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Applied and Environmental Microbiology, November 2009, p. 6876-6885, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01464-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identity of the Growth-Limiting Nutrient Strongly Affects Storage Carbohydrate Accumulation in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae{triangledown} ,{dagger} ,{ddagger}

Lucie A. Hazelwood,1,2 Michael C. Walsh,3 Marijke A. H. Luttik,1,2 Pascale Daran-Lapujade,1,2,4 Jack T. Pronk,1,2 and Jean-Marc Daran1,2*

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands,1 Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands,2 Heineken Supply Chain, Research and Innovation, Burgemeester Smeetsweg 1, 2380 BB Zoeterwoude, The Netherlands,3 Netherlands Consortium for Systems Biology, Bureau Science Park 123, 1098 XG Amsterdam, The Netherlands4

Received 22 June 2009/ Accepted 20 August 2009


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ABSTRACT
 
Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h–1) of S. cerevisiae, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2{Delta} msn4{Delta} strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures.


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INTRODUCTION
 
Glycogen and trehalose are the major storage carbohydrates in baker's yeast (Saccharomyces cerevisiae) (22). The ability to accumulate these storage carbohydrates is an important criterion for selection of industrial yeast strains because of their involvement in the cellular robustness and quality of yeast extracts (e.g., for food and feed applications) (8, 16, 61).

Based on the observation that glycogen and trehalose accumulate in batch cultures of S. cerevisiae that are starved for carbon, nitrogen, sulfur, or phosphorus, Lillie and Pringle (41) proposed that storage carbohydrate synthesis is a general response to nutrient starvation. Parrou et al. (56), who studied carbohydrate metabolism during nutrient-limited growth rather than during nutrient starvation, found that accumulation of glycogen and trehalose occurred when growth was limited by the carbon or nitrogen source. Because these workers were unable to obtain sulfur- or phosphorus-limited growth with their experimental setup (56), it remained unclear whether accumulation of glycogen and trehalose is a common response to nutrient limitation or whether, instead, nutrient limitations other than nitrogen or carbon limitation may result in different storage carbohydrate contents.

Research on the molecular mechanisms involved in the regulation of storage carbohydrates has focused mostly on the diauxic shift in aerobic, glucose-grown batch cultures (20, 41, 55) and on responses to nitrogen starvation (56) (for a review, see reference 22). Under both conditions, transcriptional activation of the glycogen and trehalose pathways (Fig. 1) is mediated by the Msn2p/Msn4p complex (51, 55, 69, 86, 88). Transcriptional regulation of GSY2 (encoding glycogen synthase) during the diauxic shift involves integration of signaling pathways involving the protein kinases Pho85p, Snf1p, and protein kinase A (PKA) (17). Glycogen synthase and glycogen phosphorylase, two central enzymes in glycogen biosynthesis and degradation, are subject to strong posttranslational regulation by phosphorylation-dephosphorylation through complex kinase and phosphatase cascades (18, 21, 30, 32-34, 58, 64).


Figure 1
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  		FIG. 1. Glycogen and trehalose metabolism in S. cerevisiae.

Besides transcriptional regulation mediated by the Msn2p/Msn4p complex, TPS1 (trehalose-6-phosphate synthase), a key gene of trehalose metabolism, has been shown to be corepressed by Mig1p and Mig2p (43). Further analysis of promoter sequences of TPS1 and NTH1 (neutral trehalase) showed the presence of several other regulatory sequences for Gcn4p (basic leucine zipper transcriptional activator of amino acid biosynthetic genes), Gcr1 (transcriptional activator of genes involved in glycolysis), and Adr1p (carbon source-responsive zinc finger transcription factor) (14). Regulation of trehalose accumulation occurs posttranslationally as well. Similar to enzymes involved in glycogen metabolism, enzymes involved in trehalose metabolism are subject to strong posttranslational regulation by phosphorylation and dephosphorylation (54, 84). Trehalase activity has been identified as a target of PKA (78). It has been proposed that Nth1p activity is controlled by Ylr20wp/Dcs1p by direct protein interaction (14).

