulation of osmostress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
6.1. Chromatin-mediated mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
International Journal of Food Microbiology 185 (2014) 140157
Abbreviations: CDRE, calcineurin dependent response element; CNV, copy number variation; CWI, cell wall integrity; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate;
HOG, high-osmolarity glycerol; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase;
P-Hog1, phosphorylated Hog 1; STRE, stress responsive element; SWI/SNF complex, switch/sucrose non-fermenting complex.
Corresponding author. Tel.: +39 0522522057; fax +39 0522522027.
E-mail address: paolo.giudici@unimore.it (P. Giudici).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.015
0168-1605/ 2014 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
International Journal of Food Microbiology
j our nal homepage: www. el sevi er . com/ l ocat e/ i j f oodmi cr o
6.2. Phenotypic heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
7. Food exploitation and biotechnological perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
1. Introduction
The high concentrations of ionic (mainly Na
+
) and non-ionic (mainly
sugars and polyols) solutes reduce water activity (a
w
) infoodandare two
of the major abiotic stressors, both limiting the yeast growth. High exter-
nal osmolarity has been used for centuries for food preservation, because
it causes water outowfromthe cell and results in a higher intracellular
concentration of ions and metabolites and in an eventual arrest of cellu-
lar activity. The yeast ability to cope with these environmental insults
determines both the success of certain food and beverage fermentation
and the thriving of food spoilage.
Since the sequencing of strain S288c (Goffeau et al., 1996), impressive
advances in genomics, proteomics, and systems biology have made
S. cerevisiae the paradigm for understanding these osmo-adaptive
mechanisms, which have been exhaustively summarized by several re-
views (Nevoigt and Stahl, 1997; Hohmann, 2002; Ario et al., 2010;
Khn and Klipp, 2012). As a result, the S. cerevisiae response to high
external solute concentrations has been described as a system-level
coordination between the extracellular environment and the genetic
make-up inside the cell. The following interconnected modules are in-
volved: (i) receiving information fromexternal environment (sensing);
(ii) conducting it to the inside (signal transduction); (iii) integrating it
with internal genetic information in order to mount an appropriate re-
sponse (effector processes) (de Nadal et al., 2011). This system-level
knowledge has been exploited in food industry to improve yeast fer-
mentations on highly salty and sugary matrices or to decrease the
food spoilage by sugar and salt resistant-yeast species. However, as
being moderately halotolerant and osmotolerant, S. cerevisiae could be
inappropriate to describe the yeast response to hypersaline and
hyperosmotic food.
Zygosaccharomyces rouxii is the osmotolerant and halotolerant yeast
most phylogenetically related to S. cerevisiae and inhabits a variety
of highly sugary and salty food, where it carries out fermentation or
determines food spoilage. It belongs to the genus Zygosaccharomyces,
which includes the highest number of salt and sugar-tolerant yeasts.
The majority of these species are osmotolerant (positive growth at
high sugar concentration up to 6070% glucose), whereas only a few
are both highly osmo and halotolerant (Table 1). Recently, the complete
genome sequences of Z. rouxii (Souciet et al., 2009) and other highly
osmo and halotolerant yeasts, such as Millerozyma farinosa (formerly
Pichia sorbitophila) (Louis et al., 2012), Debaryomyces hansenii (Kumar
et al., 2012), and Zygosaccharomyces bailii (Galeote et al., 2013), have
become available. Furthermore, omics tools and genetic manipulation
protocols have been recently employed to analyze the relationships
of osmostress phenotype to genetic and molecular determinants
(Prybilova et al., 2007a,b; Watanabe et al., 2010). From these intense
efforts, the yeast osmostress adaptation emerges as a complex mecha-
nism that integrates genes, regulatory networks, and signalling path-
ways, and that differs depending upon the species and the osmoticum
in the surrounding medium. Comparison of species with different
sugar and salt tolerance highlighted howyeasts exploit different strate-
gies to survive under osmotic and salt stress (Ramos et al., 2011). For
example, Z. rouxii resembles S. cerevisiae in extruding Na
+
cations out
of the cell or driving them into the vacuole (Ramos, 1999), while the
halotolerant yeast Debaryomyces hansenii is a sodium includer, which
accumulates intracellularly Na
+
without getting intoxicated (Ramos,
1999). Beyond the species-specic strategies, other osmostress re-
sponses, such as the osmolytes accumulation, are ubiquitous among
yeasts to avoid outow of cellular water in low a
w
environments
(Nevoigt and Sthal, 1997; Lages et al., 1999; Silva-Graa and Lucas,
2003). Another emerging issue concerns how salt and sugars elicit dis-
tinct or partial overlapping responses in yeasts. Whereas sugars and
polyols modify osmotic pressure, salts induce alterations both in osmot-
ic pressure and ion homeostasis. The result is that partially different
mechanisms become operational in response to sugar and salts. Since
halo and osmotolerance could be paired and unpaired phenotypes in
Z. rouxii and relatives, these yeasts are very attractive models for
deciphering genetic circuits and functional pathways underlying
halotolerance and osmotolerance.
Here, we review recent insights on the mechanisms that govern
halotolerance and osmotolerance in Z. rouxii and compare them to
those active in S. cerevisiae and in other osmo and halotolerant food
yeasts at genetic, metabolic, signalling, andepigenetic level. Furthermore,
we highlight how these yeasts can achieve generic and osmoticum-
specic responses to sugar and salt stresses. Finally, we point out how
the understanding of osmostress responsive mechanisms can advan-
tage microbial fermentation and food quality.
2. A matter of nomenclature
Tolerance to high ionic and non-ionic solute concentrations is a
specic cellular adaptability to sudden and severe uctuations in water
availability and a tendency of cells to restore or maintain normal physiol-
ogy, morphology and biological functions (Yancey, 2005; Klipp et al.,
2005). Microbial growth under high external osmolarity is frequently de-
scribed in terms of a
w
that is the chemical potential of free water in solu-
tion. Microorganisms able to colonize food with high osmolarity and,
consequently, low a
w
, were collectively indicated as xerotolerant (no ab-
solute requirement of low a
w
), and xerophilic (lovers of low a
w
) (Pitt
and Hocking, 2009) (Table 1). Amore appropriate microbial classication
would consider the kind of osmoticumand include the following catego-
ries: osmophilic, absolute requirement for non-ionic solutes and ability to
grow up to solute concentrations approaching saturation; osmotolerant,
no absolute requirement of non-ionic solutes for viability and ability to
tolerate a wide range of osmolarity, from hypo-osmotic to hyper-
osmotic solutions; osmosensitive, sensitive to excess concentration of
non-ionic solutes; halophilic, absolute requirement for high salt and
ability to grow up to salt concentrations approaching saturation;
halotolerant, no absolute requirement of salt for viability and ability to
tolerate a wide range of salinity, fromhypo-saline to hyper-saline solu-
tion; and halosensitive, sensitive to excess concentration of salt.
Most food yeasts can develop well at a
w
values around 0.950.90. A
cut-off of a
w
b0.70 has been frequently used to delineate osmotolerant
and halotolerant yeasts. In the past, yeasts isolated from sugary and
salty food with a
w
lower than 0.70 were referred to as osmophilic and
halophilic (Tokuoka, 1993). For instance, Debaryomyces hansenii has
been described as halophilic yeast based on the ability to grow at 1.0 M
of salt with growth rate and nal biomass close to the values obtained
without salt (Almagro et al., 2000; Gonzlez-Hernndez et al., 2004;
Aggarwal and Mondal, 2009). Other yeasts were classied as halophilic
or osmophilic, such as M. farinosa (formerly P. sorbitophila) (Rodrigues
de Miranda et al., 1980), Candida etchellsii (formerly Candida
halonitratophila), Candida versatilis (Barnett et al., 2000), and the black
yeast Hortea werneckii (Gunde-Cimerman et al., 2000). However,
differently fromhalophilic and osmophilic bacteria, none of these yeasts
satises the true denition of osmophily or halophily, because they
141 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
tolerate high osmotic conditions, without requiring high sugar or
salt amounts for their growth (Silva-Graa et al., 2003). Similarly, the
terms osmotolerant and halotolerant have been improperly used as
synonymous, but they should be reserved for yeasts that are able to
live at high sugar and ionic solutes (mainly Na
+
) concentrations,
respectively (Onishi, 1963; Tokuoka et al., 1992; Tokuoka, 1993;
Lages et al., 1999; Mareova and Sychrov, 2003; Pribylova et al.,
2007a,b). Since different solutes elicit distinct yeast stress responses,
osmotolerant and halotolerant phenotypes have been dened in
relation to the yeast ability to grow up to 5565% (w/v) sugar (lower
than ~0.88 a
w
) or at 1525% (w/v) salt concentrations (corresponding
to 0.920.85 a
w
range), respectively (Deak, 2006, 2007; Kurtzamn
et al., 2011). Based on these assays, tolerance to sugar and salt could
be differentially distributed among yeasts species. Table 1 shows the
classication of representative yeast species into four classes according
to their degree of sugar and salt tolerance.
3. Osmotolerant and halotolerant yeasts in food
Osmotolerant and halotolerant yeasts have a pivotal role in food
fermentation and spoilage (Fleet, 1992) and several of them belong to
the genus Zygosaccharomyces. The genus comprises 7 species (James
and Stratford, 2011) frequently isolated from highly sugary (honey,
jams, syrups, fruit-juices, fruit juice concentrates, chocolate candies,
and concentrated grape must) and salty food (soy sauce and miso
paste) (Dek and Beuchat, 1993; Tokuoka, 1993; Solieri et al., 2006,
2013a,b; Martorell et al., 2007; Suezawa et al., 2008). Zygosaccharomyces
sapae and Z. rouxii are the main biocatalysts of alcoholic fermentation in
high sugar and/or salt fermented food, such as traditional balsamic vin-
egar and miso, respectively (Suezawa et al., 2008; Solieri and Giudici,
2008; Solieri et al., 2013a). Furthermore, Z. rouxii andZygosaccharomyces
bailii are involved in alcoholic fermentation during kombucha
production. Zygosaccharomyces species have been also recognized as
one of the main spoilage yeasts in food industry due to their tolerances
to salt, sugar, and weak acid preservatives (Pitt and Hocking, 2009;
Fleet, 2011). In food manufacture, the yeast spoilage of products causes
severe economic loss and affects a variety of processed foods, including
bread, cereals, spices, dairy products such as cheese, spreads (marga-
rine), dressings, fondant, chocolate, fermented sauces (soy), soft drinks,
fruits, jams, and high-sugar fruit syrups (Stratford, 2006). In particular,
Z. bailii and Zygosaccharomyces lentus represent the most important
Zygosaccharomyces from the point of view of weak acid preservative
Table 1
Proposal for a classication of representative yeast species according to their halotolerance and osmotolerance behaviors.
