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Transcriptional regulation of fermentative and respiratory metabolism in Saccharomyces cerevisiae industrial bakers' strains

Rafael Dueñas-Sánchez, Gabriel Gutiérrez, Ana M. Rincón, Antonio C. Codón, Tahía Benítez
DOI: http://dx.doi.org/10.1111/j.1567-1364.2012.00813.x 625-636 First published online: 1 September 2012

Abstract

Bakers' yeast-producing companies grow cells under respiratory conditions, at a very high growth rate. Some desirable properties of bakers' yeast may be altered if fermentation rather than respiration occurs during biomass production. That is why differences in gene expression patterns that take place when industrial bakers' yeasts are grown under fermentative, rather than respiratory conditions, were examined. Macroarray analysis of V1 strain indicated changes in gene expression similar to those already described in laboratory Saccharomyces cerevisiae strains: repression of most genes related to respiration and oxidative metabolism and derepression of genes related to ribosome biogenesis and stress resistance in fermentation. Under respiratory conditions, genes related to the glyoxylate and Krebs cycles, respiration, gluconeogenesis, and energy production are activated. DOG21 strain, a partly catabolite-derepressed mutant derived from V1, displayed gene expression patterns quite similar to those of V1, although lower levels of gene expression and changes in fewer number of genes as compared to V1 were both detected in all cases. However, under fermentative conditions, DOG21 mutant significantly increased the expression of SNF1-controlled genes and other genes involved in stress resistance, whereas the expression of the HXK2 gene, involved in catabolite repression, was considerably reduced, according to the pleiotropic stress-resistant phenotype of this mutant. These results also seemed to suggest that stress-resistant genes control desirable bakers' yeast qualities.

Keywords
  • bakers' yeast biomass
  • fermentation—respiration regulation
  • macroarray analysis
  • catabolite-derepressed mutant

Introduction

In the baking industry, Saccharomyces cerevisiae yeasts are usually grown in sucrose-rich molasses, in batch cultures that consist of several stages by means of which the amount of biomass produced is gradually increased (Rehm et al., 1996). At the last stage, cells are cultivated at a specific growth rate and the added substrate is regulated, so that sucrose is rapidly hydrolyzed into glucose and fructose, but ethanol is not produced (Rehm et al., 1996; Randez-Gil et al., 1999; Verstrepen et al., 2004). Thus, the final stage in bakers' yeast industrial production takes place under respiratory conditions to optimize the conversion of substrate into biomass (Rehm et al., 1996; Randez-Gil et al., 1999).

When glucose is maintained below a certain threshold, there are a direct use of pyruvate by mitochondrial respiration and a decrease in acetic acid production (Franzen, 2003; Kaeberlein, 2010). Respiration is accompanied by decreased messenger RNA translation and above all by enhanced stress resistance (Franzen, 2003; Kennedy et al., 2009; Shima & Takagi, 2009).

However, if glucose concentration increases over that threshold, cells ferment pyruvate to ethanol (Gancedo, 2008; van den Brink et al., 2008). After glucose depletion, ethanol is metabolized to acetic acid, which is toxic to yeast cells (Kaeberlein, 2010); acetic acid is secreted to the medium, thus acidifying it, with its subsequent pH drop. In addition, acetic acid increases the production of reactive oxygen species and causes mitochondrial damage in cells (Kaeberlein, 2010), thus altering the desired properties in the final bakers' yeast produced.

Once biomass is produced, bakers' yeast undergoes additional environmental stresses, such as lack of nutrients, high concentration of sucrose, oxidative stress, and others (Attfield, 1997; Puig & Perez-Ortin, 2000; Verstrepen et al., 2004; Shima & Takagi, 2009) not only during storage but also when it is applied to ferment sweet and lean doughs. Yeasts need to induce stress-resistant components, such as proteins and protectants (Salvado et al., 2008), by regulating the expression of the corresponding genes involved (Turcotte et al., 2010), in order to maintain their appropriate baking qualities (Rehm et al., 1996).

