APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2009, p. 6876–6885 Vol. 75, No. 210099-2240/09/$12.00 doi:10.1128/AEM.01464-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identity of the Growth-Limiting Nutrient Strongly Affects StorageCarbohydrate Accumulation in Anaerobic Chemostat Cultures

of Saccharomyces cerevisiae�†‡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 Netherlands1; Kluyver Centre for

Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands2; Heineken Supply Chain, Research andInnovation, Burgemeester Smeetsweg 1, 2380 BB Zoeterwoude, The Netherlands3; and Netherlands Consortium for

Systems Biology, Bureau Science Park 123, 1098 XG Amsterdam, The Netherlands4

Received 22 June 2009/Accepted 20 August 2009

Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negativelycorrelated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation bynutrients 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 storagecarbohydrates 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, orzinc) 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 underthe other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limitedconditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures ofS. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of thegrowth-limiting nutrient. While transcriptome analysis of wild-type and msn2� msn4� strains confirmed thattranscriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p,transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrateaccumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supportedinvolvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limitedcultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higherthan the ratio in the other glucose-excess cultures.

Glycogen and trehalose are the major storage carbohydratesin baker’s yeast (Saccharomyces cerevisiae) (22). The ability toaccumulate these storage carbohydrates is an important crite-rion for selection of industrial yeast strains because of theirinvolvement in the cellular robustness and quality of yeastextracts (e.g., for food and feed applications) (8, 16, 61).

Based on the observation that glycogen and trehaloseaccumulate in batch cultures of S. cerevisiae that are starvedfor carbon, nitrogen, sulfur, or phosphorus, Lillie and Prin-gle (41) proposed that storage carbohydrate synthesis is ageneral response to nutrient starvation. Parrou et al. (56),who studied carbohydrate metabolism during nutrient-lim-ited growth rather than during nutrient starvation, found thataccumulation of glycogen and trehalose occurred when growthwas limited by the carbon or nitrogen source. Because theseworkers were unable to obtain sulfur- or phosphorus-limited

growth with their experimental setup (56), it remained unclearwhether accumulation of glycogen and trehalose is a commonresponse to nutrient limitation or whether, instead, nutrientlimitations other than nitrogen or carbon limitation may resultin different storage carbohydrate contents.

Research on the molecular mechanisms involved in the reg-ulation of storage carbohydrates has focused mostly on thediauxic 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 acti-vation of the glycogen and trehalose pathways (Fig. 1) is me-diated by the Msn2p/Msn4p complex (51, 55, 69, 86, 88). Tran-scriptional regulation of GSY2 (encoding glycogen synthase)during the diauxic shift involves integration of signaling path-ways involving the protein kinases Pho85p, Snf1p, and proteinkinase A (PKA) (17). Glycogen synthase and glycogen phos-phorylase, two central enzymes in glycogen biosynthesis anddegradation, are subject to strong posttranslational regulationby phosphorylation-dephosphorylation through complex ki-nase and phosphatase cascades (18, 21, 30, 32–34, 58, 64).

Besides transcriptional regulation mediated by the Msn2p/Msn4p complex, TPS1 (trehalose-6-phosphate synthase), a keygene of trehalose metabolism, has been shown to be core-pressed by Mig1p and Mig2p (43). Further analysis of promotersequences of TPS1 and NTH1 (neutral trehalase) showed the

* Corresponding author. Mailing address: Department of Biotech-nology, 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@tudelft.nl.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 4 September 2009.‡ The authors have paid a fee to allow immediate free access to

this article.

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presence of several other regulatory sequences for Gcn4p (ba-sic leucine zipper transcriptional activator of amino acid bio-synthetic genes), Gcr1 (transcriptional activator of genes in-volved in glycolysis), and Adr1p (carbon source-responsivezinc finger transcription factor) (14). Regulation of trehaloseaccumulation occurs posttranslationally as well. Similar to en-zymes involved in glycogen metabolism, enzymes involved intrehalose metabolism are subject to strong posttranslationalregulation 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 byYlr20wp/Dcs1p by direct protein interaction (14).

