International Journal of Food Microbiology -...

International Journal of Food Microbiology 185 (2014) 140–157

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j foodmicro

Review

Adaptive response and tolerance to sugar and salt stress in the food yeastZygosaccharomyces 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

Abbreviations: CDRE, calcineurin dependent responseHOG, high-osmolarity glycerol; MAPK, mitogen-activatedP-Hog1, phosphorylated Hog 1; STRE, stress responsive el⁎ Corresponding author. Tel.: +39 0522522057; fax +

E-mail address: [email protected] (P. Giudici).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.0150168-1605/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 14 November 2013Received in revised form 18 April 2014Accepted 4 May 2014Available online 25 May 2014

Keywords:Zygosaccharomyces rouxiiSpoilage yeastOsmotoleranceHalotoleranceGlycerol accumulation and retentionCation homeostasis

The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survivein 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 fermentation onhighosmolarity foodmatrices and leads to spoilage ofhigh sugar and high salt foods. The knowledge about the genes, themetabolic pathways, and the regulatory circuitsshaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation con-trol, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights onthe 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-specific adaptive responses under sugar and salts stresses, including regulation ofNa+ and K+-fluxes across the plasmamembrane, modulation of cell wall properties, compatible osmolyte produc-tion and accumulation, and stress signalling pathways.We suggest howan integrated and system-based knowledgeon these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control infood, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms.

© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412. A matter of nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413. Osmotolerant and halotolerant yeasts in food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1424. Gene circuits and metabolic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

4.1. Cell wall and plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1434.2. Cation homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

4.2.1. Na+ inward and outward movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1444.2.2. K+ inward and outward movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

4.3. Sugar transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1464.4. Production and accumulation of osmolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

4.4.1. Glycerol metabolic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1474.4.2. Glycerol biosynthesis in non-stressed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484.4.3. Glycerol biosynthesis in osmo-stressed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484.4.4. Glycerol retention and active transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484.4.5. Other compatible solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

5. Signal transduction and cis/trans-acting regulatory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.1. High osmolarity glycerol (HOG) pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.2. Calcineurin/Crz1 pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515.3. Ras-cAMP signalling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

6. Non genetic regulation of osmostress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1526.1. Chromatin-mediated mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

element; CNV, copy number variation; CWI, cell wall integrity; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate;protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase;ement; SWI/SNF complex, switch/sucrose non-fermenting complex.39 0522522027.

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141T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157

6.2. Phenotypic heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1527. Food exploitation and biotechnological perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1528. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

1. Introduction

The high concentrations of ionic (mainly Na+) and non-ionic (mainlysugars and polyols) solutes reducewater activity (aw) in food and are twoof the major abiotic stressors, both limiting the yeast growth. High exter-nal osmolarity has been used for centuries for food preservation, becauseit causeswater outflow from the cell and results in a higher intracellularconcentration of ions andmetabolites and in an eventual arrest of cellu-lar activity. The yeast ability to cope with these environmental insultsdetermines both the success of certain food and beverage fermentationand the thriving of food spoilage.

Since the sequencing of strain S288c (Goffeau et al., 1996), impressiveadvances in genomics, proteomics, and systems biology have madeS. cerevisiae the paradigm for understanding these osmo-adaptivemechanisms, which have been exhaustively summarized by several re-views (Nevoigt and Stahl, 1997; Hohmann, 2002; Ariño et al., 2010;Kühn and Klipp, 2012). As a result, the S. cerevisiae response to highexternal solute concentrations has been described as a system-levelcoordination between the extracellular environment and the geneticmake-up inside the cell. The following interconnected modules are in-volved: (i) receiving information from external environment (sensing);(ii) conducting it to the inside (signal transduction); (iii) integrating itwith internal genetic information in order to mount an appropriate re-sponse (effector processes) (de Nadal et al., 2011). This system-levelknowledge has been exploited in food industry to improve yeast fer-mentations on highly salty and sugary matrices or to decrease thefood spoilage by sugar and salt resistant-yeast species. However, asbeing moderately halotolerant and osmotolerant, S. cerevisiae could beinappropriate to describe the yeast response to hypersaline andhyperosmotic food.

Zygosaccharomyces rouxii is the osmotolerant and halotolerant yeastmost phylogenetically related to S. cerevisiae and inhabits a varietyof highly sugary and salty food, where it carries out fermentation ordetermines 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 athigh sugar concentration up to 60–70% glucose), whereas only a feware both highly osmo and halotolerant (Table 1). Recently, the completegenome sequences of Z. rouxii (Souciet et al., 2009) and other highlyosmo and halotolerant yeasts, such as Millerozyma farinosa (formerlyPichia sorbitophila) (Louis et al., 2012), Debaryomyces hansenii (Kumaret al., 2012), and Zygosaccharomyces bailii (Galeote et al., 2013), havebecome available. Furthermore, ‘omics’ tools and genetic manipulationprotocols have been recently employed to analyze the relationshipsof osmostress phenotype to genetic and molecular determinants(Prybilova et al., 2007a,b; Watanabe et al., 2010). From these intenseefforts, 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 osmoticumin the surrounding medium. Comparison of species with differentsugar and salt tolerance highlighted how yeasts exploit different strate-gies to survive under osmotic and salt stress (Ramos et al., 2011). Forexample, Z. rouxii resembles S. cerevisiae in extruding Na+ cations outof the cell or driving them into the vacuole (Ramos, 1999), while thehalotolerant yeast Debaryomyces hansenii is a sodium includer, whichaccumulates intracellularly Na+ without getting intoxicated (Ramos,1999). Beyond the species-specific strategies, other osmostress re-sponses, such as the osmolytes accumulation, are ubiquitous among

yeasts to avoid outflow of cellular water in low aw environments(Nevoigt and Sthal, 1997; Lages et al., 1999; Silva-Graça and Lucas,2003). Another emerging issue concerns how salt and sugars elicit dis-tinct or partial overlapping responses in yeasts. Whereas sugars andpolyolsmodify osmotic pressure, salts induce alterations both in osmot-ic pressure and ion homeostasis. The result is that partially differentmechanisms become operational in response to sugar and salts. Sincehalo and osmotolerance could be paired and unpaired phenotypes inZ. rouxii and relatives, these yeasts are very attractive models fordeciphering genetic circuits and functional pathways underlyinghalotolerance and osmotolerance.

Here, we review recent insights on the mechanisms that governhalotolerance and osmotolerance in Z. rouxii and compare them tothose active in S. cerevisiae and in other osmo and halotolerant foodyeasts at genetic,metabolic, signalling, and epigenetic level. Furthermore,we highlight how these yeasts can achieve generic and osmoticum-specific responses to sugar and salt stresses. Finally, we point out howthe 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 aspecific cellular adaptability to sudden and severe fluctuations in wateravailability and a tendency of cells to restore ormaintain 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 aw that is the chemical potential of free water in solu-tion. Microorganisms able to colonize food with high osmolarity and,consequently, low aw, were collectively indicated as xerotolerant (no ab-solute requirement of low aw), and xerophilic (“lovers of low aw”) (PittandHocking, 2009) (Table 1). Amore appropriatemicrobial classificationwould consider the kind of osmoticum and include the following catego-ries: osmophilic, absolute requirement for non-ionic solutes and ability togrow up to solute concentrations approaching saturation; osmotolerant,no absolute requirement of non-ionic solutes for viability and ability totolerate a wide range of osmolarity, from hypo-osmotic to hyper-osmotic solutions; osmosensitive, sensitive to excess concentration ofnon-ionic solutes; halophilic, absolute requirement for high salt andability to grow up to salt concentrations approaching saturation;halotolerant, no absolute requirement of salt for viability and ability totolerate a wide range of salinity, from hypo-saline to hyper-saline solu-tion; and halosensitive, sensitive to excess concentration of salt.

Most food yeasts can develop well at aw values around 0.95–0.90. Acut-off of aw b0.70 has been frequently used to delineate osmotolerantand halotolerant yeasts. In the past, yeasts isolated from sugary andsalty food with aw lower than 0.70 were referred to as “osmophilic” and“halophilic” (Tokuoka, 1993). For instance, Debaryomyces hansenii hasbeen described as halophilic yeast based on the ability to grow at 1.0 Mof salt with growth rate and final biomass close to the values obtainedwithout salt (Almagro et al., 2000; González-Hernández et al., 2004;Aggarwal and Mondal, 2009). Other yeasts were classified as halophilicor osmophilic, such asM. farinosa (formerly P. sorbitophila) (Rodriguesde Miranda et al., 1980), Candida etchellsii (formerly Candidahalonitratophila), Candida versatilis (Barnett et al., 2000), and the blackyeast Hortea werneckii (Gunde-Cimerman et al., 2000). However,differently fromhalophilic and osmophilic bacteria, none of these yeastssatisfies the true definition of osmophily or halophily, because they

Table 1Proposal for a classification of representative yeast species according to their halotolerance and osmotolerance behaviors.