Functional genomic approaches to the study of yeast cells using DNA micro- and macroarrays profiling are a suitable means to analyze metabolic adaptation (Venkatasubbarao, 2004; Shima & Takagi, 2009; Rintala et al., 2011). Laboratory strains have been analyzed by comparing growth limitation by different nutrients (Tai et al., 2005; Hazelwood et al., 2009), fermentable and respiratory carbon sources (Daran-Lapujade et al., 2004), DNA-damaging agents (Lee et al., 2007), aerobiosis and anaerobiosis (Piper et al., 2002), cold or freeze shock stress (Rodriguez-Vargas et al., 2002), high and low temperatures (Tai et al., 2007), high sugar stress (Erasmus et al., 2003), and hydric stress (Rossignol et al., 2006; Ratnakumar & Young, 2010). Wild-type strains and mutants with altered levels of catabolic regulators were also studied (Schuurmans et al., 2008; Kummel et al., 2010). However, few studies on industrial yeasts have been carried out so far. The analysis of gene expression profile in industrial bakers' yeasts seems relevant, because it may differ from that of laboratory strains. So, macroarray analyses were carried out in industrial bakers' yeast V1 and in its partly deregulated 2-deoxy-D-glucose-resistant mutant DOG21 (Rincon et al., 2001), in fermentable (glucose) and respirable (glycerol) carbon sources, to study the regulation of genes involved in fermentation–respiration shift (induction or repression) and to identify several of the genes needed for stress resistance and for the maintenance of baking properties.

Materials and methods

Strains

The S. cerevisiae strains used were bakers' strain V1 – normally used for panification of lean doughs – chosen for its higher fermentative capacity (Codon & Benitez, 1995; Codon et al., 1995) when compared to other bakers' strains (Codon et al., 1997, 1998), and DOG21 strain – which is a 2-deoxy-D-glucose-resistant mutant isolated from V1 bakers' yeast – chosen and characterized for its good qualities in the baking industry (Rincon et al., 2001; Codon et al., 2003).

Media and culture conditions

Complete YP medium (1% yeast extract – Pronadisa, 2% bacteriological peptone – USB) supplemented with 2% glucose (YPD) or 3% glycerol (YPG) was used. Yeasts were inoculated into 20-mL tubes containing 5 mL YPD and incubated at 200 r.p.m. in a New Brunswick incubator at 30 °C up to stationary phase (about 108 cells mL−1). Flasks (250-mL) with 50 mL medium were inoculated with stationary-phase culture to reach 0.1 initial optical density at OD660 and incubated at 200 r.p.m., at 30 °C. Growth was determined by measuring the increase in turbidity at OD660, using a Beckman DU640 (Brea, CA) spectrophotometer.

RNA isolation

Samples from V1 and DOG21 strains cultivated in either YPD or YPG were collected at middle exponential phase (OD660 about 0.7, c. 5 × 107 cell mL−1). Total RNA was isolated using Master Pure yeast RNA purification kit (Epicenter Biotechnology, Madison, WI), following the manufacturer's indications. Purified RNA was newly treated with RNAse-free DNase (USB, Miles Road, Cleveland, OH), and the reaction was stopped following the manufacturer's instructions. Total RNA from each strain was obtained from three independent purifications and was used in three independent macroarray hybridizations. Nearly 1 mg from each of the three cultures was sent to DNA-SCSIE service (Servicio Central de Soporte a la Investigación Experimental de la Universidad de Valencia, Valencia, Spain; http://scsie.uv.es/chipsdna) for hybridization to take place.

Data analysis

A total of 12 (three replicates in all four conditions) macroarray experiments were performed using Valencia yeast v2 chip platform developed at SCSIE (number GPL772 of GEO database). It consisted of a nylon macroarray with 6049 single-spotted yeast ORF probes comprising the whole ORF. Radioactive sample labeling and macroarray hybridization followed the protocol described in the study by Alberola et al. (2004). Conditions were named V1-YPG, V1-YPD, DOG21-YPG, and DOG21-YPD. Only four of six possible pairs of comparisons were considered relevant for this work: DOG21-YPD/DOG21-YPG, V1-YPD/DOG21-YPD, V1-YPD/V1-YPG, and V1-YPG/DOG21-YPG.

Image acquisition was performed in a Fuji Film FLA3000 Phosphorimager and quantified using arrayvision 7.0 software (Imaging Research, Inc.) taking sARM density (with its corresponding subtracted background) as signal. Transcript levels of 1.25 times over background were considered as valid data and were normalized.

Spot intensities were measured as ARM density (artifact-removed density), background and sARM density (background-corrected ARM density) using Array Vision software (Imaging Research, Canada).

The normalization process and the measurement of significance level for each ORF were carried out using ArrayStat software (Imaging Research, Inc.). sARM data were subjected to double normalization – first of all between replicates – to test reproducibility. Data were considered independent, thus allowing the program to take two as the minimum number of valid replicates in order to calculate mean values for every gene. A pooled ‘curve-fit’ random error estimate method for a proportional model was carried out by offset. Data were transformed logarithmically and then a second normalization between conditions was then carried out by iterative median, which was corrected by false discovery rate test to estimate statistical errors associated with each gene. A z-test for independent data was applied in order to detect differences in individual gene expression between conditions (P < 0.05). Genes that changed at least 2.5-fold between each pair of conditions were taken into account for further analyses and discussion. However, genes with changes of twofold that have statistical relevance were also taken into account on some occasions.