Batch cultures have some inherent drawbacks for studyingthe impact of nutrient limitation on storage carbohydrate me-tabolism. First, nutrient limitation in batch cultures is a tran-sient phenomenon that occurs only during the transition fromnutrient-excess conditions to nutrient-depleted conditions.Moreover, nutrient limitation in batch fermentations affectsthe specific growth rate, which in turn affects accumulation ofstorage carbohydrates (26, 39, 68). The specific growth ratealso has a profound impact on transcriptional responses (10,62). The stress response element (STRE) genes (stress re-sponse regulon controlled by Msn2p/Msn4p) are particularlysensitive to the specific growth rate, and their expression isnegatively correlated with this rate. Clearly, dissection of theeffects of the specific growth rate from the effects of nutrientlimitation is essential for interpreting and understanding theregulation of storage carbohydrate metabolism by nutrientavailability and for interpreting Msn2p/Msn4p-mediated in-duction of STRE genes.

Chemostat cultivation offers the unique possibility of grow-ing microorganisms at a constant specific growth rate underdifferent nutrient limitation regimens. In S. cerevisiae, this ap-proach has been used to study transcriptional responses to

various nutrient limitation regimens (e.g., limitation by differ-ent carbon and nitrogen sources, sulfate, phosphate, and zinc)(for a review, see reference 13). Chemostat cultivation allowsdissection of the effects of nutrient limitation on yeast physi-ology from the effects of a fixed specific growth rate. In a recentstudy (15), we observed accumulation of very low levels ofstorage carbohydrates and transcriptional downregulation ofthe glycogen pathway in zinc-limited chemostat cultures com-pared to glucose- and ammonia-limited cultures grown at thesame specific growth rate. These unexpected observations stressedthe need for a broader investigation of the effect of nutrientlimitation regimens on storage carbohydrate accumulation.

The goal of the present study was to systematically investi-gate the impact of the identity of the growth-limiting nutrienton storage carbohydrate metabolism in S. cerevisiae. To thisend, accumulation of glycogen and trehalose was studied usinganaerobic glucose chemostat cultures at a fixed dilution rate of0.10 h�1 under five different nutrient-limitation regimens (glu-cose, ammonia, sulfate, phosphate, and zinc). In addition, westudied regulation of storage carbohydrate metabolism at thetranscriptional and posttranscriptional levels and assessed therole of Msn2p/Msn4p in mediating transcriptional induction ofglycogen and trehalose genes.

MATERIALS AND METHODS

Yeast strain and maintenance. The strains used in this study are listed in Table1. The haploid prototrophic S. cerevisiae strain CEN.PK 113-7D (MATa) wasobtained from P. Kotter, Frankfurt, Germany. Deletion mutants were con-structed by using standard yeast media and genetic techniques and were routinelygrown at 30°C on complex (YPD) or defined media (9). Gene deletions wereintroduced into strain CEN.PK113-5D (provided by P. Kotter, Frankfurt, Ger-many), which is auxotrophic for uracil. The geneticin (G418) resistance cassettewas amplified by using the pUG6 vector as the template, and the KanMX (G418)resistance expression cassette was removed using the Cre-loxP recombinationsystem as previously described (27). For chemostat cultivation, strain IMK151

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

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(MATa MAL2-8c SUC2 ura3 msn2::loxP msn4::loxP-kanMX-loxP) was trans-formed with plasmid p426GPD carrying the URA3 gene to complement its uracilauxotrophy (49); this yielded strain IMZ066. The oligonucleotide primers used inthis study are listed in Table S1 in the supplemental material.

Media for chemostat cultivation. The synthetic medium used was based on themedium described by Verduyn et al. (81). The modifications introduced forammonium-, sulfate-, phosphate-, and zinc-limited growth have been describedpreviously (15, 70, 81) and are listed in Table 2. In all chemostats except thosein which growth was limited by glucose, the target residual glucose concentrationwas 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). Thereservoir medium was supplemented with vitamins and the anaerobic growthfactors Tween 80 and ergosterol as previously described (81).

