NaCl stress inhibits maltose fermentation by Saccharomyces cerevisiae

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1 Biotechnology Letters 23: , Kluwer Academic Publishers. Printed in the Netherlands NaCl stress inhibits maltose fermentation by Saccharomyces cerevisiae Nadir Trainotti & Boris U. Stambuk Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC , Brazil Author for correspondence (Fax: ; Received 30 July 2001; Revisions requested 3 August 2001; Revisions received 20 August 2001; Accepted 20 August 2001 Key words: active maltose transport, glycerol, maltose fermentation, Saccharomyces cerevisiae, salt stress Abstract While fermentation of 20 g glucose l 1 by Saccharomyces cerevisiae was not impaired by high NaCl concentrations, fermentation of 20 g maltose l 1 was significantly decreased by 0.7 M NaCl, and completely inhibited with 1.4 M NaCl. No glycerol was produced in response to the salt stress when yeast cells were fermenting maltose. Active maltose transport, and not intracellular hydrolysis, was the metabolic step severely impaired by the NaCl stress. Introduction Many industrial applications of Saccharomyces cerevisiae involve their exposure to high osmotic pressures during production, downstream processing, or use. Thus, their ability to withstand these stressful conditions and still function to a commercially acceptable standard is crucial to their usefulness in industry (Attfield 1997). Tolerance of S. cerevisiae to changing osmotic pressures is one of the better understood stress responses. Hyperosmolarity exerted by high salt (NaCl) or other osmolytes induces both specific and general stress responses that have been extensively studied at the physiological, genetic and transcriptional level (Attfield 1997, Posas et al. 2000). Indeed, several studies (Oda & Tonomura 1993, Bagum et al. 1998) have shown an increase in fermentation performance by yeast previously exposed or grown on hyperosmotic media (e.g., with high NaCl concentration). However, most of these studies have been performed with cells growing on or fermenting a relatively narrow set of carbon sources, including glucose, galactose, and sucrose, and little is known about the effects of high salt concentrations on the utilization of other sugars by yeasts. Most industrial applications of S. cerevisiae rely on the ability of this yeast to ferment maltose. Maltose utilization requires the active transport of the sugar across the cell membrane and, subsequently, its hydrolysis by cytoplasmic α-glucosidases. Up to now two active maltose transporters have been described in S. cerevisiae: the maltose permease, and a general α-glucoside permease able to transport maltose, trehalose, maltotriose, melezitose, α-methylglucoside and sucrose (Stambuk et al. 1999, Stambuk & de Araujo 2001). Transport of α-glucosides into the cell, besides being the first step in the metabolism of these sugars, is also the rate-limiting step in the fermentation of α-glucosides by yeast cells (Kodama et al. 1995, Zastrow et al. 2000). In the present report we analyzed the effects of high NaCl concentrations on maltose utilization by S. cerevisiae. Materials and methods Materials Media components were purchased from Difco. Glucose, maltose, p-nitrophenyl-α-d-glucopyranoside, and the enzymatic kit for ethanol determination were obtained from Sigma. Commercial enzymatic kits for glucose and glycerol determination were from Biobras (Brazil). D-[U- 14 C]Maltose (460 mci mmol 1 )was