Genes were gathered following ontological relationships obtained from Saccharomyces Genome Database (SGD; www.yeastgenome.org).

Results

Gene expression analysis of industrial bakers' strains cultivated in fermentative and respiratory conditions

To study transcriptional regulation of fermentative versus respiratory metabolism, macroarray analyses were carried out by comparing data obtained from RNA purified in strains cultivated in media with fermentable (glucose) (YPD medium) or respirable (glycerol) (YPG medium) carbon sources. Cells were in all cases collected at middle exponential phase – 5 × 107 cells mL−1 – to ensure that metabolism was completely fermentative in YPD. After comparing strain V1 versus DOG21 and their growth in YPD versus YPG, four lists of genes were obtained according to gene expression ratios when criteria described in Materials and Methods were applied; genes that increase expression in YPD versus YPG and those that do so in YPG versus YPD in either V1 or DOG21 strains were compared.

V1 and DOG21 strains displayed different gene expression patterns. Those differences in gene number may be observed after carrying out Venn diagrams (Fig. 1). All genes that shared differences in their expression levels (at least 2.5-fold in this and in all other comparisons described in this study) were listed and subjected to ontological genetic analysis; that is, they were gathered either according to their involvement in specific metabolic or structural component pathways or according to their function. The listed genes were then introduced in the GO-Slim Mapper from SGD (www.yeastgenome.org) and were classified by gene ontology (GO).

Figure 1

A comparison between gene expression patterns of strains V1 and DOG21. Empty circles represent the number of V1 genes, while black circles that of DOG21 genes. Intersection between two subsets shows the number of genes with a common pattern in both strains (gray), whereas the rest are specific of one of the strains. (a) Genes with more expression in YPD than in YPG (YPD > YPG) in both strains, (b) genes with more expression in YPG than in YPD (YPG > YPD) in both strains, (c) genes with more expression in YPD than in YPG only in V1 versus genes with more expression in YPG than in YPD in DOG21, (d) genes with more expression in YPG than in YPD in V1 versus genes with more expression in YPD than in YPG in DOG21.

The Supporting Information, Tables S1 and S2 shows such ontological analysis. Gene groups were gathered when their expression increased in YPD with regard to YPG (Table S1) and vice versa, thus increasing their expression in YPG with regard to YPD (Table S2). Only groups that include at least five genes were considered. In addition, some genes may be related to more than one pathway or cellular function. As a result, those genes were taken into account as many times as the number of functions in which they are involved. Hence, there is a discrepancy between gene number in Fig. 1 and gene number in Tables S1 and S2 (395 and 871, respectively). Figures 2 and 3 show a simplified distribution of genes listed in Tables S1 and S2, according to their involvement in different cellular functions. Function is considered only when a significant number of genes related to a specific cellular function; otherwise, genes and functions are gathered under the category ‘others'.

Figure 2

Absolute and relative number of genes involved in each of the different biological processes. Genes represented are those that have more expression in YPD than in YPG. Each process corresponds to one or more GO terms. The total number of genes involved in one or more biological processes (a) for V1 strain (black bar) and DOG21 (white bar). Percentage of genes for V1 (b) and DOG21 strains (c).

Figure 3

Absolute and relative number of genes involved in each of the different biological processes. Genes represented are those that have more expression in YPG than in YPD. Each process corresponds to one or more GO terms. The total number of genes involved in one or more biological processes (a) for V1 strain (black bar) and DOG21 (white bar). Percentage of genes for V1 (b) and DOG21 strains (c).

When the gene expression of V1 strains grown either in YPD or in YPG was compared, 292 genes showed higher expression levels in fermentable (YPD) medium, whereas 550 genes did so in respirable (YPG) medium (Fig. 1a and b). During growth in fermentable medium (YPD), V1 strain increased the expression in a significant gene group related to structure and ribosome biogenesis, transcription and translation, DNA and RNA metabolism and transport, as well as amino acid and amine metabolism. Such expression increase in all those genes indicated a high growth and cellular division rate and a very active metabolism (Fig. 2). Results are comparable to those obtained in the study of laboratory yeast strains. Under respiratory conditions (YPG), several genes related to catabolic functions, mostly those involved in carbohydrate catabolism, as well as those involved in the formation of metabolic precursors of energy production, respiration, phosphorus metabolism, stress responses, signal transduction and, in general, genes whose functions are located into the mitochondria, increased their expression levels under fermentative conditions (Fig. 3).

