Application of Genome-Wide Expression Analysis To Identify Molecular Markers Useful in Monitoring Industrial Fermentations

Abstract

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae identified the YOR387c and YGL258w homologues as highly inducible in zinc-depleted conditions. Induction was specific for zinc deficiency and was dependent on Zap1p. The results indicate that these sequences may be valuable molecular markers for detecting zinc deficiency in industrial fermentations.

Deficiency of the essential nutrient zinc is a major contributor to retarded yeast fermentation in the brewing process(4). During the processing of wort, zinc ions can form complexes with polyphenols and can also form insoluble complexes of alpha acids (12). In this form the zinc is not accessible to yeast, and direct methods of measuring zinc in wort do not always accurately predict its availability to the yeast. Molecular markers have been used as an alternative method to indirectly monitor fermentation processes through yeast gene expression. Genes encoding heat shock (HSP12) and osmotic shock (SPI1) proteins were used as predictors of stress conditions in industrial fermentations (2, 17) and the maltose genes (MAL) have been used to monitor conditions that affect yeast fermentation activity (19). However, because these genes respond to a broad range of conditions, their usefulness for the identification of the specific cause of a defective fermentation is limited. Here we identify molecular markers useful in monitoring industrial fermentations specifically for conditions of zinc deficiency.

To identify genes useful as molecular markers, genome-wide expression analysis was performed on the Saccharomyces cerevisiae industrial Lager 1 strain (6) growing in zinc-depleted and zinc-replete conditions. The low-zinc medium was essentially LZM (23) except that the carbon source was changed to maltose (LZMM), which is the most abundant sugar available to yeast in beer fermentations (21). Lager 1 seed cultures actively growing in LZMM-40 μM ZnSO₄ were harvested and washed three times with sterile distilled water before inoculation into LZMM with and without 40 μM ZnSO₄. RNA was isolated from cells at 4 h, when the rates of growth of the two cultures were beginning to diverge (Fig. 1). Radiolabeled (³³P) cDNA produced from isolated RNA was hybridized to GeneFilters microarrays (Research Genetics) as outlined by Higgins et al. (10). Genes that were induced or repressed more than fivefold in zinc-depleted conditions are listed in Table 1. The majority of the induced genes have been previously reported to be possible targets for the transcriptional activators Zap1p (14) and Msn2p-Msn4p(8) and were therefore subsequently grouped into these categories. The metalloregulatory protein Zap1p is involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae (25, 26). The ZRT1 and ZRT2 genes (induced 8.1- and 7.7-fold, respectively) encode high- and low-affinity zinc permeases, respectively, and both are Zap1p targets (23, 24). Another Zap1p target induced was the ZRT3 gene (induced 8.3-fold), which encodes the yeast vacuolar permease (15). Up-regulation of these genes would enhance the ability of the Lager 1 strain to maintain intracellular zinc levels. The HSP12 and HSP26 genes, encoding heat shock proteins, were also induced by zinc depletion (Table 1). Both of these are targets of Msn2p-Msn4p and, unlike the Zap1p targets, are induced by a broad range of conditions, including general starvation (1, 18). The presence of a general starvation response is further highlighted by the repression of genes involved in sugar utilization (MAL genes), ethanol production (ADH1), and ribosomal functions (Table 1), all of which are vital for yeast growth and metabolic activity.

FIG. 1.

YOR387c and YGL258w differential gene expression is the most rapid and responsive to zinc depletion. Northern blot kinetic analysis of YOR387c and YGL258w, ZPS1, ZRT1, and ACT1 gene expression was performed with total RNA isolated from Lager 1 cells grown in LZMM with (□; Zn⁺) and without (▵; Zn⁻) 40 μM ZnSO₄ added. Each Northern blot is a representative of a duplicate experiment, and the growth curves are the means of triplicate readings of duplicate experiments with lower than 12% standard errors. OD600nm, optical density at 600 nm.

TABLE 1.

Highly differentially expressed Lager 1 genes (more than five fold) after growth in zinc-depleted conditions compared to zinc-replete conditions

