Impact of the reg1 mutation glycocen accumulation and glucose consumption rates in Saccharomyces cerevisiae cells based on a macrokinetic model

Abstract

In S. cerevisiae, catabolite repression controls glycogen accumulation and glucose consumption. Glycogen is responsible for stress resistance, and its accumulation in derepression conditions results in a yeast with good quality. In yeast cells, catabolite repression also named glucose effect takes place at the transcriptional levels, decreasing enzyme respiration and causing the cells to enter a fermentative metabolism, low cell mass yield and yeast with poor quality. Since glucose is always present in molasses the glucose effect occurs in industrial media. A quantitative characterization of cell growth, substrate consumption and glycogen formation was undertaken based on an unstructured macrokinetic model for a reg1/hex2 mutant, capable of the respiration while growing on glucose, and its isogenic repressible strain (REG1/HEX2). The results show that the estimated value to maximum specific glycogen accumulation rate (muG,MAX) is eight times greater in the reg1/hex2 mutant than its isogenic strain, and the glucose affinity constant (K SS) is fifth times greater in reg1/hex2 mutant than in its isogenic strain with less glucose uptake by the former channeling glucose into cell mass growth and glycogen accumulation simultaneously. This approach may be one more tool to improve the glucose removal in yeast production. Thus, disruption of the REG1/HEX2 gene may constitute an important strategy for producing commercial yeast.

yeast; S. cerevisiae; catabolite repression; glycogen; macrokinetic model

Impact of the reg1 mutation glycocen accumulation and glucose consumption rates in Saccharomyces cerevisiae cells based on a macrokinetic model

M.H.M.Rocha-Leão; M.A.Z.Coelho; O.Q.F.Araújo^*

Universidade Federal do Rio de Janeiro, Escola de Química, Centro de Tecnologia, Bloco E, sala E-203, Phone: [phone], Fax:[phone], Cidade Universitária, CEP: 21949-900, Rio de Janeiro - RJ, Brasil, E-mail: ofelia@.eq.ufrj.br

ABSTRACT

In S. cerevisiae, catabolite repression controls glycogen accumulation and glucose consumption. Glycogen is responsible for stress resistance, and its accumulation in derepression conditions results in a yeast with good quality. In yeast cells, catabolite repression also named glucose effect takes place at the transcriptional levels, decreasing enzyme respiration and causing the cells to enter a fermentative metabolism, low cell mass yield and yeast with poor quality. Since glucose is always present in molasses the glucose effect occurs in industrial media. A quantitative characterization of cell growth, substrate consumption and glycogen formation was undertaken based on an unstructured macrokinetic model for a reg1/hex2 mutant, capable of the respiration while growing on glucose, and its isogenic repressible strain (REG1/HEX2). The results show that the estimated value to maximum specific glycogen accumulation rate (m_G,MAX) is eight times greater in the reg1/hex2 mutant than its isogenic strain, and the glucose affinity constant (K_SS) is fifth times greater in reg1/hex2 mutant than in its isogenic strain with less glucose uptake by the former channeling glucose into cell mass growth and glycogen accumulation simultaneously. This approach may be one more tool to improve the glucose removal in yeast production. Thus, disruption of the REG1/HEX2 gene may constitute an important strategy for producing commercial yeast.

Keywords: yeast, S. cerevisiae, catabolite repression, glycogen, macrokinetic model.