Category Denition Species name
1
D-glucose %
(w/v)
NaCl (M); % (w/v) Food spoilage
2
References
3
50 60
Moderately osmotolerant
and moderately
halotolerant
Lack of growth
at N50% (w/v)
D glucose;
lack of growth
at N2.0M NaCl
Saccharomyces cerevisiae b1.70; 10% Soft drink, fruit juice Onishi, 1963
Schizosaccharomyces
pombe
1.00; 5.8% Cheese, fruit (rarely) Lages et al., 1999;
Barnett et al., 2000
Zygosaccharomyces
orentinus
(Zygotorulaspora
orentina)
+ 1.00; 5.8% Wine Lages et al., 1999;
Barnett et al., 2000
Candida glabrata + 1.70; 10% Juice concentrate Pitt and Hocking, 2009
Osmotolerant and
moderately
halotolerant
Growth at N50%
(w/v) D glu-
cose;
lack of growth
at N2.0M NaCl
Candida tropicalis + 1.72.0; 10-11.7% Fruit juice Barnett et al., 2000;
Deak, 2007;
Pitt and Hocking, 2009
Zygosaccharomyces
mellis
+ + 1.70; 10% Juice concentrate,
honey
Kurtzman et al., 2011
Zygosaccharomyces
sapae
+ + 2.0; 11.7% n Solieri et al., 2014
Zygosaccharomyces bailii + w 1.02.0; 5.8-11.7% Juice, sauces, ciders,
wines
Lages et al., 1999;
Barnett et al., 2000
Zygosaccharomyces
bisporus
+ + 1.02.0; 5.8-11.7% Soft drink, wine James and Stratford, 2003
Moderately osmotolerant
and halotolerant
Lack of growth
at N50% (w/v)
D glucose;
growth at
N2.0M NaCl
Candida parapsilosis + 3.0; 17.5% Dairy food Pitt and Hocking, 2009
Pichia membranifaciens + 3.00; 17.5% Bread, fermented milk,
olive
Lages et al., 1999;
Barnett et al., 2000
Issatchenkia orientalis
(Pichia kudriavzevii)
+ 2.0; 11.7% Olives, pickles and
sauces (rarely)
Lages et al., 1999;
Barnett et al., 2000
Osmotolerant and
halotolerant
Growth at N50%
(w/v) D glu-
cose;
growth at
N2.0M NaCl
Pichia sorbitophila
(Millerozyma farinosa)
+ + 3.04.0; 17.5-23.4% Beer, sake, soy sauce,
Mash of rice vinegar
Lages and Lucas, 1995
Zygosaccharomyces
rouxii
(allodiploid strains)
+ + 3.0; 17.5% Juice concentrate, honey,
jams, confectionery,
dried fruits, soy sauce
Lages et al., 1999;
Solieri et al., 2014
Candida magnoliae + + 3.0; 17.5% Sugary food Barnett et al., 2000:
Martorell et al., 2007
Pichia guillermondii
(Meyerozyma
guilliermondii)
+ + 3.0; 17.5% Olive, salt meat Butinar et al., 2005
Hortaea werneckii + n 5.20; 30.8% Salt sh Butinar et al., 2005;
Lenassi et al., 2011
Debaryomyces hansenii
(Candida famata)
+ + 3.04.0; 17.5-23.4% Olive Barnett et al., 2000;
Lages et al., 1999
Candida halophila
(Candida versatilis)
+ + 4.05.0; 23.4-29.1% Cheese brines Barnett et al., 2000;
Silva-Graa and
Lucas, 2003
1
Species names are reported according to the corresponding reference; current names are reported in bracket.
2
Information about food spoilage was retrived from Pitt and Hocking, 2009.
3
References used for growth data; , variable trait; n, not reported; w, weak.
142 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
resistance (Thomas and Davenport, 1985; Steels et al., 1999; 2002),
displaying anunusually high resistance to the small number of acids ap-
proved for use as food preservatives (primarily sorbic, benzoic, acetic
and propionic acids). On the other hand, Z. rouxii and Z. mellis can toler-
ate low a
w
with different sensitivity to ionic and non-ionic solutes.
Zygosaccharomyces rouxii is well-known for osmo and halotolerance
and survives up to 0.80 a
w
in presence of ionic solutes (salt) and up
to 0.65 a
w
in presence of non-ionic solutes (sugars), while Z. mellis
survives at low a
w
values only when the solutes are sugars (Stratford,
2006). Moreover, inter-strain differences in halotolerance have been
reported within Z. rouxii, with the strains isolated from high-salt foods
tolerating better NaCl than those isolated from high-sugar foods
(Pribylova et al., 2007b; Solieri et al., 2014). The highly salt-tolerant
strains are allodiploid strains arising from putative outcrossing events,
or aneuploid strains which differ from haploid Z. rouxii for karyotype,
ploidy level and copy number variation (CNV) of housekeeping genes
(Solieri et al., 2008, 2013b). This nding suggests that osmo and
halotolerance are two distinct physiological phenotypes.
Food yeasts other than Zygosaccharomyces species have been
described as being osmotolerant and/or halotolerant. Millerozyma
farinosa (formerly P. sorbitophila) is a ubiquitous, halotolerant yeast
found mainly in food (alcoholic beverages like beer and sake, soy
sauce, miso, mash of rice vinegar etc.) and it is known for the ability to
grow in more than 4.0 M NaCl (Lages and Lucas, 1995; Silva-Graa
and Lucas, 2003; Silva-Graa et al., 2003) and to tolerate up to 70%
glucitol (Rodriques de Miranda et al., 1980). Debaryomyces hansenii
was originally isolated from saline environments, such as seawater
and concentrated brines, and it is also associated with cheese and
meat processing. It can tolerate salinity levels up to 4.0 M NaCl and
survive in high-sugar products with a
w
values as low as 0.62 (as
reviewed by Aggarwal and Mondal, 2009). The strongly halotolerant
yeast-like fungus H. werneckii was found on salty food and other low-
water-activity substrates for its ability to grow, albeit extremely slowly,
in a nearly saturated salt solution (5.2 M NaCl), or completely without
salt, with a broad growth optimum from 1.03.0 M NaCl. In addition, a
group of poorly studied osmotolerant species have been associated
with spoilage of sugary food and with insects, including Candida
davenportii (Stratfordet al., 2002), Candida stellata andCandida magnoliae
(Rosa et al., 2003). Schizosaccharomyces pombe is an osmotolerant,
preservative-resistant yeast, but it is rarely associated to food spoilage
due to its salt-sensitivity (Pitt and Hocking, 2009).
4. Gene circuits and metabolic pathways
In presence of high extra-cellular solute concentrations, yeast cell
experiences three main physiological alterations: changes in physical
and chemical structure of the cell wall and plasma membrane; increase
of intracellular solute/ion toxicity; and alterations in the osmotic
pressure and cell volume. Therefore, three systems enable yeasts to
counteract stress challenges andto restore osmotic balance: a) regulation
of morphological and structural properties of the cell wall and plasma
membrane; b) modulation of transport systems; c) production, accu-
mulation and retention of metabolically compatible osmolytes.
4.1. Cell wall and plasma membrane
Morphological and structural properties of the cell wall and plasma
membrane are important factors affecting the yeast osmo and
halotolerance. By reshaping their integrity and uidity, yeast cell estab-
lishes a balance by which the force driving water across the osmotic
gradient into the cell is counteracted by turgor pressure against the
plasma membrane and cell wall (Klis et al., 2006; Levin, 2011).
The yeast cell wall is a rigid skeleton formed by four classes of
macromolecules interconnected by covalent bonds: the mannosylated
cell wall proteins called mannoproteins, 1,3--D-glucan, 1,6--D-
glucan and chitin (a polymer of GlcNAc) (Klis et al., 2006). Early studies
on cell wall composition suggested that Z. rouxii decreases cell wall
mannans in presence of salt (Hamada et al., 1984; Hosono, 1992).
These studies rstly suggested that cell wall rigidity and integrity have
been implicated in tolerance to salt-induced stress in Z. rouxii. Strain-
specic differences have been also described in the internal layer of
-D-glucan and cell wall mannans. In particular, Z. rouxii strains having
a more rigid cell wall tend to be less halotolerant than those having a
more exible and elastic cell wall (Pribylova et al., 2007b). Although
these variations are congruent with the possible involvement of cell
wall mannans in salt tolerance, more evidences are required to reinforce
such speculations. Recently, some highly salt-tolerant Z. rouxii strains
have been found to possess an increased copy number of FLO11 gene
that encodes a glycophosphatidylinositol-anchored cell surface glyco-
protein. This copy number amplication affects positively the cell wall
hydrophobicity and enables strains with a higher copy number of
FLO11 to exhibit a tness advantage compared to a reference strain
under osmostress static culture conditions (Watanabe et al., 2013).
Interestingly, in S. cerevisiae FLO11 is responsible for lamentation,
invasive growth, and biolm formation (Fidalgo et al., 2006) and it is
regulated by at least three well-known signalling cascades, such as the
Ras-cAMP pathway, the Mitogen-activated protein kinase (MAPK)-
dependent lamentous growth pathway, and the glucose repression
pathway (Verstrepen and Klis, 2006).
A few studies dealt with the regulatory pathways employed by
Z. rouxii to maintain and modulate the cell wall integrity in the face
of environmental challenges. In the two main model yeasts
S. pombe and S. cerevisiae, the pathway mainly responsible for regu-
lating cell wall changes is known as cell wall integrity (CWI) signal-
ling pathway. Upon osmotic stress, this pathway transmits wall
stress signals from the cell surface sensors to the Rho1 GTPase,
which mobilizes a variety of effectors. Activation of CWI pathway
regulates the production of various cell wall carbohydrate polymers
and their polarized delivery to the site of cell wall. Moreover, CWI
serves different functions other than the osmotic stress response,
such as the response against mechanical stress, cell shape maintain-
ing, and scaffold for cell-surface proteins (as reviewed by Levin,
2011). A fewproteins involved in CWI pathway have been associated
to osmo and halotolerance phenotypes. In S. pombe, the MAPK Pmk1
has been implicated in cell wall integrity, cytokinesis, and ion ho-
meostasis (Sengar et al., 1997). The MAPK kinase kinase (also
known as MEK kinase, MEKK or MAPKKK) Mkh1 and the MAPK ki-
nase (also known as MEK or MAPKK) Pek1 act as the upstream sig-
nalling components in the CWI pathway cascade and are essential
for Pmk1 activation. The involvement of Mkh1, Pek1, and Pmk1 has
been demonstrated in salt stress response of S. pombe (Madrid
et al., 2006) and, recently, also of S. cerevisiae (Rodicio and
Heinisch, 2010; Levin, 2011). In S. pombe, MKH1 gene-lacking cells
re-enter the cell cycle quite slowly after a prolonged arrest in stationary
phase and in the presence of NaCl or KCl they show a reduced growth
(Sengar et al., 1997). In S. cerevisiae, Kcs1 kinase, which is involved in ino-
sitol signalling, also ensures the cell wall integrity and consequently con-
fers adaptive responses to salt stress. The search for orthologous genes in
non-conventional yeast genomes has suggested that a similar pathway
could also operate in Z. rouxii (Rodicio and Heinisch, 2010).
Inside the cell wall there is the plasma membrane, which is involved
in a variety of cellular processes such as cell adhesion, ion conductivity,
and signalling. Like prokaryotes, yeasts regulate the plasma membrane
uidity in osmostress adaptation (Turk et al., 2011). The main factors
affecting the membrane uidity are the length, branching and degree
of saturation of fatty acids, the amount of sterols, and the phospholipid
composition (Russell, 1989; Rodriguez-Vargas et al., 2007). The remod-
elling of these parameters strongly affects not only the membrane uid-
ity, but also the proper functioning of membrane-attached proteins,
such as those involved in ion homeostasis, the glycerol transport
systems (Marquez and Serrano, 1996; Kamauchi et al., 2002), and the
plasma membrane ATPase activity (Coccetti et al., 1998).
