Expression of Saccharomyces cerevisiae α-glucoside transporters under different growth conditions

BIOPROCESS ENGINEERING

Expression of Saccharomyces cerevisiae α-glucoside transporters under different growth conditions

S. L. Alves Jr.I,III,^*; J. M. Thevelein^III,IV; B. U. Stambuk^II

^IUniversidade Federal da Fronteira Sul, Campus Chapecó, Unidade Bom Pastor, Sala 1211, Phone: + (55) (49) 2049-3106, Av. Fernando Machado 108-E, CEP 89802-112, Chapecó - SC, Brazil. E-mail: [e-mail]

^IIDepartamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis - SC, Brazil

^IIILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, and

^IVDepartment of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001, Leuven-Heverlee, Flanders, Belgium

ABSTRACT

Important biotechnological processes depend on the efficient fermentation by Saccharomyces cerevisiae yeasts of starch hydrolysates rich in maltose and maltotriose. The rate-limiting step for fermentation of these α-glucosides is the transport across the plasma membrane of the cells. In order to contribute to a better understanding of maltose and maltotriose metabolism by S. cerevisiae, we analyzed the expression of the main α glucoside transporter genes in two different yeast strains grown on media with glucose, maltose or maltotriose as carbon source. Although both yeast strains have higher αglucoside transport activity during growth on maltotriose, our results show similar expression levels of the analyzed genes on either maltose or maltrotriose media. Thus, our results indicate that, although the transport capacity of maltotriose grown cells is higher than that of maltose grown cells, maltotriose cannot be considered a better inducer of αglucoside transporter genes.

Keywords: Expression; Transporters; MAL genes; Maltose; Maltotriose; S. cerevisiae.

INTRODUCTION

Humans have been using yeasts in different biotechnological processes, such as baking and brewing, for millennia. Saccharomyces cerevisiae is considered to be the predominant agent in these processes, although other close Saccharomyces species (and hybrids among them) have also been isolated from these industrial applications (Querol and Bond, 2009). Baking and brewing rely on the efficient fermentation by yeasts of starch hydrolysates rich in maltose and maltotriose. In the brewing industry, for example, these two sugars are of special importance since they are the predominant sugars in wort (generally 50-60% is maltose and 15-20% is maltotriose), followed by glucose (10-15%) and other minor carbohydrates (Willaert, 2001). Of these sugars, glucose is preferentially and rapidly utilized by yeast cells, but both process efficiency and product quality require the complete fermentation of all sugars, including maltose and maltotriose. Although maltose is easily fermented by the majority of yeast strains after glucose exhaustion, maltotriose is not only the least preferred sugar for uptake by these Saccharomyces cells, but many yeasts will not use this α-glucoside at all (Zheng et al., 1994; Yoon et al., 2003; Duval et al., 2010). Slow and incomplete yeast sugar fermentation represents a significant economic loss for these industries, and consequently most strain development programs aim to select yeasts with improved fermentation performance.

Utilization of maltose and maltotriose by yeast cells initially requires their active transport across the plasma membrane by H⁺ symporters (Han et al., 1995; Alves et al., 2007, 2008). In the cytoplasm, these sugars are hydrolyzed by αglucosidases (maltases), releasing glucose molecules which will be metabolized through glycolysis to ethanol (Zastrow et al., 2001; Novak et al., 2004). Previous genetic and biochemical studies focusing mainly on the maltose fermentation system revealed a series of five unlinked telomere-associated MAL loci: MAL1 through MAL4 and MAL6. Each locus contains at least one copy of three different genes encoding a maltose permease (MALx1, where x stands for one of the five MAL loci mentioned above) responsible for the active uptake of the sugar, an intracellular maltase or αglucosidase (encoded by MALx2), and a positive regulatory protein (encoded by MALx3) that induces the transcription of the two previous genes in the presence of maltose. In order to be able to ferment maltose, a S. cerevisiae strain needs to have only one copy of these three genes (Needleman, 1991; Novak et al., 2004). These genes present in the different MAL loci show a high degree of sequence and functional homology, but there may also be extensive variability since these genes are telomeric and several different alleles that determine distinct phenotypes (i.e., MALinducible and MALconstitutive phenotypes) have been described previously. The MAL1 locus is considered to be the progenitor locus from which all other MAL loci were derived, as all S. cerevisiae strains contain MAL1 sequences at chromosome VII. This pattern holds true even for many non-maltose-fermenting yeast strains, which may harbor partially functional mal1p (MAL13 mal11 mal12), mal1g (mal13 MAL11 MAL12), or mal1⁰ (mal13 mal11 MAL12) loci containing just a functional regulator, just a maltose permease and maltase genes, or just a maltase gene, respectively (Naumov et al., 1994, Han et al., 1995). More recently, two other highly homologous genes (MPH2 and MPH3) were described as encoding for maltose and maltotriose transporters. MPH2 and MPH3 are closely related to the MALx1 genes, and apparently also induced by maltose (Day et al., 2002), although their role in maltotriose uptake and fermentation has been a matter of debate (Alves et al., 2008; Duval et al., 2010).

