Adaptive Response and Tolerance to Sugar and Salt Stress in the Food Yeast Zygosaccharomyces Rouxii

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

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Review Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii

Tikam Chand Dakal, Lisa Solieri, Paolo Giudici ⁎

Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42122, Reggio Emilia, Italy article info abstract

Article history: The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survive Received 14 November 2013 in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na+ cations) Received in revised form 18 April 2014 solutes. This ability determines the success of fermentation on high osmolarity food matrices and leads to spoilage of Accepted 4 May 2014 high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits Available online 25 May 2014 shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation con-

Keywords: trol, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights on Zygosaccharomyces rouxii the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses. Spoilage yeast Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts inte- Osmotolerance grate general and osmoticum-speciﬁc adaptive responses under sugar and salts stresses, including regulation of Halotolerance Na+ and K+-ﬂuxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte produc- Glycerol accumulation and retention tion and accumulation, and stress signalling pathways. We suggest how an integrated and system-based knowledge Cation homeostasis on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms. © 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction...... 141 2. Amatterofnomenclature...... 141 3. Osmotolerantandhalotolerantyeastsinfood...... 142 4. Genecircuitsandmetabolicpathways...... 143 4.1. Cellwallandplasmamembrane...... 143 4.2. Cationhomeostasis...... 144 4.2.1. Na+ inwardandoutwardmovements...... 144 4.2.2. K+ inwardandoutwardmovements...... 146 4.3. Sugartransporters...... 146 4.4. Productionandaccumulationofosmolytes...... 147 4.4.1. Glycerolmetabolicpathway...... 147 4.4.2. Glycerolbiosynthesisinnon-stressedcells...... 148 4.4.3. Glycerolbiosynthesisinosmo-stressedcells...... 148 4.4.4. Glycerolretentionandactivetransport...... 148 4.4.5. Othercompatiblesolutes...... 149 5. Signal transduction and cis/trans-actingregulatoryfactors...... 150 5.1. Highosmolarityglycerol(HOG)pathway...... 150 5.2. Calcineurin/Crz1pathway...... 151 5.3. Ras-cAMPsignallingpathway...... 152 6. Nongeneticregulationofosmostresstolerance...... 152 6.1. Chromatin-mediatedmechanisms...... 152

Abbreviations: CDRE, calcineurin dependent response element; CNV, copy number variation; CWI, cell wall integrity; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; HOG, high-osmolarity glycerol; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; P-Hog1, phosphorylated Hog 1; STRE, stress responsive element; SWI/SNF complex, switch/sucrose non-fermenting complex. ⁎ Corresponding author. Tel.: +39 0522522057; fax +39 0522522027. E-mail address: [email protected] (P. Giudici).

6.2. Phenotypicheterogeneity...... 152 7. Foodexploitationandbiotechnologicalperspective...... 152 8. Concludingremarks...... 153 Acknowledgements...... 153 References...... 153

1. Introduction yeasts to avoid outflow of cellular water in low aw environments (Nevoigt and Sthal, 1997; Lages et al., 1999; Silva-Graça and Lucas, The high concentrations of ionic (mainly Na+) and non-ionic (mainly 2003). Another emerging issue concerns how salt and sugars elicit dis- sugars and polyols) solutes reduce water activity (aw)infoodandaretwo tinct or partial overlapping responses in yeasts. Whereas sugars and of the major abiotic stressors, both limiting the yeast growth. High exter- polyols modify osmotic pressure, salts induce alterations both in osmot- nal osmolarity has been used for centuries for food preservation, because ic pressure and ion homeostasis. The result is that partially different it causes water outflow from the cell and results in a higher intracellular mechanisms become operational in response to sugar and salts. Since concentration of ions and metabolites and in an eventual arrest of cellu- halo and osmotolerance could be paired and unpaired phenotypes in lar activity. The yeast ability to cope with these environmental insults Z. rouxii and relatives, these yeasts are very attractive models for determines both the success of certain food and beverage fermentation deciphering genetic circuits and functional pathways underlying and the thriving of food spoilage. halotolerance and osmotolerance. Since the sequencing of strain S288c (Goffeau et al., 1996), impressive Here, we review recent insights on the mechanisms that govern advances in genomics, proteomics, and systems biology have made halotolerance and osmotolerance in Z. rouxii and compare them to S. cerevisiae the paradigm for understanding these osmo-adaptive those active in S. cerevisiae and in other osmo and halotolerant food mechanisms, which have been exhaustively summarized by several re- yeasts at genetic, metabolic, signalling, and epigenetic level. Furthermore, views (Nevoigt and Stahl, 1997; Hohmann, 2002; Ariño et al., 2010; we highlight how these yeasts can achieve generic and osmoticum- Kühn and Klipp, 2012). As a result, the S. cerevisiae response to high specific responses to sugar and salt stresses. Finally, we point out how external solute concentrations has been described as a system-level the understanding of osmostress responsive mechanisms can advan- coordination between the extracellular environment and the genetic tage microbial fermentation and food quality. make-up inside the cell. The following interconnected modules are involved: (i) receiving information from external environment (sensing); 2. A matter of nomenclature (ii) conducting it to the inside (signal transduction); (iii) integrating it with internal genetic information in order to mount an appropriate re- Tolerance to high ionic and non-ionic solute concentrations is a sponse (effector processes) (de Nadal et al., 2011). This system-level specific cellular adaptability to sudden and severe fluctuations in water knowledge has been exploited in food industry to improve yeast fer- availability and a tendency of cells to restore or maintain normal physiol- mentations on highly salty and sugary matrices or to decrease the ogy, morphology and biological functions (Yancey, 2005; Klipp et al., food spoilage by sugar and salt resistant-yeast species. However, as 2005). Microbial growth under high external osmolarity is frequently de- being moderately halotolerant and osmotolerant, S. cerevisiae could be scribed in terms of aw that is the chemical potential of free water in solu- inappropriate to describe the yeast response to hypersaline and tion. Microorganisms able to colonize food with high osmolarity and, hyperosmotic food. consequently, low aw, were collectively indicated as xerotolerant (no ab- Zygosaccharomyces rouxii is the osmotolerant and halotolerant yeast solute requirement of low aw), and xerophilic (“lovers of low aw”)(Pitt most phylogenetically related to S. cerevisiae and inhabits a variety and Hocking, 2009)(Table 1). A more appropriate microbial classification of highly sugary and salty food, where it carries out fermentation or would consider the kind of osmoticum and include the following catego- determines food spoilage. It belongs to the genus Zygosaccharomyces, ries: osmophilic, absolute requirement for non-ionic solutes and ability to which includes the highest number of salt and sugar-tolerant yeasts. grow up to solute concentrations approaching saturation; osmotolerant, The majority of these species are osmotolerant (positive growth at no absolute requirement of non-ionic solutes for viability and ability to high sugar concentration up to 60–70% glucose), whereas only a few tolerate a wide range of osmolarity, from hypo-osmotic to hyper- are both highly osmo and halotolerant (Table 1). Recently, the complete osmotic solutions; osmosensitive, sensitive to excess concentration of genome sequences of Z. rouxii (Souciet et al., 2009) and other highly non-ionic solutes; halophilic, absolute requirement for high salt and osmo and halotolerant yeasts, such as Millerozyma farinosa (formerly ability to grow up to salt concentrations approaching saturation; Pichia sorbitophila)(Louis et al., 2012), Debaryomyces hansenii (Kumar halotolerant, no absolute requirement of salt for viability and ability to et al., 2012), and Zygosaccharomyces bailii (Galeote et al., 2013), have tolerate a wide range of salinity, from hypo-saline to hyper-saline solu- become available. Furthermore, ‘omics’ tools and genetic manipulation tion; and halosensitive, sensitive to excess concentration of salt. protocols have been recently employed to analyze the relationships Most food yeasts can develop well at aw values around 0.95–0.90. A of osmostress phenotype to genetic and molecular determinants cut-offofaw b0.70 has been frequently used to delineate osmotolerant (Prybilova et al., 2007a,b; Watanabe et al., 2010). From these intense and halotolerant yeasts. In the past, yeasts isolated from sugary and efforts, the yeast osmostress adaptation emerges as a complex mecha- salty food with aw lower than 0.70 were referred to as “osmophilic” and nism that integrates genes, regulatory networks, and signalling path- “halophilic” (Tokuoka, 1993). For instance, Debaryomyces hansenii has ways, and that differs depending upon the species and the osmoticum been described as halophilic yeast based on the ability to grow at 1.0 M in the surrounding medium. Comparison of species with different of salt with growth rate and final biomass close to the values obtained sugar and salt tolerance highlighted how yeasts exploit different strate- without salt (Almagro et al., 2000; González-Hernández et al., 2004; gies to survive under osmotic and salt stress (Ramos et al., 2011). For Aggarwal and Mondal, 2009). Other yeasts were classified as halophilic + example, Z. rouxii resembles S. cerevisiae in extruding Na cations out or osmophilic, such as M. farinosa (formerly P. sorbitophila)(Rodrigues of the cell or driving them into the vacuole (Ramos, 1999), while the de Miranda et al., 1980), Candida etchellsii (formerly Candida halotolerant yeast Debaryomyces hansenii is a sodium includer, which halonitratophila), Candida versatilis (Barnett et al., 2000), and the black + accumulates intracellularly Na without getting intoxicated (Ramos, yeast Hortea werneckii (Gunde-Cimerman et al., 2000). However, 1999). Beyond the species-specific strategies, other osmostress re- differently from halophilic and osmophilic bacteria, none of these yeasts sponses, such as the osmolytes accumulation, are ubiquitous among satisfiesthetruedefinition of osmophily or halophily, because they 142 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157

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

Category Deﬁnition Species name1 D-glucose % NaCl (M); % (w/v) Food spoilage2 References3 (w/v)