Open reading frame	Gene name	Fold induction or repressiona	Gene or protein descriptionb
Induced by zinc depletion
Putative Zap 1p target genes
YOR387c		80
YGL258w		63
YOL154w	ZPS1	28
YGL256w	ADH4	13	Alcohol dehydrogenase isoenzyme IV
YJL056c	ZAP1	10	Involved in zinc-responsive transcriptional regulation
YBL049w		8.5
YKL175w	ZRT3	8.3	Vacuolar Zn efflux
YGL255w	ZRT1	8.1	High-affinity zinc transport protein
YLR130c	ZRT2	7.7	Low-affinity zinc transport protein
YDR284c	DPP1	6.8	Diacylglycerol pyrophosphate phosphatase
YGL121c	GPG1	6.2	G protein gamma
Putative Msn2p-Msn4p target genes
YPL223c	GRE1	27	Hydrophilic polypeptide with no known homology
YGR088w	CTT1	26	Cytoplasmic catalase T
YDR070c		26
YBR072w	HSP26	25	Heat shock protein 26
YMR181c		23
YMR175w	SIP18	17	Salt-induced protein of 18 kDa
YOL053c-A	DDR2	12	Induced by heat shock, osmotic shock, and oxidative stress
YIR038c	GTT1	10	Glutathione transferase
YML128c	GIN3	9.5	Meiotic sister chromatid recombination
YFL014w	HSP12	9	12-kDa heat shock protein
YDL124w		9	NADPH-dependent reductase
YHR138c		8.6
YBL075c	SSA3	7.7	Cytosolic member of the 70-kDa heat shock protein family
YMR107w		7.5
YKL151c		7.2
YHR096c	HXT5	6.9	Hexose transporter
YGR256w	GND2	6.4	6-Phosphogluconate dehydrogenase
YDL204w		6.1
YHR104w	GRE3	5.8	α-Keto-aldose reductase
YBR116c		5.7
YER150w	SPI1	5.6	Component of cell wall
YKL163w	PIR3	5.2	Protein containing tandem internal repeats
YKL150w	MCR1	5.0	NADH-cytochrome b5 reductase
Other
YOR374w	ALD4	24	Mitochondrial aldehyde dehydrogenase
YER073w	ALD5	9.5	Mitochondrial aldehyde dehydrogenase
YNL172w	APC1	8.2	Subunit of ubiquitin-protein ligase
YNL056w		8.2
YOL082w		7.8
YMR173w	DDR48	7.3	Flocculent-specific protein
YMR118c		6.9	Induced by starvation
YLR136c	TIS11	6.7	Homologue of mammalian TIS11
YMR173w-A		6.4	Overlaps DDR48
YNL239w	LAP3	6.1	Aminopeptidase of cysteine protease family
YNR077c		5.8
YDR543c		5.6
YKL065c	YET1	5.4	Yeast endoplasmic reticulum 25-kDa transmembrane protein
YBR214w	SDS24	5.1	Nuclear protein similar to Schizosaccharomyces pombe sds23
YIR037w	HYR1	5.1	Putative glutathione peroxidase
Repressed by zinc depletion
YOL086c	ADH1	15	Alcohol dehydrogenase
YMR116c	ASC1	10	WD repeat protein (G-beta-like protein)
YLR150w	MPT4	9.2	Telomeric DNA binding activity
YBR299w	MAL32	9.1	α-Glucosidase, hydrolyses maltose
YGR292w	MAL12	8.5	α-Glucosidase, hydrolyses maltose
YBR298c	MAL31	7.2	High-affinity maltose permease
YHR183w	GND1	5.3	6-Phosphogluconate dehydrogenase/PICK>
Ribosomal protein subunits
YPL131w	RPL1
YOR063w	RPL3	9.1
YGL147c	RPL9A	7.6
YLR325c	RPL38	6.4
YHR010w	RPL27	5.8
YLR029c	RPL13A	5.8
YJL177w	RPL17B	5.7
YKR094c	RPL40B	5.7
YOR293w	RPS10A	5.6
YOR096w	RPS30	5.5
YKL180w	RPL17	5.5
YLR048w	RPS0B	5.5
YHR203c	RPS7A	5.4
YGL123w	RPS2	5.3
YLR062c		5.3
YML073c	RPL6A	5.2
YBR191w	RPL21A	5.1
YDR450w	RPS18A	5.1
YGR027c	RPS31A	5.0
YIL148w	RPL40A	5.0

^aInduction factors were calculated by dividing the expression levels in zinc-depleted conditions by those in zinc-replete conditions. Values are means of duplicate experiments for genes with less than 20% variation between the duplicate experiments.

^bGene annotations were obtained with GeneSpring (A. R. Conway, GeneSpring, version 5.0, Silicon Genetics, Redwood City, Calif., 2002).