Gene expression analysis of the partly derepressed DOG21 strain

DOG21 strain is a 2-deoxy-D-glucose-resistant mutant derived from bakers' yeast V1 (Rincon et al., 2001). This mutant possesses, under repression conditions, increased levels (between 2- to 2.5-fold) of trehalose and catabolic enzymes such as invertase, maltase, and phosphatase. In addition, DOG21 ferments better than V1 lean and sweet doughs, either fresh or frozen (Rincon et al., 2001; Codon et al., 2003). Changes in gene expression of strain DOG21 under fermentable and respirable conditions are worth studying, owing to their biotechnological use.

Under fermentable conditions (YPD), DOG21 strain increased the expression of 133 genes when compared to growth under respiratory conditions (YPG), whereas the expression of 338 genes was increased during respiration as compared to fermentative growth (Fig. 1). Once again, gene lists were introduced in GO-Slim Mapper, which generated Tables S1 and S2, with the discrepancy in gene number explained above.

Results seemed to point to slow metabolism of DOG21 strain in YPD as compared to V1 strain. Gene gathering according to the involvement in main metabolic pathways also shows a significant number of genes whose expression increases in YPD. They are related to nucleic acid metabolism, transcription, and translation, but not to carbohydrate metabolism or ribosome biogenesis (Fig. 2). Under respiratory conditions, those genes that increased expression with regard to YPD could be associated with energy generation, stress responses, respiration, carbohydrate metabolism, and generation of metabolite precursors (Fig. 3).

Comparison of gene expression in industrial bakers' strain V1 and derivative mutant DOG21 under fermentative conditions

A total amount of 292 genes in strain V1 and 133 genes in strain DOG21 increased expression under fermentable conditions, as compared to respiratory growth (Fig. 1a). Only 59 of those genes were common to both strains, whereas 233 genes were specific for V1 strain and 74 genes were specific for DOG21 strain. The small number of genes that increased expression in DOG21 as compared to V1 strains (about 30%) indicated that metabolism under fermentable conditions is slower in the mutant and, in fact, the growth rate is about 15% slower in DOG21 as compared to V1 (data not shown). As Table S1 indicates, there are few or no genes at all involved in metabolic pathways such as carbohydrate metabolism, DNA metabolism, ribosome biogenesis, or heterocyclic compound or cofactor metabolism that increase expression in YPD in strain DOG21 as compared to V1. In V1 strain, however, 22% genes that increase expression in YPD with regard to YPG are involved in translation, and of those, 21% are related to the biosynthesis of ribosome components. Those data confirm that metabolism is much slower in DOG21 than in V1 strain, despite the fact that DOG21 is a very useful industrial producer of sweet and plain bakery products. On the other hand, most of the genes that increase expression in YPD are different in V1 and DOG21 (80% and 56% are specific for V1 and DOG21, respectively), although they are involved in the same cell functions (Table S1). Actually, 20% of common genes are involved in amino acid and amine biosynthesis, which may hint at important genetic changes in mutant DOG21 apart from hydrolase deregulation and trehalose accumulation. In fact, DOG21 strain presents much higher increase than V1 (Table 1) in expression of the following genes: those involved in the utilization of carbon sources as an alternative to glucose, such as SUC2, MAL12, MAL32 genes, all of them involved in sucrose and maltose metabolism; transcriptional factors such as ADR1 and CAT8, and in consequence ADR1- and CAT8-dependent genes (Fig. 4; Table 1). DOG1 and DOG2 genes are involved in 2-deoxy-D-glucose resistance, or HSP12 and HSP26 genes are involved in stress resistance. On the contrary, DOG21 has a much lower expression than V1 of genes such as HXK2 that regulates catabolite repression (Table 1).

Figure 4

Principal regulatory network.

View this table:
Table 1

The expression pattern of particularly interesting genes is listed. Ratios are represented as Log2 (expression in V1 strain in YPD/expression in DOG21 strain in YPD)