50 60

Moderately osmotolerant Lack of growth Saccharomyces cerevisiae −−b1.70; 10% Soft drink, fruit juice Onishi, 1963 and moderately at N50% (w/v) Schizosaccharomyces −−1.00; 5.8% Cheese, fruit (rarely) Lages et al., 1999; halotolerant D glucose; pombe Barnett et al., 2000 lack of growth Zygosaccharomyces + − 1.00; 5.8% Wine Lages et al., 1999; at N2.0M NaCl florentinus Barnett et al., 2000 (Zygotorulaspora florentina) Candida glabrata + − 1.70; 10% Juice concentrate Pitt and Hocking, 2009 Osmotolerant and Growth at N50% Candida tropicalis + − 1.7–2.0; 10-11.7% Fruit juice Barnett et al., 2000; moderately (w/v) D glu- Deak, 2007; halotolerant cose; Pitt and Hocking, 2009 lack of growth Zygosaccharomyces + + 1.70; 10% Juice concentrate, Kurtzman et al., 2011 at N2.0M NaCl mellis honey Zygosaccharomyces + + 2.0; 11.7% n Solieri et al., 2014 sapae Zygosaccharomyces bailii +w1.0–2.0; 5.8-11.7% Juice, sauces, ciders, Lages et al., 1999; wines Barnett et al., 2000 Zygosaccharomyces + +§1.0–2.0; 5.8-11.7% Soft drink, wine James and Stratford, 2003 bisporus Moderately osmotolerant Lack of growth Candida parapsilosis + − 3.0; 17.5% Dairy food Pitt and Hocking, 2009 and halotolerant at N50% (w/v) Pichia membranifaciens +§ − 3.00; 17.5% Bread, fermented milk, Lages et al., 1999; D glucose; olive Barnett et al., 2000 growth at Issatchenkia orientalis +§ − 2.0; 11.7% Olives, pickles and Lages et al., 1999; N2.0M NaCl (Pichia kudriavzevii) sauces (rarely) Barnett et al., 2000 Osmotolerant and Growth at N50% Pichia sorbitophila ++3.0–4.0; 17.5-23.4% Beer, sake, soy sauce, Lages and Lucas, 1995 halotolerant (w/v) D glu- (Millerozyma farinosa) Mash of rice vinegar cose; Zygosaccharomyces + + 3.0§; 17.5% Juice concentrate, honey, Lages et al., 1999; growth at rouxii jams, confectionery, Solieri et al., 2014 N2.0M NaCl (allodiploid strains) dried fruits, soy sauce Candida magnoliae + + 3.0; 17.5% Sugary food Barnett et al., 2000: Martorell et al., 2007 Pichia guillermondii +§ +§ 3.0; 17.5% Olive, salt meat Butinar et al., 2005 (Meyerozyma guilliermondii) Hortaea werneckii + n 5.20; 30.8% Salt fish Butinar et al., 2005; Lenassi et al., 2011 Debaryomyces hansenii +§ +§3.0–4.0; 17.5-23.4% Olive Barnett et al., 2000; (Candida famata) Lages et al., 1999 Candida halophila ++4.0–5.0; 23.4-29.1% Cheese brines Barnett et al., 2000; (Candida versatilis) Silva-Graça and Lucas, 2003

1 Species names are reported according to the corresponding reference; current names are reported in bracket. 2 Information about food spoilage was retrived from Pitt and Hocking, 2009. 3 References used for growth data; §, variable trait; n, not reported; w, weak.

tolerate high osmotic conditions, without requiring high sugar or the genus Zygosaccharomyces. The genus comprises 7 species (James salt amounts for their growth (Silva-Graça et al., 2003). Similarly, the and Stratford, 2011) frequently isolated from highly sugary (honey, terms ‘osmotolerant’ and ‘halotolerant’ have been improperly used as jams, syrups, fruit-juices, fruit juice concentrates, chocolate candies, synonymous, but they should be reserved for yeasts that are able to and concentrated grape must) and salty food (soy sauce and miso live at high sugar and ionic solutes (mainly Na+) concentrations, paste) (Deák and Beuchat, 1993; Tokuoka, 1993; Solieri et al., 2006, respectively (Onishi, 1963; Tokuoka et al., 1992; Tokuoka, 1993; 2013a,b; Martorell et al., 2007; Suezawa et al., 2008). Zygosaccharomyces Lages et al., 1999; Marešova and Sychrová, 2003; Pribylova et al., sapae and Z. rouxii are the main biocatalysts of alcoholic fermentation in 2007a,b). Since different solutes elicit distinct yeast stress responses, high sugar and/or salt fermented food, such as traditional balsamic vin- osmotolerant and halotolerant phenotypes have been defined in egar and miso, respectively (Suezawa et al., 2008; Solieri and Giudici, relation to the yeast ability to grow up to 55–65% (w/v) sugar (lower 2008; Solieri et al., 2013a). Furthermore, Z. rouxii and Zygosaccharomyces than ~0.88 aw)orat15–25% (w/v) salt concentrations (corresponding bailii are involved in alcoholic fermentation during kombucha to 0.92–0.85 aw range), respectively (Deak, 2006, 2007; Kurtzamn production. Zygosaccharomyces species have been also recognized as et al., 2011). Based on these assays, tolerance to sugar and salt could one of the main spoilage yeasts in food industry due to their tolerances be differentially distributed among yeasts species. Table 1 shows the to salt, sugar, and weak acid preservatives (Pitt and Hocking, 2009; classification of representative yeast species into four classes according Fleet, 2011). In food manufacture, the yeast spoilage of products causes to their degree of sugar and salt tolerance. severe economic loss and affects a variety of processed foods, including bread, cereals, spices, dairy products such as cheese, spreads (marga- 3. Osmotolerant and halotolerant yeasts in food rine), dressings, fondant, chocolate, fermented sauces (soy), soft drinks, fruits, jams, and high-sugar fruit syrups (Stratford, 2006). In particular, Osmotolerant and halotolerant yeasts have a pivotal role in food Z. bailii and Zygosaccharomyces lentus represent the most important fermentation and spoilage (Fleet, 1992) and several of them belong to Zygosaccharomyces from the point of view of weak acid preservative T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 143 resistance (Thomas and Davenport, 1985; Steels et al., 1999; 2002), on cell wall composition suggested that Z. rouxii decreases cell wall displaying an unusually high resistance to the small number of acids ap- mannans in presence of salt (Hamada et al., 1984; Hosono, 1992). proved for use as food preservatives (primarily sorbic, benzoic, acetic These studies firstly suggested that cell wall rigidity and integrity have and propionic acids). On the other hand, Z. rouxii and Z. mellis can toler- been implicated in tolerance to salt-induced stress in Z. rouxii. Strain- ate low aw with different sensitivity to ionic and non-ionic solutes. specific differences have been also described in the internal layer of Zygosaccharomyces rouxii is well-known for osmo and halotolerance β-D-glucan and cell wall mannans. In particular, Z. rouxii strains having and survives up to 0.80 aw in presence of ionic solutes (salt) and up a more rigid cell wall tend to be less halotolerant than those having a to 0.65 aw in presence of non-ionic solutes (sugars), while Z. mellis more flexible and elastic cell wall (Pribylova et al., 2007b). Although survives at low aw values only when the solutes are sugars (Stratford, these variations are congruent with the possible involvement of cell 2006). Moreover, inter-strain differences in halotolerance have been wall mannans in salt tolerance, more evidences are required to reinforce reported within Z. rouxii, with the strains isolated from high-salt foods such speculations. Recently, some highly salt-tolerant Z. rouxii strains tolerating better NaCl than those isolated from high-sugar foods have been found to possess an increased copy number of FLO11 gene (Pribylova et al., 2007b; Solieri et al., 2014). The highly salt-tolerant that encodes a glycophosphatidylinositol-anchored cell surface glyco- strains are allodiploid strains arising from putative outcrossing events, protein. This copy number amplification affects positively the cell wall or aneuploid strains which differ from haploid Z. rouxii for karyotype, hydrophobicity and enables strains with a higher copy number of ploidy level and copy number variation (CNV) of housekeeping genes FLO11 to exhibit a fitness advantage compared to a reference strain (Solieri et al., 2008, 2013b). This finding suggests that osmo and under osmostress static culture conditions (Watanabe et al., 2013). halotolerance are two distinct physiological phenotypes. Interestingly, in S. cerevisiae FLO11 is responsible for filamentation, Food yeasts other than Zygosaccharomyces species have been invasive growth, and biofilm formation (Fidalgo et al., 2006) and it is described as being osmotolerant and/or halotolerant. Millerozyma regulated by at least three well-known signalling cascades, such as the farinosa (formerly P. sorbitophila) is a ubiquitous, halotolerant yeast Ras-cAMP pathway, the Mitogen-activated protein kinase (MAPK)- found mainly in food (alcoholic beverages like beer and sake, soy dependent filamentous growth pathway, and the glucose repression sauce, miso, mash of rice vinegar etc.) and it is known for the ability to pathway (Verstrepen and Klis, 2006). grow in more than 4.0 M NaCl (Lages and Lucas, 1995; Silva-Graça A few studies dealt with the regulatory pathways employed by and Lucas, 2003; Silva-Graça et al., 2003) and to tolerate up to 70% Z. rouxii to maintain and modulate the cell wall integrity in the face glucitol (Rodriques de Miranda et al., 1980). Debaryomyces hansenii of environmental challenges. In the two main model yeasts was originally isolated from saline environments, such as seawater S. pombe and S. cerevisiae, the pathway mainly responsible for regu- and concentrated brines, and it is also associated with cheese and lating cell wall changes is known as cell wall integrity (CWI) signal- meat processing. It can tolerate salinity levels up to 4.0 M NaCl and ling pathway. Upon osmotic stress, this pathway transmits wall survive in high-sugar products with aw values as low as 0.62 (as stress signals from the cell surface sensors to the Rho1 GTPase, reviewed by Aggarwal and Mondal, 2009). The strongly halotolerant which mobilizes a variety of effectors. Activation of CWI pathway yeast-like fungus H. werneckii was found on salty food and other low- regulates the production of various cell wall carbohydrate polymers water-activity substrates for its ability to grow, albeit extremely slowly, and their polarized delivery to the site of cell wall. Moreover, CWI in a nearly saturated salt solution (5.2 M NaCl), or completely without serves different functions other than the osmotic stress response, salt, with a broad growth optimum from 1.0–3.0 M NaCl. In addition, a such as the response against mechanical stress, cell shape maintain- group of poorly studied osmotolerant species have been associated ing, and scaffold for cell-surface proteins (as reviewed by Levin, with spoilage of sugary food and with insects, including Candida 2011). A few proteins involved in CWI pathway have been associated davenportii (Stratford et al., 2002), Candida stellata and Candida magnoliae to osmo and halotolerance phenotypes. In S. pombe,theMAPKPmk1 (Rosa et al., 2003). Schizosaccharomyces pombe is an osmotolerant, has been implicated in cell wall integrity, cytokinesis, and ion ho- preservative-resistant yeast, but it is rarely associated to food spoilage meostasis (Sengar et al., 1997).TheMAPKkinasekinase(also due to its salt-sensitivity (Pitt and Hocking, 2009). known as MEK kinase, MEKK or MAPKKK) Mkh1 and the MAPK kinase (also known as MEK or MAPKK) Pek1 act as the upstream sig- 4. Gene circuits and metabolic pathways nalling components in the CWI pathway cascade and are essential for Pmk1 activation. The involvement of Mkh1, Pek1, and Pmk1 has In presence of high extra-cellular solute concentrations, yeast cell been demonstrated in salt stress response of S. pombe (Madrid experiences three main physiological alterations: changes in physical et al., 2006)and,recently,alsoofS. cerevisiae (Rodicio and and chemical structure of the cell wall and plasma membrane; increase Heinisch, 2010; Levin, 2011). In S. pombe, MKH1 gene-lacking cells of intracellular solute/ion toxicity; and alterations in the osmotic re-enter the cell cycle quite slowly after a prolonged arrest in stationary pressure and cell volume. Therefore, three systems enable yeasts to phase and in the presence of NaCl or KCl they show a reduced growth counteract stress challenges and to restore osmotic balance: a) regulation (Sengar et al., 1997). In S. cerevisiae, Kcs1 kinase, which is involved in ino- of morphological and structural properties of the cell wall and plasma sitol signalling, also ensures the cell wall integrity and consequently con- membrane; b) modulation of transport systems; c) production, accu- fers adaptive responses to salt stress. The search for orthologous genes in mulation and retention of metabolically compatible osmolytes. non-conventional yeast genomes has suggested that a similar pathway could also operate in Z. rouxii (Rodicio and Heinisch, 2010). 4.1. Cell wall and plasma membrane Inside the cell wall there is the plasma membrane, which is involved in a variety of cellular processes such as cell adhesion, ion conductivity, Morphological and structural properties of the cell wall and plasma and signalling. Like prokaryotes, yeasts regulate the plasma membrane membrane are important factors affecting the yeast osmo and fluidity in osmostress adaptation (Turk et al., 2011). The main factors halotolerance. By reshaping their integrity and fluidity, yeast cell estab- affecting the membrane fluidity are the length, branching and degree lishes a balance by which the force driving water across the osmotic of saturation of fatty acids, the amount of sterols, and the phospholipid gradient into the cell is counteracted by turgor pressure against the composition (Russell, 1989; Rodriguez-Vargas et al., 2007). The remod- plasma membrane and cell wall (Klis et al., 2006; Levin, 2011). elling of these parameters strongly affects not only the membrane fluid- The yeast cell wall is a rigid skeleton formed by four classes of ity, but also the proper functioning of membrane-attached proteins, macromolecules interconnected by covalent bonds: the mannosylated such as those involved in ion homeostasis, the glycerol transport cell wall proteins called mannoproteins, 1,3-β-D-glucan, 1,6-β-D- systems (Marquez and Serrano, 1996; Kamauchi et al., 2002), and the glucan and chitin (a polymer of GlcNAc) (Klis et al., 2006). Early studies plasma membrane ATPase activity (Coccetti et al., 1998). 144 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157