Batch cultures have some inherent drawbacks for studying the impact of nutrient limitation on storage carbohydrate metabolism. First, nutrient limitation in batch cultures is a transient phenomenon that occurs only during the transition from nutrient-excess conditions to nutrient-depleted conditions. Moreover, nutrient limitation in batch fermentations affects the specific growth rate, which in turn affects accumulation of storage carbohydrates (26, 39, 68). The specific growth rate also has a profound impact on transcriptional responses (10, 62). The stress response element (STRE) genes (stress response regulon controlled by Msn2p/Msn4p) are particularly sensitive to the specific growth rate, and their expression is negatively correlated with this rate. Clearly, dissection of the effects of the specific growth rate from the effects of nutrient limitation is essential for interpreting and understanding the regulation of storage carbohydrate metabolism by nutrient availability and for interpreting Msn2p/Msn4p-mediated induction of STRE genes.

Chemostat cultivation offers the unique possibility of growing microorganisms at a constant specific growth rate under different nutrient limitation regimens. In S. cerevisiae, this approach has been used to study transcriptional responses to various nutrient limitation regimens (e.g., limitation by different carbon and nitrogen sources, sulfate, phosphate, and zinc) (for a review, see reference 13). Chemostat cultivation allows dissection of the effects of nutrient limitation on yeast physiology from the effects of a fixed specific growth rate. In a recent study (15), we observed accumulation of very low levels of storage carbohydrates and transcriptional downregulation of the glycogen pathway in zinc-limited chemostat cultures compared to glucose- and ammonia-limited cultures grown at the same specific growth rate. These unexpected observations stressed the need for a broader investigation of the effect of nutrient limitation regimens on storage carbohydrate accumulation.

The goal of the present study was to systematically investigate the impact of the identity of the growth-limiting nutrient on storage carbohydrate metabolism in S. cerevisiae. To this end, accumulation of glycogen and trehalose was studied using anaerobic glucose chemostat cultures at a fixed dilution rate of 0.10 h–1 under five different nutrient-limitation regimens (glucose, ammonia, sulfate, phosphate, and zinc). In addition, we studied regulation of storage carbohydrate metabolism at the transcriptional and posttranscriptional levels and assessed the role of Msn2p/Msn4p in mediating transcriptional induction of glycogen and trehalose genes.


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MATERIALS AND METHODS
 
Yeast strain and maintenance.
The strains used in this study are listed in Table 1. The haploid prototrophic S. cerevisiae strain CEN.PK 113-7D (MATa) was obtained from P. Kötter, Frankfurt, Germany. Deletion mutants were constructed by using standard yeast media and genetic techniques and were routinely grown at 30°C on complex (YPD) or defined media (9). Gene deletions were introduced into strain CEN.PK113-5D (provided by P. Kötter, Frankfurt, Germany), which is auxotrophic for uracil. The geneticin (G418) resistance cassette was amplified by using the pUG6 vector as the template, and the KanMX (G418) resistance expression cassette was removed using the Cre-loxP recombination system as previously described (27). For chemostat cultivation, strain IMK151 (MATa MAL2-8c SUC2 ura3 msn2::loxP msn4::loxP-kanMX-loxP) was transformed with plasmid p426GPD carrying the URA3 gene to complement its uracil auxotrophy (49); this yielded strain IMZ066. The oligonucleotide primers used in this study are listed in Table S1 in the supplemental material.


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  		TABLE 1. Strains used in this study

Media for chemostat cultivation.
The synthetic medium used was based on the medium described by Verduyn et al. (81). The modifications introduced for ammonium-, sulfate-, phosphate-, and zinc-limited growth have been described previously (15, 70, 81) and are listed in Table 2. In all chemostats except those in which growth was limited by glucose, the target residual glucose concentration was 17 g/liter (95 mM) in order to have the same degree of glucose repression. No significant differences in glucose consumption, biomass, and ethanol and CO2 formation were observed for the four glucose-excess conditions (15, 70). The reservoir medium was supplemented with vitamins and the anaerobic growth factors Tween 80 and ergosterol as previously described (81).