GeneRatioSGD description
Regulators
ADR1−1.7A carbon source–responsive zinc-finger transcription factor; required for the transcription of glucose-repressed gene ADH2, peroxisomal protein genes, and genes required for ethanol, glycerol, and fatty acid utilization
CAT8−2.9A zinc cluster transcriptional activator necessary for derepression in a variety of genes under nonfermentative growth conditions; active after diauxic shift; binds carbon source–responsive elements
HXK21A hexokinase isoenzyme that catalyzes the phosphorylation of glucose in cytosol; predominant hexokinase during growth on glucose
HAP40.4A subunit of heme-activated, glucose-repressed Hap2p/3p/4p/5p CCAAT-binding complex; a transcriptional activator, and global regulator of respiratory gene expression; provides main activation function in the complex
MIG10.8A transcription factor involved in glucose repression; a sequence-specific DNA-binding protein containing two Cys2His2 zinc-finger motifs; it is regulated by SNF1 kinase and GLC7 phosphatase
SNF10.4A AMP-activated serine/threonine protein kinase found in a complex containing Snf4p and members of Sip1p/Sip2p/Gal83p family; required for the transcription of glucose-repressed genes, thermotolerance, sporulation, and peroxisome biogenesis
ADR1-dependent genes
ALD4−3.15A mitochondrial aldehyde dehydrogenase; required for growth on ethanol and conversion of acetaldehyde to acetate; phosphorylated; activity is K+ dependent; utilizes NADP+ or NAD+ equally as coenzymes; expression is glucose repressed
ADH52.1An alcohol dehydrogenase isoenzyme, involved in ethanol production
GUT1−1.5A glycerol kinase; converts glycerol to glycerol-3-phosphate; glucose repression of expression is mediated by Adr1p and Ino2p-Ino4p
CTP1−2.9A mitochondrial inner membrane citrate transporter; a member of the mitochondrial carrier family
POX1−5.7A fatty-acyl coenzyme A oxidase, involved in fatty acid beta-oxidation pathway; localized in peroxisomal matrix
SPS19−3.5A peroxisomal 2,4-dienoyl-CoA reductase, auxiliary enzyme of fatty acid beta-oxidation; homodimeric enzyme required for growth and sporulation on petroselineate medium; expression induced during late sporulation and in the presence of oleate
CTA1−2.2Catalase A, breaks down hydrogen peroxide in peroxisomal matrix formed by acyl-CoA oxidase (Pox1p) during fatty acid beta-oxidation
POT1−2.43-Ketoacyl-CoA thiolase with broad chain length specificity; it cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA during beta-oxidation of fatty acids
ARO9−1.8Aromatic aminotransferase II; it catalyzes the first step of tryptophan, phenylalanine, and tyrosine catabolism
GIP2−4.9A putative regulatory subunit of protein phosphatase Glc7p, involved in glycogen metabolism
ETR1−1.5A 2-enoyl thioester reductase, a member of the medium-chain dehydrogenase/reductase family; localized in mitochondria, where it has a probable role in fatty acid synthesis
TIR13.1A cell wall mannoprotein of the Srp1p/Tip1p family of serine-/alanine-rich proteins; expression is downregulated at acidic pH and induced by cold shock and anaerobiosis
BTN21.4A v-SNARE binding protein that facilitates specific protein retrieval from a late endosome to the Golgi; it modulates arginine uptake; possible role in mediating pH homeostasis between vacuole and plasma membrane H(+)-ATPase
YML131W−3.4A putative protein of unknown function rather similar to medium-chain dehydrogenase/reductases; expression induced by stresses including osmotic shock, DNA-damaging agents, and other chemicals; GFP-fusion protein localized in cytoplasm
YGR043C−1.7A transaldolase of unknown function; transcription is repressed by Mot1p and induced by alpha-factor and also during diauxic shift
YOR389W−2.1A putative protein of unknown function; expression regulated by copper levels
CAT8-dependent genes
FBP1−2.1Fructose-1,6-bisphosphatase, a key regulatory enzyme in gluconeogenesis pathway, required for glucose metabolism; it undergoes either proteasome-mediated or autophagy-mediated degradation depending on growth conditions; it interacts with Vid30p
STL1−1.8A glycerol proton symporter of plasma membrane, subject to glucose-induced inactivation, strongly but transiently induced when cells are subjected to osmotic shock