Chemostat cultivation. Chemostat cultures were fed using synthetic mediumthat resulted in limitation of growth by glucose, ammonia, sulfate, phosphate, orzinc and that contained all other components required for growth in excess andat constant residual concentrations. The dilution rate was set at 0.10 h�1, and thepH was measured online and kept constant at 5.0 by automatic addition of 2 MKOH as previously described (70). Cultures were assumed to be in steady statewhen, after at least 5 volume changes, the culture dry weight, glucose concen-tration, and carbon dioxide production rate varied by less than 2% during oneadditional volume change (19). Steady-state samples were taken after 10 gener-ations 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 centrifugationof samples from the chemostats. For glucose determination and carbon recovery,culture supernatants and media were analyzed by high-performance liquid chro-matography using an AMINEX HPX-87H ion-exchange column and 5 mMH2SO4 as the mobile phase. Culture dry weights were determined by filtration asdescribed by Postma et al. (60). Trehalose measurement and glycogen measure-ment were adapted as described by Parrou and Francois (57), and values wereobtained in triplicate. Glucose contents were determined using the UV methodand a Roche kit (no. 0716251; Roche Applied Science, Almere, The Nether-lands).

In vitro enzyme assays. To minimize the loss of phosphate groups on proteins,cell sampling of chemostat cultures for measurement of glycogen synthase andglycogen phosphorylase activities was performed as described by Francois andHers (21). Fresh cell extracts were prepared using the fast prep method (11),

except that the extraction buffer contained the phosphatase inhibitor cocktailPhosSTOP (Roche Applied Science, Almere, The Netherlands), which was usedaccording to the manufacturer’s recommendations. Glycogen synthase activitywas 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 PerkinElmer, Groningen, The Netherlands), 10 mM Na2SO4, 2.5 mM EDTA, and 3%glycogen from rabbit liver (Sigma Aldrich, Zwijndrecht, The Netherlands) asdescribed by Francois and Hers (21). When glucose-6-phosphate was added tothe assay mixture, it was added at a concentration of 10 mM. Glycogen phos-phorylase activity was determined in the direction of glycogen synthesis bymeasuring the incorporation of glucose-1-phosphate into glycogen. The reactionmixture contained 5 mM [U-14C]glucose-1-phosphate (300 cpm/nmol; Amer-sham Perkin Elmer, Groningen, The Netherlands), 2.5 mM EDTA, 2.5% glyco-gen, and 50 mM sodium succinate (pH 5.8) (21). Both enzymes were measuredin duplicate using three independent chemostat cultures. One unit was defined asthe amount of enzyme that catalyzed the conversion of 1 �mol of substrate in 1min under the conditions of the assay. The concentration of protein was deter-mined 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 fromchemostats and extraction of total RNA were performed as previously described(15). The results for each growth condition were derived using three independentculture replicates. Acquisition and quantification of array images and data fil-tering were performed using Affymetrix GeneChip operating software, version1.2. To eliminate insignificant variations, the expression value for genes withexpression values less than 12 was defined as 12 as previously described (6).

The Significance Analysis of Microarrays (SAM) (version 1.12) (77) add-in toMicrosoft Excel was used for comparison of replicate array experiments. Fortranscriptome analysis of CEN.PK113-7D under the five different nutrient lim-itation conditions, the statistical significance of observed differences was assessedby 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� msn4�)transcriptome data to data for the isogenic reference strain CEN.PK113-7D,pairwise comparisons (SAM Microsoft excel add-in) using a threshold differenceof twofold and a false discovery rate of 1% were performed. Venn diagrams andheat map visualizations of transcript data were generated with ExpressionistAnalyst, version 3.2. Transcript data have been deposited in the Genome Ex-pression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accessionnumber GSE15465.

Hypergeometric tests and motif discovery. Groups of coresponsive genes wereconsulted 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 Se-quence Analysis Tools (79, 80). The promoters (from position �800 to position�1) of each set of coregulated genes were analyzed for overrepresented hexa-nucleotides.