2 1704 Fig. 1. Effects of salt stress on glucose utilization by yeast cells. Growth (a), production of ethanol (b), and sugar consumption from the medium (c) were determined in media containing 2% glucose (w/v) as carbon source in the absence of salt ( ), or in the presence of 0.7 ( )or1.4mnacl( ). Incubations were carried out at 28 C with shaking (160 rpm) in Erlenmeyer flasks filled to 1 5 of their volume. purchased from Amersham Pharmacia Biotech. All other chemicals were of analytical grade. Strain, media and culture conditions Strain AP69, a S. cerevisiae maltose constitutive utilization diploid (Stambuk et al. 1998), was used. Similar results were obtained with other maltose fermenting strains. Cells were grown aerobically in batch culture (28 C and 160 rpm) on YEP medium (ph 5) containing 20 g peptone l 1, 10 g yeast extract l 1, and 20 g of the indicated carbon source (glucose or Fig. 2. Effects of salt stress on maltose utilization by yeast cells. Growth (a), production of ethanol (b), and sugar consumption from the medium (c) were determined in media containing 2% maltose (w/v) as carbon source in the absence of salt ( ), or in the presence of 0.7 ( ) or 1.4 M NaCl( ). Incubations were carried out as described in Figure 1. maltose) l 1. When indicated the YEP medium was supplemented with 0.7 or 1.4 M NaCl. Growth was followed turbidometrically at 570 nm and correlated to the cell dry weight. Assays Glucose, ethanol and glycerol were determined using commercial enzymatic kits as previously described (Stambuk et al. 1998, Zastrow et al. 2000). Maltose was assayed spectrophotometrically at 540 nm after reaction with methylamine in 0.25 M NaOH as described by Cáceres et al. (2000).

3 1705 Transport of 14 C-maltose was determined as described (Stambuk et al. 1998). All assays were done at least in triplicate, with a maximum deviation of less than 10%. Rates of transport were expressed as µmol maltose transported min 1 (g dry yeast cells) 1. α-glucosidase activity was determined with p-nitrophenyl-α-d-glucopyranoside (Stambuk 1999), and activity is expressed as µmol substrate hydrolysed min 1 (g protein) 1. All assays were done in triplicate, with a maximum deviation of less than 5%. Protein was assayed as described (Cordeiro & Freire 1994) using bovine serum albumin as standard. Results The addition of increasing concentrations of NaCl to the cultures of S. cerevisiae decreased the growth rate from those obtained in the absence of salt (Figures 1a and 2a, Table 1). Although these effects were observed with both carbon sources, at high NaCl concentrations growth on maltose was more susceptible than on glucose. While the fermentation of glucose was not affected by the addition of NaCl (Figure 1b, Table 1), in the case of maltose fermentation the ethanol yield was significantly decreased when cells were grown with 0.7 M NaCl, and completely inhibited with higher salt concentrations (Figure 2b, Table 1). Indeed, the uptake of this sugar from the medium was completely inhibited with 1.4 M NaCl (Figure 2c), while this salt concentration did not impair glucose uptake by yeast cells (Figure 1c). We also analyzed the production of glycerol, the major solute produced by S. cerevisiae in response to osmotic stresses (Attfield 1997). As already reported, increasing salt concentrations induced the synthesis of glycerol when the cells were metabolizing glucose (Table 1), but no glycerol was produced when cells were exposed to NaCl with maltose as a carbon source (Table 1). In order to gain insights into the molecular basis of the observed impairment in maltose utilization induced by high NaCl concentrations, we determined the activity of the two enzymatic steps involved in maltose fermentation: sugar transport across the plasma membrane, and intracellular hydrolysis. As can be seen in Table 2, when cells were grown with maltose in the presence of NaCl the activity of the intracellular α-glucosidase increased with increasing salt concentrations (see also Oda & Tonomura 1993, Posas et al. 2000). On the other hand, the active transport of maltose decreased with the addition of 0.7 M NaCl (Table 2) in a manner similar to the observed decrease of ethanol production triggered by the osmotic stress (Table 1), and was undetectable at high salt concentrations. Thus, maltose active transport, and not intracellular hydrolysis, is the metabolic step affected by high NaCl concentrations. Discussion The deleterious effect of high salt concentration has two main components: osmotic stress and ion toxicity. In yeast, glycerol is the main osmolyte accumulated to counterbalance the external osmotic pressure. Homeostasis of intracellular ion concentrations is fundamental to several key physiological parameters, and S. cerevisiae cells respond to high extracellular NaCl concentrations by increasing both potassium uptake and sodium efflux, in order to attain an appropriate Na + /K + ratio (Attfield 1997, Rios et al. 1997, Mulet et al. 1999, Posas et al. 2000). Our results have shown that maltose-growing cells are unable to produce glycerol when exposed to salt stress. Similar results have been reported when cells that are metabolizing galactose (or other nonfermentable carbon sources such as ethanol) were exposed to salt stress (Rios et al. 1997). However, galactose-grown cells are more tolerant to salt stress, a very different situation from the pronounced inhibition of growth and fermentation observed when yeast cells are using maltose as carbon source. Our results also indicate that maltose active transport is the major biochemical parameter affected by high salt concentrations, leading to the absence of maltose fermentation under this stress condition. Although several reports have shown alterations in the expression of some glucose transporters (HXT1 is induced, whilst HXT2 and HXT4 are repressed) when yeast cells are exposed to an osmotic stress (Turkel 1999, Posas et al. 2000), the absence of maltose transport under high salt stress is probably not due to reduced expression of this transporter (see α- glucosidase expression in Table 2, and Posas et al. 2000), but rather the consequence of other parameter(s) affected by high NaCl concentrations. In this regard is important to note that other active transport activities in S. cerevisiae (e.g., amino acid transport) are severely inhibited by salt stress (Norbeck & Blomberg 1998).