Category Definition Species name1 D-glucose %(w/v)

NaCl (M); % (w/v) Food spoilage2 References3

50 60

Moderately osmotolerantand moderatelyhalotolerant

Lack of growthat N50% (w/v)D glucose;lack of growthat N2.0M NaCl

Saccharomyces cerevisiae − − b1.70; 10% Soft drink, fruit juice Onishi, 1963Schizosaccharomycespombe

− − 1.00; 5.8% Cheese, fruit (rarely) Lages et al., 1999;Barnett et al., 2000

Zygosaccharomycesflorentinus(Zygotorulasporaflorentina)

+ − 1.00; 5.8% Wine Lages et al., 1999;Barnett et al., 2000

Candida glabrata + − 1.70; 10% Juice concentrate Pitt and Hocking, 2009Osmotolerant andmoderatelyhalotolerant

Growth at N50%(w/v) D glu-cose;lack of growthat N2.0M NaCl

Candida tropicalis + − 1.7–2.0; 10-11.7% Fruit juice Barnett et al., 2000;Deak, 2007;Pitt and Hocking, 2009

Zygosaccharomycesmellis

+ + 1.70; 10% Juice concentrate,honey

Kurtzman et al., 2011

Zygosaccharomycessapae

+ + 2.0; 11.7% n Solieri et al., 2014

Zygosaccharomyces bailii + w 1.0–2.0; 5.8-11.7% Juice, sauces, ciders,wines

Lages et al., 1999;Barnett et al., 2000

Zygosaccharomycesbisporus

+ +§ 1.0–2.0; 5.8-11.7% Soft drink, wine James and Stratford, 2003

Moderately osmotolerantand halotolerant

Lack of growthat N50% (w/v)D glucose;growth atN2.0M NaCl

Candida parapsilosis + − 3.0; 17.5% Dairy food Pitt and Hocking, 2009Pichia membranifaciens +§ − 3.00; 17.5% Bread, fermented milk,

oliveLages et al., 1999;Barnett et al., 2000

Issatchenkia orientalis(Pichia kudriavzevii)

+§ − 2.0; 11.7% Olives, pickles andsauces (rarely)

Lages et al., 1999;Barnett et al., 2000

Osmotolerant andhalotolerant

Growth at N50%(w/v) D glu-cose;growth atN2.0M NaCl

Pichia sorbitophila(Millerozyma farinosa)

+ + 3.0–4.0; 17.5-23.4% Beer, sake, soy sauce,Mash of rice vinegar

Lages and Lucas, 1995

Zygosaccharomycesrouxii(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(Meyerozymaguilliermondii)

+§ +§ 3.0; 17.5% Olive, salt meat Butinar et al., 2005

Hortaea werneckii + n 5.20; 30.8% Salt fish Butinar et al., 2005;Lenassi et al., 2011

Debaryomyces hansenii(Candida famata)

+§ +§ 3.0–4.0; 17.5-23.4% Olive Barnett et al., 2000;Lages et al., 1999

Candida halophila(Candida versatilis)

+ + 4.0–5.0; 23.4-29.1% Cheese brines Barnett et al., 2000;Silva-Graça andLucas, 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) 140–157

tolerate high osmotic conditions, without requiring high sugar orsalt amounts for their growth (Silva-Graça et al., 2003). Similarly, theterms ‘osmotolerant’ and ‘halotolerant’ have been improperly used assynonymous, but they should be reserved for yeasts that are able tolive at high sugar and ionic solutes (mainly Na+) concentrations,respectively (Onishi, 1963; Tokuoka et al., 1992; Tokuoka, 1993;Lages et al., 1999; Marešova and Sychrová, 2003; Pribylova et al.,2007a,b). Since different solutes elicit distinct yeast stress responses,osmotolerant and halotolerant phenotypes have been defined inrelation to the yeast ability to grow up to 55–65% (w/v) sugar (lowerthan ~0.88 aw) or at 15–25% (w/v) salt concentrations (correspondingto 0.92–0.85 aw range), respectively (Deak, 2006, 2007; Kurtzamnet al., 2011). Based on these assays, tolerance to sugar and salt couldbe differentially distributed among yeasts species. Table 1 shows theclassification of representative yeast species into four classes accordingto their degree of sugar and salt tolerance.

3. Osmotolerant and halotolerant yeasts in food

Osmotolerant and halotolerant yeasts have a pivotal role in foodfermentation and spoilage (Fleet, 1992) and several of them belong to

the genus Zygosaccharomyces. The genus comprises 7 species (Jamesand 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 misopaste) (Deák and Beuchat, 1993; Tokuoka, 1993; Solieri et al., 2006,2013a,b;Martorell et al., 2007; Suezawa et al., 2008). Zygosaccharomycessapae and Z. rouxii are themain biocatalysts of alcoholic fermentation inhigh 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 and Zygosaccharomycesbailii are involved in alcoholic fermentation during kombuchaproduction. Zygosaccharomyces species have been also recognized asone of the main spoilage yeasts in food industry due to their tolerancesto salt, sugar, and weak acid preservatives (Pitt and Hocking, 2009;Fleet, 2011). In food manufacture, the yeast spoilage of products causessevere economic loss and affects a variety of processed foods, includingbread, 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 importantZygosaccharomyces from the point of view of weak acid preservative


resistance (Thomas and Davenport, 1985; Steels et al., 1999; 2002),displaying an unusually high resistance to the small number of acids ap-proved for use as food preservatives (primarily sorbic, benzoic, aceticand propionic acids). On the other hand, Z. rouxii and Z. mellis can toler-ate low aw with different sensitivity to ionic and non-ionic solutes.Zygosaccharomyces rouxii is well-known for osmo and halotoleranceand survives up to 0.80 aw in presence of ionic solutes (salt) and upto 0.65 aw in presence of non-ionic solutes (sugars), while Z. mellissurvives at low aw values only when the solutes are sugars (Stratford,2006). Moreover, inter-strain differences in halotolerance have beenreported within Z. rouxii, with the strains isolated from high-salt foodstolerating better NaCl than those isolated from high-sugar foods(Pribylova et al., 2007b; Solieri et al., 2014). The highly salt-tolerantstrains 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 finding suggests that osmo andhalotolerance are two distinct physiological phenotypes.

Food yeasts other than Zygosaccharomyces species have beendescribed as being osmotolerant and/or halotolerant. Millerozymafarinosa (formerly P. sorbitophila) is a ubiquitous, halotolerant yeastfound mainly in food (alcoholic beverages like beer and sake, soysauce, miso, mash of rice vinegar etc.) and it is known for the ability togrow in more than 4.0 M NaCl (Lages and Lucas, 1995; Silva-Graçaand Lucas, 2003; Silva-Graça et al., 2003) and to tolerate up to 70%glucitol (Rodriques de Miranda et al., 1980). Debaryomyces hanseniiwas originally isolated from saline environments, such as seawaterand concentrated brines, and it is also associated with cheese andmeat processing. It can tolerate salinity levels up to 4.0 M NaCl andsurvive in high-sugar products with aw values as low as 0.62 (asreviewed by Aggarwal and Mondal, 2009). The strongly halotolerantyeast-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 withoutsalt, with a broad growth optimum from 1.0–3.0 M NaCl. In addition, agroup of poorly studied osmotolerant species have been associatedwith spoilage of sugary food and with insects, including Candidadavenportii (Stratford et al., 2002),Candida stellata andCandidamagnoliae(Rosa et al., 2003). Schizosaccharomyces pombe is an osmotolerant,preservative-resistant yeast, but it is rarely associated to food spoilagedue to its salt-sensitivity (Pitt and Hocking, 2009).

4. Gene circuits and metabolic pathways

In presence of high extra-cellular solute concentrations, yeast cellexperiences three main physiological alterations: changes in physicaland chemical structure of the cell wall and plasmamembrane; increaseof intracellular solute/ion toxicity; and alterations in the osmoticpressure and cell volume. Therefore, three systems enable yeasts tocounteract stress challenges and to restore osmotic balance: a) regulationof morphological and structural properties of the cell wall and plasmamembrane; 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 plasmamembrane are important factors affecting the yeast osmo andhalotolerance. By reshaping their integrity and fluidity, yeast cell estab-lishes a balance by which the force driving water across the osmoticgradient into the cell is counteracted by turgor pressure against theplasma membrane and cell wall (Klis et al., 2006; Levin, 2011).

The yeast cell wall is a rigid skeleton formed by four classes ofmacromolecules interconnected by covalent bonds: the mannosylatedcell wall proteins called mannoproteins, 1,3-β-D-glucan, 1,6-β-D-glucan and chitin (a polymer of GlcNAc) (Klis et al., 2006). Early studies

on cell wall composition suggested that Z. rouxii decreases cell wallmannans in presence of salt (Hamada et al., 1984; Hosono, 1992).These studies firstly suggested that cell wall rigidity and integrity havebeen implicated in tolerance to salt-induced stress in Z. rouxii. Strain-specific differences have been also described in the internal layer ofβ-D-glucan and cell wall mannans. In particular, Z. rouxii strains havinga more rigid cell wall tend to be less halotolerant than those having amore flexible and elastic cell wall (Pribylova et al., 2007b). Althoughthese variations are congruent with the possible involvement of cellwallmannans in salt tolerance,more evidences are required to reinforcesuch speculations. Recently, some highly salt-tolerant Z. rouxii strainshave been found to possess an increased copy number of FLO11 genethat encodes a glycophosphatidylinositol-anchored cell surface glyco-protein. This copy number amplification affects positively the cell wallhydrophobicity and enables strains with a higher copy number ofFLO11 to exhibit a fitness advantage compared to a reference strainunder osmostress static culture conditions (Watanabe et al., 2013).Interestingly, in S. cerevisiae FLO11 is responsible for filamentation,invasive growth, and biofilm formation (Fidalgo et al., 2006) and it isregulated by at least three well-known signalling cascades, such as theRas-cAMP pathway, the Mitogen-activated protein kinase (MAPK)-dependent filamentous growth pathway, and the glucose repressionpathway (Verstrepen and Klis, 2006).

A few studies dealt with the regulatory pathways employed byZ. rouxii to maintain and modulate the cell wall integrity in the faceof environmental challenges. In the two main model yeastsS. 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 wallstress signals from the cell surface sensors to the Rho1 GTPase,which mobilizes a variety of effectors. Activation of CWI pathwayregulates the production of various cell wall carbohydrate polymersand their polarized delivery to the site of cell wall. Moreover, CWIserves 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 few proteins involved in CWI pathway have been associatedto osmo and halotolerance phenotypes. In S. pombe, the MAPK Pmk1has been implicated in cell wall integrity, cytokinesis, and ion ho-meostasis (Sengar et al., 1997). The MAPK kinase kinase (alsoknown 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 essentialfor Pmk1 activation. The involvement of Mkh1, Pek1, and Pmk1 hasbeen demonstrated in salt stress response of S. pombe (Madridet al., 2006) and, recently, also of S. cerevisiae (Rodicio andHeinisch, 2010; Levin, 2011). In S. pombe, MKH1 gene-lacking cellsre-enter the cell cycle quite slowly after a prolonged arrest in stationaryphase 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 innon-conventional yeast genomes has suggested that a similar pathwaycould also operate in Z. rouxii (Rodicio and Heinisch, 2010).