When the external salinity is shifted from low to high concentrations, and plasma membrane fluidity in response to ionic and non-ionic S. cerevisiae shortens the fatty-acid chain length (Turk et al., 2007)andin- stresses. Furthermore, it was demonstrated that certain osmolytes, es- creases their saturation level by synthesizing fatty acid desaturases that pecially trehalose, stabilize phospholipid bilayers during osmostress introduce double bonds into the fatty acids of membrane lipids (Levin, conditions (Hounsa et al., 1998; Gancedo and Flores, 2004). In prokary- 2011). Whereas many eukaryotic organisms can synthesize dienoic otes, trehalose stabilizes lipid bilayers and prevents damages derived fatty acids, S. cerevisiae can introduce only a single double bond at the from dehydration by inhibiting the fusion between vesicles and by Δ9 position (Tunblad-Johansson and Adler, 1987; Sharma et al., 1996; maintaining the membrane lipids in the fluid phase (phase transition). Rodriguez-Vargas et al., 2007). Under NaCl stress Z. rouxii increases the Similarly, in S. cerevisiae trehalose also protects plasma membrane amount of free ergosterol (decreasing the sterol-to-phospholipid ratio) against osmotic stress (Iturriaga et al., 2009 and references herein). and reduces both the lipid unsaturation index and the phospholipid- to-protein ratio (Watanabe and Takakuwa, 1984, 1987; Hosono, 1992; 4.2. Cation homeostasis Yoshikawa et al., 1995). The modifications result in a reduction of membrane fluidity. On the contrary, in the extreme halotolerant yeast High concentrations of Na+ cations are toxic to most living cells and D. hansenii high NaCl levels increase the sterol-to-phospholipid ratio are frequent in food, while K+ cations are less abundant, but they are es- and fatty acid unsaturation, without significantly affecting fluidity sential for compensating negative charges and activating key metabolic (Turk et al., 2007). In the strongly halotolerant yeast-like fungus processes, such as pyruvate synthesis and protein translation. Thus, the H. werneckii, high salinity conditions induce slight changes in the total majority of yeasts maintain a high intracellular ratio of K+/Na+,byse- sterol content, but cause a significant increase both in the phospholipid lectively accumulating K+ and actively extruding Na+. Intracellular ho- content and the fatty acid unsaturation level (Gunde-Cimerman and meostasis of these alkali metal cations affects physiological parameters Plemenitaš, 2006). Therefore, H. werneckii tends to maintain the and functioning, such as cell volume, plasma membrane potential, and sterol-to-phospholipid ratio significantly lower than other yeasts, mak- intracellular pH (Arino et al., 2010). When cation concentrations in ing the plasma membrane comparatively more fluid and offering higher the external medium are higher than the physiological range, the acclimatization to salt stress conditions (Turk et al., 2007). difference between the electrochemical potentials of the cations across Ionic and non-ionic solutes have different effects on the plasma mem- the membrane may be so high that the entrance cannot be annulled by brane. When the sunflower (Helianthus annuus)oleateΔ12 desaturases simply inhibiting the transporters mediating the uptake. To avoid an FAD2-1 and FAD2-3 genes are expressed in S. cerevisiae, they increase internal toxic cation concentration, different types of efflux systems the content of dienoic fatty acids, especially 18:2Δ9,12 and the plasma have been evolved to balance any excessive entrance. membrane unsaturation index (Rodríguez-Vargas et al., 2007). Under salt stress FAD-expressing cells display higher membrane fluidity and 4.2.1. Na+ inward and outward movements salt tolerance than the wild-type cells. In contrast, under high sorbitol Fig. 1 illustrates efflux and influx systems recruited by Z. rouxii to concentrations, the FAD-expressing cells do not differ in growth rate modulate the transport activity of the alkali metal cations across the from the wild-type cells, suggesting that the dienoic fatty acid content plasma membrane. Like S. cerevisiae, Z. rouxii has been regarded as a doesn’t affect the tolerance to non-ionic solutes. Although further sodium excluder species, for which Na+ is comparatively more cytotoxic researches are required, these evidences collectively support that at high concentrations compared to K+ (Pribylova et al., 2008). In there are distinct mechanisms in modulating the cell wall integrity sodium excluder yeasts, two main mechanisms mediate the efflux of

Fig. 1. Overview of the main plasma membrane systems mediating the alkali metal cation homeostasis and the glycerol uptake/retention in Zygosaccharomyces rouxii. T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 145 excessive cations (Na+ or K+) through the plasma membrane. The first increases the tolerance to Na+ (Bañuelos et al., 1995). Other studies one is represented by the Na+ (K+) P-type -ATPase, also known as so- reported that S. pombe Sod2 antiporter complements the Na+ sensitivity dium pump, encoded by ENA1-4 genes in S. cerevisiae, and by ZrENA1 in S. cerevisiae ena1 mutants, suggesting that antiporters or sodium in Z. rouxii. The yeast Na+ (K+)-ATPases couple the ATP hydrolysis to pumpscanbothbeusedbyS. cerevisiae to regulate the internal sodium the cation transport against electrochemical gradients, and, unlike concentration (Hahnenberger et al., 1996). In D. hansenii two ENA Na+ specific-ATPases from higher eukaryotes, they mediate both the parologs, namely DhENA1 and DhENA2, have been found to complement Na+ and K+ efflux. They are indispensable for the growth at high Na+ the salt sensitivity when heterologously expressed in S. cerevisiae ena1 and K+ concentrations in alkaline environments (Benito et al., 2002). mutants (Almago et al., 2001). Similarly, H. werneckii possesses two The second mechanism consists of Na+/H+antiporters encoded by highly salt responsive ENA genes, namely HwENA1 expressed in stressed NHA1 in S. cerevisiae, SpSOD2 and SpSOD22 in S. pombe, ZrNHA1 cells exposed to high salt concentrations, and HwENA2 that is mainly and ZrSOD2-22 (with its variants ZrSOD2 and ZrSOD22)inZ. rouxii expressed in stress-adapted cells (Gunde-Cimerman and Plemenitaš, (Watanabe et al., 1995; Hahnenberg et al., 1996; Bañuelos et al., 1998; 2006). More recently, it was demonstrated that upon initial imposition Benito et al., 2002; Papouskova and Sychrová, 2007; Pribylova et al., of NaCl stress, S. cerevisiae extrudes intracellular Na+ primarily by 2008)(Table 2). Based on the substrate specificity, the Na+/H+- Nha1, whereas the long term salt adaptation is mediated by the tran- antiporters can be further divided into two subfamilies: those recognizing scriptional up-regulation of ENA1 gene (Proft and Struhl, 2004; Ruiz both K+ and Na+ (and their respective analogues Rb+ and Li+), such as et al., 2007; Ke et al., 2013). S. cerevisiae Nha1 and SpSod22, are involved in K+ homeostasis and are In Z. rouxii, the salt tolerance has been attributed to different variants present in almost all hemiascomycetes, whereas those recognizing only of Na+/H+ antiporters and Na+-ATPase genes (Hahnenberger et al., Na+ (and its analogue Li+) as substrate, such as SpSod2, ZrSod2-22 and 1996; Pribylova et al., 2008). Like S. cerevisiae, Z. rouxii has a gene ZrSod2, determine salt detoxification in yeasts most distant from encoding Ena1-homologous protein named ZrENA1 (Watanabe et al., S. cerevisiae (Kinclová et al., 2002). Furthermore, a third less studied 1999; 2002). Transcriptional studies showed that, unlike S. cerevisiae mechanism, which operates in acute response to salt stress, entails ENA1, ZrENA1 has little relevance in Z. rouxii salt tolerance, as, under the sequestration of a surplus of toxic Na+ cations in intracellular NaCl shock, the major Na+ pumpoutactivityreliesontheNa+/H+ compartments. A plethora of proton-coupled antiporters are included antiporter ZrSod22 (and their variants) (Watanabe et al., 1995, 1999). in this process; in S. cerevisiae, the most studied ones are Nhx1, an Accordingly, under NaCl stress, Z. rouxii ena1Δ mutants and wide-type endosomal Na+/H+ exchanger (Nass et al., 1997; Nass and Rao, strains exhibit similar growth rates (Watanabe et al., 1999). Other stud- 1998), and Vnx1, a vacuolar Na+ and K+/H+ exchanger (Cagnac et al., ies pointed out that Na+-ATPases and Na+ antiporters almost serve 2007). An endosomal Na+/H+ exchanger homologous to S. cerevisiae similar functions, but they are operational at different external pH Nhx1, has been also found in the halotolerant food yeast D. hansenii levels. In most sodium excluder species, Ena1 Na+-ATPase mediates (Moniel and Ramos, 2007). the Na+ export mainly at high external pH levels. When the external Yeast salt tolerance significantly depends on the genes encoding pH is lower than the cytoplasmic pH, the function of Ena1 Na+- Na+ ATPases, plasma membrane and intracellular Na+/H+-antiporters ATPase can be replaced by electroneutral Na+/H+ antiporters, which harboured in the yeast genome (Table 2). The sodium pump Ena1 is drive the Na+ efflux by the ΔpH (Bañuelos et al., 1998). Similarly, in the most important salt tolerance determinant in S. cerevisiae (Prior Z. rouxii Na+-ATPase and Na+ antiporters have different pH sensitivity. et al., 1996; Bañuelos et al., 1998). Deletion of ENA1 gene determines Since Z. rouxii is acidophilic yeast, ZrEna1 could not be active at the the salt-sensitivity in S. cerevisiae (Haro et al., 1991; Marquez and low pH usually encountered by Z. rouxii in food (Watanabe et al., Serrano, 1996). In the salt-sensitive S. pombe, the single ENA-related 1999). More recent evidences have demonstrated that the Na+ extru- gene, called cta3, only mediates the potassium efflux (Benito et al., sion is mainly mediated by the Na+-specificNa+/H+ antiporter 2002). The expression of S. cerevisiae ENA1 in S. pombe markedly ZrSod-22 and not by the substrate-unspecificNa+/H+ antiporter

Table 2 Cation transport systems associated to halotolerance in different yeast species (adapted from Ramos et al., 2011). Number of parologs is reported in brackets.