PCK1−3.45Phosphoenolpyruvate carboxykinase, a key enzyme in gluconeogenesis; catalyzes an early reaction in carbohydrate biosynthesis; glucose represses its transcription and accelerates mRNA degradation; regulated by Mcm1p and Cat8p; located in cytosol
IDP2−3.8A cytosolic NADP-specific isocitrate dehydrogenase, catalyzes the oxidation of isocitrate to alpha-ketoglutarate; levels are increased during growth on nonfermentable carbon sources and reduced during growth on glucose
GDH2−2.1A NAD(+)-dependent glutamate dehydrogenase, degrades glutamate to ammonia and alpha-ketoglutarate; expression sensitive to nitrogen catabolite repression and intracellular ammonia levels
ODC1−1.9A mitochondrial inner membrane transporter, exports 2-oxoadipate and 2-oxoglutarate from mitochondrial matrix to cytosol for lysine and glutamate biosynthesis and lysine catabolism
YNR002C−2.18A putative transmembrane protein involved in the export of ammonia, a starvation signal that promotes cell death in aging colonies; phosphorylated in mitochondria; a member of the TC 9.B.33 YaaH family; an homolog of Ady2p and Y. lipolytica Gpr1p
GAP1+1.6A general amino acid permease; located in plasma membrane; regulated by nitrogen source
YPR021C+3.66A mitochondrial amino acid transporter; it acts both as a glutamate uniporter and as an aspartate–glutamate exchanger; involved in nitrogen metabolism and nitrogen compound biosynthesis
REG2Present only in DOG21A regulatory subunit of Glc7p type-1 protein phosphatase; it is involved together with Reg1p, Glc7p, and Snf1p, in the regulation of glucose-repressible genes; it is also involved in glucose-induced proteolysis of maltose permease
ADR1- and CAT8-dependent genes
ACS1−3.66An acetyl-coA synthetase isoform which, along with Acs2p, is the nuclear source for acetyl-coA for histone acetylation; it is expressed during growth on nonfermentable carbon sources and under aerobic conditions
Respiration genes
COX6−1.32A subunit of cytochrome c oxidase, which is the terminal member of mitochondrial inner membrane electron transport chain; expression regulated by oxygen levels
QCR8−1.54A subunit of ubiquinol–cytochrome c reductase complex, which is a component of mitochondrial inner membrane electron transport chain; oriented facing intermembrane space
QCR10−1.32A subunit of the ubiquinol–cytochrome c oxidoreductase complex that includes Cobp, Rip1p, Cyt1p, Cor1p, Qcr2p, Qcr6p, Qcr7p, Qcr8p, Qcr9p, and Qcr10p and comprises a part of mitochondrial respiratory chain
Alternative carbon source to glucose
MAL11−4.69An inducible high-affinity maltose transporter (alpha-glucoside transporter); encoded in MAL1 complex locus; broad substrate specificity that includes maltotriose; required for isomaltose utilization
MAL12−4.48Maltase (alpha-D-glucosidase), an inducible protein involved in maltose catabolism; encoded in MAL1 complex locus; it hydrolyzes disaccharides maltose, turanose, maltotriose, and sucrose
MAL31−3.29A maltose permease, high-affinity maltose transporter (alpha-glucoside transporter); encoded in MAL3 complex locus; a member of 12 transmembrane domain superfamily of sugar transporters
MAL32−5.13Maltase (alpha-D-glucosidase), an inducible protein involved in maltose catabolism; encoded in MAL3 complex locus; functional in genomic reference strain S288C; it hydrolyzes disaccharides maltose, turanose, maltotriose, and sucrose
SUC2−3.22Invertase, a sucrose-hydrolyzing enzyme; a secreted, glycosylated form is regulated by glucose repression, and an intracellular, nonglycosylated enzyme is produced constitutively
ATH1−2.23Acid trehalase required for the utilization of extracellular trehalose
Stress-response genes
HSP12−4.63A plasma membrane–localized protein that protects membranes from desiccation; induced by heat shock, oxidative stress, osmostress, stationary-phase entry, glucose depletion, oleate, and alcohol; regulated by HOG and Ras-Pka pathways
DOG1−2.23A 2-deoxyglucose-6-phosphate phosphatase, similar to Dog2p, a member of a family of low molecular weight phosphatases; it confers 2-deoxyglucose resistance when overexpressed; it has not yet been identified in vivo substrate
DOG2−1.54A 2-deoxyglucose-6-phosphate phosphatase, a member of a family of low molecular weight phosphatases, similar to Dog1p; induced by oxidative and osmotic stress; it confers 2-deoxyglucose resistance when overexpressed