143 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
When the external salinity is shifted fromlowto high concentrations,
S. cerevisiae shortens the fatty-acid chainlength (Turk et al., 2007) and in-
creases their saturation level by synthesizing fatty acid desaturases that
introduce double bonds into the fatty acids of membrane lipids (Levin,
2011). Whereas many eukaryotic organisms can synthesize dienoic
fatty acids, S. cerevisiae can introduce only a single double bond at the

9
position (Tunblad-Johansson and Adler, 1987; Sharma et al., 1996;
Rodriguez-Vargas et al., 2007). Under NaCl stress Z. rouxii increases the
amount of free ergosterol (decreasing the sterol-to-phospholipid ratio)
and reduces both the lipid unsaturation index and the phospholipid-
to-protein ratio (Watanabe and Takakuwa, 1984, 1987; Hosono, 1992;
Yoshikawa et al., 1995). The modications result in a reduction of mem-
brane uidity. On the contrary, in the extreme halotolerant yeast
D. hansenii high NaCl levels increase the sterol-to-phospholipid ratio
and fatty acid unsaturation, without signicantly affecting uidity
(Turk et al., 2007). In the strongly halotolerant yeast-like fungus
H. werneckii, high salinity conditions induce slight changes in the total
sterol content, but cause a signicant increase both in the phospholipid
content and the fatty acid unsaturation level (Gunde-Cimerman and
Plemenita, 2006). Therefore, H. werneckii tends to maintain the
sterol-to-phospholipid ratio signicantly lower than other yeasts, mak-
ing the plasma membrane comparatively more uid and offering higher
acclimatization to salt stress conditions (Turk et al., 2007).
Ionic andnon-ionic solutes have different effects onthe plasma mem-
brane. When the sunower (Helianthus annuus) oleate
12
desaturases
FAD2-1 and FAD2-3 genes are expressed in S. cerevisiae, they increase
the content of dienoic fatty acids, especially 18:2
9,12
and the plasma
membrane unsaturation index (Rodrguez-Vargas et al., 2007). Under
salt stress FAD-expressing cells display higher membrane uidity and
salt tolerance than the wild-type cells. In contrast, under high sorbitol
concentrations, the FAD-expressing cells do not differ in growth rate
from the wild-type cells, suggesting that the dienoic fatty acid content
doesnt affect the tolerance to non-ionic solutes. Although further
researches are required, these evidences collectively support that
there are distinct mechanisms in modulating the cell wall integrity
and plasma membrane uidity in response to ionic and non-ionic
stresses. Furthermore, it was demonstrated that certain osmolytes, es-
pecially trehalose, stabilize phospholipid bilayers during osmostress
conditions (Hounsa et al., 1998; Gancedo and Flores, 2004). In prokary-
otes, trehalose stabilizes lipid bilayers and prevents damages derived
from dehydration by inhibiting the fusion between vesicles and by
maintaining the membrane lipids in the uid phase (phase transition).
Similarly, in S. cerevisiae trehalose also protects plasma membrane
against osmotic stress (Iturriaga et al., 2009 and references herein).
4.2. Cation homeostasis
High concentrations of Na
+
cations are toxic to most living cells and
are frequent in food, while K
+
cations are less abundant, but they are es-
sential for compensating negative charges and activating key metabolic
processes, such as pyruvate synthesis and protein translation. Thus, the
majority of yeasts maintain a high intracellular ratio of K
+
/Na
+
, by se-
lectively accumulating K
+
and actively extruding Na
+
. Intracellular ho-
meostasis of these alkali metal cations affects physiological parameters
and functioning, such as cell volume, plasma membrane potential, and
intracellular pH (Arino et al., 2010). When cation concentrations in
the external medium are higher than the physiological range, the
difference between the electrochemical potentials of the cations across
the membrane may be so high that the entrance cannot be annulled by
simply inhibiting the transporters mediating the uptake. To avoid an
internal toxic cation concentration, different types of efux systems
have been evolved to balance any excessive entrance.
4.2.1. Na
+
inward and outward movements
Fig. 1 illustrates efux and inux systems recruited by Z. rouxii to
modulate the transport activity of the alkali metal cations across the
plasma membrane. Like S. cerevisiae, Z. rouxii has been regarded as a
sodiumexcluder species, for whichNa
+
is comparatively more cytotoxic
at high concentrations compared to K
+
(Pribylova et al., 2008). In
sodium excluder yeasts, two main mechanisms mediate the efux of
Fig. 1. Overview of the main plasma membrane systems mediating the alkali metal cation homeostasis and the glycerol uptake/retention in Zygosaccharomyces rouxii.
144 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
excessive cations (Na
+
or K
+
) through the plasma membrane. The rst
one is represented by the Na
+
(K
+
) P-type -ATPase, also known as so-
dium pump, encoded by ENA1-4 genes in S. cerevisiae, and by ZrENA1
in Z. rouxii. The yeast Na
+
(K
+
)-ATPases couple the ATP hydrolysis to
the cation transport against electrochemical gradients, and, unlike
Na
+
specic-ATPases from higher eukaryotes, they mediate both the
Na
+
and K
+
efux. They are indispensable for the growth at high Na
+
and K
+
concentrations in alkaline environments (Benito et al., 2002).
The second mechanism consists of Na
+
/H
+
antiporters encoded by
NHA1 in S. cerevisiae, SpSOD2 and SpSOD22 in S. pombe, ZrNHA1
and ZrSOD2-22 (with its variants ZrSOD2 and ZrSOD22) in Z. rouxii
(Watanabe et al., 1995; Hahnenberg et al., 1996; Bauelos et al., 1998;
Benito et al., 2002; Papouskova and Sychrov, 2007; Pribylova et al.,
2008) (Table 2). Based on the substrate specicity, the Na
+
/H
+
-
antiporters canbe further dividedintotwosubfamilies: those recognizing
both K
+
and Na
+
(and their respective analogues Rb
+
and Li
+
), such as
S. cerevisiae Nha1 and SpSod22, are involved in K
+
homeostasis and are
present in almost all hemiascomycetes, whereas those recognizing only
Na
+
(and its analogue Li
+
) as substrate, such as SpSod2, ZrSod2-22 and
ZrSod2, determine salt detoxication in yeasts most distant from
S. cerevisiae (Kinclov et al., 2002). Furthermore, a third less studied
mechanism, which operates in acute response to salt stress, entails
the sequestration of a surplus of toxic Na
+
cations in intracellular
compartments. A plethora of proton-coupled antiporters are included
in this process; in S. cerevisiae, the most studied ones are Nhx1, an
endosomal Na
+
/H
+
exchanger (Nass et al., 1997; Nass and Rao,
1998), and Vnx1, a vacuolar Na
+
and K
+
/H
+
exchanger (Cagnac et al.,
2007). An endosomal Na
+
/H
+
exchanger homologous to S. cerevisiae
Nhx1, has been also found in the halotolerant food yeast D. hansenii
(Moniel and Ramos, 2007).
Yeast salt tolerance signicantly depends on the genes encoding
Na
+
ATPases, plasma membrane and intracellular Na
+
/H
+
-antiporters
harboured in the yeast genome (Table 2). The sodium pump Ena1 is
the most important salt tolerance determinant in S. cerevisiae (Prior
et al., 1996; Bauelos et al., 1998). Deletion of ENA1 gene determines
the salt-sensitivity in S. cerevisiae (Haro et al., 1991; Marquez and
Serrano, 1996). In the salt-sensitive S. pombe, the single ENA-related
gene, called cta3, only mediates the potassium efux (Benito et al.,
2002). The expression of S. cerevisiae ENA1 in S. pombe markedly
increases the tolerance to Na
+
(Bauelos et al., 1995). Other studies
reported that S. pombe Sod2 antiporter complements the Na
+
sensitivity
in S. cerevisiae ena1 mutants, suggesting that antiporters or sodium
pumps can both be used by S. cerevisiae to regulate the internal sodium
concentration (Hahnenberger et al., 1996). In D. hansenii two ENA
parologs, namely DhENA1 and DhENA2, have been foundto complement
the salt sensitivity when heterologously expressed in S. cerevisiae ena1
mutants (Almago et al., 2001). Similarly, H. werneckii possesses two
highly salt responsive ENA genes, namely HwENA1 expressed in stressed
cells exposed to high salt concentrations, and HwENA2 that is mainly
expressed in stress-adapted cells (Gunde-Cimerman and Plemenita,
2006). More recently, it was demonstrated that upon initial imposition
of NaCl stress, S. cerevisiae extrudes intracellular Na
+
primarily by
Nha1, whereas the long term salt adaptation is mediated by the tran-
scriptional up-regulation of ENA1 gene (Proft and Struhl, 2004; Ruiz
et al., 2007; Ke et al., 2013).
In Z. rouxii, the salt tolerance has beenattributed to different variants
of Na
+
/H
+
antiporters and Na
+
-ATPase genes (Hahnenberger et al.,
1996; Pribylova et al., 2008). Like S. cerevisiae, Z. rouxii has a gene
encoding Ena1-homologous protein named ZrENA1 (Watanabe et al.,
1999; 2002). Transcriptional studies showed that, unlike S. cerevisiae
ENA1, ZrENA1 has little relevance in Z. rouxii salt tolerance, as, under
NaCl shock, the major Na
+
pumpout activity relies on the Na
+
/H
+
antiporter ZrSod22 (and their variants) (Watanabe et al., 1995, 1999).
Accordingly, under NaCl stress, Z. rouxii ena1 mutants and wide-type
strains exhibit similar growth rates (Watanabe et al., 1999). Other stud-
ies pointed out that Na
+
-ATPases and Na
+
antiporters almost serve
similar functions, but they are operational at different external pH
levels. In most sodium excluder species, Ena1 Na
+
-ATPase mediates
the Na
+
export mainly at high external pH levels. When the external
pH is lower than the cytoplasmic pH, the function of Ena1 Na
+
-
ATPase can be replaced by electroneutral Na
+
/H
+
antiporters, which
drive the Na
+
efux by the pH (Bauelos et al., 1998). Similarly, in
Z. rouxii Na
+
-ATPase and Na
+
antiporters have different pH sensitivity.
Since Z. rouxii is acidophilic yeast, ZrEna1 could not be active at the
low pH usually encountered by Z. rouxii in food (Watanabe et al.,
1999). More recent evidences have demonstrated that the Na
+
extru-
sion is mainly mediated by the Na
+
-specic Na
+
/H
+
antiporter
ZrSod-22 and not by the substrate-unspecic Na
+
/H
+
antiporter
Table 2
Cation transport systems associated to halotolerance in different yeast species (adapted from Ramos et al., 2011). Number of parologs is reported in brackets.