RESULTS

Storage carbohydrate accumulation is not a general re-sponse to nutrient limitation. In aerobic, glucose-grown che-mostat cultures of S. cerevisiae in which glucose is not thegrowth-limiting nutrient (e.g., sulfate- or phosphate-limitedcultures), glucose dissimilation is respirofermentative. As aconsequence, such glucose-excess cultures have significantly

TABLE 1. Strains used in this study

Strain Genotype Source orreference

CEN.PK113-7D MATa MAL2-8c SUC2 P. Kottera

CEN.PK113-5D MATa MAL2-8c SUC2 ura3 P. Kottera

IMK140 MATa MAL2-8c SUC2 ura3msn2::loxP-kanMX-loxP

This study

IMK149 MATa MAL2-8c SUC2 ura3 msn2::loxP This studyIMK151 MATa MAL2-8c SUC2 ura3 msn2::loxP

msn4::loxP-kanMX-loxPThis study

IMZ066 MATa MAL2-8c SUC2 ura3 msn2::loxPmsn4::loxP-kanMX-loxP p426GPD(URA3)

This study

a Institut fur Mikrobiologie des J.W. Goethe Universiteit.

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

Limiting nutrientin medium

Concn (g � liter�1) ofa:

Glucose (NH4)2SO4 NH4Cl K2SO4 KH2PO4 MgSO4 � 7H2O MgCl2 ZnSO4 � 7H2O Reference

Carbon 25 5 3 0.5 0.0045 70Nitrogen 46 1 5.3 3 0.5 0.0045 70Sulfur 59 4 3 0.05 0.4 0.0045 70Phosphorus 66 5 1.9 0.12 0.5 0.0045 70Zinc 58 5 3 0.5 0 15

a Bold type indicates the modifications made to the synthetic media described by Di Nicola et al. (15), Tai et al. (70), and Verduyn et al. (81) in order to obtain therelevant nutrient limitation conditions.

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lower biomass yields and higher glucose uptake rates thanglucose-limited cultures (6, 15, 70). Glycolytic flux has previ-ously been shown to be negatively correlated with storagecarbohydrate accumulation (26, 40). Under anaerobic condi-tions, glucose is dissimilated via alcoholic fermentation irre-spective of the nutrient limitation. To minimize possible indirecteffects of glycolytic flux on storage carbohydrate metabolism, thepresent study was performed with anaerobic chemostat cul-tures.

An approximately 20% lower biomass yield and a corre-sponding higher glucose consumption rate (P � 1.0E�05 forboth measurements, as determined by a t test) were found inthe four glucose-excess scenarios than in the anaerobic glu-cose-limited cultures, indicating that there was partial uncou-pling of glucose dissimilation and biomass formation underglucose-excess conditions (Table 3). These modest differencesare unlikely to contribute to a substantial influence of glyco-lytic flux on storage carbohydrate accumulation. The produc-tion rates of minor metabolites derived from central carbonmetabolism, such as glycerol, pyruvate, and succinate, weresimilar under all nutrient limitation regimens (Table 3). How-ever, the intracellular levels of glycogen and trehalose werevery different for the five nutrient-limitation regimens (Fig.2A). When both excess glucose and excess ammonia werepresent (sulfate-, phosphate-, and zinc-limited cultures), the

cellular contents of both glycogen and trehalose were less than3 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 threeglucose- and ammonia-excess conditions. The trehalose levelswere specifically higher in ammonia-limited cultures (at leastfourfold higher than the levels for the other conditions)(Fig. 2A).

Transcriptional responses of glycogen- and trehalose-re-lated genes to different nutrient limitation regimens. Statisticalanalysis of the genome-wide transcriptional responses of S.cerevisiae CEN.PK113-7D to the five different nutrient limita-tion regimens revealed that 945 genes were differentially ex-pressed under at least one of the five nutrient limitation con-ditions (see Table S2 in the supplemental material). Analysisof the transcript levels of the subsets of genes encoding theenzymes of the glycogen and trehalose pathways revealed aclear correlation with cellular glycogen contents (compare Fig.2 and Fig. 3). In cultures grown with excess glucose and am-monia (i.e., cultures in which growth was limited by sulfate,phosphate, or zinc), almost all genes of the glycogen biosyn-thesis pathway were expressed at a significantly lower level thanthey were in the glucose- and ammonia-limited cultures (Fig. 3).For example, the level of expression of GAC1, which encodes theregulatory subunit of Glc7p, the activator of glycogen synthase

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

Limiting nutrient Residual glucoseconcn (g � liter�1)

Biomass yield on glucose(g of biomass �dry wt�/gof glucose consumed)

Biomass-specific rate (mmol � g �dry wt��1 � h�1)

