Emerging relevance of cell wall components from non-conventional yeasts as functional ingredients for the food and feed industry.

Non-conventional yeast species, or non-Saccharomyces yeasts, are increasingly recognized for their involvement in fermented foods. Many of them exhibit probiotic characteristics that are mainly due to direct contacts with other cell types through various molecular components of their cell wall. The biochemical composition and/or the molecular structure of the cell wall components are currently considered the primary determinant of their probiotic properties. Here we first present the techniques that are used to extract and analyze the cell wall components of food industry-related non-Saccharomyces yeasts. We then review the current understanding of the cell wall composition and structure of each polysaccharide from these yeasts. Finally, the data exploring the potential beneficial role of their cell wall components, which could be a source of innovative functional ingredients, are discussed. Such research would allow the development of high value-added products and provide the food industry with novel inputs beyond the well-established S. cerevisiae.

Toxic effect and inability of L-homoserine to be a nitrogen source for growth of Escherichia coli resolved by a combination of in vivo evolution engineering and omics analyses.

L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of E. coli. However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of this bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid. Sequencing of this evolved strain identified only four genomic modifications, including a 49 bp truncation starting from the stop codon of thrL. This mutation resulted in a modified thrL locus carrying a thrL* allele encoding a polypeptide 9 amino acids longer than the thrL encoded leader peptide. Remarkably, the replacement of thrL with thrL* in the original strain MG1655 alleviated L-homoserine inhibition to the same extent as strain 4E, but did not allow growth with this amino acid as a nitrogen source. The loss of L-homoserine toxic effect could be explained by the rapid conversion of L-homoserine into threonine via the thrL*-dependent transcriptional activation of the threonine operon thrABC. On the other hand, the growth of E. coli on a mineral medium with L-homoserine required an activation of the threonine degradation pathway II and glycine cleavage system, resulting in the release of ammonium ions that were likely recaptured by NAD(P)-dependent glutamate dehydrogenase. To infer about the direct molecular targets of L-homoserine toxicity, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which notably identified a potent repression of locomotion-motility-chemotaxis process and of branched-chain amino acids synthesis. Since the magnitude of these effects was lower in a ΔthrL mutant, concomitant with a twofold lower sensitivity of this mutant to L-homoserine, it could be argued that growth inhibition by L-homoserine is due to the repression of these biological processes. In addition, L-homoserine induced a strong upregulation of genes in the sulfate reductive assimilation pathway, including those encoding its transport. How this non-canonical amino acid triggers these transcriptomic changes is discussed.

Carbon sources and XlnR-dependent transcriptional landscape of CAZymes in the industrial fungus Talaromyces versatilis: when exception seems to be the rule.

BACKGROUND: Research on filamentous fungi emphasized the remarkable redundancy in genes encoding hydrolytic enzymes, the similarities but also the large differences in their expression, especially through the role of the XlnR/XYR1 transcriptional activator. The purpose of this study was to evaluate the specificities of the industrial fungus Talaromyces versatilis, getting clues into the role of XlnR and the importance of glucose repression at the transcriptional level, to provide further levers for cocktail production. RESULTS: By studying a set of 62 redundant genes representative of several categories of enzymes, our results underlined the huge plasticity of transcriptional responses when changing nutritional status. As a general trend, the more heterogeneous the substrate, the more efficient to trigger activation. Genetic modifications of xlnR led to significant reorganisation of transcriptional patterns. Just a minimal set of genes actually fitted in a simplistic model of regulation by a transcriptional activator, and this under specific substrates. On the contrary, the diversity of xlnR+ versus ΔxlnR responses illustrated the existence of complex and unpredicted patterns of co-regulated genes that were highly dependent on the culture condition, even between genes that encode members of a functional category of enzymes. They notably revealed a dual, substrate-dependant repressor-activator role of XlnR, with counter-intuitive transcripts regulations that targeted specific genes. About glucose, it appeared as a formal repressive sugar as we observed a massive repression of most genes upon glucose addition to the mycelium grown on wheat straw. However, we also noticed a positive role of this sugar on the basal expression of a few genes, (notably those encoding cellulases), showing again the strong dependence of these regulatory mechanisms upon promoter and nutritional contexts. CONCLUSIONS: The diversity of transcriptional patterns appeared to be the rule, while common and stable behaviour, both within gene families and with fungal literature, the exception. The setup of a new biotechnological process to reach optimized, if not customized expression patterns of enzymes, hence appeared tricky just relying on published data that can lead, in the best scenario, to approximate trends. We instead encourage preliminary experimental assays, carried out in the context of interest to reassess gene responses, as a mandatory step before thinking in (genetic) strategies for the improvement of enzyme production in fungi.