Further expression analyses were performed on highly induced candidate genes from the listed Zap1p targets in Table 1. These were chosen for their likely specificity for zinc depletion since those activated by the Msn2p-Msn4p complex are subject to a wide range of stresses (16, 20). The Lager 1 genes most highly induced in response to zinc-depleted conditions were YOR387c and YGL258w (Table 1), both of which have no known cell function (11). They are highly homologous genes, with over 92% DNA identity in their coding regions; this homology probably results in cross-hybridization during expression analyses. Since the promoter regions of these genes are almost identical (99.6% over 1 kb of upstream sequence), both transcripts were regarded as one in subsequent tests. The other genes selected for analysis were ZRT1, based on its specificity to zinc-depleted conditions (23), and the highly induced gene ZPS1 (Table 1), whose product has weak homology to zinc metalloproteinases (11). The kinetics and degrees of induction of the selected genes were measured by Northern analyses (10) during a 10-h incubation in zinc-depleted and zinc-replete medium. By the fourth hour all four genes were induced in zinc-depleted conditions (Fig. 1), validating the expression patterns seen in the genome-wide expression analysis (Table 1). YOR387c and YGL258w were rapidly induced, with a very clear increase in transcripts by the second hour of exposure to zinc depletion (Fig. 1). This analysis shows that the highly differential nature of expression of YOR387c and YGL258w and of ZPS1 (Table 1) can be attributed not only to very high levels of expression in zinc-depleted conditions but also to very low expression levels when zinc was present. This is in contrast to what was found for ZRT1, which, although it has increased expression in zinc-depleted conditions, has a relatively high basal level, effectively decreasing its apparent induction. This was surprising since an analysis of ZRT1 expression in a laboratory yeast strain did not show high basal levels (23). This characteristic may be unique to the Lager 1 strain, or other mechanisms may be involved, a possibility supported by observations that changes in nitrogen source affect ZRT1 expression levels (5). Although highly induced by zinc depletion, ZPS1 expression was also found to be affected by changes in pH (13). This is unlikely with YOR387c and YGL258w since an investigation of microarray data using the Yeast Microarray Global Viewer (Laboratoire de Genetique Moleculaire, Ecole Normale Superieure, Paris, France [http://transcriptome.ens.fr/ymgv/who.php]) did not identify any other conditions that significantly affected its expression.

YOR387c and YGL258w were identified in a genome-wide characterization of the zinc-responsive regulon (14) as possible Zap1p targets. To verify this, YOR387c and YGL258w expression in a zap1Δ mutant was measured. The YOR387c and YGL258w transcripts were not evident in the zap1Δ mutant when the mutant was grown in zinc-depleted conditions, whereas they were characteristically present at high levels in the wild type (Fig. 2). This confirmed that YOR387c and YGL258w expression is induced through the Zap1p transcriptional activator in response to zinc deficiency.

FIG. 2.

Induction of YOR387c and YGL258w in zinc-depleted conditions is dependent on Zap1p. YOR387c and YGL258w and ACT1 expression in wild-type (BY4743) and zap1Δ mutant cells grown in Chelex-treated synthetic defined (CSD) medium (14) with (Zn⁺) and without (Zn⁻) 10 μM ZnSO₄ added was measured.

To further analyze the specificity of YOR387c and YGL258w induction, their expression in a Lager 1 strain exposed to oxidative stress and carbon starvation, two conditions known to impact yeast during industrial fermentations (3), was measured. Figure 3 shows that YOR387c and YGL258w expression was not induced in these conditions. A similar pattern of induction was observed with the ZRT1 transcript; however, the basal level of expression in unstressed conditions was higher. The integrity of the stress conditions was confirmed by the induction of the Msn2p-Msn4p-activated gene, HSP12, in all stress conditions compared to induction in the control medium (Fig. 3).

FIG. 3.

YOR387c and YGL258w expression is not induced by oxidative stress or carbon starvation. YOR387c and YGL258w, ZRT1, HSP12, and ACT1 expression was measured in Lager 1 cells grown in modified LZMM with (Zn⁺) or without (Zn⁻) 40 μM ZnSO₄ added, LZMM (Zn⁺) with 2 mM H₂O₂ added (H₂O₂), and LZMM (Zn⁺) with no added maltose (C⁻).

Iron, another trace element in yeast, is, like zinc, essential for yeast growth and metabolic activity (22). To determine the effect of iron depletion on YOR387c and YGL258w expression, transcript levels in the Lager 1 strain growing in low-iron medium (7) were measured by genome-wide expression analysis. From Fig. 4 it is clear that YOR387c and YGL258w were not induced in response to iron-depleted conditions and therefore are not part of a general response to depletion of divalent cations. ZRT1 on the other hand showed some induction. The induction of the ARN2 gene, which encodes a siderochrome iron transporter (9), confirmed the iron-depleted condition (Fig. 4).

FIG. 4.

YOR387c and YGL258w expression is not induced by iron depletion. Shown are YOR387c and YGL258w, ZRT1, ARN2, and ACT1 expression patterns from GeneFilters microarray analysis of Lager 1 cells grown for 4 h in low-iron medium with (Fe⁺) and without (Fe⁻) 25 μM FeCl₃ added.

Genome-wide expression analysis was useful in identifying YOR387c and YGL258w as molecular markers of zinc-deficient conditions. The expression of these genes was highly inducible from a virtually undetectable basal level, making them easily discernible targets for detection of differential expression by rapid methods of gene transcript analysis such as real-time PCR. These experiments and publicly available microarray information confirmed the specificity of YOR387c and YGL258w induction for zinc-depleted conditions and show that they are induced 2 h before zinc depletion is seen to affect growth of the culture to a significant level. They are therefore not only effective predictors of defective fermentation but also identifiers of the actual cause so that subsequent preventative measures can be carried out.