Inside the cell wall there is the plasmamembrane, which is involvedin a variety of cellular processes such as cell adhesion, ion conductivity,and signalling. Like prokaryotes, yeasts regulate the plasma membranefluidity in osmostress adaptation (Turk et al., 2011). The main factorsaffecting the membrane fluidity are the length, branching and degreeof saturation of fatty acids, the amount of sterols, and the phospholipidcomposition (Russell, 1989; Rodriguez-Vargas et al., 2007). The remod-elling of these parameters strongly affects not only themembrane fluid-ity, but also the proper functioning of membrane-attached proteins,such as those involved in ion homeostasis, the glycerol transportsystems (Marquez and Serrano, 1996; Kamauchi et al., 2002), and theplasma membrane ATPase activity (Coccetti et al., 1998).


When the external salinity is shifted from low to high concentrations,S. cerevisiae shortens the fatty-acid chain length (Turk et al., 2007) and in-creases their saturation level by synthesizing fatty acid desaturases thatintroduce double bonds into the fatty acids of membrane lipids (Levin,2011). Whereas many eukaryotic organisms can synthesize dienoicfatty acids, S. cerevisiae can introduce only a single double bond at theΔ9 position (Tunblad-Johansson and Adler, 1987; Sharma et al., 1996;Rodriguez-Vargas et al., 2007). Under NaCl stress Z. rouxii increases theamount 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). Themodifications result in a reduction of mem-brane fluidity. On the contrary, in the extreme halotolerant yeastD. hansenii high NaCl levels increase the sterol-to-phospholipid ratioand fatty acid unsaturation, without significantly affecting fluidity(Turk et al., 2007). In the strongly halotolerant yeast-like fungusH. werneckii, high salinity conditions induce slight changes in the totalsterol content, but cause a significant increase both in the phospholipidcontent and the fatty acid unsaturation level (Gunde-Cimerman andPlemenitaš, 2006). Therefore, H. werneckii tends to maintain thesterol-to-phospholipid ratio significantly lower than other yeasts, mak-ing the plasmamembrane comparativelymore fluid and offering higheracclimatization to salt stress conditions (Turk et al., 2007).

Ionic andnon-ionic solutes have different effects on the plasmamem-brane. When the sunflower (Helianthus annuus) oleate Δ12 desaturasesFAD2-1 and FAD2-3 genes are expressed in S. cerevisiae, they increasethe content of dienoic fatty acids, especially 18:2Δ9,12 and the plasmamembrane unsaturation index (Rodríguez-Vargas et al., 2007). Undersalt stress FAD-expressing cells display higher membrane fluidity andsalt tolerance than the wild-type cells. In contrast, under high sorbitolconcentrations, the FAD-expressing cells do not differ in growth ratefrom the wild-type cells, suggesting that the dienoic fatty acid contentdoesn’t affect the tolerance to non-ionic solutes. Although furtherresearches are required, these evidences collectively support thatthere are distinct mechanisms in modulating the cell wall integrity

Fig. 1. Overview of the main plasma membrane systems mediating the alkali metal ca

and plasma membrane fluidity in response to ionic and non-ionicstresses. Furthermore, it was demonstrated that certain osmolytes, es-pecially trehalose, stabilize phospholipid bilayers during osmostressconditions (Hounsa et al., 1998; Gancedo and Flores, 2004). In prokary-otes, trehalose stabilizes lipid bilayers and prevents damages derivedfrom dehydration by inhibiting the fusion between vesicles and bymaintaining the membrane lipids in the fluid phase (phase transition).Similarly, in S. cerevisiae trehalose also protects plasma membraneagainst osmotic stress (Iturriaga et al., 2009 and references herein).

4.2. Cation homeostasis

High concentrations of Na+ cations are toxic to most living cells andare frequent in food,while K+ cations are less abundant, but they are es-sential for compensating negative charges and activating key metabolicprocesses, such as pyruvate synthesis and protein translation. Thus, themajority 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 parametersand functioning, such as cell volume, plasma membrane potential, andintracellular pH (Arino et al., 2010). When cation concentrations inthe external medium are higher than the physiological range, thedifference between the electrochemical potentials of the cations acrossthe membrane may be so high that the entrance cannot be annulled bysimply inhibiting the transporters mediating the uptake. To avoid aninternal toxic cation concentration, different types of efflux systemshave been evolved to balance any excessive entrance.

4.2.1. Na+ inward and outward movementsFig. 1 illustrates efflux and influx systems recruited by Z. rouxii to

modulate the transport activity of the alkali metal cations across theplasma membrane. Like S. cerevisiae, Z. rouxii has been regarded as asodiumexcluder species, forwhichNa+ is comparativelymore cytotoxicat high concentrations compared to K+ (Pribylova et al., 2008). Insodium excluder yeasts, two main mechanisms mediate the efflux of

tion homeostasis and the glycerol uptake/retention in Zygosaccharomyces rouxii.


excessive cations (Na+ or K+) through the plasmamembrane. The firstone 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 ZrENA1in Z. rouxii. The yeast Na+ (K+)-ATPases couple the ATP hydrolysis tothe cation transport against electrochemical gradients, and, unlikeNa+ specific-ATPases from higher eukaryotes, they mediate both theNa+ and K+ efflux. 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 byNHA1 in S. cerevisiae, SpSOD2 and SpSOD22 in S. pombe, ZrNHA1and ZrSOD2-22 (with its variants ZrSOD2 and ZrSOD22) in Z. rouxii(Watanabe et al., 1995; Hahnenberg et al., 1996; Bañuelos et al., 1998;Benito et al., 2002; Papouskova and Sychrová, 2007; Pribylova et al.,2008) (Table 2). Based on the substrate specificity, the Na+/H+-antiporters canbe further divided into two subfamilies: those recognizingboth K+ and Na+ (and their respective analogues Rb+ and Li+), such asS. cerevisiaeNha1 and SpSod22, are involved in K+ homeostasis and arepresent in almost all hemiascomycetes, whereas those recognizing onlyNa+ (and its analogue Li+) as substrate, such as SpSod2, ZrSod2-22 andZrSod2, determine salt detoxification in yeasts most distant fromS. cerevisiae (Kinclová et al., 2002). Furthermore, a third less studiedmechanism, which operates in acute response to salt stress, entailsthe sequestration of a surplus of toxic Na+ cations in intracellularcompartments. A plethora of proton-coupled antiporters are includedin this process; in S. cerevisiae, the most studied ones are Nhx1, anendosomal 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. cerevisiaeNhx1, has been also found in the halotolerant food yeast D. hansenii(Moniel and Ramos, 2007).

Yeast salt tolerance significantly depends on the genes encodingNa+ ATPases, plasma membrane and intracellular Na+/H+-antiportersharboured in the yeast genome (Table 2). The sodium pump Ena1 isthe most important salt tolerance determinant in S. cerevisiae (Prioret al., 1996; Bañuelos et al., 1998). Deletion of ENA1 gene determinesthe salt-sensitivity in S. cerevisiae (Haro et al., 1991; Marquez andSerrano, 1996). In the salt-sensitive S. pombe, the single ENA-relatedgene, called cta3, only mediates the potassium efflux (Benito et al.,2002). The expression of S. cerevisiae ENA1 in S. pombe markedly

Table 2Cation transport systems associated to halotolerance in different yeast species (adapted from R

Category Gene§ Function

Systems for K+ influx TRK1 plasma membrane K+ transporter belongwith a main role in K + homeostasis

TRK2 plasma membrane K+ transporter belongwith a minor role in K+ homeostasis

HAK1 High Affinity K+-H+ symporter belonginACU1 K+-Na+ P-type ATPase functionally simil

Systems of K+ efflux TOK1 membrane depolarization activated K+ cSystems for Na+ and K+ efflux NHA1 antiporter which uses a proton-motive fo

membrane H+-ATPase to mediate the efflthrough the plasma membrane

SOD2 antiporter which uses a proton-motive fomembrane H+-ATPase to mediate the efflthe plasma membrane

ENA1-4 P-type ATPase sodium pump; involved inIntracellular cations/H + transporters NHX1 endosomal Na+(K+)/H+ antiporter whic

of cytosol and vacuole lumenKHA1 Putative K+/H+ antiporter from Golgi wi

cation homeostasisVNX1 Vacuolar Na+ (K+)/H+ exchanger localiz

reticulummembrane

§Nomenclature according to S. cerevisiae genome, with the exception of HAK1, ACU1, and SOD2carried out to identify homologues of K+, Na+ channel subunits, using the following querie(DAA08707); D. hansenii Hak1 (ABI37006);M. farinosa Acu1 (CAF22247.1); S. cerevisiae Ena1Nhx1 (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 (formerinter-strains variability.

increases the tolerance to Na+ (Bañuelos et al., 1995). Other studiesreported that S. pombe Sod2 antiporter complements theNa+ sensitivityin S. cerevisiae ena1 mutants, suggesting that antiporters or sodiumpumps can both be used by S. cerevisiae to regulate the internal sodiumconcentration (Hahnenberger et al., 1996). In D. hansenii two ENAparologs, namelyDhENA1 andDhENA2, have been found to complementthe salt sensitivity when heterologously expressed in S. cerevisiae ena1mutants (Almago et al., 2001). Similarly, H. werneckii possesses twohighly salt responsive ENA genes, namelyHwENA1 expressed in stressedcells exposed to high salt concentrations, and HwENA2 that is mainlyexpressed in stress-adapted cells (Gunde-Cimerman and Plemenitaš,2006). More recently, it was demonstrated that upon initial impositionof NaCl stress, S. cerevisiae extrudes intracellular Na+ primarily byNha1, whereas the long term salt adaptation is mediated by the tran-scriptional up-regulation of ENA1 gene (Proft and Struhl, 2004; Ruizet al., 2007; Ke et al., 2013).