Maltose transport into the cell is required for full induction of MAL genes, and several reports have shown that maltose uptake is also the rate-limiting step for fermentation (Kodama et al., 1995; Wang et al., 2002; Rautio and Londesborough, 2003). In the presence of maltose, the MALx3 regulatory protein binds to DNA at the promoter region between MALx1 and MALx2 genes (the so-called UAS_MAL sequence), inducing the transcription of the MALx1 transporter and MALx2 maltase genes present at any MAL locus (Michels and Needleman, 1984; Needleman et al., 1984; Kim and Michels, 1988; Levine et al., 1992; Sirenko et al., 1995). In the case of maltose, an increased flow of this sugar into the cells and its subsequent accumulation in the cytoplasm can act as a signal to induce higher expression of MAL genes and to allow fermentation of this carbon source. Indeed, it was shown that both transport activity and levels of intracellular maltose are directly related to levels of induction of MAL genes (Wang et al., 2002).

On the other hand, the presence of glucose can repress the expression of MAL genes in two different ways: (1) inhibiting the activation of the regulatory protein MALx3 by maltose and (2) activating the MIG1 repressor, which prevents the transcription of the three MAL genes by binding at their promoter regions (Hu et al., 1995, 2000; Santangelo, 2006). In addition to its role in regulating transcription of MAL genes, glucose also exerts a strong control on the fermentation of other carbon sources by promoting the inactivation of their respective transport systems. This catabolic inactivation involves the phosphorylation and ubiquitination of the transporters, followed by their endocytosis and vacuolar degradation (Riballo et al., 1995; Medintz et al., 1996).

Nevertheless, some yeast strains express their MAL genes with no need of the induction by maltose. This phenotype is found in the so-called MAL constitutive (MAL^C) strains. The MAL^C alleles are usually the result of mutations on the genes that encode the regulatory MALx3 protein. It is known that, when maltose binds to MALx3, this regulatory protein changes its structure and then is able to bind at the UAS_MAL region of MAL genes. However, in the case of constitutive MALx3 alleles, the mutations promote a different structure of the protein, allowing it to bind at the UAS_MAL region even in the absence of maltose (Wang and Needleman, 1996; Gibson et al., 1997). In addition, MAL constitutive strains can either be sensitive or insensitive to glucose repression (Charron and Michels, 1987; Gibson et al., 1997; Higgins et al., 1999).

Regarding the utilization of maltotriose by S. cerevisiae, little is known about its transport and regulation compared to the level of molecular detail described for the maltose transporter in yeast cells. Maltotriose is transported by a different transport system from the well-characterized maltose transport carried out through MALx1 permeases. The AGT1 permease, which is able to transport not only maltotriose, but also maltose, trehalose and sucrose (Han et al., 1995; Stambuk et al., 1999; 2000; Stambuk and de Araújo, 2001; Zastrow et al., 2000; 2001; Alves et al., 2007), is found in a mal1g locus on chromosome VII of S. cerevisiae. It is probably regulated through the same regulatory mechanisms as the others MAL genes, since it has an UAS_MAL sequence at its promoter region (Han et al., 1995; Alves et al., 2007), although it was recently shown that some industrial yeast strains may have divergent sequences at the promoter region of their AGT1 gene, which have been shown not to be induced by maltose (Vidgren et al., 2011).

Some reports have recently suggested that the expression of αglucoside transporters in yeasts is higher in maltotriose-grown cells than in maltose-grown cells (Dietvorst et al., 2005; Salema-oom et al., 2005; Alves et al., 2007; 2008). Thus, due to the relevance of maltose and maltotriose transport in yeasts for the development of biotechnological processes that depend on the efficient fermentation of starch hydrolysates, in the present study we have analyzed the expression of the genes that encode maltose and maltotriose transporters in order to better understand the uptake of these α-glucosides by S. cerevisiae cells.