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  		TABLE 2. Composition of the media used to perform carbon, nitrogen, sulfur, phosphorus, and zinc limitation experiments

Chemostat cultivation.
Chemostat cultures were fed using synthetic medium that resulted in limitation of growth by glucose, ammonia, sulfate, phosphate, or zinc and that contained all other components required for growth in excess and at constant residual concentrations. The dilution rate was set at 0.10 h–1, and the pH was measured online and kept constant at 5.0 by automatic addition of 2 M KOH as previously described (70). Cultures were assumed to be in steady state when, after at least 5 volume changes, the culture dry weight, glucose concentration, and carbon dioxide production rate varied by less than 2% during one additional volume change (19). Steady-state samples were taken after 10 generations at the latest to avoid strain adaptation due to long-term cultivation (35). The experiment for each cultivation condition was performed in triplicate.

Analytical methods.
Culture supernatants were obtained after centrifugation of samples from the chemostats. For glucose determination and carbon recovery, culture supernatants and media were analyzed by high-performance liquid chromatography using an AMINEX HPX-87H ion-exchange column and 5 mM H2SO4 as the mobile phase. Culture dry weights were determined by filtration as described by Postma et al. (60). Trehalose measurement and glycogen measurement were adapted as described by Parrou and Francois (57), and values were obtained in triplicate. Glucose contents were determined using the UV method and a Roche kit (no. 0716251; Roche Applied Science, Almere, The Netherlands).

In vitro enzyme assays.
To minimize the loss of phosphate groups on proteins, cell sampling of chemostat cultures for measurement of glycogen synthase and glycogen phosphorylase activities was performed as described by Francois and Hers (21). Fresh cell extracts were prepared using the fast prep method (11), except that the extraction buffer contained the phosphatase inhibitor cocktail PhosSTOP (Roche Applied Science, Almere, The Netherlands), which was used according to the manufacturer's recommendations. Glycogen synthase activity was assayed at 30°C for 20 min using a mixture containing 50 mM glycylglycine (pH 7.4), 0.25 mM UDP-[U-14C]glucose (900 cpm/nmol; Amersham Perkin Elmer, Groningen, The Netherlands), 10 mM Na2SO4, 2.5 mM EDTA, and 3% glycogen from rabbit liver (Sigma Aldrich, Zwijndrecht, The Netherlands) as described by Francois and Hers (21). When glucose-6-phosphate was added to the assay mixture, it was added at a concentration of 10 mM. Glycogen phosphorylase activity was determined in the direction of glycogen synthesis by measuring the incorporation of glucose-1-phosphate into glycogen. The reaction mixture contained 5 mM [U-14C]glucose-1-phosphate (300 cpm/nmol; Amersham Perkin Elmer, Groningen, The Netherlands), 2.5 mM EDTA, 2.5% glycogen, and 50 mM sodium succinate (pH 5.8) (21). Both enzymes were measured in duplicate using three independent chemostat cultures. One unit was defined as the amount of enzyme that catalyzed the conversion of 1 µmol of substrate in 1 min under the conditions of the assay. The concentration of protein was determined by the method of Lowry et al. with bovine serum albumin (Sigma Aldrich, Zwijndrecht, The Netherlands) as the standard (42).

Microarrays, data acquisition, and statistical analysis.
Sampling of cells from chemostats and extraction of total RNA were performed as previously described (15). The results for each growth condition were derived using three independent culture replicates. Acquisition and quantification of array images and data filtering were performed using Affymetrix GeneChip operating software, version 1.2. To eliminate insignificant variations, the expression value for genes with expression values less than 12 was defined as 12 as previously described (6).

The Significance Analysis of Microarrays (SAM) (version 1.12) (77) add-in to Microsoft Excel was used for comparison of replicate array experiments. For transcriptome analysis of CEN.PK113-7D under the five different nutrient limitation conditions, the statistical significance of observed differences was assessed by SAM multiclass analysis, using an expected false discovery rate of 0.02%. K-means clustering was performed with Expressionist Analyst, version 3.2 (Genedata, Basel, Switzerland). For comparison of IMZ066 (msn2{Delta} msn4{Delta}) transcriptome data to data for the isogenic reference strain CEN.PK113-7D, pairwise comparisons (SAM Microsoft excel add-in) using a threshold difference of twofold and a false discovery rate of 1% were performed. Venn diagrams and heat map visualizations of transcript data were generated with Expressionist Analyst, version 3.2. Transcript data have been deposited in the Genome Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE15465.