In Z. rouxii, the salt tolerance has been attributed to different variantsof Na+/H+ antiporters and Na+-ATPase genes (Hahnenberger et al.,1996; Pribylova et al., 2008). Like S. cerevisiae, Z. rouxii has a geneencoding Ena1-homologous protein named ZrENA1 (Watanabe et al.,1999; 2002). Transcriptional studies showed that, unlike S. cerevisiaeENA1, ZrENA1 has little relevance in Z. rouxii salt tolerance, as, underNaCl 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 ena1Δ mutants and wide-typestrains exhibit similar growth rates (Watanabe et al., 1999). Other stud-ies pointed out that Na+-ATPases and Na+ antiporters almost servesimilar functions, but they are operational at different external pHlevels. In most sodium excluder species, Ena1 Na+-ATPase mediatesthe Na+ export mainly at high external pH levels. When the externalpH is lower than the cytoplasmic pH, the function of Ena1 Na+-ATPase can be replaced by electroneutral Na+/H+ antiporters, whichdrive the Na+ efflux by the ΔpH (Bañuelos et al., 1998). Similarly, inZ. rouxii Na+-ATPase and Na+ antiporters have different pH sensitivity.Since Z. rouxii is acidophilic yeast, ZrEna1 could not be active at thelow 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+-specific Na+/H+ antiporterZrSod-22 and not by the substrate-unspecific Na+/H+ antiporter

amos et al., 2011). Number of parologs is reported in brackets.

Sc Sp Zr Dh Mf Ca Hw

ing to HKT–TRK family, + + + + + + +(8)

ing to HKT–TRK family, + + − − + − nd

g to the HAK–KUP family − − − + + + −ar to plant HKT transporters − − − − + ps −hannel + − + − − + +(4)rce generated by the plasmaux of Na+, Li+, K+, and Rb+

+ + + + +(2) + +(8)

rce generated by the plasmaux of Na+, and Li+ through

− + + (v) nd +(2) − nd

Na+, K+ Li+ efflux +(v) + + +(2) +(2) + +(4)h it regulates the acidification + + + + + +(2) +(2)

th a probable role in intracellular + + + + +(2) + +(2)

ed to the endoplasmic + + + + +(2) +(2) +(2)

genes. When no functional data have been available, BLASTP and TBLASTN analyses weres: S. cerevisiae Trk1 (DAA08672.1); S. cerevisiae Trk2 (DAA09201.1); S. cerevisiae Tok1(DAA09449.1); S. cerevisiae Nha DAA11888.1); S. pombe Sod2 (CAB.69632.1), S. cerevisiae

ly P. sorbitophila); Ca, C. albicans; Hw, H. werneckii; nd, not determined; ps, pseudogene; v,


ZrNha1 (Prybilova et al., 2008). Synteny analysis demonstrated thatthese two genes arose from a duplication event. Furthermore, Z. rouxiigenome experienced a CNV in ZrSOD genes. The Z. rouxii type strainCBS 732T possesses one gene encoding the Na+/H+antiporter, namelyZrSOD2-22, while the highly halotolerant allodiploid strains ATCC42981, CBS 4838 and CBS 4837 have two copies, namely ZrSOD2 andZrSOD22 (Watanabe et al., 1995; Iwaki et al., 1998; Kinclová 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 repertoireencoding alkali metal cation transporters has been related to the ex-treme salt tolerance of H. werneckii (Lenassi et al., 2011). Although theCNV in Na+/H+antiporter-encoding genes has been supposed to con-tribute to the high halotolerance of Z. rouxii allodiploid strains(Gordon andWolfe, 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), whereasZrSOD22 is a poorly transcribed gene and its disruption doesn’t affectsalt-tolerant phenotype (Iwaki et al., 1998).

In S. cerevisiae, the plasma membrane proton-pumping ATPasePma1 generates the electrochemical gradient required for nutrientuptake and ionic homeostasis (Serrano et al., 1986). The PMA1 genetranscription is tightly regulated and the Pma1 activity is controlled bypH andglucose via phosphorylation of its C-terminal domain. Transcrip-tional studies demonstrated that, under salt stress, Z. rouxii drives theNa+ efflux via Na+/H+ antiporter through the H+ gradient created byZrPma1 H+-ATPase (Watanabe et al., 1995; Iwaki et al., 1998). TheZrATP2 gene, which encodes a mitochondrial F1 ATPase β subunit, isalso involved in the ATP production and salt tolerance (Watanabeet al., 2003). The disruption of this gene is lethal in Z. rouxii but notin S. cerevisiae, suggesting that ZrATP2 is essential for maintainingZ. rouxii viability and functioning (Watanabe et al., 2003). Based onthese evidences, it has been hypothesized that Z. rouxii is more efficientin Na+ extrusion than S. cerevisiae due to the cooperative action ofefficient H+-ATPase systems and Na+/H+ antiporters with high Na+-specificity (Watanabe et al., 2005).

4.2.2. K+ inward and outward movementsPotassium is an absolutely essential element for the living organisms

and, although the external K+ concentration can greatly vary dependingon the natural environment, it is in food much lower than whatmetabolically required inside the cells (200–300 mM). Therefore,besides the Na+ efflux, yeast cells also require efficient systems for K+

uptake and,when necessary, extrusion (Table 2). In S. cerevisiae, K+ cat-ions are continually taken up and extruded: the membrane potentialincreases when the potassium influx is crippled and decreases in cellsdefective for K+ efflux (Kinclova-Zimmermannova et al., 2006). TheK+ uptake occurs mainly by facilitated diffusion through the high-affinity transporters Trk1 and Trk2, and it is driven by the electrochem-ical H+ gradient across the plasmamembrane generated by H+-ATPasePma1 (Michel et al., 2006). The homologous gene encoding a putativepotassium transporter, namely ZrTRK1, has been also characterized inZ. rouxii (Stříbný and Sychrová, 2011) (Fig. 1). The functional expressionof the ZrTRK1 gene in S. cerevisiae trk1Δ trk2Δ mutants restores theability to grow at micromolar potassium concentrations, whereasthe Z. rouxii trk1Δ mutant grows more slowly than the wild-typestrain at low K+ concentrations. Other non-conventional yeasts, suchas D. hansenii, possess the high affinity K+ transport Hak1, whichworks both as K+-H+ symporter and K+-Na+ symporter dependingupon the extracellular K+/Na+ concentrations (Martínez et al., 2011)(Table 2). The extremely halotolerant M. farinosa (formerlyP. sorbitophila) possesses a peculiar p-type ATPase encoded by theACU gene, which mediates the Na+ and K+ uptake at high affinity(Benito et al., 2004).

In S. cerevisiae, the extrusion of K+ cation surplus is mediated bythe Ena Na+ (K+)-ATPase, the antiporter Nha1, and the voltage-gatechannel Tok1. While Nha1 and Ena ATPase are also involved in theNa+ detoxification, Tok1 represents the main system for the exclusiveK+ extrusion in S. cerevisiae (Ahmed et al., 1999). Tok1 is activated bythe plasma membrane depolarization and contributes to regeneratethe membrane potential by releasing intracellular K+ outside the cell.Tok1 is also involved in the short term response to salt stress via theHOG pathaway. Phosphorylated Hog1 (P-Hog1) has been recently pre-dicted to inhibit theK+ extrusionmediated by Tok1, leading to a plasmamembrane depolarization and a Na+ influx reduction (Ke et al., 2013).The plasmamembrane depolarization could thus be a short term adap-tation to osmotic (sorbitol) and ionic (Na+) stress, because it reducestransporter activities and consequently the molecular import of thecell. Similarly, the phosphorylation of Nha1 by P-Hog1 increases theNa+ cations effluxunder salt stress (Proft and Struhl, 2004), and inhibitsthe K+ efflux under sorbitol stress (Kinclová-Zimmermannova andSychrová, 2006). In D. hansenii, the K+ efflux 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 cellsfrom 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 expressedin S. cerevisiae lacking alkali metal cation efflux systems (ena1–4Δnha1Δ), ZrNha1 was less effective than S. cerevisiae Nha1 in restoringthe tolerance to K+ excess (Prybilova et al., 2008). Therefore, differentlyfromorthologs in S. cerevisiae andD. hansenii, ZrEna1 doesn’t seem to beinvolved in potassium homeostasis.

4.3. Sugar transporters

In yeasts hyperosmotic stimuli trigger a variety of regulatorymechanisms, which modulate the glucose uptake rate (Horak, 2013).When glucose is available at high concentrations, S. cerevisiae uptakeshexoses via facilitated diffusion, and only when glucose is scarce, ituses the H+ gradient and high-affinity symporters. In S. cerevisiae 17HXT genes encode facilitated diffusion carriers (Boles and Hollenberg,I997), but only 4 (HXT1-HXT4) are regulated in response to extracellularglucose concentrations (Boles and Hollenberg, 1997; Ozcan andJohnston, 1999). The HXT2 and HXT4 genes encode high affinity and in-termediate affinity glucose transporters, respectively, which are up-regulated at low glucose concentrations and down-regulated underhyperosmotic 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 uponthe 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 HXT1mRNAtranscripts, indicating that both transcription and mRNA turnover areregulated in yeast osmo-adaptation (Greatrix and van Vuuren, 2005).