Development of a Metabolite Sensor for High-Throughput Detection of Aldehydes in Escherichia Coli.

We have developed a fluorescence-based metabolite sensor enabling in vivo detection of various aldehydes of biotechnological interest in Escherichia coli. YqhC is a transcriptional regulator that is known to be involved in the upregulation of the yqhD-dgkA operon in the presence of aldehydes. We took advantage of this property by constructing a bi-modular biosensor, in which a sensing module constitutively expresses yqhC while a reporter module drives the expression of the syfp2 reporter gene that is put under control of the yqhD promoter. The sensitivity of the sensor has been optimized by engineering the 5'-UTRs of both the sensing and the reporter modules resulting in a 70-fold gain of fluorescence in response to the model compound glycolaldehyde at 5 mM. The optimized sensor further responded to other aldehydes when supplemented to the cultivation medium at concentrations of 1-10 mM. We furthermore showed that this metabolite sensor was functional in vivo as it responded to the presence of glycoladehyde that is specifically produced upon induction of a synthetic xylulose-1-phosphate pathway expressed in E. coli. This bi-modular sensor can therefore be employed as an exquisite tool for FACS-based ultra-high-throughput screening of aldehyde (over) producing enzymes.

Integration of Biochemical, Biophysical and Transcriptomics Data for Investigating the Structural and Nanomechanical Properties of the Yeast Cell Wall.

The yeast cell is surrounded by a cell wall conferring protection and resistance to environmental conditions that can be harmful. Identify the molecular cues (genes) which shape the biochemical composition and the nanomechanical properties of the cell wall and the links between these two parameters represent a major issue in the understanding of the biogenesis and the molecular assembly of this essential cellular structure, which may have consequences in diverse biotechnological applications. We addressed this question in two ways. Firstly, we compared the biochemical and biophysical properties using atomic force microscopy (AFM) methods of 4 industrial strains with the laboratory sequenced strain BY4743 and used transcriptome data of these strains to infer biological hypothesis about differences of these properties between strains. This comparative approach showed a 4-6-fold higher hydrophobicity of industrial strains that was correlated to higher expression of genes encoding adhesin and adhesin-like proteins and not to their higher mannans content. The second approach was to employ a multivariate statistical analysis to identify highly correlated variables among biochemical, biophysical and genes expression data. Accordingly, we found a tight association between hydrophobicity and adhesion events that positively correlated with a set of 22 genes in which the main enriched GO function was the sterol metabolic process. We also identified a strong association of ß-1,3-glucans with contour length that corresponds to the extension of mannans chains upon pulling the mannosyl units with the lectin-coated AFM tips. This association was positively correlated with a group of 27 genes in which the seripauperin multigene family was highly documented and negatively connected with a set of 23 genes whose main GO biological process was sulfur assimilation/cysteine biosynthetic process. On the other hand, the elasticity modulus was found weakly associated with levels of ß-1,6-glucans, and this biophysical variable was positively correlated with a set of genes implicated in microtubules polymerization, tubulin folding and mitotic organization.

In vivo evolutionary engineering for ethanol-tolerance of Saccharomyces cerevisiae haploid cells triggers diploidization.

Microbial ethanol production is an important alternative energy resource to replace fossil fuels, but at high level, this product is highly toxic, which hampers its efficient production. Towards increasing ethanol-tolerance of Saccharomyces cerevisiae, the so far best industrial ethanol-producer, we evaluated an in vivo evolutionary engineering strategy based on batch selection under both constant (5%, v v-1) and gradually increasing (5-11.4%, v v-1) ethanol concentrations. Selection under increasing ethanol levels yielded evolved clones that could tolerate up to 12% (v v-1) ethanol and had cross-resistance to other stresses. Quite surprisingly, diploidization of the yeast population took place already at 7% (v v-1) ethanol level during evolutionary engineering, and this event was abolished by the loss of MKT1, a gene previously identified as being implicated in ethanol tolerance (Swinnen et al., Genome Res., 22, 975-984, 2012). Transcriptomic analysis confirmed diploidization of the evolved clones with strong down-regulation in mating process, and in several haploid-specific genes. We selected two clones exhibiting the highest viability on 12% ethanol, and found productivity and titer of ethanol significantly higher than those of the reference strain under aerated fed-batch cultivation conditions. This higher fermentation performance could be related with a higher abundance of glycolytic and ribosomal proteins and with a relatively lower respiratory capacity of the evolved strain, as revealed by a comparative transcriptomic and proteomic analysis between the evolved and the reference strains. Altogether, these results emphasize the efficiency of the in vivo evolutionary engineering strategy for improving ethanol tolerance, and the link between ethanol tolerance and diploidization.