Hypergeometric tests and motif discovery.
Groups of coresponsive genes were consulted for enrichment of functional annotation (MIPS [47] and KEGG [36]) and significant transcription factor binding (28) as previously described (37, 38). Promoter analysis was performed using the web-based software Regulatory Sequence Analysis Tools (79, 80). The promoters (from position –800 to position –1) of each set of coregulated genes were analyzed for overrepresented hexanucleotides.


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RESULTS
 
Storage carbohydrate accumulation is not a general response to nutrient limitation.
In aerobic, glucose-grown chemostat cultures of S. cerevisiae in which glucose is not the growth-limiting nutrient (e.g., sulfate- or phosphate-limited cultures), glucose dissimilation is respirofermentative. As a consequence, such glucose-excess cultures have significantly lower biomass yields and higher glucose uptake rates than glucose-limited cultures (6, 15, 70). Glycolytic flux has previously been shown to be negatively correlated with storage carbohydrate accumulation (26, 40). Under anaerobic conditions, glucose is dissimilated via alcoholic fermentation irrespective of the nutrient limitation. To minimize possible indirect effects of glycolytic flux on storage carbohydrate metabolism, the present study was performed with anaerobic chemostat cultures.

An approximately 20% lower biomass yield and a corresponding higher glucose consumption rate (P < 1.0E–05 for both measurements, as determined by a t test) were found in the four glucose-excess scenarios than in the anaerobic glucose-limited cultures, indicating that there was partial uncoupling of glucose dissimilation and biomass formation under glucose-excess conditions (Table 3). These modest differences are unlikely to contribute to a substantial influence of glycolytic flux on storage carbohydrate accumulation. The production rates of minor metabolites derived from central carbon metabolism, such as glycerol, pyruvate, and succinate, were similar under all nutrient limitation regimens (Table 3). However, the intracellular levels of glycogen and trehalose were very different for the five nutrient-limitation regimens (Fig. 2A). When both excess glucose and excess ammonia were present (sulfate-, phosphate-, and zinc-limited cultures), the cellular contents of both glycogen and trehalose were less than 3 mg glucose equivalents g (dry weight) biomass–1. In glucose- and ammonia-limited cultures, the glycogen levels were 10- and 14-fold higher, respectively, than the levels under the three glucose- and ammonia-excess conditions. The trehalose levels were specifically higher in ammonia-limited cultures (at least fourfold higher than the levels for the other conditions) (Fig. 2A).


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  		TABLE 3. Physiological characteristics of the CEN.PK113-7D strain grown under anaerobic chemostat fermentation conditions at a dilution rate of 0.10 h–1 and under five different nutrient limitation conditions (carbon, nitrogen, sulfur, phosphorus, zinc)a


Figure 2
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  		FIG. 2. Glycogen and trehalose contents in anaerobic chemostat cultures of CEN.PK113-7D (A) and IMZ066 (msn2{Delta} msn4{Delta}) (B) limited by carbon, nitrogen, sulfur, phosphorus, or zinc at a dilution rate of 0.10 h–1. Glycogen (G) and trehalose (T) contents are expressed in mgglucose equivalent·g (dry weight)–1. Measurement was performed in triplicate, and measurements were obtained from three independent chemostat cultures.

Transcriptional responses of glycogen- and trehalose-related genes to different nutrient limitation regimens.
Statistical analysis of the genome-wide transcriptional responses of S. cerevisiae CEN.PK113-7D to the five different nutrient limitation regimens revealed that 945 genes were differentially expressed under at least one of the five nutrient limitation conditions (see Table S2 in the supplemental material). Analysis of the transcript levels of the subsets of genes encoding the enzymes of the glycogen and trehalose pathways revealed a clear correlation with cellular glycogen contents (compare Fig. 2 and Fig. 3). In cultures grown with excess glucose and ammonia (i.e., cultures in which growth was limited by sulfate, phosphate, or zinc), almost all genes of the glycogen biosynthesis pathway were expressed at a significantly lower level than they were in the glucose- and ammonia-limited cultures (Fig. 3). For example, the level of expression of GAC1, which encodes the regulatory subunit of Glc7p, the activator of glycogen synthase (87), was significantly higher under carbon and nitrogen limitation conditions than under the other three conditions.