Category Gene Function Sc Sp Zr Dh Mf Ca Hw
Systems for K
+
inux TRK1 plasma membrane K
+
transporter belonging to HKTTRK family,
with a main role in K + homeostasis
+ + + + + + +(8)
TRK2 plasma membrane K
+
transporter belonging to HKTTRK family,
with a minor role in K
+
homeostasis
+ + + nd
HAK1 High Afnity K
+
-H
+
symporter belonging to the HAKKUP family + + +
ACU1 K
+
-Na
+
P-type ATPase functionally similar to plant HKT transporters + ps
Systems of K
+
efux TOK1 membrane depolarization activated K
+
channel + + + +(4)
Systems for Na
+
and K
+
efux NHA1 antiporter which uses a proton-motive force generated by the plasma
membrane H
+
-ATPase to mediate the efux of Na
+
, Li
+
, K
+
, and Rb
+
through the plasma membrane
+ + + + +(2) + +(8)
SOD2 antiporter which uses a proton-motive force generated by the plasma
membrane H
+
-ATPase to mediate the efux of Na
+
, and Li
+
through
the plasma membrane
+ + (v) nd +(2) nd
ENA1-4 P-type ATPase sodium pump; involved in Na
+
, K
+
Li
+
efux +(v) + + +(2) +(2) + +(4)
Intracellular cations/H + transporters NHX1 endosomal Na
+
(K
+
)/H
+
antiporter which it regulates the acidication
of cytosol and vacuole lumen
+ + + + + +(2) +(2)
KHA1 Putative K
+
/H
+
antiporter from Golgi with a probable role in intracellular
cation homeostasis
+ + + + +(2) + +(2)
VNX1 Vacuolar Na
+
(K
+
)/H
+
exchanger localized to the endoplasmic
reticulum membrane
+ + + + +(2) +(2) +(2)
Nomenclature according to S. cerevisiae genome, with the exception of HAK1, ACU1, and SOD2 genes. When no functional data have been available, BLASTP and TBLASTN analyses were
carried out to identify homologues of K
+
, Na
+
channel subunits, using the following queries: S. cerevisiae Trk1 (DAA08672.1); S. cerevisiae Trk2 (DAA09201.1); S. cerevisiae Tok1
(DAA08707); D. hansenii Hak1 (ABI37006); M. farinosa Acu1 (CAF22247.1); S. cerevisiae Ena1 (DAA09449.1); S. cerevisiae Nha DAA11888.1); S. pombe Sod2 (CAB.69632.1), S. cerevisiae
Nhx1 (DAA12290.1); S. cerevisiae Kha1 (DAA08706.1); S. cerevisiae Vnx1 (NP_014078.1).
Abbreviations: Sc, S. cerevisiae; Sp, S. pombe; Zr, Z. rouxii; Dh, D. hansenii; Mf, M. farinosa (formerly P. sorbitophila); Ca, C. albicans; Hw, H. werneckii; nd, not determined; ps, pseudogene; v,
inter-strains variability.
145 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
ZrNha1 (Prybilova et al., 2008). Synteny analysis demonstrated that
these two genes arose from a duplication event. Furthermore, Z. rouxii
genome experienced a CNV in ZrSOD genes. The Z. rouxii type strain
CBS 732
T
possesses one gene encoding the Na
+
/H
+
antiporter, namely
ZrSOD2-22, while the highly halotolerant allodiploid strains ATCC
42981, CBS 4838 and CBS 4837 have two copies, namely ZrSOD2 and
ZrSOD22 (Watanabe et al., 1995; Iwaki et al., 1998; Kinclov et al.,
2001; Solieri et al., 2013b). In the related species Z. sapae, two gene cop-
ies, ZrSOD2-22 and ZrSOD22, have been found (Solieri et al., 2013a).
Generally, CNV can induce important adaptive phenotypes in yeast
(Conrad et al., 2010). For instance, the expansion of gene repertoire
encoding alkali metal cation transporters has been related to the ex-
treme salt tolerance of H. werneckii (Lenassi et al., 2011). Although the
CNV in Na
+
/H
+
antiporter-encoding genes has been supposed to con-
tribute to the high halotolerance of Z. rouxii allodiploid strains
(Gordon and Wolfe, 2008), no experimental evidences support this hy-
pothesis. The ZrSOD2 gene encodes a functionally active antiporter in-
volved in salt stress response (Watanabe et al., 1995), whereas
ZrSOD22 is a poorly transcribed gene and its disruption doesnt affect
salt-tolerant phenotype (Iwaki et al., 1998).
In S. cerevisiae, the plasma membrane proton-pumping ATPase
Pma1 generates the electrochemical gradient required for nutrient
uptake and ionic homeostasis (Serrano et al., 1986). The PMA1 gene
transcription is tightly regulated and the Pma1 activity is controlled by
pHandglucose via phosphorylationof its C-terminal domain. Transcrip-
tional studies demonstrated that, under salt stress, Z. rouxii drives the
Na
+
efux via Na
+
/H
+
antiporter through the H
+
gradient created by
ZrPma1 H
+
-ATPase (Watanabe et al., 1995; Iwaki et al., 1998). The
ZrATP2 gene, which encodes a mitochondrial F
1
ATPase subunit, is
also involved in the ATP production and salt tolerance (Watanabe
et al., 2003). The disruption of this gene is lethal in Z. rouxii but not
in S. cerevisiae, suggesting that ZrATP2 is essential for maintaining
Z. rouxii viability and functioning (Watanabe et al., 2003). Based on
these evidences, it has been hypothesized that Z. rouxii is more efcient
in Na
+
extrusion than S. cerevisiae due to the cooperative action of
efcient H
+
-ATPase systems and Na
+
/H
+
antiporters with high Na
+
-
specicity (Watanabe et al., 2005).
4.2.2. K
+
inward and outward movements
Potassiumis anabsolutely essential element for the living organisms
and, althoughthe external K
+
concentration cangreatly vary depending
on the natural environment, it is in food much lower than what
metabolically required inside the cells (200300 mM). Therefore,
besides the Na
+
efux, yeast cells also require efcient systems for K
+
uptake and, when necessary, extrusion (Table 2). In S. cerevisiae, K
+
cat-
ions are continually taken up and extruded: the membrane potential
increases when the potassium inux is crippled and decreases in cells
defective for K
+
efux (Kinclova-Zimmermannova et al., 2006). The
K
+
uptake occurs mainly by facilitated diffusion through the high-
afnity transporters Trk1 and Trk2, and it is driven by the electrochem-
ical H
+
gradient across the plasma membrane generated by H
+
-ATPase
Pma1 (Michel et al., 2006). The homologous gene encoding a putative
potassium transporter, namely ZrTRK1, has been also characterized in
Z. rouxii (Stbn andSychrov, 2011) (Fig. 1). The functional expression
of the ZrTRK1 gene in S. cerevisiae trk1 trk2 mutants restores the
ability to grow at micromolar potassium concentrations, whereas
the Z. rouxii trk1 mutant grows more slowly than the wild-type
strain at low K
+
concentrations. Other non-conventional yeasts, such
as D. hansenii, possess the high afnity K
+
transport Hak1, which
works both as K
+
-H
+
symporter and K
+
-Na
+
symporter depending
upon the extracellular K
+
/Na
+
concentrations (Martnez et al., 2011)
(Table 2). The extremely halotolerant M. farinosa (formerly
P. sorbitophila) possesses a peculiar p-type ATPase encoded by the
ACU gene, which mediates the Na
+
and K
+
uptake at high afnity
(Benito et al., 2004).
In S. cerevisiae, the extrusion of K
+
cation surplus is mediated by
the Ena Na
+
(K
+
)-ATPase, the antiporter Nha1, and the voltage-gate
channel Tok1. While Nha1 and Ena ATPase are also involved in the
Na
+
detoxication, Tok1 represents the main system for the exclusive
K
+
extrusion in S. cerevisiae (Ahmed et al., 1999). Tok1 is activated by
the plasma membrane depolarization and contributes to regenerate
the membrane potential by releasing intracellular K
+
outside the cell.
Tok1 is also involved in the short term response to salt stress via the
HOG pathaway. Phosphorylated Hog1 (P-Hog1) has been recently pre-
dicted to inhibit the K
+
extrusion mediated by Tok1, leading to a plasma
membrane depolarization and a Na
+
inux reduction (Ke et al., 2013).
The plasma membrane depolarization could thus be a short term adap-
tation to osmotic (sorbitol) and ionic (Na
+
) stress, because it reduces
transporter activities and consequently the molecular import of the
cell. Similarly, the phosphorylation of Nha1 by P-Hog1 increases the
Na
+
cations efux under salt stress (Proft andStruhl, 2004), and inhibits
the K
+
efux under sorbitol stress (Kinclov-Zimmermannova and
Sychrov, 2006). In D. hansenii, the K
+
efux is mediated by the Na
+
/
H
+
antiporter DhNha1 (Velkova and Sychrova, 2006) and by the Na
+
(K
+
) pumps DhEna1 and DhEna2, which seem able to protect the cells
from sodium or potassium stress at alkaline pH (Almagro et al., 2001).
In Z. rouxii, the response to potassium surplus has been poorly investi-
gated. When ZrNha1 and S. cerevisiae Nha1 have been expressed
in S. cerevisiae lacking alkali metal cation efux systems (ena14
nha1), ZrNha1 was less effective than S. cerevisiae Nha1 in restoring
the tolerance to K
+
excess (Prybilova et al., 2008). Therefore, differently
fromorthologs in S. cerevisiae and D. hansenii, ZrEna1 doesnt seemto be
involved in potassium homeostasis.
4.3. Sugar transporters
In yeasts hyperosmotic stimuli trigger a variety of regulatory
mechanisms, which modulate the glucose uptake rate (Horak, 2013).
When glucose is available at high concentrations, S. cerevisiae uptakes
hexoses via facilitated diffusion, and only when glucose is scarce, it
uses the H
+
gradient and high-afnity symporters. In S. cerevisiae 17
HXT genes encode facilitated diffusion carriers (Boles and Hollenberg,
I997), but only 4 (HXT1-HXT4) are regulated in response to extracellular
glucose concentrations (Boles and Hollenberg, 1997; Ozcan and
Johnston, 1999). The HXT2 and HXT4 genes encode high afnity and in-
termediate afnity glucose transporters, respectively, which are up-
regulated at low glucose concentrations and down-regulated under
hyperosmotic stress (Wendell and Bisson, 1994). On the contrary,
HXT1 expression increases during the exposure to 1.0 M salt, 1.5 M sor-
bitol (Hirayama et al., 1995) or high sugar (40% w/v) (Erasmus et al.,
2003). The osmotic stress-induced HXT1 transcription depends upon
the HOG pathway (Rep et al., 2000), and it has been suggested to pro-
vide additional glucose for the glycerol synthesis (Hirayama et al.,
1995). Furthermore, high glucose concentrations stabilize HXT1 mRNA
transcripts, indicating that both transcription and mRNA turnover are
regulated in yeast osmo-adaptation (Greatrix and van Vuuren, 2005).
Zygosaccharomyces rouxii is fructophilic yeast which prefers to
consume fructose over glucose. Therefore, hexose transporters mediating
glucose facilitate diffusion have been poorly investigated. The search
for HXT orthologs in the Z. rouxii genome showed just one ORF
(ZYRO0D13310g) similar to S. cerevisiae HXT10 (http://genolevures.
org). In contrast, sugar facilitators for the fructose uptake, such as Ffz
proteins (fructose facilitator of Zygosaccharomyces) have been exten-
sively characterized in Z. bailii (Pina et al., 2004) and Z. rouxii (Leandro
et al., 2011). In particular, Z. rouxii has two low-afnity high-capacity
facilitators, ZrFfz1 and ZrFfz2, whichtransport bothfructose and glucose
when their external concentration is high (Fig. 2). More recently,
Leandro et al. (2013) characterized the high-afnity low-capacity
fructose-H
+
symporter ZrFsy1, which is up-regulated during the
growth of Z. rouxii at low extracellular sugar concentrations.
146 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
4.4. Production and accumulation of osmolytes
Compatible osmolytes are produced by yeasts to favour the cell
adaptation to osmotic stress. They signicantly maintain the water
balance, stabilize enzyme systems without interfering with the cellular
metabolism, and restore the original cell volume (Brown, 1990). To
assure high intracellular osmolyte concentrations, yeast cells regulate
the cell-cycle progression and take action against the requirement of
redox elements and energy demand.