Category Gene§ Function Sc Sp Zr Dh Mf Ca Hw

Systems for K+ influx TRK1 plasma membrane K+ transporter belonging to HKT–TRK family, +++++++(8) with a main role in K + homeostasis TRK2 plasma membrane K+ transporter belonging to HKT–TRK family, ++−−+ − nd with a minor role in K+ homeostasis HAK1 High Affinity K+-H+ symporter belonging to the HAK–KUP family −−− +++− ACU1 K+-Na+ P-type ATPase functionally similar to plant HKT transporters −−−−+ps− Systems of K+ efflux TOK1 membrane depolarization activated K+ channel + − + −−++(4) Systems for Na+ and K+ efflux NHA1 antiporter which uses a proton-motive force generated by the plasma + + + + +(2) + +(8) membrane H+-ATPase to mediate the efflux of Na+,Li+,K+,andRb+ through the plasma membrane SOD2 antiporter which uses a proton-motive force generated by the plasma − ++(v)nd+(2)− nd membrane H+-ATPase to mediate the efflux of Na+,andLi+ through the plasma membrane ENA1-4 P-type ATPase sodium pump; involved in Na+,K+ Li+ efflux +(v) + + +(2) +(2) + +(4) Intracellular cations/H + transporters NHX1 endosomal Na+(K+)/H+ antiporter which it regulates the acidification + + + + + +(2) +(2) of cytosol and vacuole lumen KHA1 Putative K+/H+ antiporter from Golgi with a probable role in intracellular + + + + +(2) + +(2) cation homeostasis VNX1 Vacuolar Na+ (K+)/H+ exchanger localized to the endoplasmic + + + + +(2) +(2) +(2) reticulum membrane

§Nomenclature according to S. cerevisiae genome, with the exception of HAK1, ACU1,andSOD2 genes. When no functional data have been available, BLASTP and TBLASTN analyses were carried out to identify homologues of K+,Na+ channel subunits, using the following queries: S. cerevisiae Trk1 (DAA08672.1); S. cerevisiae Trk2 (DAA09201.1); S. cerevisiae Tok1 (DAA08707); D. hansenii Hak1 (ABI37006); M. farinosa Acu1 (CAF22247.1); S. cerevisiae Ena1 (DAA09449.1); S. cerevisiae Nha DAA11888.1); S. pombe Sod2 (CAB.69632.1), S. cerevisiae Nhx1 (DAA12290.1); S. cerevisiae Kha1 (DAA08706.1); S. cerevisiae Vnx1 (NP_014078.1). Abbreviations: Sc, S. cerevisiae;Sp,S. pombe;Zr,Z. rouxii;Dh,D. hansenii;Mf,M. farinosa (formerly P. sorbitophila); Ca, C. albicans;Hw,H. werneckii; nd, not determined; ps, pseudogene; v, inter-strains variability. 146 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157

ZrNha1 (Prybilova et al., 2008). Synteny analysis demonstrated that In S. cerevisiae, the extrusion of K+ cation surplus is mediated by these two genes arose from a duplication event. Furthermore, Z. rouxii the Ena Na+ (K+)-ATPase, the antiporter Nha1, and the voltage-gate genome experienced a CNV in ZrSOD genes. The Z. rouxii type strain channel Tok1. While Nha1 and Ena ATPase are also involved in the CBS 732T possesses one gene encoding the Na+/H+antiporter, namely Na+ detoxification, Tok1 represents the main system for the exclusive ZrSOD2-22, while the highly halotolerant allodiploid strains ATCC K+ extrusion in S. cerevisiae (Ahmed et al., 1999). Tok1 is activated by 42981, CBS 4838 and CBS 4837 have two copies, namely ZrSOD2 and the plasma membrane depolarization and contributes to regenerate ZrSOD22 (Watanabe et al., 1995; Iwaki et al., 1998; Kinclová et al., the membrane potential by releasing intracellular K+ outside the cell. 2001; Solieri et al., 2013b). In the related species Z. sapae, two gene cop- Tok1 is also involved in the short term response to salt stress via the ies, ZrSOD2-22 and ZrSOD22, have been found (Solieri et al., 2013a). HOG pathaway. Phosphorylated Hog1 (P-Hog1) has been recently pre- Generally, CNV can induce important adaptive phenotypes in yeast dicted to inhibit the K+ extrusion mediated by Tok1, leading to a plasma (Conrad et al., 2010). For instance, the expansion of gene repertoire membrane depolarization and a Na+ influx reduction (Ke et al., 2013). encoding alkali metal cation transporters has been related to the ex- The plasma membrane depolarization could thus be a short term adap- treme salt tolerance of H. werneckii (Lenassi et al., 2011). Although the tation to osmotic (sorbitol) and ionic (Na+) stress, because it reduces CNV in Na+/H+antiporter-encoding genes has been supposed to con- transporter activities and consequently the molecular import of the tribute to the high halotolerance of Z. rouxii allodiploid strains cell. Similarly, the phosphorylation of Nha1 by P-Hog1 increases the (Gordon and Wolfe, 2008), no experimental evidences support this hy- Na+ cations efflux under salt stress (Proft and Struhl, 2004), and inhibits pothesis. The ZrSOD2 gene encodes a functionally active antiporter in- the K+ efflux under sorbitol stress (Kinclová-Zimmermannova and volved in salt stress response (Watanabe et al., 1995), whereas Sychrová, 2006). In D. hansenii, the K+ efflux is mediated by the Na+/ ZrSOD22 is a poorly transcribed gene and its disruption doesn’t affect H+ antiporter DhNha1 (Velkova and Sychrova, 2006) and by the Na+ salt-tolerant phenotype (Iwaki et al., 1998). (K+)pumpsDhEna1 and DhEna2, which seem able to protect the cells In S. cerevisiae, the plasma membrane proton-pumping ATPase from sodium or potassium stress at alkaline pH (Almagro et al., 2001). Pma1 generates the electrochemical gradient required for nutrient In Z. rouxii, the response to potassium surplus has been poorly investi- uptake and ionic homeostasis (Serrano et al., 1986). The PMA1gene gated. When ZrNha1 and S. cerevisiae Nha1 have been expressed transcription is tightly regulated and the Pma1 activity is controlled by in S. cerevisiae lacking alkali metal cation efflux systems (ena1–4Δ pH and glucose via phosphorylation of its C-terminal domain. Transcrip- nha1Δ), ZrNha1 was less effective than S. cerevisiae Nha1 in restoring tional studies demonstrated that, under salt stress, Z. rouxii drives the the tolerance to K+ excess (Prybilova et al., 2008). Therefore, differently Na+ efflux via Na+/H+ antiporter through the H+ gradient created by from orthologs in S. cerevisiae and D. hansenii, ZrEna1 doesn’tseemtobe ZrPma1 H+-ATPase (Watanabe et al., 1995; Iwaki et al., 1998). The involved in potassium homeostasis.

ZrATP2 gene, which encodes a mitochondrial F1 ATPase β subunit, is also involved in the ATP production and salt tolerance (Watanabe et al., 2003). The disruption of this gene is lethal in Z. rouxii but not 4.3. Sugar transporters in S. cerevisiae, suggesting that ZrATP2 is essential for maintaining Z. rouxii viability and functioning (Watanabe et al., 2003). Based on In yeasts hyperosmotic stimuli trigger a variety of regulatory these evidences, it has been hypothesized that Z. rouxii is more efficient mechanisms, which modulate the glucose uptake rate (Horak, 2013). in Na+ extrusion than S. cerevisiae due to the cooperative action of When glucose is available at high concentrations, S. cerevisiae uptakes efficient H+-ATPase systems and Na+/H+ antiporters with high Na+- hexoses via facilitated diffusion, and only when glucose is scarce, it specificity (Watanabe et al., 2005). uses the H+ gradient and high-affinity symporters. In S. cerevisiae 17 HXT genes encode facilitated diffusion carriers (Boles and Hollenberg, I997), but only 4 (HXT1-HXT4) are regulated in response to extracellular 4.2.2. K+ inward and outward movements glucose concentrations (Boles and Hollenberg, 1997; Ozcan and Potassium is an absolutely essential element for the living organisms Johnston, 1999). The HXT2 and HXT4 genes encode high affinity and in- and, although the external K+ concentration can greatly vary depending termediate affinity glucose transporters, respectively, which are upon the natural environment, it is in food much lower than what regulated at low glucose concentrations and down-regulated under metabolically required inside the cells (200–300 mM). Therefore, hyperosmotic stress (Wendell and Bisson, 1994). On the contrary, besides the Na+ efflux, yeast cells also require efficient systems for K+ HXT1 expression increases during the exposure to 1.0 M salt, 1.5 M sor- uptake and, when necessary, extrusion (Table 2). In S. cerevisiae,K+ cat- bitol (Hirayama et al., 1995) or high sugar (40% w/v) (Erasmus et al., ions are continually taken up and extruded: the membrane potential 2003). The osmotic stress-induced HXT1 transcription depends upon increases when the potassium influx is crippled and decreases in cells the HOG pathway (Rep et al., 2000), and it has been suggested to pro- defective for K+ efflux (Kinclova-Zimmermannova et al., 2006). The vide additional glucose for the glycerol synthesis (Hirayama et al., K+ uptake occurs mainly by facilitated diffusion through the high- 1995). Furthermore, high glucose concentrations stabilize HXT1 mRNA affinity transporters Trk1 and Trk2, and it is driven by the electrochem- transcripts, indicating that both transcription and mRNA turnover are ical H+ gradient across the plasma membrane generated by H+-ATPase regulated in yeast osmo-adaptation (Greatrix and van Vuuren, 2005). Pma1 (Michel et al., 2006). The homologous gene encoding a putative Zygosaccharomyces rouxii is fructophilic yeast which prefers to potassium transporter, namely ZrTRK1, has been also characterized in consume fructose over glucose. Therefore, hexose transporters mediating Z. rouxii (Stříbný and Sychrová, 2011)(Fig. 1). The functional expression glucose facilitate diffusion have been poorly investigated. The search of the ZrTRK1 gene in S. cerevisiae trk1Δ trk2Δ mutants restores the for HXT orthologs in the Z. rouxii genome showed just one ORF ability to grow at micromolar potassium concentrations, whereas (ZYRO0D13310g) similar to S. cerevisiae HXT10 (http://genolevures. the Z. rouxii trk1Δ mutant grows more slowly than the wild-type org). In contrast, sugar facilitators for the fructose uptake, such as Ffz strain at low K+ concentrations. Other non-conventional yeasts, such proteins (fructose facilitator of Zygosaccharomyces) have been exten- as D. hansenii, possess the high affinity K+ transport Hak1, which sively characterized in Z. bailii (Pina et al., 2004)andZ. rouxii (Leandro works both as K+-H+ symporter and K+-Na+ symporter depending et al., 2011). In particular, Z. rouxii has two low-affinity high-capacity upon the extracellular K+/Na+ concentrations (Martínez et al., 2011) facilitators, ZrFfz1 and ZrFfz2, which transport both fructose and glucose (Table 2). The extremely halotolerant M. farinosa (formerly when their external concentration is high (Fig. 2). More recently, P. sorbitophila) possesses a peculiar p-type ATPase encoded by the Leandro et al. (2013) characterized the high-affinity low-capacity ACU gene, which mediates the Na+ and K+ uptake at high affinity fructose-H+ symporter ZrFsy1, which is up-regulated during the (Benito et al., 2004). growth of Z. rouxii at low extracellular sugar concentrations. T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 147