INTRODUCTION

It was suggested that in S. cerevisiae cells glycogen provides survival advantages for cell proliferation and stress resistance and that glycogen turnover may function as glycolytic safety valves to avoid "substrate-accelerated death" when cells are in conditions of stress (François and Parrou, 2001). The nutritional status of the cells controls glycogen accumulation through an intimate connection between glucose-repression mechanism, responses to nutrient deprivation (Hardy et al., 1994; Lillie and Pringle, 1980) and specific growth rate (Rocha-Leão, 1987). Glycogen synthase isoforms (Gsy1 and Gsy2) occur in phosphorylated (less active) and dephosphorylated (active) forms, and both are subjected to allosteric control (Farkas et al., 1991; Cabib and Rothman-Denes, 1971; François et al., 1988; Hardy and Roach, 1993; Huang and Cabib, 1974; Huang et al., 1997; Toda et al., 1985). Gsy2 is a predominant form, which occurs under catabolite derepression, heat shock and nitrogen starvation. Glucose-6-P, which is a direct activator of glycogen synthase, controls the phosphorylation state of this enzyme by inhibiting a glycogen synthase kinase (Huang et al., 1997).

It has been observed that some genes are simultaneously involved in catabolite repression and glycogen metabolism (Gancedo, 1998). Those of the significant interest are the GLC7 (encoding protein phosphatase 1 catalytic subunit), REG1/HEX2 (encoding the regulatory subunit, which modulates phosphatase 1 protein) and SNF1/CAT1 (encoding a protein kinase important to the glucose repression mechanism). Glc7 controls a variety of proteins and sustains both Gsy2 and Gsy1 in an active state, forming a complex with Gac1 that favors glycogen accumulation (Feng et al, 1991; François et al, 1992; Huang et al, 1996). The Reg1-Glc7 phosphatase complex is a major cytoplasmic component of the glucose repression pathway and is responsible for reducing Snf1 kinase activity levels that establish glucose repression. Furthermore, Bisson (1988) demonstrated that the reg1/hex2 mutant resulting in constitutive expression of glucose-repressible functions also resulted in constitutive expression of high-affinity glucose uptake. On the other hand, derepressed yeast cells consuming glucose control the glucose transport rate via inhibition by intracellular glucose (Teusink et al., 1998). There is also evidence that Reg1/Hex2 interacts with the Snf1 catalytic domain (Ludin et al., 1998) and its state of phosphorylation is also regulated by hexokinase PII (Sanz et al., 2000). Expressed constitutively, the SNF1 gene is absolutely required in derepression of glucose-repressed genes (Celenza and Carlson, 1986). Furthermore, interactions between CAMP-PK (protein kinase cyclic AMP dependent), which appears to play a central role in the control of glycogen metabolism (Hardy et al., 1994; Toda et al., 1985), and Snf1 protein kinase control glycogen accumulation (Hardy et al., 1994). It is important to note that in the Snf1 mutant, the Gsy-2 enzyme was blocked both in the inactive and the phosphorylated forms. Wu et al. (2001) show that overexpression of Gac1 affects glucose repression, suggesting that multiple regulatory subunits compete for Glc7 binding in vivo increasing the complexity of the regulation of the yeast metabolism. On the other hand, the SMC1B/3 strain, a hex2-3 mutant, currently referred to as reg1, shows a high level of glucose phosphorylation and an increased amount of glucose-6-P and frutose-6-P when in glucose medium (Entian and Zimmermann, 1980). Nevertheless, when this strain was introduced in nitrogen-limited medium, glycogen accumulation was much more pronounced than it was for its isogenic SMC-1B strain (Costa-Carvalho et al., 1986).

Bisson (1988) obtained results supporting the conclusion that high-affinity glucose uptake in S. cerevisiae is under general glucose repression control. Reifemberg et al. (1997) showed that the triggering of glucose repression was not dependent on a specific hexose transporter protein, but rather was correlated with glucose uptake activity of the cells and glycolytic flux.

In the present work, the relationship between catabolite repression, glycogen accumulation and glucose uptake was quantitatively characterized based on an unstructured macrokinetic model for cell growth, substrate consumption and glycogen formation using the repressed SMC-1B and the derepressed SMC-1B/3 strains. Moreover no reports on quantitative determination of the specific rate of glucose consumption by repressed and derepressed S. cerevisiae strains had been shown previously. Model parameters for both strains were estimated from experimental data.