Comparison of gene expression in industrial bakers' strain V1 and derivative mutant DOG21 cultivated in respirable conditions

A total amount of 550 genes in V1 strain and 338 genes in DOG21 mutant increase the expression in YPG as compared to fermentable conditions. Of those genes, 239 were common to V1 and DOG21 strains, 311 were specific for V1 (56%), and 99 were specific for DOG21 strain (30%; Fig. 1b).

Such an increase in 338 genes in DOG21 versus 550 genes in V1 strains (almost 60%) seems to indicate that, under respiratory conditions, behavior of DOG21 strain is quite similar to V1 strain, but not so much under fermentable conditions. Such a suggestion is supported by a higher percentage of genes that increase expression in both V1 and DOG21. There is in both strains an increase in genes involved in cell functions such as respiration, energy metabolism, stress response, or catabolism and modification of proteins (Table S2; Fig. 3). A significant percentage of genes that are specific to the DOG21 strain are associated with hexose transport. However, there are few or no genes at all involved in metabolic pathways, such as aromatic compound metabolism, or cell structure formation, such as cytoskeleton, that increase the expression in YPG and in DOG21 strain as compared to V1.

Genes with opposite expression levels in V1 and DOG21 strains in fermentative and respirable conditions

Figure 1c shows three genes that increased expression in YPD with regard to YPG in strain V1 but decreased expression in strain DOG21. Those genes are FYV1, RAD59, and TIR4. According to SGD, FYV1 is an ORF with a doubtful function, not present in other Saccharomyces species, whose deletion results in hypersensitivity to K1 killer toxin and in some defects during growth in fermentable carbon sources; Rad59p, homologous to Rad52p, is related to reparation of double breaks in DNA; TIR4 encodes a mannoprotein that is expressed under anaerobic conditions and required for anaerobic growth.

In addition, two genes increased expression in YPD with regard to YPG in strain DOG21 but decreased expression in strain V1 (Fig. 1d). Those genes are YBL100C and RCK1. YBL100C possesses a doubtful function, although no mutants display any defects when they are grown in respirable carbon sources. RCK1 encodes a kinase related to oxidative stress, which interacts with YAP2 transcriptional factor.

Discussion

Bakers' yeast has been subjected to strong selective adaptation to specific industrial conditions (Codon et al., 1997, 1998) and that adaptation may be noticed in different gene regulation patterns under conditions such as fermentative versus respiratory metabolism (Codon et al., 1997).

Macroarray studies allow analyses of genes according to their related functions or to their expression patterns, thus providing information on gene expression patterns along a growth curve (DeRisi et al., 1997) or allowing comparison of gene expression between a mutant strain and its parental strain (Haurie et al., 2001; Buschlen et al., 2003; Lascaris et al., 2003, 2004; Schuurmans et al., 2008; Ratnakumar & Young, 2010). Global information on gene expression changes occurring throughout cell growth can be obtained in the first case, whereas more specific information on the expression of some particular genes after their overexpression or lack of function can be obtained in the second case.

In this study, four macroarrays were carried out, which made it possible to compare a specific industrial bakers' strain, V1, growing under fermentative versus respiratory conditions, and to obtain information on genes regulated by a carbon source. On the other hand, a comparison of V1 behavior versus its DOG21 mutant allowed to establish which genes are involved and are responsible for the different behavior detected in DOG21 strain with regard to its V1 parental bakers' strain (Rincon et al., 2001; Codon et al., 2003).

There are a large number of genes whose expression is higher in respirable (YPG) as compared to fermentable (YPD) media, in both V1 and DOG21 strains. Even so, the expression of genes related to ribosome biogenesis and initiation and elongation of translation, as well as genes that encode proteins involved in tRNA synthesis, increased in V1 and in YPD. An increase in the expression of genes related to translation in laboratory strains, as well as their repression when glucose or any other easily assimilable fermentable carbon sources are exhausted, or when cells grow in glycerol, has already been described (DeRisi et al., 1997; Roberts & Hudson, 2006). That increase has been associated with a rapid growth rate occurring in fermentable rich media such as YPD. Together with genes related to translation, other genes such as those related to incorporation of ammonium (GDH1 and GDH3) or sulfate (MET3, MET14, and MET16), to carbon residues, or to synthesis of amino acids such as arginine from glutamic acid (all except ARG2 and ARG8), asparagine from aspartic acid (ASN1 and ASN2), or methionine or cysteine (MET17, STR3, CYS3, SAM2) also increased their expression in YPD with regard to YPG.

In YPG, however, the number of genes that increased their expression is much higher than in YPD both in V1 and in DOG21 strains. The genes included those related to ethanol (ADH2), glycerol (GUT1 and GUT2), or acetate utilization (ACS1), the ones involved in the use of alternative carbon sources such as sucrose (SUC2) or maltose (MAL13, MAL12, MAL32), pyruvate or lactate uptake (JEN1), or genes related to Krebs cycle (ACO1, IDH1, KGD1, KGD2, LSC2, SDH2, SDH4, and MDH1), electron transport chains (NDI1, NDE2, SDH1, COR1, RIP1, QCRx, CYC7, and COXx), oxidative phosphorylation (ATPx), glyoxylate synthesis (CIT2, MDH2, ICL1, and IDP2), gluconeogenesis (PCK1, PYC1, FDP1), and glycogen synthesis (PGM2, UGP1, GYS1, and GLC3; DeRisi et al., 1997; Roberts & Hudson, 2006). In addition, genes involved in pathways related to energy generation such as β-oxidation of fatty acids (FAA2, POX1, FOX2, POT1, ECL1, and DCL1) are also derepressed in YPG as compared to YPD.