Glycerol is the main osmolyte that is produced and accumulated
intracellularly in response to hyperosmotic stress, and also the most
studied one. The role of glycerol under varied osmotic conditions will
be discussed in detail. However, other less studied osmoprotective
osmolytes, such as trehalose, arabitol, mannitol and erythritol, also get
accumulate under certain conditions and therefore will be mentioned.
4.4.1. Glycerol metabolic pathway
Yeasts possess two well-established pathways for glycerol biosyn-
thesis, bothinitiating fromthe glycolytic intermediate dihydroxyacetone
phosphate (DHAP) and ending up with the production of glycerol:
(1) the Gcy-Dak pathway includes the dephosphorylation of DHAP to
dihydroxyacetone (DHA) by dihydroxyacetone phosphate kinase
(Dak) followed by the production of glycerol from DHA in a reaction
catalyzed by glycerol dehydrogenase (Gcy); (2) the Gpd-Gpp pathway
comprises the conversion of DHAP to glycerol 3-phosphate mediated
by glycerol 3-phosphate dehydrogenase (Gpd), and subsequently,
the dephosphorylation of glycerol 3-phosphate by glycerol-3-
phosphatase enzyme (Gpp), resulting in glycerol production (Fig. 2).
Even if the Gcy-Dak pathway seems to be active under a range of stress
conditions, under osmotic stress glycerol is mainly synthesized by Gpd-
Gpp pathway (Larsson et al., 1993; Albertyn et al., 1994; Eriksson et al.,
1995).
InS. cerevisiae, genes implicatedinglycerol productionare duplicated
in differentially regulated paralogs, namely GPD1 and GPD2 (Albertyn
et al., 1994; Eriksson et al., 1995), GPP1 and GPP2 (Norbeck et al., 1996;
Phlman et al., 2001a), and DAK1 and DAK2 (Norbeck and Blomberg,
1997; Rep et al., 2000). These genes arose from either single gene
duplication or whole genome duplication events, and show frequently
divergence and functional differentiation (Kondrashov et al., 2002;
Conant and Wolfe, 2008). For example, while GPP2 is mainly involved
in osmoadaptation, GPP1 has a role both in osmoadaptation and growth
under anaerobic conditions (Phlman et al., 2001a,b). Similarly, Gpd1
and Gpd2 enzymes have similar kinetic characteristics, but differ
with respect to cellular distribution and transcriptional regulation
(Albertyn et al., 1994; Eriksson et al., 1995). They are located in the
cytosol, but Gpd1 possesses a peroxisome-targeting sequence, while
Gpd2 is partly translocated into the mitochondria of non-respiring
cells (Valadi et al., 2004; Jung et al., 2010). The single deletion of GPD1
resulted in strains sensitive to osmotic stress (Albertyn et al., 1994),
while the deletion of GPD2 reduced growth under anaerobiosis (Rep
et al., 1999). Therefore, Gpd1 has a major role in osmoadaptation. How-
ever, neither the deletion of GPD1 nor the deletion of GPD2 resulted in a
noticeable change in glycerol yield. GPD1 and GPD2 genes could thus
have roles which parlty overlap to compensate compromised functions,
as recently shown for GPD2, which is up-regulated in response to GPD1
deletion (DeLuna et al., 2010).
The Gpd-Gpp pathway is the main metabolic route leading from
DHAP to glycerol also in Z. rouxii (Fig. 2). Accordingly, the heterologous
expression of ZrGCY1, but not of ZrGPD1, restores the glycerol produc-
tion and the salt tolerance in S. cerevisiae gpd1gpd2 mutants unable
to synthesize glycerol (Watanabe et al., 2004). Furthermore, it was
found that both the glycerol production and salt tolerance increase
when ZrGPD1 is expressed along with ScGPP2 (Watanabe et al.,
2004). Like in S. cerevisiae, in Z. rouxii allodiploid strain ATCC 42981
two isoforms of some glycerol synthesis genes have been found, such
Fig. 2. Interactions among redox balance, glycolysis, and glycerol production during the Zygosaccharomyces rouxii ethanol fermentation. Black, green and orange lines indicate the
enzymatic steps in glycolysis, Gpd-Gpp and Gcy-Dak pathways, respectively. Blue line indicates the relationship among glycolysis, Gpd-Gpp and Gcy-Dak pathways. Red lines represent
the remodelling inmetabolic uxes under bisulphite andosmostress conditions. All the dottedlines represent omittedsteps. The roles of glycerol metabolisminPi recycling, redox balance,
gluconeogenesis and fatty acid biosynthesis are reported in gray boxes.
147 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
as ZrGPD1 and ZrGPD2, ZrGPP1 and ZrGPP2, ZrGCY1 and ZrGCY2 (Iwaki
et al., 2001; Wang et al., 2002; Watanabe et al., 2004). On the basis
of their deduced amino-acid sequence, these proteins have a close
homology with the S. cerevisiae orthologs. ZrGPD1/ZrGPD2 and ZrGCY1/
ZrGCY2 are constitutively expressed in Z. rouxii cells, but their differen-
tial roles have not yet been investigated (Iwaki et al., 1999, 2001;
Watanabe et al., 2004).
4.4.2. Glycerol biosynthesis in non-stressed cells
Under anaerobic conditions, sugars are reduced to glycerol for main-
taining the cytosolic redox balance and consuming the excess of NADH
produced during the glycolytic pathway, amino acid biosynthesis and
organic acids anabolic routes (van Dijken and Scheffers, 1986; Prior
and Hohmann, 1997; Medina et al., 2010). The glycerol production is
also pivotal in lipid biosynthesis and recycling the inorganic phosphate
consumed during glycolysis via the dephosphorylation step catalyzed
by glycerol phosphatase (Nevoigt and Stahl, 1997) (Fig. 2).
The glycerol production was rstly studied in some non-
Saccharomyces yeasts that are naturally unable to produce glycerol
and grow on glucose under anaerobic conditions. It was observed that
their ability to produce glycerol can be restored by introducing oxygen
or another external electron acceptor in the culture medium. This effect
is called Custers effect (Nevoigt and Stahl, 1997). In subsequent
studies, it was found that a S. cerevisiae gpd2 mutant grows poorly in
anaerobic conditions without acetoin as an external electron acceptor
(Ansell et al., 1997; Bjrkqvist et al., 1997; Valadi et al., 2004). When
acetoin is added in anaerobic conditions, it is converted to butanediol
by NAD
+
-dependent butanediol dehydrogenase (Nevoigt and Stahl,
1997; Bjrkqvist et al., 1997). The GPD2 and GPP1 expression is stimu-
lated under anaerobic conditions, when the glycerol production
becomes essential for the redox balance and ethanol production
(Albertyn et al., 1994; Eriksson et al., 1995; Nevoigt and Stahl, 1997).
Under aerobic conditions, bisulphite ions induce the GPD2 transcription
andinhibit the nal reductive step inethanol fermentation, avoiding the
accumulation of excessive NADH (Ansell et al., 1997). Bisulphite ions
form a complex with acetaldehyde that limits the ethanol production
and promotes the reoxidation of glycolytically formed NADH by the
glycerol synthesis (Fig. 2). The effect can be reversed upon addition of
acetaldehyde (Ansell et al., 1997). All these evidences pointed out that
the function of GPD2 is mainly linked to redox imbalance, and not to
osmotolerance.
4.4.3. Glycerol biosynthesis in osmo-stressed cells
The cellular redox balance is pivotal for several aspects of cellular
physiology and its perturbation is implicated in the cellular adaptation
to sugar and salt-stress conditions. Under high external osmolarity, an
equimolar increase in cytoplasmic NADH is required to enhance the
glycerol production. This requirement seems to be partially fullled by
decreasing the acetaldehyde reduction to ethanol and increasing the
acetaldehyde oxidation to acetate (Fig. 2).
In S. cerevisiae, high external osmolarity induces the glycerol
production both under aerobic and anaerobic conditions. Depending
on the strain, medium, and process parameters, 4 to 10% of the carbon
source may be converted to glycerol. Under stress conditions, the
GPD1 and GPP2 genes are positively regulated by the HOG pathway to
produce glycerol (Albertyn et al., 1994; Eriksson et al., 1995). Besides
GPD1 and GPP2 genes, S. cerevisiae positively regulates the expression
of DAK1 gene encoding the Dha1 kinase (Rep et al., 2000). Accordingly,
glycolytic pathway enzymes are slightly repressed in cells exposed to
saline medium(Akhtar et al., 1997). Unlike S. cerevisiae, Z. rouxii doesnt
increase the expression of neither ZrGPD1 or ZrGPP2 in response to salt
stress (Iwaki et al., 2001) and the specic activity of the corresponding
enzymes remains unaltered (van Zyl et al., 1991). Although ZrGPD1 and
ZrGPP2 have a main role in glycerol production (Watanabe et al., 2004),
these results suggest that these genes are constitutively expressed in
Z. rouxii and by-pass the HOG pathway control. In contrast, ZrGCY1
and ZrGCY2 are up-regulated by salt stress, indicating that these genes
are target candidates of the HOG pathway (Iwaki et al., 2001). More-
over, differently from ZrGpd1, glycerol dehydrogenase ZrGcy1 and ki-
nase Zrdak1 increase the activities under hyperosmotic conditions (a
w
b0.96) (van Zyl et al., 1991).
Extreme halotolerant yeasts produce and accumulate large amounts
of osmo-protective metabolites. Although this feature has been widely
exploited in industrial bioprocesses (Nevoigt and Stahl, 1997), its
molecular mechanism has been poorly investigated. In the black
yeast H. werneckii, a set of 95 salt-responsive genes have been identied
and most of them have not previously been related to halotolerance
and HOG pathway in any other halotolerant yeasts (Vaupoti and
Plemenita, 2007). Furthermore, in H. werneckii the adaptation to high
amounts of NaCl and sorbitol involves the differential expression
of mitochondria-related genes (Vaupotic et al., 2008). Mitochondria
preferentially accumulate energy metabolism-related enzymes in
hypersaline medium, and chaperones and heat shock proteins, such as
Kar2 and Hsp60, in medium supplemented with sorbitol. Live-cell
imaging showed that the mitochondria condense differentally in
response to different osmolytes. In hypersaline medium, the mitochon-
drial condensation is accompanied by an increasing in ATP synthesis
and oxidative damage protection, whereas in presence of non-ionic
osmolytes it is accompanied by a decreasing both in ATP synthesis and
lipid peroxidation level (Vaupotic et al., 2008).
4.4.4. Glycerol retention and active transport
Besides de novo biosynthesis, the glycerol retention is effective to
prevent the massive outow of water from cells in response to an
osmotic stress. Being a liposoluble molecule, glycerol has the tendency
to be leaked out through the plasma membrane, and its retention has
thus to be an active response by the cell. Under osmotic conditions,
S. cerevisiae synthesizes a considerable glycerol amount most of which
leaks out of the plasma membrane (Hohmann, 2002). In contrast,
Z. rouxii synthesizes a smaller glycerol quantity than S. cerevisiae, and
changes membrane phospholipid and fatty acid compositions to
decrease the membrane uidity and permeability. This strategy entails
Z. rouxii to effectively retain polyols and maintain a very high gradient
between the intra and extracellular environments (van Zyl et al.,
1990; Pribylova et al., 2007a).
In S. cerevisiae, the glycerol enters into the cell by two different
mechanisms: a low afnity transport system (facilitated diffusion) and
a high afnity proton symport system (active transport) (Table 3).