Zygosaccharomyces rouxii is fructophilic yeast which prefers toconsume fructose over glucose. Therefore, hexose transportersmediatingglucose facilitate diffusion have been poorly investigated. The searchfor 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 Ffzproteins (fructose facilitator of Zygosaccharomyces) have been exten-sively characterized in Z. bailii (Pina et al., 2004) and Z. rouxii (Leandroet al., 2011). In particular, Z. rouxii has two low-affinity high-capacityfacilitators, ZrFfz1 and ZrFfz2,which transport both fructose and glucosewhen their external concentration is high (Fig. 2). More recently,Leandro et al. (2013) characterized the high-affinity low-capacityfructose-H+ symporter ZrFsy1, which is up-regulated during thegrowth of Z. rouxii at low extracellular sugar concentrations.

http://genolevures.org

http://genolevures.org

Fig. 2. Interactions among redox balance, glycolysis, and glycerol production during the Zygosaccharomyces rouxii ethanol fermentation. Black, green and orange lines indicate theenzymatic 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 representthe remodelling inmetabolicfluxes under bisulphite and osmostress conditions. All the dotted lines represent omitted steps. The roles of glycerolmetabolism inPi recycling, redox balance,gluconeogenesis and fatty acid biosynthesis are reported in gray boxes.


4.4. Production and accumulation of osmolytes

Compatible osmolytes are produced by yeasts to favour the celladaptation to osmotic stress. They significantly maintain the waterbalance, stabilize enzyme systems without interfering with the cellularmetabolism, and restore the original cell volume (Brown, 1990). Toassure high intracellular osmolyte concentrations, yeast cells regulatethe cell-cycle progression and take action against the requirement ofredox elements and energy demand.

Glycerol is the main osmolyte that is produced and accumulatedintracellularly in response to hyperosmotic stress, and also the moststudied one. The role of glycerol under varied osmotic conditions willbe discussed in detail. However, other less studied osmoprotectiveosmolytes, such as trehalose, arabitol, mannitol and erythritol, also getaccumulate under certain conditions and therefore will be mentioned.

4.4.1. Glycerol metabolic pathwayYeasts possess two well-established pathways for glycerol biosyn-

thesis, both initiating from the glycolytic intermediate dihydroxyacetonephosphate (DHAP) and ending up with the production of glycerol:(1) the Gcy-Dak pathway includes the dephosphorylation of DHAP todihydroxyacetone (DHA) by dihydroxyacetone phosphate kinase(Dak) followed by the production of glycerol from DHA in a reactioncatalyzed by glycerol dehydrogenase (Gcy); (2) the Gpd-Gpp pathwaycomprises the conversion of DHAP to glycerol 3-phosphate mediatedby 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 stressconditions, under osmotic stress glycerol is mainly synthesized by Gpd-Gpp pathway (Larsson et al., 1993; Albertyn et al., 1994; Eriksson et al.,1995).

In S. cerevisiae, genes implicated in glycerol production are duplicatedin differentially regulated paralogs, namely GPD1 and GPD2 (Albertynet al., 1994; Eriksson et al., 1995), GPP1 and GPP2 (Norbeck et al., 1996;Påhlman et al., 2001a), and DAK1 and DAK2 (Norbeck and Blomberg,1997; Rep et al., 2000). These genes arose from either single geneduplication or whole genome duplication events, and show frequentlydivergence and functional differentiation (Kondrashov et al., 2002;Conant and Wolfe, 2008). For example, while GPP2 is mainly involvedin osmoadaptation,GPP1 has a role both in osmoadaptation and growthunder anaerobic conditions (Påhlman et al., 2001a,b). Similarly, Gpd1and Gpd2 enzymes have similar kinetic characteristics, but differwith respect to cellular distribution and transcriptional regulation(Albertyn et al., 1994; Eriksson et al., 1995). They are located in thecytosol, but Gpd1 possesses a peroxisome-targeting sequence, whileGpd2 is partly translocated into the mitochondria of non-respiringcells (Valadi et al., 2004; Jung et al., 2010). The single deletion of GPD1resulted in strains sensitive to osmotic stress (Albertyn et al., 1994),while the deletion of GPD2 reduced growth under anaerobiosis (Repet al., 1999). Therefore, Gpd1 has amajor role in osmoadaptation. How-ever, neither the deletion of GPD1 nor the deletion of GPD2 resulted in anoticeable change in glycerol yield. GPD1 and GPD2 genes could thushave roles which parlty overlap to compensate compromised functions,as recently shown for GPD2, which is up-regulated in response to GPD1deletion (DeLuna et al., 2010).

The Gpd-Gpp pathway is the main metabolic route leading fromDHAP to glycerol also in Z. rouxii (Fig. 2). Accordingly, the heterologousexpression of ZrGCY1, but not of ZrGPD1, restores the glycerol produc-tion and the salt tolerance in S. cerevisiae gpd1Δgpd2Δ mutants unableto synthesize glycerol (Watanabe et al., 2004). Furthermore, it wasfound that both the glycerol production and salt tolerance increasewhen ZrGPD1 is expressed along with ScGPP2 (Watanabe et al.,2004). Like in S. cerevisiae, in Z. rouxii allodiploid strain ATCC 42981two isoforms of some glycerol synthesis genes have been found, such

image of Fig.�2


as ZrGPD1 and ZrGPD2, ZrGPP1 and ZrGPP2, ZrGCY1 and ZrGCY2 (Iwakiet al., 2001; Wang et al., 2002; Watanabe et al., 2004). On the basisof their deduced amino-acid sequence, these proteins have a closehomology 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 cellsUnder anaerobic conditions, sugars are reduced to glycerol formain-

taining the cytosolic redox balance and consuming the excess of NADHproduced during the glycolytic pathway, amino acid biosynthesis andorganic acids anabolic routes (van Dijken and Scheffers, 1986; Priorand Hohmann, 1997; Medina et al., 2010). The glycerol production isalso pivotal in lipid biosynthesis and recycling the inorganic phosphateconsumed during glycolysis via the dephosphorylation step catalyzedby glycerol phosphatase (Nevoigt and Stahl, 1997) (Fig. 2).

The glycerol production was firstly studied in some non-Saccharomyces yeasts that are naturally unable to produce glyceroland grow on glucose under anaerobic conditions. It was observed thattheir ability to produce glycerol can be restored by introducing oxygenor another external electron acceptor in the culturemedium. This effectis called ‘Custers effect’ (Nevoigt and Stahl, 1997). In subsequentstudies, it was found that a S. cerevisiae gpd2Δ mutant grows poorly inanaerobic conditions without acetoin as an external electron acceptor(Ansell et al., 1997; Björkqvist et al., 1997; Valadi et al., 2004). Whenacetoin is added in anaerobic conditions, it is converted to butanediolby NAD+-dependent butanediol dehydrogenase (Nevoigt and Stahl,1997; Björkqvist et al., 1997). The GPD2 and GPP1 expression is stimu-lated under anaerobic conditions, when the glycerol productionbecomes 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 transcriptionand inhibit thefinal reductive step in ethanol fermentation, avoiding theaccumulation of excessive NADH (Ansell et al., 1997). Bisulphite ionsform a complex with acetaldehyde that limits the ethanol productionand promotes the reoxidation of glycolytically formed NADH by theglycerol synthesis (Fig. 2). The effect can be reversed upon addition ofacetaldehyde (Ansell et al., 1997). All these evidences pointed out thatthe function of GPD2 is mainly linked to redox imbalance, and not toosmotolerance.

4.4.3. Glycerol biosynthesis in osmo-stressed cellsThe cellular redox balance is pivotal for several aspects of cellular

physiology and its perturbation is implicated in the cellular adaptationto sugar and salt-stress conditions. Under high external osmolarity, anequimolar increase in cytoplasmic NADH is required to enhance theglycerol production. This requirement seems to be partially fulfilled bydecreasing the acetaldehyde reduction to ethanol and increasing theacetaldehyde oxidation to acetate (Fig. 2).

In S. cerevisiae, high external osmolarity induces the glycerolproduction both under aerobic and anaerobic conditions. Dependingon the strain, medium, and process parameters, 4 to 10% of the carbonsource may be converted to glycerol. Under stress conditions, theGPD1 and GPP2 genes are positively regulated by the HOG pathway toproduce glycerol (Albertyn et al., 1994; Eriksson et al., 1995). BesidesGPD1 and GPP2 genes, S. cerevisiae positively regulates the expressionof DAK1 gene encoding the Dha1 kinase (Rep et al., 2000). Accordingly,glycolytic pathway enzymes are slightly repressed in cells exposed tosalinemedium (Akhtar et al., 1997). Unlike S. cerevisiae, Z. rouxii doesn’tincrease the expression of neither ZrGPD1 or ZrGPP2 in response to saltstress (Iwaki et al., 2001) and the specific activity of the correspondingenzymes remains unaltered (van Zyl et al., 1991). Although ZrGPD1 andZrGPP2 have a main role in glycerol production (Watanabe et al., 2004),these results suggest that these genes are constitutively expressed inZ. rouxii and by-pass the HOG pathway control. In contrast, ZrGCY1

and ZrGCY2 are up-regulated by salt stress, indicating that these genesare 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 (awb0.96) (van Zyl et al., 1991).

Extreme halotolerant yeasts produce and accumulate large amountsof osmo-protective metabolites. Although this feature has been widelyexploited in industrial bioprocesses (Nevoigt and Stahl, 1997), itsmolecular mechanism has been poorly investigated. In the blackyeastH. werneckii, a set of 95 salt-responsive genes have been identifiedand most of them have not previously been related to halotoleranceand HOG pathway in any other halotolerant yeasts (Vaupotič andPlemenitaš, 2007). Furthermore, in H. werneckii the adaptation to highamounts of NaCl and sorbitol involves the differential expressionof mitochondria-related genes (Vaupotic et al., 2008). Mitochondriapreferentially accumulate energy metabolism-related enzymes inhypersaline medium, and chaperones and heat shock proteins, such asKar2 and Hsp60, in medium supplemented with sorbitol. Live-cellimaging showed that the mitochondria condense differentally inresponse to different osmolytes. In hypersaline medium, the mitochon-drial condensation is accompanied by an increasing in ATP synthesisand oxidative damage protection, whereas in presence of non-ionicosmolytes it is accompanied by a decreasing both in ATP synthesis andlipid peroxidation level (Vaupotic et al., 2008).