MATERIALS AND METHODS

Yeast Strains

SMC-1B (a his4 MAL2-8^c MAL3 SUC3 CAT1-2d), which is sensible to catabolite repression, and SMC-1B/3 (a his4 MAL2-8^c MAL3 SUC3 CAT1-2d hex2-3), which shows simultaneous fermentation and respiration of glucose due to partial derepression of enzymes linked to aerobic metabolism (Entian and Zimmermann, 1980), were used.

Culture Conditions

As described in Lillie and Pringle (1980) and Costa-Carvalho et al. (1986), cells were grown aerobically at 28ºC, pH 6.9 and agitation of the 160 oscillations/min, in 2 L flasks containing 400 mL of slow-poor medium (2 mg/mL, bacto peptone from DIFCO, 10 mg/mL glucose), where growth is nitrogen-limited followed by glucose removal from the medium; the bacto peptone containing 15% of the total nitrogen was choose because it contains a wide molecular weight distribution of peptides, which keep within all amino acids that occur in the nature; catabolite repression effects are present but less severe than in fast-rich medium with high glucose and nitrogen concentrations. The spectra of the cytochromes, attained between 500 and 640 nm, show whole derepression of the respiratory chain for SMC1B/3 strain growing in slow-poor medium highlighting that the agitation of the medium was sufficient to dissolve oxygen to produce cells with high respiratory capability.

Analytical Methods

Cell mass concentration was determined by dry weight. Glycogen was extracted from 15 mg dry weight of cells with 0.25M Na₂CO₃ solution (Becker, 1978) and after hydrolysis with Rhizopus amyloglucosidase at 37ºC and pH 4.8 for 22 hours, the amount of glucose was determined by the glucose oxidase method (Raabo and Terkildsen, 1960).

Macrokinetic Model

For continuous grow, a constant supply of resources that can be converted into all the constituents needed for cell propagation is required. In many cases, cell transport capability exceeds cell growth. For instance, when too much uptake occurs in a well-regulated cell, the growth machinery must affect the uptake systems (Koch, 1997). In the present work, the most important overall reaction is cell growth on a single limiting substrate. From a reaction engineering point of view, this reaction is autocatalytic since cell mass catalyzes its formation and is not consumed by the growth reaction (Zhao and Skogestad, 1997). The rate of cell mass growth is usually proportional to cell concentration. Furthermore, it is frequently assumed that specific growth rate (m(S)) is given by Monod-type model. Therefore, the model employed is

Rate of cell mass growth:

Rate of substrate consumption:

Rate of glycogen formation:

Model Parameter Estimation

Model parameters (m_X,MAX, K_SX, m_S,MAX, K_SS, m_G,MAX and K_G) were estimated using MATLAB 5.0 (The Mathworks Inc.) and its Optimization Toolbox (fminu) to solve the nonlinear least-squares problem:

Statistical Analysis

In the statistical analysis the following assumptions were made: the model was correct, experimental measurements were independent, errors were normally distributed and experimental variances were proportional to the fundamental variance. The model was formulated taking into consideration the measurement errors e_i

Using linear statistics and defining S_R² as a nontendentious and coherent estimator of fundamental variance, the corresponding variance-covariance matrix for the estimated parameters is given by

where is the Jacobi matrix for the state variables with respect to the parameters.

Validity of the parameters can be verified by analyzing their confidence intervals as determined by the t-Student distribution (Box et al., 1978), assuming statistical decoupling on parameter estimation. With a 95% confidence interval and n degrees of freedom, confidence intervals were calculated according to

Statistical analysis of confidence intervals was used to study the correlation between pairs of estimated parameters with no assumption of statistical decoupling on parameter estimation. The characteristic equation for the parameter confidence interval is given by

where

F_1–a was obtained by Fisher's analysis for n degrees of freedom and a 95% confidence interval.