Glucoseconsumption

Glycerolproduction

Pyruvateproduction

Succinateproduction

Carbon BDLb 0.09 � 0.0 6.0 � 0.0 0.81 � 0.06 0.0 � 0.00 0.03 � 0.01Nitrogen 17.9 � 0.3 0.07 � 0.0 8.2 � 0.2 0.85 � 0.07 0.03 � 0.00 0.04 � 0.01Sulfur 16.3 � 1.9 0.07 � 0.1 8.4 � 1.3 0.97 � 0.02 0.04 � 0.01 0.03 � 0.00Phosphorus 19.1 � 2.2 0.06 � 0.0 8.7 � 0.2 0.97 � 0.01 0.02 � 0.00 0.00 � 0.00Zinc 19.9 � 0.6 0.07 � 0.0 8.4 � 0.1 1.07 � 0.00 0.03 � 0.01 0.04 � 0.01

a The data are the averages and standard deviations of data from at least three independent steady-state chemostat cultivations.b BDL, below the detection limit, which was 0.1 mM.

FIG. 2. Glycogen and trehalose contents in anaerobic chemostat cultures of CEN.PK113-7D (A) and IMZ066 (msn2� msn4�) (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 (dryweight)�1. Measurement was performed in triplicate, and measurements were obtained from three independent chemostat cultures.

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(87), was significantly higher under carbon and nitrogen limita-tion conditions than under the other three conditions.

The transcript levels of all but one (ATH1) of the trehalosemetabolism genes were specifically and significantly (P � 0.05)higher in the nitrogen-limited cultures, which matched theassociated phenotype. The transcript levels of TPS1, TPS 2,and TPS 3 (encoding the trehalose-6-phosphate synthase andphosphatase [3, 4, 86]), TSL1 (encoding the regulatory com-ponent of the trehalose-6-phosphate synthase [83]), and NTH2(encoding a putative neutral trehalase [53]) were up to twofoldhigher under nitrogen limitation conditions than under theother conditions. Except for PGM2 (encoding phosphoglu-comutase), GLC3 (encoding the glycogen branching enzyme)(76), GPH1 (encoding glycogen phosphorylase), and UGP1(encoding UDP-glucose pyrophosphorylase [12]), whose tran-script levels were higher under nitrogen limitation conditions,the genes of the glycogen biosynthesis pathway were not dif-ferentially expressed under the carbon and nitrogen limitationconditions. This indicated that mechanisms other than tran-scriptional regulation are involved in the increased glycogenaccumulation by nitrogen-limited cultures.

Role of Msn2p/Msn4p in transcriptional induction of stor-age carbohydrate metabolism under glucose and ammonialimitation conditions. The coordinated transcriptional induc-tion of genes involved in storage carbohydrate metabolism inresponse to both carbon- and nitrogen-limited growth condi-tions suggested involvement of a common transcriptional ac-

tivator. The transcriptional induction of storage carbohydratebiosynthesis upon nutrient depletion in batch cultures is de-pendent on Msn2p/Msn4p (56). Several studies have supportedthe notion that transcription of the STRE regulon, which iscontrolled by Msn2p/Msn4p, is predominantly and negativelycorrelated with the specific growth rate (10, 62). We thereforereinvestigated the role of Msn2p/Msn4p in storage carbohy-drate metabolism by growing an msn2� msn4� mutant(IMZ066) in anaerobic glucose- and ammonia-limited chemo-stat cultures at a fixed specific growth rate of 0.10 h�1.

Although the biomass yield of IMZ066 (msn2� msn4� mu-tant) was the same as that of the isogenic reference strainCEN.PK113-7D, the trehalose content of IMZ066 in nitrogen-limited cultures was 50% lower (Fig. 2B). Surprisingly, undercarbon-limited conditions, the trehalose content of IMZ066was higher than that of the isogenic reference strain. The glycogencontent was reduced by 50% in IMZ066 (msn2� msn4� 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 ofstorage carbohydrate metabolism.