MATERIALS AND METHODS

Yeast Strains and Growth Conditions

The S. cerevisiae strains used in the present study are described in Table 1. Cells were routinely grown on rich YP medium (1% yeast extract and 2% peptone) supplemented with 2% of glucose, maltose or maltotriose as the carbon source. The pH of each medium was adjusted to 5.0 with HCl. Cells were grown aerobically at 28 °C with shaking (160 rpm) in cotton-plugged Erlenmeyer flasks filled to 1/5 of the volume with the medium. Cellular growth was followed by turbidity measurements at 570 nm.

α-Glucosidase Assays

The α-glucosidase activity in yeast cells collected at the exponential phase of growth was determined in situ with permeabilized cells as described previously (Stambuk, 1999) using 100 mM maltose or maltotriose in 100 mM MOPS (morpholinepropanesulfonic acid)-NaOH (pH 6.8) buffer. The glucose produced during maltose or maltotriose hydrolysis was determined by the glucose oxidase and peroxidase method using a commercial kit (BioDiagnostica-Laborclin). All assays were done in triplicate, and controls with previously boiled yeast cells were used. All activities were expressed as nmol of glucose produced by hydrolysis per milligram (dry weight) of cells per minute.

Transport Assays

Since yeast a-glucoside transporters are H⁺-symporters with a known stoichiometry of one proton co-transported with each sugar molecule (Serrano, 1977; Stambuk et al., 1996; 1998), the rates of active H⁺-maltose or H⁺-maltotriose symport were determined as previously described (Stambuk and de Araújo, 2001; Alves et al., 2008) using a PHM84 research pHmeter attached to a TT1 Servograph (Radiometer, Copenhagen). Cells were suspended at a cellular density of about 12-15 g (dry weight) L^-1 in water, without any buffer, and placed in a conical water-jacketed vessel in a total volume of 3 mL. The suspension was mixed with a magnetic stirrer and the temperature of the circulating water was regulated at 30 °C. The pH of the cellular suspension was adjusted to 5.0 with HCl, and the initial rates of sugar-induced proton uptake were calculated from the slope of the initial (<10 s) part of the curve obtained on the recorder by subtracting the basal rate of proton uptake observed before the addition of 0.3 to 100 mM of the sugar. To calculate the rate of H+ uptake, a calibration curve was obtained by addition of 50-100 nmol of HCl to the cell suspension. Kinetic constants were obtained as previously described (Stambuk et al., 2000; Stambuk and de Araujo, 2001). Experimental points are represented by the symbols used in the figures while lines represent the best fit obtained for the data set. All activities were expressed as nmol of substrate transported per milligram (dry weight) of cells per minute.

Quantitative RT-PCR (qRT-PCR)

Yeast cells were grown on rich media supplemented with 2% glucose, maltose or maltotriose, collected, rapidly frozen in liquid nitrogen, and stored at -80 °C. The total RNA was extracted with TRIzol (Invitrogen), and 1 µg of extracted RNA was treated with DNAse (Roche) and used for cDNA synthesis with a Reverse Transcription System kit (Promega). For real-time PCR, the KAPA SYBR FAST qPCR kit was used with 20 ng of cDNA, according to the manufacturer's instructions. The pairs of primers AGT11347F and AGT11397R; MALx1-1352F and MALx11403R; MPH2-1615F and MPH2-1665R, and ACT1904F and ACT1962R (Table 1) were used to evaluate the expression of the AGT1, MALx1, MPH2/MPH3 and ACT1 genes, respectively. The reaction was performed with a StepOnePlus Real-Time PCR System (Applied Biosystem). A dissociation curve was generated for each assay in order to confirm the amplification of only one product. All samples were tested in triplicate and the results presented are means and standard deviation obtained from two independent experiments. The expression values represent a relative quantification obtained by dividing the amount of mRNA from each analyzed gene by the mRNA from the house-keeping gene ACT1.

RESULTS AND DISCUSSION

The two yeast strains analyzed in the present work (strains 1403-7A and CEN.PK2-1C) have unrelated genetic backgrounds, but all are able to grow on, consume and ferment both maltose and maltotriose efficiently (Alves et al., 2008). These strains have in their genomes not only the AGT1 and MPH2/MPH3 genes, but also at least two copies of MALx1 genes: MAL21 and MAL31 (both strains) and, in the case of strain 1403-7A, also the MAL41 gene (Alves et al., 2008; Duval et al., 2010). The results presented in Table 2 indicate that, in both strains, the activity of αglucoside transporters is higher in maltotriose-grown cells than in maltose-grown cells, as already described in the literature (Dietvorst et al., 2005; Salema-oom et al., 2005; Alves et al., 2007; 2008), while the aglucosidase activity is practically the same in cells of each strain grown on either carbon source, except for maltotriose hydrolysis by strain 1403-7A, which was two-fold higher in maltose-grown cells.