Figure 3
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  		FIG. 3. Heat map of the transcript levels of genes encoding members of the glycogen and trehalose pathways in CEN.PK113-7D grown in anaerobic chemostat cultures with carbon, nitrogen, sulfur, phosphorus, or zinc limitation at a dilution rate of 0.10 h–1. The transcript levels of the IMZ066 (msn2{Delta} msn4{Delta}) mutant grown under carbon and nitrogen limitation conditions are also indicated. The transcript levels were determined with YG-98 Affymettrix GeneChips and are the averages of three independent experiments. The numbers indicate P values obtained with a two-tailed, unequal-variance Student's t test in which the carbon-limited conditions were used as the reference.

The transcript levels of all but one (ATH1) of the trehalose metabolism genes were specifically and significantly (P < 0.05) higher in the nitrogen-limited cultures, which matched the associated phenotype. The transcript levels of TPS1, TPS 2, and TPS 3 (encoding the trehalose-6-phosphate synthase and phosphatase [3, 4, 86]), TSL1 (encoding the regulatory component of the trehalose-6-phosphate synthase [83]), and NTH2 (encoding a putative neutral trehalase [53]) were up to twofold higher under nitrogen limitation conditions than under the other conditions. Except for PGM2 (encoding phosphoglucomutase), GLC3 (encoding the glycogen branching enzyme) (76), GPH1 (encoding glycogen phosphorylase), and UGP1 (encoding UDP-glucose pyrophosphorylase [12]), whose transcript levels were higher under nitrogen limitation conditions, the genes of the glycogen biosynthesis pathway were not differentially expressed under the carbon and nitrogen limitation conditions. This indicated that mechanisms other than transcriptional regulation are involved in the increased glycogen accumulation by nitrogen-limited cultures.

Role of Msn2p/Msn4p in transcriptional induction of storage carbohydrate metabolism under glucose and ammonia limitation conditions.
The coordinated transcriptional induction of genes involved in storage carbohydrate metabolism in response to both carbon- and nitrogen-limited growth conditions suggested involvement of a common transcriptional activator. The transcriptional induction of storage carbohydrate biosynthesis upon nutrient depletion in batch cultures is dependent on Msn2p/Msn4p (56). Several studies have supported the notion that transcription of the STRE regulon, which is controlled by Msn2p/Msn4p, is predominantly and negatively correlated with the specific growth rate (10, 62). We therefore reinvestigated the role of Msn2p/Msn4p in storage carbohydrate metabolism by growing an msn2{Delta} msn4{Delta} mutant (IMZ066) in anaerobic glucose- and ammonia-limited chemostat cultures at a fixed specific growth rate of 0.10 h–1.

Although the biomass yield of IMZ066 (msn2{Delta} msn4{Delta} mutant) was the same as that of the isogenic reference strain CEN.PK113-7D, the trehalose content of IMZ066 in nitrogen-limited cultures was 50% lower (Fig. 2B). Surprisingly, under carbon-limited conditions, the trehalose content of IMZ066 was higher than that of the isogenic reference strain. The glycogen content was reduced by 50% in IMZ066 (msn2{Delta} msn4{Delta} strain) under both glucose- and ammonia-limited conditions (Fig. 2B). These results indicate that there is specific growth rate-independent involvement of Msn2p/Msn4p in the regulation of storage carbohydrate metabolism.

To assess the impact of Msn2p and Msn4p on transcriptional regulation, the transcriptomes of IMZ066 (msn2{Delta} msn4{Delta} strain) and the reference strain CENPK113-7D grown in glucose- and ammonia-limited chemostat cultures were compared. In the glucose-limited cultures, 55 genes were upregulated and 53 genes were downregulated in IMZ066 (msn2{Delta} msn4{Delta} strain), while under nitrogen-limited conditions, 56 genes were upregulated and 145 genes were downregulated (see Table S3 in the supplemental material). When the responses for the two nutrient limitation regimens were overlaid, the transcript levels for three genes were consistently higher and the transcript levels for 26 genes were consistently lower in the mutant strain. The large majority of the latter 26 genes could be grouped in functional categories related to the Msn2p/Msn4p regulon (Fig. 4), and the promoters of 24 of these genes contained the STRE. Eight of these STRE genes were involved in glycogen and trehalose metabolism, and the remaining genes had a role in sugar metabolism or oxidative stress or had an unknown function (Fig. 4).