4 1706 Table 1. Specific growth rate (µ) and biomass, ethanol, and glycerol yields during growth of yeast cells on YEP media containing 2% (w/v) glucose or maltose as carbon sources and the indicated concentrations of salt. Incubations were carried out in Erlenmeyer flasks, filled to 1 5 of their volume, with shaking at 160 rpm and 28 C. Carbon source µ (h 1 ) Maximum yield [g (g sugar) 1 ] Biomass Ethanol Glycerol Glucose Glucose M NaCl Glucose M NaCl Maltose Maltose M NaCl Maltose M NaCl Table 2. Effects of salt stress on maltose transport and hydrolysis by yeast cells. The rates of maltose transport and p-nitrophenyl-α-d-glucopyranoside hydrolysis were determined in cells grown on YEP medium containing 2% (w/v) maltose and the indicated concentrations of salt. Growth condition Activity (µmol min 1 g 1 ) a Maltose permease α-glucosidase Maltose 4.7 ± Maltose M NaCl 2.9 ± Maltose M NaCl a Maltose permease activity is expressed as µmol maltose transported min 1 (g dry yeast cells) 1, and α-glucosidase activity as µmol p-nitrophenyl-α-d-glucopyranoside hydrolysed min 1 (g protein) 1. Secondary active transporters depend on the electrochemical proton gradient across the plasma membrane, created by the H + -ATPase, even for downhill uptake. However, the electrical membrane potential is also determined by the activity of the K + transport system, the major modulator of membrane potential in S. cerevisiae (Mulet et al. 1999). Thus, maltose active transport is probably inhibited by high salt concentrations as a consequence of a collapse of the membrane potential due to increased K + (and probably also H + ) uptake triggered by the salt stress (Mulet et al. 1999). In accordance with this hypothesis, glucose transport and fermentation are not affected by high NaCl concentrations because this sugar is transported into the cell by a facilitated diffusion mechanism. Another biophysical parameter that must be considered is the lipid environment at the plasma membrane. It is well known that salt stress induces changes in the lipid composition of yeast cells, with an increase in the relative proportion of phosphatidycholine and a decrease of phosphatidylethanolamine and phosphatidylserine (Sharma et al. 1996). It has been recently demonstrated that in yeast cells depleted of phosphatidylethanolamine the active transport of amino acids is drastically decreased, while the uptake of glucose is unaffected (Robl et al. 2001). At the moment it is not clear if these lipid effects on active transport are related to the changes in membrane potential, or whether they are a consequence of a direct effect of the membrane lipids on the transporter proteins. Further research efforts will be directed toward the identification of the molecular mechanism(s) involved in the inhibition of maltose transport and fermentation triggered by high NaCl concentrations. Acknowledgements N.T. was recipient of a fellowship from CNPq. This work was supported by grants from FAPESP

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