Fps1 is an aquaglyceroporin belonging to the MIP family that is mainly
involved in the glycerol transport by facilitated diffusion (Sutherland
et al., 1997; Karlgren et al., 2005; Hohmann et al., 2007). FPS1 is consti-
tutively expressed in a salt-independent manner and mutants lacking a
region in the Fps1 N-terminal domain (amino acid residues from150 to
231) constitutively release glycerol (Tams et al., 2003). A shift from
low to high external osmolarity induces the Fps1 closure, whereas a
decrease in osmolarity causes the channel opening, both within seconds
after the change in external osmolarity (Luyten et al., 1995; Tams et al.,
1999). In the absence of osmotic stress, Fps1 is opened by the binding
of Rgc2 (regulator of the glycerol channel 2) to the Fps1 C-terminal
cytoplasmic domain. In response to osmostress Fps1 is closed by
P-Hog1, which binds the N-terminal cytoplasmic domain of Fps1 and
phosphorilates the positive regulator Rgc2 (Beese et al., 2009; Lee
et al., 2013; Petelenz-Kurdziel et al., 2013). Furthermore, Fps1 affects
the glycolipid and phospholipid composition of the plasma membrane
(Sutherland et al., 1997; Toh et al., 2001).
Several evidences show that the Z. rouxii ortholog to Fps1, namely
ZrFps1, conserves structural features and regulatory mechanisms.
ZrFps1 is a 692 aa-long protein characterized by long hydrophilic N
(228-LHQNPQTPTVLP-239) and C-terminal (537-HESPVNWPIATY-548)
domains, both sharing high homology with their S. cerevisiae counter-
parts (Tang et al., 2005). Since Z. rouxii fps1 mutants retain the ability
to growon glycerol as the sole carbon source, ZrFPS1 is not required for
148 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
the glycerol uptake but it is mainly involved in polyol efux, as reported
in S. cerevisiae (Luytenet al., 1995; Tang et al., 2005). Recently, Wei et al.
(2013) exploited the genome shufing to gain a highly salt-tolerant
Z. rouxii mutant strain, which, under a salt surplus, enhances the
ZrGPD1 transcription and reduces that of ZrFPS1. This nding supports
that in Z. rouxii salt adaptation relies both on the induction of glycerol
production and the inhibition of facilitated glycerol diffusion through
the plasma membrane (Hou et al., 2013). In other halotolerant yeasts,
like M. farinosa (P. sorbitophila) and D. hansenii, Fps1-like channels
have not been documented, suggesting that the glycerol diffusion
occurs in these yeasts through other membrane proteins (Table 3).
Active transport systems for glycerol have been documented in
several yeasts. Yet, little is known about their genetics. In a pivotal
study involving 42 yeast species, Lages et al. (1999) suggested that the
active glycerol uptake is essential in yeast halotolerance. These authors
found that only the most salt-tolerant yeasts show a constitutive active
glycerol uptake, which is highly efcient in the intracellular glycerol
accumulation against the gradient. The analysis of glycerol uptake in
presence of salts has identied two different types of constitutive active
glycerol transport systems, namely Na
+
-glycerol and H
+
-glycerol
symporters (Adler et al., 1985; Mareov and Sychrov, 2003) (Table 3).
In S. cerevisiae, glycerol is actively transported inside the cell by the
H
+
symporter (Lages and Lucas, 1997) encoded by STL1 (Ferreira
et al., 2005). The STL1 gene expression is repressed by glucose and
induced by nonfermentable carbon sources and by osmotic stresses in
a Hog1-dependent manner (Lages and Lucas, 1997; Rep et al., 2000;
Ferreira and Lucas, 2007). In S. cerevisiae, the glycerol transport is also
strongly inuenced by the GUP1 gene. The protein Gup1 has twelve
predicted transmembrane domains, which are compatible with a
transporter function, and the GUP1 gene disruption induces an osmo-
sensitive phenotype in S. cerevisiae (Holst et al., 2000). Based on these
evidences, Gup1 was rstly proposed as glycerol transport. Further
studies, however, have demonstrated that Gup1 and the paralog Gup2
are not active glycerol transporters, but regulatory elements with pleio-
tropic effects on cell wall phenotypes (Neves et al., 2004 and references
herein) (Table 3).
Inthe halotolerant Z. rouxii andD. hansenii, the expressionof glycerol-
Na
+
symporters requires salt, suggesting that NaCl is the driving force
for the glycerol accumulation under osmostress (Lucas et al., 1990;
van Zyl et al., 1990; Lages et al., 1999). More recently, two glycerol-
H
+
symporters orthologous to S. cerevisiae Stl1, namely ZrStl1 and
ZrStl2, have been documentedin Z. rouxii to mediate the glycerol uptake
under high salt concentrations (Neves et al., 2004; Bubnov and
Sychrov, 2011). The Z. rouxii genome harbours also a gene homologous
to GUP1, but so far no studies have established the role in glycerol trans-
port. Five polyol-H
+
symporters have been found in D. hansenii, with
different specicities and afnities for polyols (Pereira et al., 2014). In
M. farinosa (formerly P. sorbitophila), specic glycerol-H
+
symporters
are constitutively expressed and unresponsive to NaCl (Neves et al.,
2004) (Table 3).
4.4.5. Other compatible solutes
In response to hyperosmotic stress, compatible solute production is
regulated by a complex process depending upon the growth phase,
Table 3
Comparative overview of the main genes involved in glycerol transport in Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and other non-conventional yeasts.
Species Glycerol
dissimilation
Glycerol transport References
Type Gene Description Role in osmostress protein
(% identity)*
S. cerevisiae + AGT STL1 Main glycerol-H
+
glycerol symporter in
glycerol dissimilation; repressed by glucose
+ NP_010825.3, (/) Ferreira et al., 2005
AGT GUP1-2 Putative glycerol symporter involved in
glycerol uptake when glycerol is unique
carbon source; pleiotrophic effect on
wall-related phenotypes
NI NP_011431.1, (/) Holst et al., 2000; Ferreira et al.,
2006; Ferreira and Lucas, 2008
PGC FPS1 Aquaglyceroporin mediates glycerol diffusion
in presence of glucose; closed in response to
osmotic stress
NP_013057.1, (/) Luyten et al., 1995; Oliveira
et al., 2003
Z. rouxii + AGT ND Putative glycerol-Na
+
symporter mediates
glycerol accumulation as a function of
extracellular NaCl
+ / van Zyl et al., 1990; Lages et al.,
1999
AGT Putative
ZrSTL1/2
Putative glycerol-H
+
symporter important for
growth under hyperosmotic condtions
XP_002498998.1 (62%)
XP_002498999.1 (64%)
Bubnov and Sychrov, 2011
PGC ZrFPS1 Aquaglyceroporin closed in response to
osmotic stress
AAR29350.1 (51%) Neves et al., 2004; Tang et al.,
2005
D. hansenii + AGT ND Putative glycerol-Na
+
symporter mediates
glycerol uptake as a function of extracellular
NaCl
ND Lucas et al., 1990; Oliveria et al.,
1996; Lages et al., 1999
AGT DhSTL1 Glycerol/H
+
symporter + XP_459387.2 (62%) Lucas et al., 1990; Gonzlez-
Hernndez, 2010
PGC Absent / / / Prista et al., 2005
M. farinosa (formerly
P. sorbitophila)
+ AGT ND Putative glycerol-H
+
symporter constitutively
expressed
NI XP_004204191.1 (62%) Lages and Lucas, 1995
S. pombe - AGT SpGUP1 Membrane bound O-acyltransferase ND NP_592951.3 (39%) Neves et al., 2004
PGC SpFPS1 Aquaglyceroporin + NP_592788.1
Spac977.17p (52%)
Kayingo et al., 2004
C. halophila + AGT ND Glycerol-H
+
symporter constitutively
expressed
NI / Silva-Graa and Lucas, 2003
PGC Absent / / / Silva-Graa and Lucas, 2003
C. albicans + AGT CaSTL1 Glycerol-H
+
symporter mediates glycerol
uptake
+ CaO19.5753 (60%)
XP_718089.1
Kayingo et al., 2009
CaSTL2 No direct role in glycerol uptake + XP_720384.1 (38%)
PGC CaAQY1 Acquaporin for the passive diffusion of
glycerol in presence of glucose
NI XP_715831.1 (53%) Carbrey et al., 2001; Tang et al.,
2005
, ability to grow on glycerol as unique carbon source; *, protein identity was calculated with respect to orthologous protein in S. cerevisiae; AGT, active glycerol transporter; PGC, passive
glycerol channel; +, gene/protein positively regulated by hyperosmotic stress; , gene/protein negatively regulated by hyperosmotic stress; NI, gene/protein mot involved in
osmoadaptation; ND, not determined; /, not applicable; Sc, S. cerevisiae; Zr, Z. rouxii; Dh, D. hansenii; Sp, S. pombe; Ca, C. albicans.
149 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
carbon source, concentration and kind of stress agent (Yancey, 2005).
During the course of yeast growth and/or at increasing osmotic stress,
glycerol decreases in concentration and the osmoadaptation is mainly
conferred by other osmolytes, such as D-arabitol, erythritol, and treha-
lose (Tokuoka et al., 1992; Petrovi et al., 1999, 2002; Liu et al., 2006).
It has been speculated that the salt stress induces mainly C-3 (glycerol)
and C-4 (erythritol) polyols (Onishi and Suzuki, 1968; Plemenita et al.,
2008), while the sugar stress mainly C-3 (glycerol), C-5 (arabitol), and
C-6 polyols (mannitol and sorbitol) (van Eck et al., 1993). Under salt
stress, D. hansenii accumulates DHAP and increases glycerol production
by increasing the proteins involved in the rst steps of glycolysis and by
decreasing those involved in the Krebs-cycle (Gori et al., 2007). How-
ever, when both salt and polyols, such as erythritol and mannitol,
are present in medium, D. hansenii accumulates mannitol (during
exponential phase) and erythritol (during stationary phase) instead of
glycerol (Nobre and da Costa, 1985; Prista et al., 2005).
The disaccharide trehalose is one of the most effective
osmoprotectants that, under osmotic stress, prevents phase transition
events in the lipid bilayer, reduces the membrane permeability, and
ensures a proper protein folding (Elbein et al., 2003). Yeasts are able
to accumulate trehalose up to 15% of the cell dry mass when submitted
to a stress. The trehalose synthesis is catalyzed by a multimeric protein
complex (trehalose synthase complex), composed of four subunits
encoded by TPS1, TPS2, TPS3 and TSL1 genes (Franois and Parrou,
2001). The trehalose hydrolysis is catalysed mainly by the neutral
trehalase Nth1 (Kopp et al., 1994; Nwaka et al., 1995). It was demon-
strated that several yeast species synthetize trehalose under high
sugar and salt conditions following the same trend of the cell growth
and reaching the highest trehalose concentrations during the stationary
phase of cultivation (Tokuoka et al., 1992; Tokuoka, 1993). Hounsa et al.
(1998) also indicated that S. cerevisiae is more tolerant to osmotic
conditions during stationary than exponential phase, when it mainly
produces trehalose instead of glycerol.