To assess the impact of Msn2p and Msn4p on transcriptionalregulation, the transcriptomes of IMZ066 (msn2� msn4�strain) and the reference strain CENPK113-7D grown in glu-cose- and ammonia-limited chemostat cultures were com-pared. In the glucose-limited cultures, 55 genes were upregu-lated and 53 genes were downregulated in IMZ066 (msn2�

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

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msn4� strain), while under nitrogen-limited conditions, 56genes were upregulated and 145 genes were downregulated(see Table S3 in the supplemental material). When the re-sponses for the two nutrient limitation regimens were overlaid,the transcript levels for three genes were consistently higherand the transcript levels for 26 genes were consistently lower inthe mutant strain. The large majority of the latter 26 genescould be grouped in functional categories related to theMsn2p/Msn4p regulon (Fig. 4), and the promoters of 24 of

these genes contained the STRE. Eight of these STRE geneswere involved in glycogen and trehalose metabolism, and theremaining genes had a role in sugar metabolism or oxidativestress or had an unknown function (Fig. 4).

Qualitatively consistent with the intracellular glycogen andtrehalose measurements for IMZ066 (msn2� msn4� strain),the genes of the glycogen and trehalose pathways were down-regulated at the transcriptional level. However, the decreasesin the transcript levels were much more pronounced (up to

FIG. 4. Global transcriptome responses to MSN2 and MSN4 deletion in anaerobic carbon- and nitrogen-limited chemostats. (A) Venn diagramsshowing the number of genes up- or downregulated in IMZ066 (msn2� msn4�) compared to the isogenic reference strain under both carbon and nitrogenlimitation conditions. Of the 26 genes that were downregulated irrespective of the nutrient limitation, the 24 underlined genes were found to contain theSTRE 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).

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20-fold, resulting in levels below those observed for the refer-ence strain under glucose- and ammonia-excess conditions[Fig. 3]) than the decreases in the levels of glycogen and tre-halose, which decreased by only ca. 50%. This provided clearevidence that posttranscriptional regulation mechanisms makean important contribution to the regulation of storage carbo-hydrate metabolism under these conditions.

Posttranscriptional regulation of glycogen synthase undercarbon and nitrogen limitation conditions. The two main en-zymes of the glycogen biosynthesis and degradation pathways,glycogen synthase and glycogen phosphorylase (22), are sub-ject to strong posttranslational control by phosphorylation anddephosphorylation. This regulation is reciprocal for these twoenzymes; phosphorylation yields an inactive glycogen synthasebut an active glycogen phosphorylase (18, 21, 30, 32–34, 58,64). The inactive, phosphorylated glycogen synthase can beactivated in vitro by inclusion of glucose-6-phosphate in theassay mixture. Hence, measuring the glycogen synthase activityin the absence and presence of glucose-6-phosphate allowsreliable estimation of the proportion of active enzyme relativeto the total amount (21, 30, 33).

To assess whether the glycogen synthase and glycogen phos-phorylase enzymes were subject to posttranscriptional and/orposttranslational regulation in response to nutrient availability,in vitro activity assays were performed with cell extracts fromglucose-limited, ammonia-limited, and glucose- and ammonia-excess cultures (sulfate-limited cultures). While glycogen syn-thase activity was clearly present in extracts from the sulfate-limited cultures, no active form of glycogen synthase wasdetected (Fig. 5). The high activity of glycogen phosphorylaseunder these conditions (11.5 mU � mg�1) was consistent withlow net glycogen accumulation. In cell extracts of the glucose-limited chemostat cultures, 20% of the total glycogen synthasewas found to be in the active form (Fig. 5), thus explaining thehigher rate of glycogen synthesis and the net accumulation of

glycogen in these cultures. While the fraction of active glyco-gen synthase was also 20% in the nitrogen-limited cultures, itstotal activity was threefold higher than that in the glucose-limited cultures (Fig. 5). In addition, the lower glycogen phos-phorylase activity in the nitrogen-limited cultures suggestedthat the rate of glycogen degradation was lower (Fig. 5). Theratio of active glycogen synthase to active glycogen phosphor-ylase was eightfold higher in the nitrogen-limited cultures thanin the glucose-limited cultures, thus enabling an increased fluxtoward glycogen biosynthesis.