RESULTS

Parameter Estimation

Experimental results were used to adjust model parameters, for SMC-1B and SMC-1B/3 strains. The optimized parameter values are shown in Table 1 and the 2-D projection of confidence intervals for most correlated parameters obtained for strain the SMC-1B/3 strain is shown in Figure 1 for parameter pairs with good correlations. The same procedure was conducted for the SMC-1B strain. Results indicate that K_SX, K_SS and K_G are the parameters for which the estimates are likely to be inaccurate, whereas the corresponding parameters for the SMC-1B/3 strain are likely to be more accurate, and hence the model structure proposed can be used. Figures 2 and 3 contain experimental data and model predictions. Despite of the results presented in Figures 2 and 3 were related to one set of experiments, replicates were carried and similar profiles could be obtained. This reproducible behavior makes possible to incorporate in the statistical analysis all measurement errors as well as to determine quantitatively the difference between both studied strains.

DISCUSSION

Estimated macrokinetic model parameters show the affinity constant (K_SS) value for the derepressed strain to be as much as fifty times greater than that obtained for the repressed strain. These results show that the repressed strain has a greater affinity for glucose than the reg1/hex2 mutant. Although SMC-1B/3 might have both a high-affinity glucose transport system and a high level of glucose phosphorylation (Entian and Zimmermann, 1980), Figure 4 shows that the specific glucose uptake rate, (dS/dt)/X, given by Equation 2-a, is lower than the rate obtained by SMC-1B in accordance with constant (K_SS) estimated values for the two strains. The previous statement may be assumed to be true for this strain since there are references in the literature showing that this transport system is among others controlled by glucose repression (Bisson, 1988). Significant differences in glucose uptake rate can be observed in the range of 2 to 5 mg/mL of glucose, but when glucose concentrations are higher than 10 mg/mL, both strains reach similar rate values (Figure 4). Quantitative determination of glucose uptake from the medium highlights that glucose consumption is not the pacemaker step in glucose consumption under the conditions studied. We can assume that slow growth due to the composition of the medium and the intrinsic respiratory capacity of the derepressed cells also results in an increased ATP and glucose-6-P levels, and thus the UDPG level could be increased. The immediate conversion of these metabolites to glycogen, a biosynthetic process, which consumes ATP and glucose-6-P, sustains intracellular metabolite concentrations, avoiding "substrate-accelerated death." The excess energy generated by the imbalance during slow growth between ATP produced in oxidative catabolism of the carbon source and ATP consumed in anabolism, due to low nitrogen content in the medium, leads to slow protein biosynthesis and clearly favors glycogen biosynthesis. In accordance, the results estimated by the model to maximum specific glycogen accumulation rates (m_G,MAX ) is eight times greater for SMC1B/3 strain than for SMC1B strain. Furthermore, the glycogen synthase, which shows a Km for UDPG in the range of its intracellular concentration, works rapidly as UDPG increases under these conditions. Also in this case, the literature highlights the GSY2 expression as well as the Gsy2 isoform activity in derepression conditions. Note that the strains have similar specific growth rates as shown in Figure 5, and that the SMC-1B/3 enzyme must be more abundant in its isoform (Gsy2) and its more active form becomes completely activated to sustain glycogen biosynthesis (Figure 6). Results also suggest that glycogen is a repository of glucose when this carbon source is transported into cells under derepressed conditions and slow growth association. It should be pointed out that, in nitrogen nonlimited medium, which is a fast-rich medium because glucose and nitrogen sources are in high concentrations (20 mg/mL glucose, 20mg/mL bacto peptone, 10mg/mL yeast extract), SMC-1B/3 grows faster and glycogen accumulation occurs only at the onset of the stationary phase (Costa-Carvalho et al., 1986). Thus, at a slow growth rate, glycogen retains the intracellular nonoxidized glucose inside the cells. Nevertheless, the results obtained herein show that this organism has a great ability to accumulate glycogen when the cells are not exposed to catabolite repression due to the imbalance between glucose uptake and slow glucose catabolic flux. Under the impact of the reg1/hex2 mutation, the derepressed mutant strain, which might have high-affinity glucose transport expressed but inhibited by intracellular glucose, regulates the glycogen accumulation rate through slow catabolic flux to remove the excess of intracellular glucose under these circumstances. Thus, the glycogen accumulation is a bypass pathway for intracellular glucose that exceeds the requirements of catabolism. This conclusion was reinforced when the SMC1B/3 strain was grown in fast-rich medium. Under this condition the strain showed a high specific growth rate and glycogen accumulation very similar to those of its isogenic strain (Costa-Carvalho et al., 1986). Because glycogen content and its turnover are linked to stress resistance and cell viability, its accumulation and slow degradation is an advantageous strategy for cells in nutrient-poor environments and fasting. Thus, when both strains were incubated in 0.05 M phosphate buffer, pH 6.0, after growth in the slow-poor media, the derepressed strain was highly viable when compared with its isogenic strain (Costa-Carvalho et al., 1986). Moreover, both strains showed also higher viability under these conditions when they had been previously grown in poor medium than when they had been grown in rich medium. For derepressed strain the growth in slow-poor medium somehow leads to a more efficient utilization of the glycogen during prolonged starvation, a fact that is metabolically expressed as a greater capacity of surviving in the absence of external nutrients. Yeast cells with this ability are quoted as good quality (Cahil et al., 2000).