The compatible osmolyte production depends also upon the carbon
source. In media with non-repressing galactose as the carbon source,
S. cerevisiae reduces the glycerol production, increases sensitivity to
osmotic stress, and mainly utilizes trehalose as osmolyte (Elbein et al.,
2003). Furthermore, under severe osmotic stress (0.866 a
w
), S. cerevisiae
reduces the intracellular glycerol content, indicating that this osmolyte
is essential for the yeast growth under moderate but not severe osmotic
stress (Albertyn et al., 1994; Hounsa et al., 1998). Hyperosmotic stress
also reduces the glycerol consumption and ethanol production rate,
but increases the intra-cellular content of trehalose (Norbeck and
Blomberg, 1997). Congruently, when the genes involved in trehalose
synthesis are deleted, the survival under NaCl stress of S. cerevisiae
mutants is considerably reduced compared to the wild-type cells
(Hounsa et al., 1998).
Arabitol and mannitol are other important compatible osmolytes,
but their role in osmoregulation is still unclear. In Z. rouxii, salt and
sugar stresses induce production of glycerol, arabitol or both, depending
upon the osmoticum which has been used to reduce the a
w
. If sugar is
used as stress agent instead of salt, D-arabitol is highly produced and
accumulated, whereas the glycerol concentration remains invariable
(van Zyl and Prior, 1990). Fructose and glucose-containing media have
been associated to mannitol production (Tomaszewska et al., 2012),
which is inhibited by salt (Onishi and Suzuki, 1968; van Eck et al.,
1993; Tomaszewska et al., 2012). The salt-sensitivity of S. cerevisiae
gpd1 and gpd2 mutants is complemented by the expression of
mannitol dehydrogenase gene (MDH) involved in the mannitol biosyn-
thesis (Watanabe et al., 2006).
5. Signal transduction and cis/trans-acting regulatory factors
The yeast adaptive osmostress response is mostly controlled by the
activation of signal transduction pathways, which in turn regulate the
dynamic interactions between transcription factors and specic
promoter binding sites (Posas et al., 2000; Rep et al., 2000; Hohmann
et al., 2000; Gasch et al., 2000; Causton et al., 2001). Reversible protein
phosphorylation catalyzed by protein kinases represents a universal
regulatory mechanismof multiple physiological andmetabolic functions
in the Eukarya, Bacteria and Archaea domains. Three molecular signal-
ling pathways up- and down-regulate several subsets of osmostress-
responsive genes and are highly conserved in different yeast species:
the HOG pathway, one of the ve MAPK pathways known in yeasts
(de Nadal et al., 2002; Hohmann, 2002); the calcineurin/Crz1 pathway,
which is specically involved in adaptation to high-salt conditions
(Clapham, 2007); and the Ras-cAMP signalling pathway (Thevelein
and de Winde, 1999; Norbeck and Blomberg, 2000) (Fig. 3). These path-
ways are well-characterized in S. cerevisiae, while they are only partially
studied in Z. rouxii and other halotolerant and osmotolerant yeasts.
5.1. High osmolarity glycerol (HOG) pathway
Osmostress adaptation involves the activation of the HOG signalling
cascade, which transduces osmosensory signals in yeast species
(Brewster et al., 1993). The cascade consists of three consecutively
activated kinases: MAPKKK, MAPKK and MAPK. In S. cerevisiae, the
osmotic stress signals perceived by the Slnl osmosensor is, in turn,
transmitted from Ypd1 to Sskl to Ssk2/22 (MAP kinase kinase kinase)
to Pbs2 (MAP kinase kinase) and nally to the MAPK Hogl (Posas
et al., 1996). On the other hand, Shol regulates the action of Hogl via
Stell (MAPKKK) (Maeda et al., 1995) and Pbs2 (MAPKK) (Posas and
Saito, 1997). Phosphorylationof Hog1by Pbs2is done at the neighbouring
Thr and Tyr residues, respectively at position 174 and 176 (TGY motif).
Once phosphorylated, active P-Hogl elicits both the immediate and
long-term adaptation to ionic stress (Alepuz et al., 2001; Ruiz and
Ario, 2007; Ke et al., 2013). As mentioned before, long-term adapta-
tion involves transcriptional and translational regulationof the genome,
whereas short-term adaptation is accomplished by changes in glycerol
accumulation (Albertyn et al., 1994) and the reestablishment of ionic
balance (Proft and Struhl, 2004).
Starting from the rst minute after the induction of salt stress,
P-Hogl directly phosphorylates some membrane ion transporters, such
as Nha1 and Tok1, in order to rapidly readjust the transmembrane
uxes of Na
+
and K
+
in osmo-stressed cells (Proft and Struhl, 2004).
Furthermore, cytoplasmic P-Hog1 regulates enzymatic activities that
are necessary to rapidly produce and accumulate glycerol (Klipp et al.,
2005). Such direct metabolic adjustments entail the cell to redirect
carbon resources toward the glycerol production. Finally Hog1 induces
a temporary arrest of cell-cycle progression in G
1
phase (as reviewed
by Saito and Posas, 2012).
During the long-termcellular adaptation to salt stress, P-Hog1 trans-
locates to the nucleus, where, by the phosphorylation of at least three
separate transcription factors (Msn2/Msn4 and Hot1) (Schmitt and
McEntee, 1996), it can modulate the regulation of more than 10% of
the total yeast genome (ORourke and Herskowitz, 2004). The Hog1-
regulated genes possess stress regulatory elements (STRE) with a core
sequence CCCCT or AGGGG in their promoter region or sometimes in
the coding region (Schller et al., 1994; Martinez-Pastor et al., 1996;
Wang et al., 2002; Tang et al., 2005). These cis-acting factors are variable
in number and orientation with respect to TATA box and they are nec-
essary for stress-induced gene expression both in S. cerevisiae and
Z. rouxii (Albertyn et al., 1994; Schller et al., 1994; Akhtar et al., 1997;
Norbeck and Blomberg, 1997; Wang et al., 2002; Tang et al., 2005). Al-
though a single copy of STRE is sufcient to bind a transcription factor
and activate the expression of a reporter gene, two or more
STRE copies induce a greater expression of stress-responsive genes
(Kobayashi and McEntee, 1993).
The transduction pathway of osmotic stress signals has not yet been
fully elucidated in Z. rouxii. Like in S. cerevisiae, in Z. rouxii the HOG
pathway comprises two operationally redundant plasma membrane
osmosensors Sln1 and Sho1 (Maeda et al., 1994, 1995), and from one
150 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
to two putative homologs to S. cerevisiae HOG1, namely ZrHOG1 and
ZrHOG2 (Iwaki et al., 1999; Kinclov et al., 2001). A TGY motif similar
to that of S. cerevisiae Hog1 has also been found in ZrHogl and ZrHog2,
suggesting that a putative ZrPbs2 MAPK kinase similar to S. cerevisiae
Pbs2 could exist also in Z. rouxii (Iwaki et al., 1999). Although some
Z. rouxii allodiploid strains possess two ZrHOG gene copies, namely
ZrHOG1 and ZrHOG2, there is no relationship between this gene redun-
dancy and their osmotolerance (Kinclov et al., 2001). This could mean
that ZrHOG1 and ZrHOG2 are expressed differentially, or that one of
these paralogs is transcriptionally silent, as it happens for ZrSOD22.
ZrHOG1 and ZrHOG2 genes are functional as MAP kinase and are
able to complement the salt-sensitivity in S. cerevisiae hogl null mu-
tants (Iwaki et al., 1999). However, their overexpression in wild-type
S. cerevisiae doesnt improve the glycerol synthesis, indicating that the
amount of ZrHog1 andZrHog2is not a limiting factor inthe glycerol pro-
duction. These studies collectively supported that Z. rouxii possesses a
HOG pathway functionally equivalent to that of S. cerevisiae.
STRE motifs are species-specically distributed in the yeast genome,
so that each species has peculiar sets of osmo-responsive genes that
contribute to functional diversities in response to osmotic cues. Under
high osmolarity conditions, S. cerevisiae produces much more glycerol
than Z. rouxii, by up-regulating genes involved in glycerol synthesis via
HOG pathway (Pribylova et al., 2007a). Accordingly, the S. cerevisiae
GPD1 gene exhibits four STRE elements in its promoter region
(Albertyn et al., 1994) and encodes a Gpd1 enzyme with higher activity
than ZrGpd1 (Akhtar et al., 1997; Norbeck and Blomberg, 1997). Like
S. cerevisiae P-Hog1, P-ZrHogl also translocates into the nuclear
compartment and activates transcription factors homologous to
Msn2p/Msn4p and Hot1, thus leading to the transcription of STRE-
controlled genes. However, STREs were not found in the ZrGPD1-2 pro-
moter regions, indicating that their expression is salt and HOG
pathway-independent. The lack of STREs may account for the moderate
glycerol amount produced by Z. rouxii to cope high external osmolarity.
On the contrary, a putative STRE motif with core CCCCT sequence has
been identied in the upstream region of ZrFPS1, which is missing in
S. cerevisiae FPS1 (Tang et al., 2005). Therefore, under high salt concen-
trations, only the ZrFPS1 gene transcription is regulated via the HOG
signalling pathway, resulting in the higher ability of Z. rouxii to intracel-
lularly retain glycerol compared to S. cerevisiae. In contrast, S. cerevisiae
P-Hog1 transiently induces the closure of Fps1 channel during the short
term salt response, but it cannot induce the FPS1 transcription during
the long term salt adaptation.
5.2. Calcineurin/Crz1 pathway
The calcineurin/Crz1 signal transduction pathway has an important
role in cation homeostasis and salt stress adaptation, but it is relatively
less investigated compared to HOG pathway (Matsumoto et al., 2002;
Ke et al., 2013). Calcineurin, the Ca
2+
/calmodulin-regulated protein
phosphatase 2B, is a heterodimer containing a catalytic (A) subunit
complexed with an essential regulatory (B) subunit and requires Ca
2+
and calmodulin for activity (Cyert et al., 1991). Calcineurin controls
Crz1 activity by regulating its subcellular localization (Stathopoulos-
Gerontides et al., 1999). When calcineurin-dependent signalling is
Fig. 3. Integrated overviewof signalling transmission pathways involved in osmoadaptive gene regulation. Genes are referred to as Saccharomyces cerevisiae genome, with the exception
of ZrFPS1 and ZrGCY1/2. Genes regulated by calcineurin pathway are reported in bold.
151 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
low, Crz1 is phosphorylated and resides primarily in the cytosol. Upon
dephosphorylation by calcineurin, Crz1 enters into the nucleus and
here it regulates the expression of target genes with Calcineurin
Dependent Response Elements (CDREs) in the promoter region. These
cis-elements consist of a common sequence motif, 5-GAGGCTG-3,
which Crz1 binds through a C2H2 zinc nger motif (Stathopoulos and
Cyert, 1997; Matheos et al., 1997). Around 163 genes have been found
under control of Calcineuirn/Crz1 pathway (Yoshimoto et al., 2002).
Among them, the GSC2 gene encodes a subunit of beta-1, 3 glucan
synthase, which is responsible for the synthesis of 1, 3-beta-D-glucan
(Levin, 2005), and it is up-regulated after exposure to high sugar
concentrations (Erasmus et al., 2003). CDRE motif was also found
in the anking region of ENA1 (Kafadar and Cyert, 2004). High cytosolic
Ca
2+
levels activate calcineurin in response to extracellular hyperionic
stress and increase Na
+
efux by up-regulating the ENA gene
(Matsumoto et al., 2002; Ruiz and Arino, 2007). Ke et al. (2013) demon-
strated that S. cerevisiae coordinates the HOG and the calcineurin
pathways to achieve both immediate and longer-term adaptation to
NaCl stress, respectively. In Torulaspora delbrueckii, a species close to
Z. rouxii, crz1-null cells were insensitive to high Na
+
concentrations,
indicating that TdCrz1is not requiredfor the salt-induced transcriptional
activation of TdENA1 gene (Hernandez-Lopez et al., 2006). This evi-
dence suggests that yeast species could differ in regulating salt response
via calcineurin/Crz1 pathway.