Respiratory conditions increase free radical oxygen species (ROS) so that genes involved in adaptation to ROS presence, such as MGA2 involved in DNA repair, also increase their expression in YPG (Kelley & Ideker, 2009). According to the YEASTRACT database (Teixeira et al., 2006), 11% of the genes that increased their expression in YPG were related to Rox1p and Mga2p, involved in an early adaptation to oxidative stress, 29% are related to yap1p and 10% to Skn7p, involved in a late adaptation to such oxidative stress. Genes such as CTT1, GAC1, HSP26, and HSP12, whose increased expression in YPG seems to be related to the activation of TOR pathway, involved in mechanisms of general stress responses, are also found (Gagiano et al., 2002; Swiegers et al., 2006; Erkina et al., 2008).

In YPG and in bakers' and laboratory strains, there is also an increase in the expression of genes involved in respiration, and so there is an increase in biomass yield under respiratory conditions (Dejean et al., 2002; Swiegers et al., 2006), as well as in genes involved in hexose transport (HXT2, HXT3, HXT6, and HXT7) and hexose uptake sensors (SNF3). Transcriptional factors described as elements involved in the control of central carbon metabolism, such as MIG1, CAT8, ADR1, and HAP4, display gene expression patterns in industrial bakers' strain V1 similar to that of laboratory strains.

With regard to the DOG21 strain specifically, there is a general gene expression pattern similar to V1 strain, although there is no increase in YPD in the expression of genes related to ribosome biogenesis. DOG21 seems to grow slower and to possess a less active metabolism than V1 strain, which may explain a lower number of genes induced in YPD as compared to the parental V1.

In YPG, strain DOG21 displayed, as occurs with V1, an increase in the expression of genes related to the utilization of alternative carbon sources, Krebs cycle, glyoxylate cycle, or oxidative phosphorylation, although the number of genes induced is slightly lower than in V1. There is no substantial increase in the DOG21 strain and in YPG, in the expression of genes controlled by Msn2/4p or involved in the Ras/AMPc/PKA pathway, as compared to V1. The pattern of genes involved in the change from fermentative to respiratory metabolism, however, is similar in both strains. On the other hand, the DOG21 mutant in YPD, as compared to V1 parental strain, presents a higher level of expression of genes mostly related to stress-response functions, utilization of carbon sources alternative to glucose, respiratory functions and above all, regulatory functions such as ADR1, CAT8, HAP4, MIG1, and SNF1 genes, and ADR1-, CAT8- and ADR1/CAT8-dependent genes (Fig. 4). Snf1p activates Cat8p and Adr1p. Activation via Snf1p is needed to bind Adr1p to promoters regulated by this protein (Turcotte et al., 2010).

With regard to V1, the DOG21 strain shows increased expression in YPD, in genes related to alternative carbon source utilization such as SUC2, MAL12, MAL32, MDH2 or transcriptional factors such as MGA2, CAT8, and ADR1, and in genes involved in fatty acid β-oxidation and gluconeogenesis such as FBP1 and PCK1. Higher expression of both MIG1 and SUC2 or MAL12 genes may indicate a partially functional Mig1p (Diderich et al., 2001; Schuurmans et al., 2008). However, an increase in the expression of other genes involved in respiration, such as HAP4, is not found. In addition, the increase in the expression of genes such as CAT8 may be related to resistance to stress conditions via Snf1p (Ahuatzi et al., 2007), as indicated above. Other genes related to stress responses and regulated by Mig1p, such as DOG1, DOG2, and HSP12 (Randez-Gil et al., 1995), are also partly derepressed in DOG21 strain in YPD. DOG21 displays a 2.5-fold increase in invertase and maltase and 1.5-fold rise in trehalose; in addition, the strain is more resistant to freezing than V1. Such physiological evidence might indicate that in DOG21, Snf1p could be active by a misinterpretation of a stress stimulus. Consequently, activation of Cat8p and Adr1p, as well as that of some genes regulated by them, also takes place. Increase in the expression of CAT8 could be the result of deregulation of Mig1p, due in turn to the scarce presence of Hxk2p, which prevents the SNF1-mediated inactivation of Mig1p.

Data may thus indicate that behavior of DOG21 strain derives from a decrease in the expression of the HXK2 gene and an increase in genes controlled by Snf1p, which results in an increase in stress resistance and a partial derepression of genes related to alternative carbon source utilization.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Genes with higher expression in YPD as compared to YPG, gathered according to their ontological group in V1 and DOG21 strains. Only GO with more than five genes are shown.

Table S2. Genes with higher expression in YPG than in YPD, gathered according to their ontological group in V1 and DOG21 strains. Only GO with more than five genes are shown.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Acknowledgements

This research was supported by CICYT projects AGL2006-03947, PETRI-95-1010.90.01, and TRACE PET2008_0283, and Junta de Andalucía PAI CVI-107, 2009/BIO-167, and P06-CVI-01546. R.D-S. was the recipient of a grant from Institute DANONE Barcelona, Spain.

Footnotes

  • Editor: Isak Pretorius

References

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