Figure 4
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  		FIG. 4. Global transcriptome responses to MSN2 and MSN4 deletion in anaerobic carbon- and nitrogen-limited chemostats. (A) Venn diagrams showing the number of genes up- or downregulated in IMZ066 (msn2{Delta} msn4{Delta}) compared to the isogenic reference strain under both carbon and nitrogen limitation conditions. Of the 26 genes that were downregulated irrespective of the nutrient limitation, the 24 underlined genes were found to contain the STRE and are thus considered part of the Msn2p/Msn4p regulon. (B) Comparison of the genes affected by deletion of MSN2 and MSN4 during carbon- and nitrogen-limited growth (this study) with the environmental stress response (ESR) genes (23) and growth rate-responsive genes (10, 62).

Qualitatively consistent with the intracellular glycogen and trehalose measurements for IMZ066 (msn2{Delta} msn4{Delta} strain), the genes of the glycogen and trehalose pathways were downregulated at the transcriptional level. However, the decreases in the transcript levels were much more pronounced (up to 20-fold, resulting in levels below those observed for the reference strain under glucose- and ammonia-excess conditions [Fig. 3]) than the decreases in the levels of glycogen and trehalose, which decreased by only ca. 50%. This provided clear evidence that posttranscriptional regulation mechanisms make an important contribution to the regulation of storage carbohydrate metabolism under these conditions.

Posttranscriptional regulation of glycogen synthase under carbon and nitrogen limitation conditions.
The two main enzymes of the glycogen biosynthesis and degradation pathways, glycogen synthase and glycogen phosphorylase (22), are subject to strong posttranslational control by phosphorylation and dephosphorylation. This regulation is reciprocal for these two enzymes; phosphorylation yields an inactive glycogen synthase but an active glycogen phosphorylase (18, 21, 30, 32-34, 58, 64). The inactive, phosphorylated glycogen synthase can be activated in vitro by inclusion of glucose-6-phosphate in the assay mixture. Hence, measuring the glycogen synthase activity in the absence and presence of glucose-6-phosphate allows reliable estimation of the proportion of active enzyme relative to the total amount (21, 30, 33).

To assess whether the glycogen synthase and glycogen phosphorylase enzymes were subject to posttranscriptional and/or posttranslational regulation in response to nutrient availability, in vitro activity assays were performed with cell extracts from glucose-limited, ammonia-limited, and glucose- and ammonia-excess cultures (sulfate-limited cultures). While glycogen synthase activity was clearly present in extracts from the sulfate-limited cultures, no active form of glycogen synthase was detected (Fig. 5). The high activity of glycogen phosphorylase under these conditions (11.5 mU·mg–1) was consistent with low net glycogen accumulation. In cell extracts of the glucose-limited chemostat cultures, 20% of the total glycogen synthase was found to be in the active form (Fig. 5), thus explaining the higher rate of glycogen synthesis and the net accumulation of glycogen in these cultures. While the fraction of active glycogen synthase was also 20% in the nitrogen-limited cultures, its total activity was threefold higher than that in the glucose-limited cultures (Fig. 5). In addition, the lower glycogen phosphorylase activity in the nitrogen-limited cultures suggested that the rate of glycogen degradation was lower (Fig. 5). The ratio of active glycogen synthase to active glycogen phosphorylase was eightfold higher in the nitrogen-limited cultures than in the glucose-limited cultures, thus enabling an increased flux toward glycogen biosynthesis.


Figure 5
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  		FIG. 5. Glycogen synthase and glycogen phosphorylase activities in cell extracts from anaerobic chemostat cultures of CEN.PK113-7D limited by carbon, nitrogen, or sulfur. One unit was defined as the amount of enzyme that catalyzed the conversion of 1 µmol substrate in 1 min under the conditions of the assay. The percentage of active glycogen synthase relative to the total amount under each nutrient limitation condition is also indicated. TGS, total glycogen synthase; AGS, active glycogen synthase; AGP, active glycogen phosphorylase. For each culture condition measurements were obtained in triplicate from three independent chemostat cultures.