5.3. Ras-cAMP signalling pathway
The yeast cAMP-dependent protein kinase (PKA) is the effector
kinase of the Ras-cAMP signalling pathway. It is a conserved serine/
threonine protein kinase, which, through the phosphorylation of differ-
ent targets, has pleiotropic effects onthe cell growth, trehalose andglyco-
gen metabolism, dimorphic shift, and stress adaptation (Smith et al.,
1998). Proteomic approaches were exploited to study the inuence of
PKA on protein expression during the exponential growth of
S. cerevisiae under osmotic stress (Boy-Marcotte et al., 1998). Proteins
up-regulated under NaCl stress are grouped into three classes as regards
the PKA activity: i) PKA-independent proteins (Gpd1, Gpp2 and Dak1);
ii) fully PKA-dependent proteins (Tps1 and Gcy1); iii) partly PKA-
dependent proteins (Eno1, Tdh1, Ald3, and Ctt1) (Boy-Marcotte et al.,
1998). Osmo-response seems to be mediated by PKA both at transcrip-
tion level through the inhibition of STRE-dependent genes expression
(Thevelein and de Winde, 1999), and at post-translational level through
the regulation of trehalose synthesis (Kobayashi and McEntee, 1993). In
particular, Ras-cyclic AMP pathway negatively regulates Msn2/Msn4
that are cytoplasmically accumulated, leading to the inhibition of
osmostress response (Grner et al., 1998) (Fig. 3).
6. Non genetic regulation of osmostress tolerance
Understanding the non-genetic regulationof cellular stress response
is an important biological question and it has received considerable
attention in recent years. In relation to yeast osmotic tolerance, two
non-genetic mechanisms have been implicated so far. The rst one
includes epigenetic alterations in the chromatin structure that induce
genome-wide and local changes in gene transcription. The second one
is the non-genetic cell-to-cell phenotypic heterogeneity within an
isogenic cell population under osmotic stress conditions.
6.1. Chromatin-mediated mechanisms
Post-translational modication of nucleosomal histone proteins and
DNA methylation are two extensively characterized epigenetic mecha-
nisms that regulate gene expression in plants grown under osmotic
stress conditions (Chinnusamy and Zhu, 2009; Grativol et al., 2012).
On the contrary, few studies have dealt with this topic in yeasts. The
rst evidence about the epigenetic control of osmo-responsive genes
in yeasts was provided for the protein Sgd1, which is homologous in
its N-terminal domain to Spt7, a subunit of the nucleosomal Spt-Ada-
Gcn acetyltransferase (SAGA) histone acetylation complex (Roberts
and Winston, 1997). The SGD1 overexpression is able to partially
complement growth defects in S. cerevisiae hog1 and pbs2 mutants
and to increase their glycerol production (Akhtar et al., 2000). Other
studies suggested that changes in the chromatin structure contribute
to the osmostress-stimulated expression of GPD1 gene. Under osmotic
stimuli, the transcriptional repressor activator protein Rap1 binds the
GPD1 promoter and induced the GPD1 transcription. On the contrary,
the specic inactivation of all Rap1 binding sites completely abolishes
the osmostress-induced transcription of GPD1 (Eriksson et al., 2000;
Morse, 2000).
A third evidence for the epigenetic regulation of cellular osmo-
adaptiation involves the HOG pathway. Under osmotic shock, Hog1
activates the transcription factors Hot1 and Msn2/4 which mediate
the recruitment of Rpd3-Sin3 histone deacetylase (de Nadal et al.,
2004). Active Rpd3-Sin3 complex binds to specic promoters leading
to histone deacetylation, entry of RNA polymerase II, and transcription
initiation of osmoresponsive genes (Alepuz et al., 2003; de Nadal et al.,
2004). Upon unstressed conditions, the transcription factor Sko1,
which is related to bZIP/ATF family of transcriptional regulators,
represses the ENA1 transcription by binding CDRE (Proft and Serrano,
1999). Under hypertonic stress, Sko1 is phosphorylated by Hog1 and
recruits the SAGA histone deacetylase and the switch/sucrose non-
fermenting (SWI/SNF) complex. The latters are transcription activators
which promote chromatin remodelling (Prot and Sturhl, 2002) and
induce the ENA1 expression in conjunction with Calcineurin/Crz1
mediated pathway (Prot and Serrano, 1999). Yeast mutants lacking a
functional SWI/SNF complex are less tolerant to NaCl than wild-type
cells (Prot and Sturhl, 2002). Further studies are required to establish
whether similar epigenetic regulatory mechanisms also exist in
Zygosaccharomyces yeasts.
6.2. Phenotypic heterogeneity
Phenotypic heterogeneity is a super-organismfeature of prokaryotes
andyeasts that provides a dynamic source of diversity andadaptive phe-
notypes and increases the microbial tness in stressful environments
(Avery, 2006). Within an isogenic (genetically uniform) microbial pop-
ulation in a homogenous environment, individual cells can still exhibit
differences in phenotype. The precise mechanisms of such cell-to-cell
heterogeneity are elusive and few studies have linked variations in
yeast morphology to molecular effectors. Hsieh et al. (2013) have
highlighted that reduction in the intracellular amount of chaperon
protein Hsp90 triggers morphological heterogeneity in Z. rouxii clonal
populations. Under standard conditions high Hsp90 levels assure the
stability of Cla4, a key regulator of spectrin formation that inhibits
morphological switching of the cell from budding to lamentous
growth. Under salt stress, low Hsp90 levels reduce the Cla4 stability
and stimulate the cellular switching towards a lamentous form.
Additionally, the stress-induced Hsp90 inhibition is known to favour
chromosomal instability and aneuploidy, which in turn potentiate the
cellular adaption to stressful environments (Chen et al., 2012). Interest-
ingly, aneuploidy has been also frequently found in highly salt-tolerant
Z. rouxii strains (Solieri et al., 2013b, 2014).
7. Food exploitation and biotechnological perspective
The knowledge on the genetic and regulatory networks underlying
important phenotypic outcomes is a prerequisite for the successful
exploitation and control of microorganisms in food (Giudici et al.,
2005). In the last decade, researches in food science and microbiology
moved from classical methodologies to more advanced strategies,
and usually borrowed well-established methods in medical, phar-
macological, and/or biotechnology research. As a result, omics
152 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140157
approaches and bioinformatics have been recently applied to identify:
1) candidate genes to use in genetic improvement of industrial yeast
strains; 2) molecular targets for new food preservation technology;
3) biomarkers for the early prediction of food process outcomes.
In particular, new perspectives have been opened by the identica-
tion of genetic mechanisms that shape the osmostress response in
Z. rouxii and other osmotolerant and halotolerant food spoilage yeasts.
To prevent their growth in food, it is crucial to understand what causes
yeast stress tolerance. This knowledge could be exploited to set up
quantitative prediction methods for the growth of food spoilage yeasts
and, consequently, to reduce the economical loss derived from food
spoilage (Loureiro and Malfeito-Ferreira, 2003; Plemenita et al.,
2008). On the other hand, genetic loci that determine yeast sugar and
salt-tolerance could be cloned and expressed in industrial strains to im-
prove their growth and fermentation performance in stressful condi-
tions. For example, the ZrSod22 expression in S. cerevisiae increases
the expulsion of toxic Na
+
and Li
+
cations from cells at external acidic
pH values and enhances the salt tolerance (Iwaki et al., 1998;
Watanabe et al., 2005). Similarly, salt stress-responsive genes from
halotolerant fungi and bacteria can be expressed in plants to alleviate
problems caused by soil salinization in agriculture (Gostinar et al.,
2012). A similar strategy was successful to reduce stuck fermentation
in the bioethanol production (Zhao and Bai, 2009).
The ability to grow at very low a
w
makes the Zygosaccharomyces
strains very attractive as microbial cell factories both for the expression
of heterologous proteins and metabolite production (Branduardi et al.,
2004, 2006; Vigentini et al., 2005). Zygosaccharomyces bailii has been
used as host for the production of interleukin-1 due to its ability to
withstand hyper-osmotic, hyper-thermic, and acidic environments
(Vigentini et al., 2005). Zygosacchaomyces rouxii strains have been
exploited for producing glutaminase (Kashyap et al., 2002; Iyer and
Singhal, 2008), chiral compounds (Hauck et al., 2003), andheterologous
proteins (Ogawa et al., 1990). Taking advantage of their ability to pro-
duce polyols, osmotolerant and halotolerant yeasts, such as M. farinosa
(formerly P. sorbitophila) and C. tropicalis, have been employed on a
large-scale fermentation processes for the glycerol production (Liu
et al., 2006). Owing to the problems associated with the chemical pro-
duction, halotolerant and osmotolerant yeasts became valuable for the
fermentation-based production of sweet-tasting bodying and texturing
agents, such as mannitol, D-arbitol, and xylitol (Saha et al., 2007; Saha
and Racine, 2011).
Zygosaccharomyces species have been exploited as biocatalysts in
food and beverage fermentation. Zygosacchaomyces rouxii plays a
central role in the avour formation of soy sauce (Sluis et al., 2001;
Cao et al., 2010; Wei et al., 2013). Flavour components, such as
4-hydroxyfuranone derivatives, 4-hydroxy-2 (or 5) -ethyl-5 (or 2) -
methyl-3 (2H) -furanone (HEMF), are produced from D-fructose-1,6-
bisphosphate by Z. rouxii and positively affect soy sauce avour and
quality (Hauck et al., 2003). A highly salt-tolerant Z. rouxii mutant con-
structed by whole genome shufing grows better in soy sauce than the
parental strain and produces higher amounts of aroma compounds,
such as ethyl acetate, HEMF, and 4-ethylguaiacol (Cao et al., 2010).
The novel species Z. sapae and Zygosaccharomyces gambellarensis were
isolated from Italian traditional balsamic vinegar (Solieri et al., 2007;
Solieri et al., 2013a) and highly sugary wine (Torriani et al., 2011)
respectively, where they have a role in the fermentation performance
and in tailoring the sensory characteristics of these products (Solieri
and Giudici, 2008).
8. Concluding remarks
A critical reviewon the mechanisms underlying sugar and salt stress
tolerance in non-conventional yeasts unveils the complex nature of the
cellular adaptation to lowa
w
environments. Inthe past, most researches
about this topic have been carried out using S. cerevisiae as model
organism. Recently, the availability of transformation tools and the
genome sequences fromosmo andhalotolerant food species highlighted
how these yeasts combine conserved and species-specic strategies to
counteract better sugar and salt stress than S. cerevisiae. These differ-
ences are operational at genetic, metabolic, and regulatory levels. In
particular, new insights on Z. rouxii functional biology and genomics
point out how sugar and salt tolerance arise from distinct adaptive
mechanisms, which integrate solute-unspecic and solute-specic
routes. Future investigations will benet by the application of systems
biology tools. They will contribute to complete the detailed map of
osmo and halotolerance determinants in Z. rouxii. All these efforts hold
promise for i) rational strain engineering for biotechnological and food
exploitation; ii) prevention of yeast food spoilage; iii) understanding
the yeast adaptation to adverse environments.
Acknowledgements
This work has beensupportedby a grant of Regione Toscana (Misura
124 Acetoscana: PSF 2007/2013).
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