Fig. 2. Interactions among redox balance, glycolysis, and glycerol production during the Zygosaccharomyces rouxii ethanol fermentation. Black, green and orange lines indicate the enzymatic steps in glycolysis, Gpd-Gpp and Gcy-Dak pathways, respectively. Blue line indicates the relationship among glycolysis, Gpd-Gpp and Gcy-Dak pathways. Red lines represent the remodelling in metabolic ﬂuxes under bisulphite and osmostress conditions. All the dotted lines represent omitted steps. The roles of glycerol metabolism in Pi recycling, redox balance, gluconeogenesis and fatty acid biosynthesis are reported in gray boxes.

4.4. Production and accumulation of osmolytes In S. cerevisiae, genes implicated in glycerol production are duplicated in differentially regulated paralogs, namely GPD1 and GPD2 (Albertyn Compatible osmolytes are produced by yeasts to favour the cell et al., 1994; Eriksson et al., 1995), GPP1 and GPP2 (Norbeck et al., 1996; adaptation to osmotic stress. They significantly maintain the water Påhlman et al., 2001a), and DAK1 and DAK2 (Norbeck and Blomberg, balance, stabilize enzyme systems without interfering with the cellular 1997; Rep et al., 2000). These genes arose from either single gene metabolism, and restore the original cell volume (Brown, 1990). To duplication or whole genome duplication events, and show frequently assure high intracellular osmolyte concentrations, yeast cells regulate divergence and functional differentiation (Kondrashov et al., 2002; the cell-cycle progression and take action against the requirement of Conant and Wolfe, 2008). For example, while GPP2 is mainly involved redox elements and energy demand. in osmoadaptation, GPP1 has a role both in osmoadaptation and growth Glycerol is the main osmolyte that is produced and accumulated under anaerobic conditions (Påhlman et al., 2001a,b). Similarly, Gpd1 intracellularly in response to hyperosmotic stress, and also the most and Gpd2 enzymes have similar kinetic characteristics, but differ studied one. The role of glycerol under varied osmotic conditions will with respect to cellular distribution and transcriptional regulation be discussed in detail. However, other less studied osmoprotective (Albertyn et al., 1994; Eriksson et al., 1995). They are located in the osmolytes, such as trehalose, arabitol, mannitol and erythritol, also get cytosol, but Gpd1 possesses a peroxisome-targeting sequence, while accumulate under certain conditions and therefore will be mentioned. Gpd2 is partly translocated into the mitochondria of non-respiring cells (Valadi et al., 2004; Jung et al., 2010). The single deletion of GPD1 resulted in strains sensitive to osmotic stress (Albertyn et al., 1994), 4.4.1. Glycerol metabolic pathway while the deletion of GPD2 reduced growth under anaerobiosis (Rep Yeasts possess two well-established pathways for glycerol biosyn- et al., 1999). Therefore, Gpd1 has a major role in osmoadaptation. How- thesis, both initiating from the glycolytic intermediate dihydroxyacetone ever, neither the deletion of GPD1 nor the deletion of GPD2 resulted in a phosphate (DHAP) and ending up with the production of glycerol: noticeable change in glycerol yield. GPD1 and GPD2 genes could thus (1) the Gcy-Dak pathway includes the dephosphorylation of DHAP to have roles which parlty overlap to compensate compromised functions, dihydroxyacetone (DHA) by dihydroxyacetone phosphate kinase as recently shown for GPD2, which is up-regulated in response to GPD1 (Dak) followed by the production of glycerol from DHA in a reaction deletion (DeLuna et al., 2010). catalyzed by glycerol dehydrogenase (Gcy); (2) the Gpd-Gpp pathway The Gpd-Gpp pathway is the main metabolic route leading from comprises the conversion of DHAP to glycerol 3-phosphate mediated DHAP to glycerol also in Z. rouxii (Fig. 2). Accordingly, the heterologous by glycerol 3-phosphate dehydrogenase (Gpd), and subsequently, expression of ZrGCY1, but not of ZrGPD1, restores the glycerol produc- the dephosphorylation of glycerol 3-phosphate by glycerol-3- tion and the salt tolerance in S. cerevisiae gpd1Δgpd2Δ mutants unable phosphatase enzyme (Gpp), resulting in glycerol production (Fig. 2). to synthesize glycerol (Watanabe et al., 2004). Furthermore, it was Even if the Gcy-Dak pathway seems to be active under a range of stress found that both the glycerol production and salt tolerance increase conditions, under osmotic stress glycerol is mainly synthesized by Gpd- when ZrGPD1 is expressed along with ScGPP2 (Watanabe et al., Gpp pathway (Larsson et al., 1993; Albertyn et al., 1994; Eriksson et al., 2004). Like in S. cerevisiae,inZ. rouxii allodiploid strain ATCC 42981 1995). two isoforms of some glycerol synthesis genes have been found, such 148 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 as ZrGPD1 and ZrGPD2, ZrGPP1 and ZrGPP2, ZrGCY1 and ZrGCY2 (Iwaki and ZrGCY2 are up-regulated by salt stress, indicating that these genes et al., 2001; Wang et al., 2002; Watanabe et al., 2004). On the basis are target candidates of the HOG pathway (Iwaki et al., 2001). More- of their deduced amino-acid sequence, these proteins have a close over, differently from ZrGpd1, glycerol dehydrogenase ZrGcy1 and ki- homology with the S. cerevisiae orthologs. ZrGPD1/ZrGPD2 and ZrGCY1/ nase Zrdak1 increase the activities under hyperosmotic conditions (aw ZrGCY2 are constitutively expressed in Z. rouxii cells, but their differen- b0.96) (van Zyl et al., 1991). tial roles have not yet been investigated (Iwaki et al., 1999, 2001; Extreme halotolerant yeasts produce and accumulate large amounts Watanabe et al., 2004). of osmo-protective metabolites. Although this feature has been widely exploited in industrial bioprocesses (Nevoigt and Stahl, 1997), its 4.4.2. Glycerol biosynthesis in non-stressed cells molecular mechanism has been poorly investigated. In the black Under anaerobic conditions, sugars are reduced to glycerol for main- yeast H. werneckii, a set of 95 salt-responsive genes have been identified taining the cytosolic redox balance and consuming the excess of NADH and most of them have not previously been related to halotolerance produced during the glycolytic pathway, amino acid biosynthesis and and HOG pathway in any other halotolerant yeasts (Vaupotič and organic acids anabolic routes (van Dijken and Scheffers, 1986; Prior Plemenitaš,2007). Furthermore, in H. werneckii the adaptation to high and Hohmann, 1997; Medina et al., 2010). The glycerol production is amounts of NaCl and sorbitol involves the differential expression also pivotal in lipid biosynthesis and recycling the inorganic phosphate of mitochondria-related genes (Vaupotic et al., 2008). Mitochondria consumed during glycolysis via the dephosphorylation step catalyzed preferentially accumulate energy metabolism-related enzymes in by glycerol phosphatase (Nevoigt and Stahl, 1997)(Fig. 2). hypersaline medium, and chaperones and heat shock proteins, such as The glycerol production was firstly studied in some non- Kar2 and Hsp60, in medium supplemented with sorbitol. Live-cell Saccharomyces yeasts that are naturally unable to produce glycerol imaging showed that the mitochondria condense differentally in and grow on glucose under anaerobic conditions. It was observed that response to different osmolytes. In hypersaline medium, the mitochon- their ability to produce glycerol can be restored by introducing oxygen drial condensation is accompanied by an increasing in ATP synthesis or another external electron acceptor in the culture medium. This effect and oxidative damage protection, whereas in presence of non-ionic is called ‘Custers effect’ (Nevoigt and Stahl, 1997). In subsequent osmolytes it is accompanied by a decreasing both in ATP synthesis and studies, it was found that a S. cerevisiae gpd2Δ mutant grows poorly in lipid peroxidation level (Vaupotic et al., 2008). anaerobic conditions without acetoin as an external electron acceptor (Ansell et al., 1997; Björkqvist et al., 1997; Valadi et al., 2004). When 4.4.4. Glycerol retention and active transport acetoin is added in anaerobic conditions, it is converted to butanediol Besides de novo biosynthesis, the glycerol retention is effective to by NAD+-dependent butanediol dehydrogenase (Nevoigt and Stahl, prevent the massive outflow of water from cells in response to an 1997; Björkqvist et al., 1997). The GPD2 and GPP1 expression is stimu- osmotic stress. Being a liposoluble molecule, glycerol has the tendency lated under anaerobic conditions, when the glycerol production to be leaked out through the plasma membrane, and its retention has becomes essential for the redox balance and ethanol production thus to be an active response by the cell. Under osmotic conditions, (Albertyn et al., 1994; Eriksson et al., 1995; Nevoigt and Stahl, 1997). S. cerevisiae synthesizes a considerable glycerol amount most of which Under aerobic conditions, bisulphite ions induce the GPD2 transcription leaks out of the plasma membrane (Hohmann, 2002). In contrast, and inhibit the final reductive step in ethanol fermentation, avoiding the Z. rouxii synthesizes a smaller glycerol quantity than S. cerevisiae, and accumulation of excessive NADH (Ansell et al., 1997). Bisulphite ions changes membrane phospholipid and fatty acid compositions to form a complex with acetaldehyde that limits the ethanol production decrease the membrane fluidity and permeability. This strategy entails and promotes the reoxidation of glycolytically formed NADH by the Z. rouxii to effectively retain polyols and maintain a very high gradient glycerol synthesis (Fig. 2). The effect can be reversed upon addition of between the intra and extracellular environments (van Zyl et al., acetaldehyde (Ansell et al., 1997). All these evidences pointed out that 1990; Pribylova et al., 2007a). the function of GPD2 is mainly linked to redox imbalance, and not to In S. cerevisiae, the glycerol enters into the cell by two different osmotolerance. mechanisms: a low affinity transport system (facilitated diffusion) and a high affinity proton symport system (active transport) (Table 3). 4.4.3. Glycerol biosynthesis in osmo-stressed cells Fps1 is an aquaglyceroporin belonging to the MIP family that is mainly The cellular redox balance is pivotal for several aspects of cellular involved in the glycerol transport by facilitated diffusion (Sutherland physiology and its perturbation is implicated in the cellular adaptation et al., 1997; Karlgren et al., 2005; Hohmann et al., 2007). FPS1 is consti- to sugar and salt-stress conditions. Under high external osmolarity, an tutively expressed in a salt-independent manner and mutants lacking a equimolar increase in cytoplasmic NADH is required to enhance the region in the Fps1 N-terminal domain (amino acid residues from 150 to glycerol production. This requirement seems to be partially fulfilled by 231) constitutively release glycerol (Tamás et al., 2003). A shift from decreasing the acetaldehyde reduction to ethanol and increasing the low to high external osmolarity induces the Fps1 closure, whereas a acetaldehyde oxidation to acetate (Fig. 2). decrease in osmolarity causes the channel opening, both within seconds In S. cerevisiae, high external osmolarity induces the glycerol after the change in external osmolarity (Luyten et al., 1995; Tamás et al., production both under aerobic and anaerobic conditions. Depending 1999). In the absence of osmotic stress, Fps1 is opened by the binding on the strain, medium, and process parameters, 4 to 10% of the carbon of Rgc2 (regulator of the glycerol channel 2) to the Fps1 C-terminal source may be converted to glycerol. Under stress conditions, the cytoplasmic domain. In response to osmostress Fps1 is closed by GPD1 and GPP2 genes are positively regulated by the HOG pathway to P-Hog1, which binds the N-terminal cytoplasmic domain of Fps1 and produce glycerol (Albertyn et al., 1994; Eriksson et al., 1995). Besides phosphorilates the positive regulator Rgc2 (Beese et al., 2009; Lee GPD1 and GPP2 genes, S. cerevisiae positively regulates the expression et al., 2013; Petelenz-Kurdziel et al., 2013). Furthermore, Fps1 affects of DAK1 gene encoding the Dha1 kinase (Rep et al., 2000). Accordingly, the glycolipid and phospholipid composition of the plasma membrane glycolytic pathway enzymes are slightly repressed in cells exposed to (Sutherland et al., 1997; Toh et al., 2001). saline medium (Akhtar et al., 1997). Unlike S. cerevisiae, Z. rouxii doesn’t Several evidences show that the Z. rouxii ortholog to Fps1, namely increase the expression of neither ZrGPD1 or ZrGPP2 in response to salt ZrFps1, conserves structural features and regulatory mechanisms. stress (Iwaki et al., 2001) and the specific activity of the corresponding ZrFps1 is a 692 aa-long protein characterized by long hydrophilic N enzymes remains unaltered (van Zyl et al., 1991). Although ZrGPD1 and (228-LHQNPQTPTVLP-239) and C-terminal (537-HESPVNWPIATY-548) ZrGPP2 have a main role in glycerol production (Watanabe et al., 2004), domains, both sharing high homology with their S. cerevisiae counter- these results suggest that these genes are constitutively expressed in parts (Tang et al., 2005). Since Z. rouxii fps1Δ mutants retain the ability Z. rouxii and by-pass the HOG pathway control. In contrast, ZrGCY1 to grow on glycerol as the sole carbon source, ZrFPS1 is not required for T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 149