4.4.4. Glycerol retention and active transportBesides de novo biosynthesis, the glycerol retention is effective to

prevent the massive outflow of water from cells in response to anosmotic stress. Being a liposoluble molecule, glycerol has the tendencyto be leaked out through the plasma membrane, and its retention hasthus to be an active response by the cell. Under osmotic conditions,S. cerevisiae synthesizes a considerable glycerol amount most of whichleaks out of the plasma membrane (Hohmann, 2002). In contrast,Z. rouxii synthesizes a smaller glycerol quantity than S. cerevisiae, andchanges membrane phospholipid and fatty acid compositions todecrease the membrane fluidity and permeability. This strategy entailsZ. rouxii to effectively retain polyols and maintain a very high gradientbetween 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 differentmechanisms: a low affinity transport system (facilitated diffusion) anda high affinity proton symport system (active transport) (Table 3).Fps1 is an aquaglyceroporin belonging to the MIP family that is mainlyinvolved in the glycerol transport by facilitated diffusion (Sutherlandet al., 1997; Karlgren et al., 2005; Hohmann et al., 2007). FPS1 is consti-tutively expressed in a salt-independent manner and mutants lacking aregion in the Fps1 N-terminal domain (amino acid residues from 150 to231) constitutively release glycerol (Tamás et al., 2003). A shift fromlow to high external osmolarity induces the Fps1 closure, whereas adecrease in osmolarity causes the channel opening, bothwithin secondsafter the change in external osmolarity (Luyten et al., 1995; Tamás et al.,1999). In the absence of osmotic stress, Fps1 is opened by the bindingof Rgc2 (regulator of the glycerol channel 2) to the Fps1 C-terminalcytoplasmic domain. In response to osmostress Fps1 is closed byP-Hog1, which binds the N-terminal cytoplasmic domain of Fps1 andphosphorilates the positive regulator Rgc2 (Beese et al., 2009; Leeet al., 2013; Petelenz-Kurdziel et al., 2013). Furthermore, Fps1 affectsthe 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, namelyZrFps1, 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 fps1Δ mutants retain the abilityto grow on glycerol as the sole carbon source, ZrFPS1 is not required for

Table 3Comparative overview of the main genes involved in glycerol transport in Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and other non-conventional yeasts.

Species Glyceroldissimilation§

Glycerol transport References

Type Gene Description Role in osmostress protein(% identity)*

S. cerevisiae + AGT STL1 Main glycerol-H+ glycerol symporter inglycerol dissimilation; repressed by glucose

+ NP_010825.3, (/) Ferreira et al., 2005

AGT GUP1-2 Putative glycerol symporter involved inglycerol uptake when glycerol is uniquecarbon source; pleiotrophic effect onwall-related phenotypes

NI NP_011431.1, (/) Holst et al., 2000; Ferreira et al.,2006; Ferreira and Lucas, 2008

PGC FPS1 Aquaglyceroporin mediates glycerol diffusionin presence of glucose; closed in response toosmotic stress

− NP_013057.1, (/) Luyten et al., 1995; Oliveiraet al., 2003

Z. rouxii + AGT ND Putative glycerol-Na+ symporter mediatesglycerol accumulation as a function ofextracellular NaCl

+ / van Zyl et al., 1990; Lages et al.,1999

AGT PutativeZrSTL1/2

Putative glycerol-H+ symporter important forgrowth under hyperosmotic condtions

XP_002498998.1 (62%)XP_002498999.1 (64%)

Bubnová and Sychrová, 2011

PGC ZrFPS1 Aquaglyceroporin closed in response toosmotic stress

− AAR29350.1 (51%) Neves et al., 2004; Tang et al.,2005

D. hansenii + AGT ND Putative glycerol-Na+ symporter mediatesglycerol uptake as a function of extracellularNaCl

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; González-Hernández, 2010

PGC Absent / / / Prista et al., 2005M. farinosa (formerlyP. sorbitophila)

+ AGT ND Putative glycerol-H+ symporter constitutivelyexpressed

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., 2004PGC SpFPS1 Aquaglyceroporin + NP_592788.1

Spac977.17p (52%)Kayingo et al., 2004

C. halophila + AGT ND Glycerol-H+ symporter constitutivelyexpressed

NI / Silva-Graça and Lucas, 2003

PGC Absent / / / Silva-Graça and Lucas, 2003C. albicans + AGT CaSTL1 Glycerol-H+ symporter mediates glycerol

uptake+ CaO19.5753 (60%)

XP_718089.1Kayingo 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 glucoseNI 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, passiveglycerol channel; +, gene/protein positively regulated by hyperosmotic stress; −, gene/protein negatively regulated by hyperosmotic stress; NI, gene/protein mot involved inosmoadaptation; ND, not determined; /, not applicable; Sc, S. cerevisiae; Zr, Z. rouxii; Dh, D. hansenii; Sp, S. pombe; Ca, C. albicans.


the glycerol uptake but it ismainly involved in polyol efflux, as reportedin S. cerevisiae (Luyten et al., 1995; Tang et al., 2005). Recently,Wei et al.(2013) exploited the genome shuffling to gain a highly salt-tolerantZ. rouxii mutant strain, which, under a salt surplus, enhances theZrGPD1 transcription and reduces that of ZrFPS1. This finding supportsthat in Z. rouxii salt adaptation relies both on the induction of glycerolproduction and the inhibition of facilitated glycerol diffusion throughthe plasma membrane (Hou et al., 2013). In other halotolerant yeasts,like M. farinosa (P. sorbitophila) and D. hansenii, Fps1-like channelshave not been documented, suggesting that the glycerol diffusionoccurs in these yeasts through other membrane proteins (Table 3).

Active transport systems for glycerol have been documented inseveral yeasts. Yet, little is known about their genetics. In a pivotalstudy involving 42 yeast species, Lages et al. (1999) suggested that theactive glycerol uptake is essential in yeast halotolerance. These authorsfound that only the most salt-tolerant yeasts show a constitutive activeglycerol uptake, which is highly efficient in the intracellular glycerolaccumulation against the gradient. The analysis of glycerol uptake inpresence of salts has identified two different types of constitutive activeglycerol transport systems, namely Na+-glycerol and H+-glycerolsymporters (Adler et al., 1985; Marešová and Sychrová, 2003) (Table 3).In S. cerevisiae, glycerol is actively transported inside the cell by theH+ symporter (Lages and Lucas, 1997) encoded by STL1 (Ferreiraet al., 2005). The STL1 gene expression is repressed by glucose andinduced by nonfermentable carbon sources and by osmotic stresses ina Hog1-dependent manner (Lages and Lucas, 1997; Rep et al., 2000;Ferreira and Lucas, 2007). In S. cerevisiae, the glycerol transport is also

strongly influenced by the GUP1 gene. The protein Gup1 has twelvepredicted transmembrane domains, which are compatible with atransporter function, and the GUP1 gene disruption induces an osmo-sensitive phenotype in S. cerevisiae (Holst et al., 2000). Based on theseevidences, Gup1 was firstly proposed as glycerol transport. Furtherstudies, however, have demonstrated that Gup1 and the paralog Gup2are not active glycerol transporters, but regulatory elements with pleio-tropic effects on cell wall phenotypes (Neves et al., 2004 and referencesherein) (Table 3).

In the halotolerant Z. rouxii andD. hansenii, the expression of glycerol-Na+ symporters requires salt, suggesting that NaCl is the driving forcefor 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 andZrStl2, have been documented in Z. rouxii tomediate the glycerol uptakeunder high salt concentrations (Neves et al., 2004; Bubnová andSychrová, 2011). The Z. rouxii genomeharbours also a gene homologousto GUP1, but so far no studies have established the role in glycerol trans-port. Five polyol-H+ symporters have been found in D. hansenii, withdifferent specificities and affinities for polyols (Pereira et al., 2014). InM. farinosa (formerly P. sorbitophila), specific glycerol-H+ symportersare constitutively expressed and unresponsive to NaCl (Neves et al.,2004) (Table 3).

4.4.5. Other compatible solutesIn response to hyperosmotic stress, compatible solute production is

regulated by a complex process depending upon the growth phase,


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 mainlyconferred by other osmolytes, such as D-arabitol, erythritol, and treha-lose (Tokuoka et al., 1992; Petrovič 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; Plemenitaš et al.,2008), while the sugar stress mainly C-3 (glycerol), C-5 (arabitol), andC-6 polyols (mannitol and sorbitol) (van Eck et al., 1993). Under saltstress, D. hansenii accumulates DHAP and increases glycerol productionby increasing the proteins involved in the first steps of glycolysis and bydecreasing those involved in the Kreb’s-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 (duringexponential phase) and erythritol (during stationary phase) instead ofglycerol (Nobre and da Costa, 1985; Prista et al., 2005).

The disaccharide trehalose is one of the most effectiveosmoprotectants that, under osmotic stress, prevents phase transitionevents in the lipid bilayer, reduces the membrane permeability, andensures a proper protein folding (Elbein et al., 2003). Yeasts are ableto accumulate trehalose up to 15% of the cell dry mass when submittedto a stress. The trehalose synthesis is catalyzed by a multimeric proteincomplex (trehalose synthase complex), composed of four subunitsencoded by TPS1, TPS2, TPS3 and TSL1 genes (François and Parrou,2001). The trehalose hydrolysis is catalysed mainly by the neutraltrehalase Nth1 (Kopp et al., 1994; Nwaka et al., 1995). It was demon-strated that several yeast species synthetize trehalose under highsugar and salt conditions following the same trend of the cell growthand reaching the highest trehalose concentrations during the stationaryphase of cultivation (Tokuoka et al., 1992; Tokuoka, 1993). Hounsa et al.(1998) also indicated that S. cerevisiae is more tolerant to osmoticconditions during stationary than exponential phase, when it mainlyproduces trehalose instead of glycerol.