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DISCUSSION
 
Impact of growth-limiting nutrient on storage carbohydrate levels.
The use of nutrient-limited chemostat cultures enabled us to investigate the impact of the identity of the growth-limiting nutrient on the accumulation of storage carbohydrates by S. cerevisiae at a fixed specific growth rate and, for the non-glucose-limited growth conditions, at a fixed residual glucose concentration in the cultures. This prevented indirect effects on storage carbohydrate metabolism due to the specific growth rate or to glucose signaling. The results show that, in contrast to previous hypotheses (41, 56), storage carbohydrate accumulation by S. cerevisiae is not a general response to nutrient limitation or to glucose-excess scenarios.

Of the five nutrient limitation regimens investigated in this study, only glucose limitation and ammonia limitation led to increased levels of storage carbohydrates. When both carbon and nitrogen sources were present in excess, the storage carbohydrate levels remained low. These observations enabled more precise interpretation of the impact of nutrient limitation on storage carbohydrates. For example, the high levels of accumulated glycogen and trehalose observed upon sulfate or phosphate starvation described previously (41) were likely to have been caused primarily by a decreased specific growth rate rather than by the identity of the depleted nutrient. Furthermore, the low glycogen and trehalose contents observed in a previous study of zinc-limited cultures of S. cerevisiae (15) cannot be interpreted as a specific response to zinc limitation.

Trehalose accumulation and glycogen accumulation showed subtly different responses to nutrient limitation; trehalose accumulation occurred specifically during ammonia-limited growth, while glycogen accumulation was greatest under ammonia-limited conditions but was also substantial in glucose-limited cultures. In future research, the cultivation conditions studied here can be expanded further to define the impact of the growth-limiting nutrient on storage carbohydrate limitation. For example, it would be very interesting to see to what extent the identity of the nitrogen source (e.g., amino acids instead of ammonia) and aeration affect storage carbohydrate metabolism.

The correlations between storage carbohydrate metabolism and the nutrient limitation regimen suggest that design of media and feed regimens may be used successfully to control storage carbohydrate metabolism. In general, for fermentation processes in which high storage carbohydrate levels are desirable (e.g., repitching of brewer's yeast [7, 46, 59]) workers should avoid growth limitation by nutrients other than the sugar or nitrogen source. Conversely, when a low storage carbohydrate level is desirable, such as during the production of yeast extract (82), use of phosphate or sulfate limitation in the final phases of a fed-batch process may be beneficial.

Transcriptional regulation: involvement of Msn2p and Msn4p.
Transcriptome analysis showed that in anaerobic glucose- and ammonia-limited chemostat cultures, Msn2p and Msn4p were involved in the increased levels of storage carbohydrates observed under these conditions. However, of the 266 STRE-containing genes that show increased transcript levels upon environmental stress or nutrient starvation ("environmental stress response" genes [23]) in batch cultures, only 16 were found to be transcriptionally downregulated upon deletion of MSN2 and MSN4 in the glucose- and ammonia-limited conditions. These 16 environmental stress response genes showed strong overrepresentation of genes involved in storage carbohydrate metabolism (Fig. 4B). This observation strongly suggests that the impact of Msn2p and Msn4p on transcriptional regulation is context dependent. In the absence of strong environmental stress and at a fixed specific growth rate, the impact of Msn2p and Msn4p on transcriptional regulation appears to be focused on genes involved in storage carbohydrate metabolism.

The activity of Msn2p and Msn4p is controlled by their Bmh1/Bmh2-mediated cytosolic anchoring in response to the PKA (24, 25, 69) and target of rapamycin (TOR) (2, 45) signaling pathways. Release from this PKA or TOR signaling leads to translocation of Msn2p and Msn4p to the nucleus and transcription of their target genes (2, 69). Indeed, treatment of S. cerevisiae with rapamycin results in transcriptional induction of glycogen and trehalose biosynthesis genes and accumulation of both carbohydrates (1, 67, 75).

The PKA pathway is activated by high glucose concentrations (72), whereas the TOR pathway is activated by a range of nitrogen and carbon sources (1, 52, 74). This is consistent with our observation that the transcript levels of glycogen- and trehalose-related genes were low in the presence of excess glucose and ammonia (i.e., in sulfate-, phosphate-, and zinc-limited cultures), since the PKA and TOR signaling pathways are expected to be active under these conditions and Msn2p and Msn4p should thus be retained in the cytosol.