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

Species Glycerol Glycerol transport References dissimilation§ Type Gene Description Role in osmostress protein (% identity)*

S. cerevisiae +AGTSTL1 Main glycerol-H+ glycerol symporter in + NP_010825.3, (/) Ferreira et al., 2005 glycerol dissimilation; repressed by glucose AGT GUP1-2 Putative glycerol symporter involved in NI NP_011431.1, (/) Holst et al., 2000; Ferreira et al., glycerol uptake when glycerol is unique 2006; Ferreira and Lucas, 2008 carbon source; pleiotrophic effect on wall-related phenotypes PGC FPS1 Aquaglyceroporin mediates glycerol diffusion − NP_013057.1, (/) Luyten et al., 1995; Oliveira in presence of glucose; closed in response to et al., 2003 osmotic stress Z. rouxii + AGT ND Putative glycerol-Na+ symporter mediates +/ van Zyl et al., 1990; Lages et al., glycerol accumulation as a function of 1999 extracellular NaCl AGT Putative Putative glycerol-H+ symporter important for XP_002498998.1 (62%) Bubnová and Sychrová, 2011 ZrSTL1/2 growth under hyperosmotic condtions XP_002498999.1 (64%) PGC ZrFPS1 Aquaglyceroporin closed in response to − AAR29350.1 (51%) Neves et al., 2004; Tang et al., osmotic stress 2005 D. hansenii + AGT ND Putative glycerol-Na+ symporter mediates ND Lucas et al., 1990; Oliveria et al., glycerol uptake as a function of extracellular 1996; Lages et al., 1999 NaCl AGT DhSTL1 Glycerol/H+ symporter + XP_459387.2 (62%) Lucasetal.,1990;González- Hernández, 2010 PGC Absent / / / Prista et al., 2005 M. farinosa (formerly + AGT ND Putative glycerol-H+ symporter constitutively NI XP_004204191.1 (62%) Lages and Lucas, 1995 P. sorbitophila) expressed S. pombe -AGTSpGUP1 Membrane bound O-acyltransferase ND NP_592951.3 (39%) Neves et al., 2004 PGC SpFPS1 Aquaglyceroporin + NP_592788.1 Kayingo et al., 2004 Spac977.17p (52%) C. halophila +AGTNDGlycerol-H+ symporter constitutively NI / Silva-Graça and Lucas, 2003 expressed PGC Absent / / / Silva-Graça and Lucas, 2003 C. albicans +AGTCaSTL1 Glycerol-H+ symporter mediates glycerol + CaO19.5753 (60%) Kayingo et al., 2009 uptake XP_718089.1 CaSTL2 No direct role in glycerol uptake + XP_720384.1 (38%) PGC CaAQY1 Acquaporin for the passive diffusion of NI XP_715831.1 (53%) Carbrey et al., 2001; Tang et al., glycerol in presence of glucose 2005