The compatible osmolyte production depends also upon the carbonsource. In media with non-repressing galactose as the carbon source,S. cerevisiae reduces the glycerol production, increases sensitivity toosmotic stress, and mainly utilizes trehalose as osmolyte (Elbein et al.,2003). Furthermore, under severe osmotic stress (0.866 aw), S. cerevisiaereduces the intracellular glycerol content, indicating that this osmolyteis essential for the yeast growth undermoderate but not severe osmoticstress (Albertyn et al., 1994; Hounsa et al., 1998). Hyperosmotic stressalso reduces the glycerol consumption and ethanol production rate,but increases the intra-cellular content of trehalose (Norbeck andBlomberg, 1997). Congruently, when the genes involved in trehalosesynthesis are deleted, the survival under NaCl stress of S. cerevisiaemutants 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 andsugar stresses induce production of glycerol, arabitol or both, dependingupon the osmoticum which has been used to reduce the aw. If sugar isused as stress agent instead of salt, D-arabitol is highly produced andaccumulated, whereas the glycerol concentration remains invariable(van Zyl and Prior, 1990). Fructose and glucose-containing media havebeen 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. cerevisiaegpd1Δ and gpd2Δ mutants is complemented by the expression ofmannitol dehydrogenase gene (MDH) involved in themannitol biosyn-thesis (Watanabe et al., 2006).

5. Signal transduction and cis/trans-acting regulatory factors

The yeast adaptive osmostress response is mostly controlled by theactivation of signal transduction pathways, which in turn regulate thedynamic interactions between transcription factors and specific

promoter binding sites (Posas et al., 2000; Rep et al., 2000; Hohmannet al., 2000; Gasch et al., 2000; Causton et al., 2001). Reversible proteinphosphorylation catalyzed by protein kinases represents a universalregulatorymechanismofmultiple physiological andmetabolic functionsin 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 five MAPK pathways known in yeasts(de Nadal et al., 2002; Hohmann, 2002); the calcineurin/Crz1 pathway,which is specifically involved in adaptation to high-salt conditions(Clapham, 2007); and the Ras-cAMP signalling pathway (Theveleinand deWinde, 1999; Norbeck and Blomberg, 2000) (Fig. 3). These path-ways arewell-characterized in S. cerevisiae, while they are only partiallystudied in Z. rouxii and other halotolerant and osmotolerant yeasts.

5.1. High osmolarity glycerol (HOG) pathway

Osmostress adaptation involves the activation of the HOG signallingcascade, which transduces osmosensory signals in yeast species(Brewster et al., 1993). The cascade consists of three consecutivelyactivated kinases: MAPKKK, MAPKK and MAPK. In S. cerevisiae, theosmotic 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 finally to the MAPK Hogl (Posaset al., 1996). On the other hand, Shol regulates the action of Hogl viaStell (MAPKKK) (Maeda et al., 1995) and Pbs2 (MAPKK) (Posas andSaito, 1997). Phosphorylation ofHog1by Pbs2 is done at the neighbouringThr and Tyr residues, respectively at position 174 and 176 (TGY motif).Once phosphorylated, active P-Hogl elicits both the immediate andlong-term adaptation to ionic stress (Alepuz et al., 2001; Ruiz andAriño, 2007; Ke et al., 2013). As mentioned before, long-term adapta-tion involves transcriptional and translational regulation of the genome,whereas short-term adaptation is accomplished by changes in glycerolaccumulation (Albertyn et al., 1994) and the reestablishment of ionicbalance (Proft and Struhl, 2004).

Starting from the first minute after the induction of salt stress,P-Hogl directly phosphorylates some membrane ion transporters, suchas Nha1 and Tok1, in order to rapidly readjust the transmembranefluxes of Na+ and K+ in osmo-stressed cells (Proft and Struhl, 2004).Furthermore, cytoplasmic P-Hog1 regulates enzymatic activities thatare necessary to rapidly produce and accumulate glycerol (Klipp et al.,2005). Such direct metabolic adjustments entail the cell to redirectcarbon resources toward the glycerol production. Finally Hog1 inducesa temporary arrest of cell-cycle progression in G1 phase (as reviewedby Saito and Posas, 2012).

During the long-term cellular adaptation to salt stress, P-Hog1 trans-locates to the nucleus, where, by the phosphorylation of at least threeseparate transcription factors (Msn2/Msn4 and Hot1) (Schmitt andMcEntee, 1996), it can modulate the regulation of more than 10% ofthe total yeast genome (O’Rourke and Herskowitz, 2004). The Hog1-regulated genes possess stress regulatory elements (STRE) with a coresequence CCCCT or AGGGG in their promoter region or sometimes inthe coding region (Schüller et al., 1994; Martinez-Pastor et al., 1996;Wang et al., 2002; Tang et al., 2005). These cis-acting factors are variablein number and orientation with respect to TATA box and they are nec-essary for stress-induced gene expression both in S. cerevisiae andZ. rouxii (Albertyn et al., 1994; Schüller 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 sufficient to bind a transcription factorand activate the expression of a reporter gene, two or moreSTRE copies induce a greater expression of stress-responsive genes(Kobayashi and McEntee, 1993).

The transduction pathway of osmotic stress signals has not yet beenfully elucidated in Z. rouxii. Like in S. cerevisiae, in Z. rouxii the HOGpathway comprises two operationally redundant plasma membraneosmosensors Sln1 and Sho1 (Maeda et al., 1994, 1995), and from one

Fig. 3. Integrated overview of signalling transmission pathways involved in osmoadaptive gene regulation. Genes are referred to as Saccharomyces cerevisiae genome, with the exceptionof ZrFPS1 and ZrGCY1/2. Genes regulated by calcineurin pathway are reported in bold.


to two putative homologs to S. cerevisiae HOG1, namely ZrHOG1 andZrHOG2 (Iwaki et al., 1999; Kinclová et al., 2001). A TGY motif similarto that of S. cerevisiae Hog1 has also been found in ZrHogl and ZrHog2,suggesting that a putative ZrPbs2 MAPK kinase similar to S. cerevisiaePbs2 could exist also in Z. rouxii (Iwaki et al., 1999). Although someZ. rouxii allodiploid strains possess two ZrHOG gene copies, namelyZrHOG1 and ZrHOG2, there is no relationship between this gene redun-dancy and their osmotolerance (Kinclová et al., 2001). This could meanthat ZrHOG1 and ZrHOG2 are expressed differentially, or that one ofthese paralogs is transcriptionally silent, as it happens for ZrSOD22.ZrHOG1 and ZrHOG2 genes are functional as MAP kinase and areable to complement the salt-sensitivity in S. cerevisiae hoglΔ null mu-tants (Iwaki et al., 1999). However, their overexpression in wild-typeS. cerevisiae doesn’t improve the glycerol synthesis, indicating that theamount of ZrHog1 and ZrHog2 is not a limiting factor in the glycerol pro-duction. These studies collectively supported that Z. rouxii possesses aHOG pathway functionally equivalent to that of S. cerevisiae.

STRE motifs are species-specifically distributed in the yeast genome,so that each species has peculiar sets of osmo-responsive genes thatcontribute to functional diversities in response to osmotic cues. Underhigh osmolarity conditions, S. cerevisiae produces much more glycerolthan Z. rouxii, by up-regulating genes involved in glycerol synthesis viaHOG pathway (Pribylova et al., 2007a). Accordingly, the S. cerevisiaeGPD1 gene exhibits four STRE elements in its promoter region(Albertyn et al., 1994) and encodes a Gpd1 enzymewith higher activitythan ZrGpd1 (Akhtar et al., 1997; Norbeck and Blomberg, 1997). LikeS. cerevisiae P-Hog1, P-ZrHogl also translocates into the nuclear

compartment and activates transcription factors homologous toMsn2p/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 HOGpathway-independent. The lack of STREs may account for themoderateglycerol amount produced by Z. rouxii to cope high external osmolarity.On the contrary, a putative STRE motif with core CCCCT sequence hasbeen identified in the upstream region of ZrFPS1, which is missing inS. cerevisiae FPS1 (Tang et al., 2005). Therefore, under high salt concen-trations, only the ZrFPS1 gene transcription is regulated via the HOGsignalling pathway, resulting in the higher ability of Z. rouxii to intracel-lularly retain glycerol compared to S. cerevisiae. In contrast, S. cerevisiaeP-Hog1 transiently induces the closure of Fps1 channel during the shortterm salt response, but it cannot induce the FPS1 transcription duringthe long term salt adaptation.

5.2. Calcineurin/Crz1 pathway

The calcineurin/Crz1 signal transduction pathway has an importantrole in cation homeostasis and salt stress adaptation, but it is relativelyless investigated compared to HOG pathway (Matsumoto et al., 2002;Ke et al., 2013). Calcineurin, the Ca2+/calmodulin-regulated proteinphosphatase 2B, is a heterodimer containing a catalytic (A) subunitcomplexed with an essential regulatory (B) subunit and requires Ca2+

and calmodulin for activity (Cyert et al., 1991). Calcineurin controlsCrz1 activity by regulating its subcellular localization (Stathopoulos-Gerontides et al., 1999). When calcineurin-dependent signalling is

image of Fig.�3


low, Crz1 is phosphorylated and resides primarily in the cytosol. Upondephosphorylation by calcineurin, Crz1 enters into the nucleus andhere it regulates the expression of target genes with CalcineurinDependent Response Elements (CDREs) in the promoter region. Thesecis-elements consist of a common sequence motif, 5’-GAGGCTG-3’,which Crz1 binds through a C2H2 zinc finger motif (Stathopoulos andCyert, 1997; Matheos et al., 1997). Around 163 genes have been foundunder control of Calcineuirn/Crz1 pathway (Yoshimoto et al., 2002).Among them, the GSC2 gene encodes a subunit of beta-1, 3 glucansynthase, which is responsible for the synthesis of 1, 3-beta-D-glucan(Levin, 2005), and it is up-regulated after exposure to high sugarconcentrations (Erasmus et al., 2003). CDRE motif was also foundin the flanking region of ENA1 (Kafadar and Cyert, 2004). High cytosolicCa2+ levels activate calcineurin in response to extracellular hyperionicstress and increase Na+ efflux 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 calcineurinpathways to achieve both immediate and longer-term adaptation toNaCl stress, respectively. In Torulaspora delbrueckii, a species close toZ. rouxii, crz1-null cells were insensitive to high Na+ concentrations,indicating that TdCrz1 is not required for the salt-induced transcriptionalactivation of TdENA1 gene (Hernandez-Lopez et al., 2006). This evi-dence suggests that yeast species could differ in regulating salt responsevia calcineurin/Crz1 pathway.