Since glycogen metabolism is associated with cell viability during all stresses that current occur in the industrial production of yeast cell mass, its biosynthesis is associated with slow growth in derepressed strain, and triggering of glucose repression is correlated with cell's glucose uptake and glycolytic flux, the cell culture must be conducted in such a way to regulate this biosynthesis under derepressed conditions. Thus, a fed-batch bioreactor operating in optimized conditions through the estimated parameters may be therefore a statement to an optimal control for glucose feed rate permitting a regulation of glycogen metabolism. The parameters herein presented may be useful in optimization studies of fed-batch yeast cultures to produce good-quality glycogen-rich cell mass of the diploide commercial strain. Although commercial yeasts are diploids, in this work it was used two haploide strains because the effect of the genes on the cell metabolism is currently studied using haploide laboratory strains.

CONCLUSIONS

A macrokinetic model was used and model parameters were estimated from experimental data (Figures 3 and 4) for both strains. Results suggest that glycogen is a repository of glucose when this carbon source is transported into cells under derepression conditions in a limited nitrogen medium. We may propose that this organism has a great ability to accumulate glycogen in a pathway primarily regulated by catabolic flux but not by the affinity of the glucose transporter systems present in cells under derepression conditions. Although further studies are necessary to consolidate the proposed hypothesis, the results presented herein show that the impact of the reg1/hex2 mutation, observed in derepressed strain, allows simultaneous coordination of production of cell mass and accumulation of glycogen. This control can be useful in the production of. S. cerevisiae with a good quality for both baker's yeast and bread production. In these bioprocesses, the glucose repression constitutes currently a problem, because in industrial media like molasses, sugars are sequentially consumed and some times are not metabolized, resulting in prolonged production time and overload. Moreover, in the industrial processes if the ethanol produced is not consumed the cell mass yield is reduced. A direct genetic change, such as disruption of the REG1HEX2 gene in domesticated S. cerevisiae currently used in industrial process following oxidative growth, is a potential strategy, witch may offers economical benefits in these specific applications.

ACKNOWLEDGMENTS

This work was supported by PRONEX/CNPq.

NOMENCLATURE

Received: June 13, 2002

Accepted: April 7, 2003

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Publication Dates

• Publication in this collection
  01 Sept 2003
• Date of issue
  Sept 2003

History

• Accepted
  07 Apr 2003
• Received
  13 June 2002