§, ability to grow on glycerol as unique carbon source; *, protein identity was calculated with respect to orthologous protein in S. cerevisiae; AGT, active glycerol transporter; PGC, passive glycerol channel; +, gene/protein positively regulated by hyperosmotic stress; −, gene/protein negatively regulated by hyperosmotic stress; NI, gene/protein mot involved in osmoadaptation; ND, not determined; /, not applicable; Sc, S. cerevisiae;Zr,Z. rouxii;Dh,D. hansenii;Sp,S. pombe;Ca,C. albicans. the glycerol uptake but it is mainly involved in polyol efflux, as reported strongly influenced by the GUP1 gene. The protein Gup1 has twelve in S. cerevisiae (Luyten et al., 1995; Tang et al., 2005). Recently, Wei et al. predicted transmembrane domains, which are compatible with a (2013) exploited the genome shuffling to gain a highly salt-tolerant transporter function, and the GUP1 gene disruption induces an osmo- Z. rouxii mutant strain, which, under a salt surplus, enhances the sensitive phenotype in S. cerevisiae (Holst et al., 2000). Based on these ZrGPD1 transcription and reduces that of ZrFPS1.Thisfinding supports evidences, Gup1 was firstly proposed as glycerol transport. Further that in Z. rouxii salt adaptation relies both on the induction of glycerol studies, however, have demonstrated that Gup1 and the paralog Gup2 production and the inhibition of facilitated glycerol diffusion through are not active glycerol transporters, but regulatory elements with pleio- the plasma membrane (Hou et al., 2013). In other halotolerant yeasts, tropic effects on cell wall phenotypes (Neves et al., 2004 and references like M. farinosa (P. sorbitophila)andD. hansenii, Fps1-like channels herein) (Table 3). have not been documented, suggesting that the glycerol diffusion In the halotolerant Z. rouxii and D. hansenii, the expression of glycerol- occurs in these yeasts through other membrane proteins (Table 3). Na+ symporters requires salt, suggesting that NaCl is the driving force Active transport systems for glycerol have been documented in for the glycerol accumulation under osmostress (Lucas et al., 1990; several yeasts. Yet, little is known about their genetics. In a pivotal van Zyl et al., 1990; Lages et al., 1999). More recently, two glycerol- study involving 42 yeast species, Lages et al. (1999) suggested that the H+ symporters orthologous to S. cerevisiae Stl1, namely ZrStl1 and active glycerol uptake is essential in yeast halotolerance. These authors ZrStl2, have been documented in Z. rouxii to mediate the glycerol uptake found that only the most salt-tolerant yeasts show a constitutive active under high salt concentrations (Neves et al., 2004; Bubnová and glycerol uptake, which is highly efficient in the intracellular glycerol Sychrová, 2011). The Z. rouxii genome harbours also a gene homologous accumulation against the gradient. The analysis of glycerol uptake in to GUP1, but so far no studies have established the role in glycerol trans- presence of salts has identified two different types of constitutive active port. Five polyol-H+ symporters have been found in D. hansenii, with glycerol transport systems, namely Na+-glycerol and H+-glycerol different specificities and affinities for polyols (Pereira et al., 2014). In symporters (Adler et al., 1985; Marešová and Sychrová, 2003)(Table 3). M. farinosa (formerly P. sorbitophila), specific glycerol-H+ symporters In S. cerevisiae, glycerol is actively transported inside the cell by the are constitutively expressed and unresponsive to NaCl (Neves et al., H+ symporter (Lages and Lucas, 1997) encoded by STL1 (Ferreira 2004)(Table 3). et al., 2005). The STL1 gene expression is repressed by glucose and induced by nonfermentable carbon sources and by osmotic stresses in 4.4.5. Other compatible solutes a Hog1-dependent manner (Lages and Lucas, 1997; Rep et al., 2000; In response to hyperosmotic stress, compatible solute production is Ferreira and Lucas, 2007). In S. cerevisiae, the glycerol transport is also regulated by a complex process depending upon the growth phase, 150 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 carbon source, concentration and kind of stress agent (Yancey, 2005). promoter binding sites (Posas et al., 2000; Rep et al., 2000; Hohmann During the course of yeast growth and/or at increasing osmotic stress, et al., 2000; Gasch et al., 2000; Causton et al., 2001). Reversible protein glycerol decreases in concentration and the osmoadaptation is mainly phosphorylation catalyzed by protein kinases represents a universal conferred by other osmolytes, such as D-arabitol, erythritol, and treha- regulatory mechanism of multiple physiological and metabolic functions lose (Tokuoka et al., 1992; Petrovič et al., 1999, 2002; Liu et al., 2006). in the Eukarya, Bacteria and Archaea domains. Three molecular signal- It has been speculated that the salt stress induces mainly C-3 (glycerol) ling pathways up- and down-regulate several subsets of osmostress- and C-4 (erythritol) polyols (Onishi and Suzuki, 1968; Plemenitaš et al., responsive genes and are highly conserved in different yeast species: 2008), while the sugar stress mainly C-3 (glycerol), C-5 (arabitol), and the HOG pathway, one of the five MAPK pathways known in yeasts C-6 polyols (mannitol and sorbitol) (van Eck et al., 1993). Under salt (de Nadal et al., 2002; Hohmann, 2002); the calcineurin/Crz1 pathway, stress, D. hansenii accumulates DHAP and increases glycerol production which is specifically involved in adaptation to high-salt conditions by increasing the proteins involved in the first steps of glycolysis and by (Clapham, 2007); and the Ras-cAMP signalling pathway (Thevelein decreasing those involved in the Kreb’s-cycle (Gori et al., 2007). How- and de Winde, 1999; Norbeck and Blomberg, 2000)(Fig. 3). These path- ever, when both salt and polyols, such as erythritol and mannitol, ways are well-characterized in S. cerevisiae, while they are only partially are present in medium, D. hansenii accumulates mannitol (during studied in Z. rouxii and other halotolerant and osmotolerant yeasts. exponential phase) and erythritol (during stationary phase) instead of glycerol (Nobre and da Costa, 1985; Prista et al., 2005). 5.1. High osmolarity glycerol (HOG) pathway The disaccharide trehalose is one of the most effective osmoprotectants that, under osmotic stress, prevents phase transition Osmostress adaptation involves the activation of the HOG signalling events in the lipid bilayer, reduces the membrane permeability, and cascade, which transduces osmosensory signals in yeast species ensures a proper protein folding (Elbein et al., 2003). Yeasts are able (Brewster et al., 1993). The cascade consists of three consecutively to accumulate trehalose up to 15% of the cell dry mass when submitted activated kinases: MAPKKK, MAPKK and MAPK. In S. cerevisiae,the to a stress. The trehalose synthesis is catalyzed by a multimeric protein osmotic stress signals perceived by the Slnl osmosensor is, in turn, complex (trehalose synthase complex), composed of four subunits transmitted from Ypd1 to Sskl to Ssk2/22 (MAP kinase kinase kinase) encoded by TPS1, TPS2, TPS3 and TSL1 genes (François and Parrou, to Pbs2 (MAP kinase kinase) and finally to the MAPK Hogl (Posas 2001). The trehalose hydrolysis is catalysed mainly by the neutral et al., 1996). On the other hand, Shol regulates the action of Hogl via trehalase Nth1 (Kopp et al., 1994; Nwaka et al., 1995). It was demon- Stell (MAPKKK) (Maeda et al., 1995) and Pbs2 (MAPKK) (Posas and strated that several yeast species synthetize trehalose under high Saito, 1997). Phosphorylation of Hog1 by Pbs2 is done at the neighbouring sugar and salt conditions following the same trend of the cell growth Thr and Tyr residues, respectively at position 174 and 176 (TGY motif). and reaching the highest trehalose concentrations during the stationary Once phosphorylated, active P-Hogl elicits both the immediate and phase of cultivation (Tokuoka et al., 1992; Tokuoka, 1993). Hounsa et al. long-term adaptation to ionic stress (Alepuz et al., 2001; Ruiz and (1998) also indicated that S. cerevisiae is more tolerant to osmotic Ariño, 2007; Ke et al., 2013). As mentioned before, long-term adapta- conditions during stationary than exponential phase, when it mainly tion involves transcriptional and translational regulation of the genome, produces trehalose instead of glycerol. whereas short-term adaptation is accomplished by changes in glycerol The compatible osmolyte production depends also upon the carbon accumulation (Albertyn et al., 1994) and the reestablishment of ionic source. In media with non-repressing galactose as the carbon source, balance (Proft and Struhl, 2004). S. cerevisiae reduces the glycerol production, increases sensitivity to Starting from the first minute after the induction of salt stress, osmotic stress, and mainly utilizes trehalose as osmolyte (Elbein et al., P-Hogl directly phosphorylates some membrane ion transporters, such

2003). Furthermore, under severe osmotic stress (0.866 aw), S. cerevisiae as Nha1 and Tok1, in order to rapidly readjust the transmembrane reduces the intracellular glycerol content, indicating that this osmolyte ﬂuxes of Na+ and K+ in osmo-stressed cells (Proft and Struhl, 2004). is essential for the yeast growth under moderate but not severe osmotic Furthermore, cytoplasmic P-Hog1 regulates enzymatic activities that stress (Albertyn et al., 1994; Hounsa et al., 1998). Hyperosmotic stress are necessary to rapidly produce and accumulate glycerol (Klipp et al., also reduces the glycerol consumption and ethanol production rate, 2005). Such direct metabolic adjustments entail the cell to redirect but increases the intra-cellular content of trehalose (Norbeck and carbon resources toward the glycerol production. Finally Hog1 induces

Blomberg, 1997). Congruently, when the genes involved in trehalose a temporary arrest of cell-cycle progression in G1 phase (as reviewed synthesis are deleted, the survival under NaCl stress of S. cerevisiae by Saito and Posas, 2012). mutants is considerably reduced compared to the wild-type cells During the long-term cellular adaptation to salt stress, P-Hog1 trans- (Hounsa et al., 1998). locates to the nucleus, where, by the phosphorylation of at least three Arabitol and mannitol are other important compatible osmolytes, separate transcription factors (Msn2/Msn4 and Hot1) (Schmitt and but their role in osmoregulation is still unclear. In Z. rouxii,saltand McEntee, 1996), it can modulate the regulation of more than 10% of sugar stresses induce production of glycerol, arabitol or both, depending the total yeast genome (O’Rourke and Herskowitz, 2004). The Hog1- upon the osmoticum which has been used to reduce the aw. If sugar is regulated genes possess stress regulatory elements (STRE) with a core used as stress agent instead of salt, D-arabitol is highly produced and sequence CCCCT or AGGGG in their promoter region or sometimes in accumulated, whereas the glycerol concentration remains invariable the coding region (Schüller et al., 1994; Martinez-Pastor et al., 1996; (van Zyl and Prior, 1990). Fructose and glucose-containing media have Wang et al., 2002; Tang et al., 2005). These cis-acting factors are variable been associated to mannitol production (Tomaszewska et al., 2012), in number and orientation with respect to TATA box and they are nec- which is inhibited by salt (Onishi and Suzuki, 1968; van Eck et al., essary for stress-induced gene expression both in S. cerevisiae and 1993; Tomaszewska et al., 2012). The salt-sensitivity of S. cerevisiae Z. rouxii (Albertyn et al., 1994; Schüller et al., 1994; Akhtar et al., 1997; gpd1Δ and gpd2Δ mutants is complemented by the expression of Norbeck and Blomberg, 1997; Wang et al., 2002; Tang et al., 2005). Al- mannitol dehydrogenase gene (MDH) involved in the mannitol biosyn- though a single copy of STRE is sufﬁcient to bind a transcription factor thesis (Watanabe et al., 2006). and activate the expression of a reporter gene, two or more STRE copies induce a greater expression of stress-responsive genes 5. Signal transduction and cis/trans-acting regulatory factors (Kobayashi and McEntee, 1993). The transduction pathway of osmotic stress signals has not yet been The yeast adaptive osmostress response is mostly controlled by the fully elucidated in Z. rouxii. Like in S. cerevisiae,inZ. rouxii the HOG activation of signal transduction pathways, which in turn regulate the pathway comprises two operationally redundant plasma membrane dynamic interactions between transcription factors and speciﬁc osmosensors Sln1 and Sho1 (Maeda et al., 1994, 1995), and from one T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 151