The PKA and TOR pathways control common targets via parallel routes (66, 89), but their interactions remain largely unknown (63). Understanding these interactions is important for understanding the transcriptional induction of storage carbohydrate metabolism under glucose- and ammonia-limited conditions. Moreover, other key regulators may be involved in nitrogen regulation of storage carbohydrate metabolism. Ten glycogen and trehalose biosynthesis genes have been identified as targets of Gcn4p, the transcriptional activator of the general amino acid control response (50). A Gcn4p-mediated response to amino acid starvation has been proposed to lead to a net breakdown of glycogen (31). In addition, the glycogen and trehalose levels in a leu3{Delta} mutant (Leu3p is a transcriptional activator of the branched-chain amino acid biosynthesis pathway), grown in an aerobic nitrogen-limited chemostat culture, were fivefold lower than those in the isogenic reference strain (5). This decrease in storage carbohydrate accumulation was accompanied by transcriptional downregulation of the glycogen and trehalose genes and of IRA1 and IRA2 (GTPase-activating proteins for Ras1p and Ras2p [48, 71]), which indicated that hyperactivity of PKA might have been the cause of repression of glycogen and trehalose synthesis.

Posttranscriptional regulation.
In vitro studies with cell extracts of cultures grown under glucose, ammonia, and sulfate limitation conditions, as well as cell extracts of cultures of IMZ066 (msn2{Delta} msn4{Delta} strain), demonstrated that posttranscriptional regulation plays a major role in adapting storage carbohydrate metabolism to the different nutrient limitation regimens. This posttranscriptional regulation could be partially attributed to previously described mechanisms. First, apart from its role in transcriptional downregulation of glycogen and trehalose biosynthesis, active PKA deactivates glycogen synthase and activates glycogen phosphorylase by phosphorylation (73). Consistent with our observations, high PKA activities in the sulfate-limited cultures should result in the absence of active glycogen synthase. Second, glucose-limited cultivation conditions lead to activation of the Snf1p kinase, which activates glycogen synthase, possibly via the Gac1p-Glc7p phosphatase complex (29, 30). The cyclin-dependent kinase Pho85p, in association with the cyclins Pcl8p and Pcl10p, has been implicated in signaling cascades leading to deactivation of glycogen synthase and is negatively regulated by Snf1p (85). Hence, under glucose-limited conditions, activation of Snf1p (and thereby deactivation of Pho85p) would result in the presence of active glycogen synthase.

The two mechanisms mentioned above cannot contribute to posttranscriptional upregulation of storage carbohydrate metabolism in the ammonia-limited, glucose-excess cultures. Cross talk between the TOR and PKA signaling pathways, affecting the phosphorylation state of the glycogen synthase and phosphorylase, offers an attractive hypothesis. Activation of PKA by TOR has previously been shown for transcriptional induction of ribosomal protein genes via Yak1p (44). Furthermore, it should be realized that other phosphorylases and kinases (including Psk2p [65], Sch9p, and Pho85p) may be involved and that not all kinases and phosphatases regulating the activity of the glycogen phosphorylase have been identified (22). The threefold-higher total (active and inactive) levels of glycogen synthase in the ammonia-limited cultures than in the glucose-limited cultures were not matched by a similar difference in transcript levels. This suggests that, additionally, storage carbohydrate metabolism may be controlled at the level of translation and/or protein turnover. Storage carbohydrate accumulation by S. cerevisiae cultures is the net result of an intricate network of multilevel, multi-input combinatorial regulation mechanisms which is highly relevant for many industrial applications. Chemostat fermentation offers an attractive platform for systematic dissection of this regulation network.


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ACKNOWLEDGMENTS
 
We thank Erwin Suir for his technical assistance.

The research group of J.T.P. is part of the Kluyver Centre for Genomics of Industrial Fermentation, which is supported by The Netherlands Genomics Initiative.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31 15 278 24 10. Fax: 31 15 278 23 55. E-mail: j.g.daran{at}tudelft.nl Back

{triangledown} Published ahead of print on 4 September 2009. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back

{ddagger} The authors have paid a fee to allow immediate free access to this article. Back


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Applied and Environmental Microbiology, November 2009, p. 6876-6885, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01464-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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