DISCUSSION

Impact of growth-limiting nutrient on storage carbohydratelevels. The use of nutrient-limited chemostat cultures enabledus to investigate the impact of the identity of the growth-limiting nutrient on the accumulation of storage carbohydratesby S. cerevisiae at a fixed specific growth rate and, for thenon-glucose-limited growth conditions, at a fixed residual glu-cose concentration in the cultures. This prevented indirecteffects on storage carbohydrate metabolism due to the specificgrowth rate or to glucose signaling. The results show that, incontrast to previous hypotheses (41, 56), storage carbohydrateaccumulation by S. cerevisiae is not a general response to nu-trient limitation or to glucose-excess scenarios.

Of the five nutrient limitation regimens investigated in thisstudy, only glucose limitation and ammonia limitation led toincreased levels of storage carbohydrates. When both carbonand nitrogen sources were present in excess, the storage car-bohydrate levels remained low. These observations enabledmore precise interpretation of the impact of nutrient limitationon storage carbohydrates. For example, the high levels of ac-cumulated glycogen and trehalose observed upon sulfate orphosphate starvation described previously (41) were likely tohave been caused primarily by a decreased specific growth raterather than by the identity of the depleted nutrient. Further-more, the low glycogen and trehalose contents observed in aprevious study of zinc-limited cultures of S. cerevisiae (15)cannot be interpreted as a specific response to zinc limitation.

Trehalose accumulation and glycogen accumulation showedsubtly different responses to nutrient limitation; trehaloseaccumulation occurred specifically during ammonia-limitedgrowth, while glycogen accumulation was greatest under am-monia-limited conditions but was also substantial in glucose-limited cultures. In future research, the cultivation conditionsstudied here can be expanded further to define the impact ofthe growth-limiting nutrient on storage carbohydrate limita-tion. For example, it would be very interesting to see to whatextent the identity of the nitrogen source (e.g., amino acidsinstead of ammonia) and aeration affect storage carbohydratemetabolism.

The correlations between storage carbohydrate metabolismand the nutrient limitation regimen suggest that design ofmedia and feed regimens may be used successfully to controlstorage carbohydrate metabolism. In general, for fermentationprocesses in which high storage carbohydrate levels are desir-able (e.g., repitching of brewer’s yeast [7, 46, 59]) workersshould avoid growth limitation by nutrients other than thesugar or nitrogen source. Conversely, when a low storage car-bohydrate level is desirable, such as during the production of

FIG. 5. Glycogen synthase and glycogen phosphorylase activities incell extracts from anaerobic chemostat cultures of CEN.PK113-7Dlimited by carbon, nitrogen, or sulfur. One unit was defined as theamount of enzyme that catalyzed the conversion of 1 �mol substrate in1 min under the conditions of the assay. The percentage of activeglycogen synthase relative to the total amount under each nutrientlimitation condition is also indicated. TGS, total glycogen synthase;AGS, active glycogen synthase; AGP, active glycogen phosphorylase.For each culture condition measurements were obtained in triplicatefrom three independent chemostat cultures.

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yeast extract (82), use of phosphate or sulfate limitation in thefinal phases of a fed-batch process may be beneficial.

Transcriptional regulation: involvement of Msn2p and Msn4p.Transcriptome analysis showed that in anaerobic glucose- andammonia-limited chemostat cultures, Msn2p and Msn4p wereinvolved in the increased levels of storage carbohydrates ob-served under these conditions. However, of the 266 STRE-containing genes that show increased transcript levels uponenvironmental stress or nutrient starvation (“environmentalstress response” genes [23]) in batch cultures, only 16 werefound to be transcriptionally downregulated upon deletion ofMSN2 and MSN4 in the glucose- and ammonia-limited condi-tions. These 16 environmental stress response genes showedstrong overrepresentation of genes involved in storage carbo-hydrate metabolism (Fig. 4B). This observation strongly sug-gests that the impact of Msn2p and Msn4p on transcriptionalregulation is context dependent. In the absence of strong en-vironmental stress and at a fixed specific growth rate, the im-pact of Msn2p and Msn4p on transcriptional regulation ap-pears to be focused on genes involved in storage carbohydratemetabolism.