Fig. 3. Integrated overview of signalling transmission pathways involved in osmoadaptive gene regulation. Genes are referred to as Saccharomyces cerevisiae genome, with the exception of ZrFPS1 and ZrGCY1/2. Genes regulated by calcineurin pathway are reported in bold. to two putative homologs to S. cerevisiae HOG1, namely ZrHOG1 and compartment and activates transcription factors homologous to ZrHOG2 (Iwaki et al., 1999; Kinclová et al., 2001). A TGY motif similar Msn2p/Msn4p and Hot1, thus leading to the transcription of STRE- to that of S. cerevisiae Hog1 has also been found in ZrHogl and ZrHog2, controlled genes. However, STREs were not found in the ZrGPD1-2pro- suggesting that a putative ZrPbs2 MAPK kinase similar to S. cerevisiae moter regions, indicating that their expression is salt and HOG Pbs2 could exist also in Z. rouxii (Iwaki et al., 1999). Although some pathway-independent. The lack of STREs may account for the moderate Z. rouxii allodiploid strains possess two ZrHOG gene copies, namely glycerol amount produced by Z. rouxii to cope high external osmolarity. ZrHOG1 and ZrHOG2, there is no relationship between this gene redun- On the contrary, a putative STRE motif with core CCCCT sequence has dancy and their osmotolerance (Kinclová et al., 2001). This could mean been identified in the upstream region of ZrFPS1, which is missing in that ZrHOG1 and ZrHOG2 are expressed differentially, or that one of S. cerevisiae FPS1 (Tang et al., 2005). Therefore, under high salt concen- these paralogs is transcriptionally silent, as it happens for ZrSOD22. trations, only the ZrFPS1 gene transcription is regulated via the HOG ZrHOG1 and ZrHOG2 genes are functional as MAP kinase and are signalling pathway, resulting in the higher ability of Z. rouxii to intracel- able to complement the salt-sensitivity in S. cerevisiae hoglΔ null mu- lularly retain glycerol compared to S. cerevisiae. In contrast, S. cerevisiae tants (Iwaki et al., 1999). However, their overexpression in wild-type P-Hog1 transiently induces the closure of Fps1 channel during the short S. cerevisiae doesn’t improve the glycerol synthesis, indicating that the term salt response, but it cannot induce the FPS1 transcription during amount of ZrHog1 and ZrHog2 is not a limiting factor in the glycerol pro- the long term salt adaptation. duction. These studies collectively supported that Z. rouxii possesses a HOG pathway functionally equivalent to that of S. cerevisiae. 5.2. Calcineurin/Crz1 pathway STRE motifs are species-specifically distributed in the yeast genome, so that each species has peculiar sets of osmo-responsive genes that The calcineurin/Crz1 signal transduction pathway has an important contribute to functional diversities in response to osmotic cues. Under role in cation homeostasis and salt stress adaptation, but it is relatively high osmolarity conditions, S. cerevisiae produces much more glycerol less investigated compared to HOG pathway (Matsumoto et al., 2002; than Z. rouxii, by up-regulating genes involved in glycerol synthesis via Ke et al., 2013). Calcineurin, the Ca2+/calmodulin-regulated protein HOG pathway (Pribylova et al., 2007a). Accordingly, the S. cerevisiae phosphatase 2B, is a heterodimer containing a catalytic (A) subunit GPD1 gene exhibits four STRE elements in its promoter region complexed with an essential regulatory (B) subunit and requires Ca2+ (Albertyn et al., 1994) and encodes a Gpd1 enzyme with higher activity and calmodulin for activity (Cyert et al., 1991). Calcineurin controls than ZrGpd1 (Akhtar et al., 1997; Norbeck and Blomberg, 1997). Like Crz1 activity by regulating its subcellular localization (Stathopoulos- S. cerevisiae P-Hog1, P-ZrHogl also translocates into the nuclear Gerontides et al., 1999). When calcineurin-dependent signalling is 152 T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 low, Crz1 is phosphorylated and resides primarily in the cytosol. Upon in yeasts was provided for the protein Sgd1, which is homologous in dephosphorylation by calcineurin, Crz1 enters into the nucleus and its N-terminal domain to Spt7, a subunit of the nucleosomal Spt-Ada- here it regulates the expression of target genes with Calcineurin Gcn acetyltransferase (SAGA) histone acetylation complex (Roberts Dependent Response Elements (CDREs) in the promoter region. These and Winston, 1997). The SGD1 overexpression is able to partially cis-elements consist of a common sequence motif, 5’-GAGGCTG-3’, complement growth defects in S. cerevisiae hog1Δ and pbs2Δ mutants which Crz1 binds through a C2H2 zinc finger motif (Stathopoulos and and to increase their glycerol production (Akhtar et al., 2000). Other Cyert, 1997; Matheos et al., 1997). Around 163 genes have been found studies suggested that changes in the chromatin structure contribute under control of Calcineuirn/Crz1 pathway (Yoshimoto et al., 2002). to the osmostress-stimulated expression of GPD1 gene. Under osmotic Among them, the GSC2 gene encodes a subunit of beta-1, 3 glucan stimuli, the transcriptional repressor activator protein Rap1 binds the synthase, which is responsible for the synthesis of 1, 3-beta-D-glucan GPD1 promoter and induced the GPD1 transcription. On the contrary, (Levin, 2005), and it is up-regulated after exposure to high sugar the specific inactivation of all Rap1 binding sites completely abolishes concentrations (Erasmus et al., 2003). CDRE motif was also found the osmostress-induced transcription of GPD1 (Eriksson et al., 2000; in the flanking region of ENA1 (Kafadar and Cyert, 2004). High cytosolic Morse, 2000). Ca2+ levels activate calcineurin in response to extracellular hyperionic A third evidence for the epigenetic regulation of cellular osmostress and increase Na+ efflux by up-regulating the ENA gene adaptiation involves the HOG pathway. Under osmotic shock, Hog1 (Matsumoto et al., 2002; Ruiz and Arino, 2007). Ke et al. (2013) demon- activates the transcription factors Hot1 and Msn2/4 which mediate strated that S. cerevisiae coordinates the HOG and the calcineurin the recruitment of Rpd3-Sin3 histone deacetylase (de Nadal et al., pathways to achieve both immediate and longer-term adaptation to 2004). Active Rpd3-Sin3 complex binds to specific promoters leading NaCl stress, respectively. In Torulaspora delbrueckii, a species close to to histone deacetylation, entry of RNA polymerase II, and transcription Z. rouxii, crz1-null cells were insensitive to high Na+ concentrations, initiation of osmoresponsive genes (Alepuz et al., 2003; de Nadal et al., indicating that TdCrz1 is not required for the salt-induced transcriptional 2004). Upon unstressed conditions, the transcription factor Sko1, activation of TdENA1 gene (Hernandez-Lopez et al., 2006). This evi- which is related to bZIP/ATF family of transcriptional regulators, dence suggests that yeast species could differ in regulating salt response represses the ENA1 transcription by binding CDRE (Proft and Serrano, via calcineurin/Crz1 pathway. 1999). Under hypertonic stress, Sko1 is phosphorylated by Hog1 and recruits the SAGA histone deacetylase and the switch/sucrose non- 5.3. Ras-cAMP signalling pathway fermenting (SWI/SNF) complex. The latters are transcription activators which promote chromatin remodelling (Profit and Sturhl, 2002) and The yeast cAMP-dependent protein kinase (PKA) is the effector induce the ENA1 expression in conjunction with Calcineurin/Crz1 kinase of the Ras-cAMP signalling pathway. It is a conserved serine/ mediated pathway (Profit and Serrano, 1999). Yeast mutants lacking a threonine protein kinase, which, through the phosphorylation of differ- functional SWI/SNF complex are less tolerant to NaCl than wild-type ent targets, has pleiotropic effects on the cell growth, trehalose and glyco- cells (Profit and Sturhl, 2002). Further studies are required to establish gen metabolism, dimorphic shift, and stress adaptation (Smith et al., whether similar epigenetic regulatory mechanisms also exist in 1998). Proteomic approaches were exploited to study the influence of Zygosaccharomyces yeasts. PKA on protein expression during the exponential growth of S. cerevisiae under osmotic stress (Boy-Marcotte et al., 1998). Proteins 6.2. Phenotypic heterogeneity up-regulated under NaCl stress are grouped into three classes as regards the PKA activity: i) PKA-independent proteins (Gpd1, Gpp2 and Dak1); Phenotypic heterogeneity is a super-organism feature of prokaryotes ii) fully PKA-dependent proteins (Tps1 and Gcy1); iii) partly PKA- and yeasts that provides a dynamic source of diversity and adaptive phe- dependent proteins (Eno1, Tdh1, Ald3, and Ctt1) (Boy-Marcotte et al., notypes and increases the microbial fitness in stressful environments 1998). Osmo-response seems to be mediated by PKA both at transcrip- (Avery, 2006). Within an isogenic (genetically uniform) microbial pop- tion level through the inhibition of STRE-dependent genes expression ulation in a homogenous environment, individual cells can still exhibit (Thevelein and de Winde, 1999), and at post-translational level through differences in phenotype. The precise mechanisms of such cell-to-cell the regulation of trehalose synthesis (Kobayashi and McEntee, 1993). In heterogeneity are elusive and few studies have linked variations in particular, Ras-cyclic AMP pathway negatively regulates Msn2/Msn4 yeast morphology to molecular effectors. Hsieh et al. (2013) have that are cytoplasmically accumulated, leading to the inhibition of highlighted that reduction in the intracellular amount of chaperon osmostress response (Görner et al., 1998)(Fig. 3). protein Hsp90 triggers morphological heterogeneity in Z. rouxii clonal populations. Under standard conditions high Hsp90 levels assure the 6. Non genetic regulation of osmostress tolerance stability of Cla4, a key regulator of spectrin formation that inhibits morphological switching of the cell from budding to filamentous Understanding the non-genetic regulation of cellular stress response growth. Under salt stress, low Hsp90 levels reduce the Cla4 stability is an important biological question and it has received considerable and stimulate the cellular switching towards a filamentous form. attention in recent years. In relation to yeast osmotic tolerance, two Additionally, the stress-induced Hsp90 inhibition is known to favour non-genetic mechanisms have been implicated so far. The first one chromosomal instability and aneuploidy, which in turn potentiate the includes epigenetic alterations in the chromatin structure that induce cellular adaption to stressful environments (Chen et al., 2012). Interest- genome-wide and local changes in gene transcription. The second one ingly, aneuploidy has been also frequently found in highly salt-tolerant is the non-genetic cell-to-cell phenotypic heterogeneity within an Z. rouxii strains (Solieri et al., 2013b, 2014). isogenic cell population under osmotic stress conditions. 7. Food exploitation and biotechnological perspective 6.1. Chromatin-mediated mechanisms The knowledge on the genetic and regulatory networks underlying Post-translational modification of nucleosomal histone proteins and important phenotypic outcomes is a prerequisite for the successful DNA methylation are two extensively characterized epigenetic mecha- exploitation and control of microorganisms in food (Giudici et al., nisms that regulate gene expression in plants grown under osmotic 2005). In the last decade, researches in food science and microbiology stress conditions (Chinnusamy and Zhu, 2009; Grativol et al., 2012). moved from classical methodologies to more advanced strategies, On the contrary, few studies have dealt with this topic in yeasts. The and usually borrowed well-established methods in medical, phar- first evidence about the epigenetic control of osmo-responsive genes macological, and/or biotechnology research. As a result, “omics” T.C. Dakal et al. / International Journal of Food Microbiology 185 (2014) 140–157 153 approaches and bioinformatics have been recently applied to identify: genome sequences from osmo and halotolerant food species highlighted 1) candidate genes to use in genetic improvement of industrial yeast how these yeasts combine conserved and species-specificstrategiesto strains; 2) molecular targets for new food preservation technology; counteract better sugar and salt stress than S. cerevisiae. These differ- 3) biomarkers for the early prediction of food process outcomes. ences are operational at genetic, metabolic, and regulatory levels. In In particular, new perspectives have been opened by the identifica- particular, new insights on Z. rouxii functional biology and genomics tion of genetic mechanisms that shape the osmostress response in point out how sugar and salt tolerance arise from distinct adaptive Z. rouxii and other osmotolerant and halotolerant food spoilage yeasts. mechanisms, which integrate solute-unspecific and solute-specific To prevent their growth in food, it is crucial to understand what causes routes. Future investigations will benefit by the application of systems yeast stress tolerance. This knowledge could be exploited to set up biology tools. They will contribute to complete the detailed map of quantitative prediction methods for the growth of food spoilage yeasts osmo and halotolerance determinants in Z. rouxii. All these efforts hold and, consequently, to reduce the economical loss derived from food promise for i) rational strain engineering for biotechnological and food spoilage (Loureiro and Malfeito-Ferreira, 2003; Plemenitaš et al., exploitation; ii) prevention of yeast food spoilage; iii) understanding 2008). On the other hand, genetic loci that determine yeast sugar and the yeast adaptation to adverse environments. salt-tolerance could be cloned and expressed in industrial strains to improve their growth and fermentation performance in stressful condi- Acknowledgements tions. For example, the ZrSod22 expression in S. cerevisiae increases + + the expulsion of toxic Na and Li cations from cells at external acidic This work has been supported by a grant of Regione Toscana (Misura pH values and enhances the salt tolerance (Iwaki et al., 1998; 124 Acetoscana: PSF 2007/2013). Watanabe et al., 2005). 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