5.3. Ras-cAMP signalling pathway

The yeast cAMP-dependent protein kinase (PKA) is the effectorkinase 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 on the cell growth, trehalose and glyco-gen metabolism, dimorphic shift, and stress adaptation (Smith et al.,1998). Proteomic approaches were exploited to study the influence ofPKA on protein expression during the exponential growth ofS. cerevisiae under osmotic stress (Boy-Marcotte et al., 1998). Proteinsup-regulated under NaCl stress are grouped into three classes as regardsthe 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 deWinde, 1999), and at post-translational level throughthe regulation of trehalose synthesis (Kobayashi andMcEntee, 1993). Inparticular, Ras-cyclic AMP pathway negatively regulates Msn2/Msn4that are cytoplasmically accumulated, leading to the inhibition ofosmostress response (Görner et al., 1998) (Fig. 3).

6. Non genetic regulation of osmostress tolerance

Understanding the non-genetic regulation of cellular stress responseis an important biological question and it has received considerableattention in recent years. In relation to yeast osmotic tolerance, twonon-genetic mechanisms have been implicated so far. The first oneincludes epigenetic alterations in the chromatin structure that inducegenome-wide and local changes in gene transcription. The second oneis the non-genetic cell-to-cell phenotypic heterogeneity within anisogenic cell population under osmotic stress conditions.

6.1. Chromatin-mediated mechanisms

Post-translational modification of nucleosomal histone proteins andDNA methylation are two extensively characterized epigenetic mecha-nisms that regulate gene expression in plants grown under osmoticstress conditions (Chinnusamy and Zhu, 2009; Grativol et al., 2012).On the contrary, few studies have dealt with this topic in yeasts. Thefirst evidence about the epigenetic control of osmo-responsive genes

in yeasts was provided for the protein Sgd1, which is homologous inits N-terminal domain to Spt7, a subunit of the nucleosomal Spt-Ada-Gcn acetyltransferase (SAGA) histone acetylation complex (Robertsand Winston, 1997). The SGD1 overexpression is able to partiallycomplement growth defects in S. cerevisiae hog1Δ and pbs2Δ mutantsand to increase their glycerol production (Akhtar et al., 2000). Otherstudies suggested that changes in the chromatin structure contributeto the osmostress-stimulated expression of GPD1 gene. Under osmoticstimuli, the transcriptional repressor activator protein Rap1 binds theGPD1 promoter and induced the GPD1 transcription. On the contrary,the specific inactivation of all Rap1 binding sites completely abolishesthe 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, Hog1activates the transcription factors Hot1 and Msn2/4 which mediatethe recruitment of Rpd3-Sin3 histone deacetylase (de Nadal et al.,2004). Active Rpd3-Sin3 complex binds to specific promoters leadingto histone deacetylation, entry of RNA polymerase II, and transcriptioninitiation 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 andrecruits the SAGA histone deacetylase and the switch/sucrose non-fermenting (SWI/SNF) complex. The latters are transcription activatorswhich promote chromatin remodelling (Profit and Sturhl, 2002) andinduce the ENA1 expression in conjunction with Calcineurin/Crz1mediated pathway (Profit and Serrano, 1999). Yeast mutants lacking afunctional SWI/SNF complex are less tolerant to NaCl than wild-typecells (Profit and Sturhl, 2002). Further studies are required to establishwhether similar epigenetic regulatory mechanisms also exist inZygosaccharomyces yeasts.

6.2. Phenotypic heterogeneity

Phenotypic heterogeneity is a super-organism feature of prokaryotesandyeasts that provides a dynamic source of diversity andadaptive phe-notypes and increases the microbial fitness in stressful environments(Avery, 2006). Within an isogenic (genetically uniform)microbial pop-ulation in a homogenous environment, individual cells can still exhibitdifferences in phenotype. The precise mechanisms of such cell-to-cellheterogeneity are elusive and few studies have linked variations inyeast morphology to molecular effectors. Hsieh et al. (2013) havehighlighted that reduction in the intracellular amount of chaperonprotein Hsp90 triggers morphological heterogeneity in Z. rouxii clonalpopulations. Under standard conditions high Hsp90 levels assure thestability of Cla4, a key regulator of spectrin formation that inhibitsmorphological switching of the cell from budding to filamentousgrowth. Under salt stress, low Hsp90 levels reduce the Cla4 stabilityand stimulate the cellular switching towards a filamentous form.Additionally, the stress-induced Hsp90 inhibition is known to favourchromosomal instability and aneuploidy, which in turn potentiate thecellular adaption to stressful environments (Chen et al., 2012). Interest-ingly, aneuploidy has been also frequently found in highly salt-tolerantZ. rouxii strains (Solieri et al., 2013b, 2014).

7. Food exploitation and biotechnological perspective

The knowledge on the genetic and regulatory networks underlyingimportant phenotypic outcomes is a prerequisite for the successfulexploitation and control of microorganisms in food (Giudici et al.,2005). In the last decade, researches in food science and microbiologymoved 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”


approaches and bioinformatics have been recently applied to identify:1) candidate genes to use in genetic improvement of industrial yeaststrains; 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 identifica-tion of genetic mechanisms that shape the osmostress response inZ. rouxii and other osmotolerant and halotolerant food spoilage yeasts.To prevent their growth in food, it is crucial to understand what causesyeast stress tolerance. This knowledge could be exploited to set upquantitative prediction methods for the growth of food spoilage yeastsand, consequently, to reduce the economical loss derived from foodspoilage (Loureiro and Malfeito-Ferreira, 2003; Plemenitaš et al.,2008). On the other hand, genetic loci that determine yeast sugar andsalt-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 increasesthe expulsion of toxic Na+ and Li+ cations from cells at external acidicpH values and enhances the salt tolerance (Iwaki et al., 1998;Watanabe et al., 2005). Similarly, salt stress-responsive genes fromhalotolerant fungi and bacteria can be expressed in plants to alleviateproblems caused by soil salinization in agriculture (Gostinčar et al.,2012). A similar strategy was successful to reduce stuck fermentationin the bioethanol production (Zhao and Bai, 2009).

The ability to grow at very low aw makes the Zygosaccharomycesstrains very attractive as microbial cell factories both for the expressionof heterologous proteins and metabolite production (Branduardi et al.,2004, 2006; Vigentini et al., 2005). Zygosaccharomyces bailii has beenused as host for the production of interleukin-1β due to its ability towithstand hyper-osmotic, hyper-thermic, and acidic environments(Vigentini et al., 2005). Zygosacchaomyces rouxii strains have beenexploited for producing glutaminase (Kashyap et al., 2002; Iyer andSinghal, 2008), chiral compounds (Hauck et al., 2003), and heterologousproteins (Ogawa et al., 1990). Taking advantage of their ability to pro-duce polyols, osmotolerant and halotolerant yeasts, such asM. farinosa(formerly P. sorbitophila) and C. tropicalis, have been employed on alarge-scale fermentation processes for the glycerol production (Liuet al., 2006). Owing to the problems associated with the chemical pro-duction, halotolerant and osmotolerant yeasts became valuable for thefermentation-based production of sweet-tasting bodying and texturingagents, such as mannitol, D-arbitol, and xylitol (Saha et al., 2007; Sahaand Racine, 2011).

Zygosaccharomyces species have been exploited as biocatalysts infood and beverage fermentation. Zygosacchaomyces rouxii plays acentral role in the flavour formation of soy sauce (Sluis et al., 2001;Cao et al., 2010; Wei et al., 2013). Flavour components, such as4-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 flavour andquality (Hauck et al., 2003). A highly salt-tolerant Z. rouxiimutant con-structed by whole genome shuffling grows better in soy sauce than theparental 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 wereisolated 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 performanceand in tailoring the sensory characteristics of these products (Solieriand Giudici, 2008).

8. Concluding remarks

A critical review on themechanisms underlying sugar and salt stresstolerance in non-conventional yeasts unveils the complex nature of thecellular adaptation to low aw environments. In the past, most researchesabout this topic have been carried out using S. cerevisiae as modelorganism. Recently, the availability of transformation tools and the

genome sequences fromosmo andhalotolerant food species highlightedhow these yeasts combine conserved and species-specific strategies tocounteract better sugar and salt stress than S. cerevisiae. These differ-ences are operational at genetic, metabolic, and regulatory levels. Inparticular, new insights on Z. rouxii functional biology and genomicspoint out how sugar and salt tolerance arise from distinct adaptivemechanisms, which integrate solute-unspecific and solute-specificroutes. Future investigations will benefit by the application of systemsbiology tools. They will contribute to complete the detailed map ofosmo and halotolerance determinants in Z. rouxii. All these efforts holdpromise for i) rational strain engineering for biotechnological and foodexploitation; ii) prevention of yeast food spoilage; iii) understandingthe yeast adaptation to adverse environments.

Acknowledgements

Thiswork has been supported by a grant of Regione Toscana (Misura124 Acetoscana: PSF 2007/2013).

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