The activity of Msn2p and Msn4p is controlled by theirBmh1/Bmh2-mediated cytosolic anchoring in response to thePKA (24, 25, 69) and target of rapamycin (TOR) (2, 45) sig-naling pathways. Release from this PKA or TOR signalingleads to translocation of Msn2p and Msn4p to the nucleus andtranscription of their target genes (2, 69). Indeed, treatment ofS. cerevisiae with rapamycin results in transcriptional inductionof glycogen and trehalose biosynthesis genes and accumulationof both carbohydrates (1, 67, 75).

The PKA pathway is activated by high glucose concentra-tions (72), whereas the TOR pathway is activated by a range ofnitrogen and carbon sources (1, 52, 74). This is consistent withour observation that the transcript levels of glycogen- andtrehalose-related genes were low in the presence of excessglucose and ammonia (i.e., in sulfate-, phosphate-, and zinc-limited cultures), since the PKA and TOR signaling pathwaysare expected to be active under these conditions and Msn2pand Msn4p should thus be retained in the cytosol.

The PKA and TOR pathways control common targets viaparallel routes (66, 89), but their interactions remain largelyunknown (63). Understanding these interactions is importantfor understanding the transcriptional induction of storage car-bohydrate metabolism under glucose- and ammonia-limitedconditions. Moreover, other key regulators may be involved innitrogen regulation of storage carbohydrate metabolism. Tenglycogen and trehalose biosynthesis genes have been identifiedas targets of Gcn4p, the transcriptional activator of the generalamino acid control response (50). A Gcn4p-mediated responseto amino acid starvation has been proposed to lead to a netbreakdown of glycogen (31). In addition, the glycogen andtrehalose levels in a leu3� mutant (Leu3p is a transcriptionalactivator of the branched-chain amino acid biosynthesis path-way), 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 wasaccompanied by transcriptional downregulation of the glyco-gen and trehalose genes and of IRA1 and IRA2 (GTPase-activating proteins for Ras1p and Ras2p [48, 71]), which indi-

cated that hyperactivity of PKA might have been the cause ofrepression of glycogen and trehalose synthesis.

Posttranscriptional regulation. In vitro studies with cell ex-tracts of cultures grown under glucose, ammonia, and sulfatelimitation conditions, as well as cell extracts of cultures ofIMZ066 (msn2� msn4� strain), demonstrated that posttran-scriptional regulation plays a major role in adapting storagecarbohydrate metabolism to the different nutrient limitationregimens. This posttranscriptional regulation could be partiallyattributed to previously described mechanisms. First, apartfrom its role in transcriptional downregulation of glycogen andtrehalose biosynthesis, active PKA deactivates glycogen syn-thase and activates glycogen phosphorylase by phosphorylation(73). Consistent with our observations, high PKA activities inthe sulfate-limited cultures should result in the absence ofactive glycogen synthase. Second, glucose-limited cultivationconditions lead to activation of the Snf1p kinase, which acti-vates glycogen synthase, possibly via the Gac1p-Glc7p phos-phatase complex (29, 30). The cyclin-dependent kinasePho85p, in association with the cyclins Pcl8p and Pcl10p, hasbeen implicated in signaling cascades leading to deactivation ofglycogen 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 pres-ence of active glycogen synthase.

The two mechanisms mentioned above cannot contribute toposttranscriptional upregulation of storage carbohydrate me-tabolism in the ammonia-limited, glucose-excess cultures. Crosstalk between the TOR and PKA signaling pathways, affectingthe phosphorylation state of the glycogen synthase and phos-phorylase, offers an attractive hypothesis. Activation of PKAby TOR has previously been shown for transcriptional induc-tion 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 involvedand that not all kinases and phosphatases regulating the activ-ity of the glycogen phosphorylase have been identified (22).The threefold-higher total (active and inactive) levels of gly-cogen synthase in the ammonia-limited cultures than in theglucose-limited cultures were not matched by a similar differ-ence in transcript levels. This suggests that, additionally, stor-age carbohydrate metabolism may be controlled at the level oftranslation and/or protein turnover. Storage carbohydrate ac-cumulation by S. cerevisiae cultures is the net result of anintricate network of multilevel, multi-input combinatorial reg-ulation mechanisms which is highly relevant for many indus-trial applications. Chemostat fermentation offers an attractiveplatform for systematic dissection of this regulation network.

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 TheNetherlands Genomics Initiative.

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