Since an increase in transport activity could be the consequence of a change in affinity of the transporter(s) present in the plasma membrane (Smit et al., 2007), we performed a kinetic analysis of the active maltotriose-H⁺ symport activity by these strains after growth on maltose or maltotriose (Figure 1). These two strains have a low affinity (K_m 36 ± 2 mM) maltotriose-H⁺ symport activity, and the only difference between maltose-grown and maltotriose-grown cells is the higher transport capacity of the yeast cells grown in this last carbon source (Figure 1, A and B).

This relatively low affinity for maltotriose is a known characteristic of the AGT1 permease, indicating that this transporter is probably responsible for maltotriose uptake in both strains. In order to better characterize the reason for these results, we quantified the expression of mRNA transcripts from the MALx1, MPH2/MPH3 and AGT1 genes through quantitative RT-PCR (qRT-PCR). To this end, the yeast strains were grown on rich media containing 2% glucose, maltose or maltotriose, and cells were collected at the middle of the exponential phase of growth for subsequent RNA extraction.

Figure 2 (parts A and B) shows that, when the MAL-constitutive strains 1403-7A and CEN.PK2-1C are metabolizing maltotriose, the expression levels of the AGT1 and MALx1 genes are similar to the ones observed in maltose growing cells, indicating that the higher transport activity found in maltotriose-growing cells is not a consequence of higher induction of MAL genes by this carbon source. Figure 2 also shows that, while strain 1403-7A is not repressed by glucose, strain CEN.PK2-1C was fully repressed by this monosaccharide. Our results are in agreement with previous characterization of the MALx3 regulators, which indicated that the MAL43^C mutant allele is insensitive to glucose repression (Charron and Michels, 1987; Wang and Needleman, 1996), while the constitutive MAL23-8^Cregulator present in the CEN.PK2 strains is glucose repressible (Rodicio and Zimmermann, 1984; Kopetzki et al., 1989). In agreement with the above results, there was also no difference in the expression levels of MPH2/MPH3 genes between maltose- and maltotriose-grown cells (Figure 2C). It is interesting to note, however, that although it has been suggested that these genes have their expression induced by MALx3 regulators (Day et al., 2002), our results showed no repression of such genes by glucose, indicating that expression of MPH2/MPH3 genes is probably not controlled exactly like the MALx1 or AGT1 genes.

While the kinetic parameters of sugar transport are a property of the transporter protein, the transport capacity (V_max) depends upon how many transporters are expressed and present (in an active form) in the cell membrane. Unfortunately studies using a MAL^C strain with its AGT1 permease tagged with green-fluorescent-protein (GFP) were also inconclusive regarding differences in the expression of the protein by the cells grown on maltose or maltotriose (data not shown). Probably other factors, including post-translational regulation of the transporters (Hu et al., 2000; Jiang et al., 2000; Gadura et al., 2006), or in the case of active transporters, also the energetic state of the cells (Guimarães and Londesborough, 2008; Guimarães et al., 2008), can explain the differences in transport rates observed in these MAL^C yeast strains. Since both the repression and catabolic inactivation of transporters is trigged not only by external glucose, but also by glucose molecules generated inside the cells due to high rates of maltose hydrolysis (Hu et al., 1995; 2000; Gibson et al., 1997; Jiang et al., 1997; 2000), our results of lack of correlation between the expression of the genes and the transport activity measured in the cells could also be explained by the different rates of αglucoside hydrolysis by the yeast cells, where maltose hydrolysis is, at least, two-fold higher than maltotriose hydrolysis (see Table 2 and Zastrow et al., 2000; 2001; Alves et al., 2007; 2008), thus generating more glucose molecules during growth in maltose, when compared with growth on maltotriose.

ACKNOWLEDGMENTS

This work was supported in part by grants from the Brazilian agencies CNPq and CAPES. We thank R. Needleman (Wayne State University) and A. Kruckeberg (University of Amsterdam) for providing yeast strains, and H. Tournu for technical collaboration during the real time PCR experiments.

Submitted: February 28, 2012

Revised: April 17, 2013

Accepted: June 12, 2013

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To whom correspondence should be addressed

This is an extended version of the manuscript presented at the SINAFERM 2011 - 18th National Symposium on Bioprocesses 2011, Caxias do Sul, Brazil.

Publication Dates

• Publication in this collection
  20 Mar 2014
• Date of issue
  Mar 2014

History

• Received
  28 Feb 2012
• Accepted
  12 June 2013
• Reviewed
  17 Apr 2013