RETRACTED:A new function for the yeast trehalose-6P synthase (Tps1) protein, as key pro-survival factor during growth, chronological ageing, and apoptotic stress.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of Marie-Ange Teste, Isabelle Léger-Silvestre, Jean M François and Jean-Luc Parrou. Marjorie Petitjean could not be reached. The corresponding author identified major issues and brought them to the attention of the Journal. These issues span from significant errors in the Material and Methods section of the article and major flaws in cytometry data analysis to data fabrication on the part of one of the authors. Given these errors, the retracting authors state that the only responsible course of action would be to retract the article, to respect scientific integrity and maintain the standards and rigor of literature from the retracting authors' group as well as the Journal. The retracting authors sincerely apologize to the readers and editors.

Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress.

UNLABELLED: A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells. IMPORTANCE: Ethanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties.

Yeast Tolerance to Various Stresses Relies on the Trehalose-6P Synthase (Tps1) Protein, Not on Trehalose.

Trehalose is a stable disaccharide commonly found in nature, from bacteria to fungi and plants. For the model yeast Saccharomyces cerevisiae, claims that trehalose is a stress protectant were based indirectly either on correlation between accumulation of trehalose and high resistance to various stresses or on stress hypersensitivity of mutants deleted for TPS1, which encodes the first enzyme in trehalose biosynthetic pathway. Our goal was to investigate more directly which one, between trehalose and/or the Tps1 protein, may serve yeast cells to withstand exposure to stress. By employing an original strategy that combined the use of mutant strains expressing catalytically inactive variants of Tps1, with MAL(+) yeast strains able to accumulate trehalose from an exogenous supply, we bring for the first time unbiased proof that trehalose does not protect yeast cells from dying and that the stress-protecting role of trehalose in this eukaryotic model was largely overestimated. Conversely, we identified the Tps1 protein as a key player for yeast survival in response to temperature, oxidative, and desiccation stress. We also showed by robust RT-quantitative PCR and genetic interaction analysis that the role of Tps1 in thermotolerance is not dependent upon Hsf1-dependent transcription activity. Finally, our results revealed that the Tps1 protein is essential to maintain ATP levels during heat shock. Altogether, these findings supported the idea that Tps1 is endowed with a regulatory function in energy homeostasis, which is essential to withstand adverse conditions and maintain cellular integrity.

A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts.

A reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of ß-(1,3) and ß-(1,6)-glucan in the total ß-glucan of the cell wall. In the first step, chitin and ß-(1,3)-glucan were hydrolysed into their corresponding monomers N-acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo-ß-(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo-ß-(1,6)-glucanase from Trichoderma harzianum with ß-glucosidase from Aspergillus niger to hydrolyse the remaining ß-glucan. This latter component corresponded to a highly branched ß-(1,6)-glucan that contained about 75-80% of linear ß-(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of ß-(1,3), ß-(1,6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42 °C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of ß-(1,3)-glucan accompanied by an increase of ß-(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.

Molecular and biochemical characterization of three GH62 α-l-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum.

Penicillium funiculosum is an industrial fungus exploited for its capacity to secrete a wide array of glycosyl hydrolases (GHs) and glycosyl transferases (GTs). These enzymes are part of an enzymatic cocktail that is commercialized under the name RovabioExcel(®), which is used as feed additive in animal nutrition. The genome sequence of this filamentous fungus has revealed a remarkable richness in several accessory enzymes, and notably in α-l-arabinofuranosidases (α-l-AFases) that participate in the hydrolysis of arabinoxylans (AX) in corn/wheat fibers used in poultry feed. Here, we report on the molecular and biochemical characterization of three GH62 family α-l-AFases encoding genes in this filamentous fungus. Amino acids sequences showed strong similarities (>65%) between them, as well with GH62 enzymes from other filamentous fungi. Interestingly, one of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1) of 37 amino acids length, which was shown to help the protein to bind to microcrystalline cellulose. Also, this PfABF62c showed optimal pH and temperature of 2.8 and 50°C, respectively, whereas optimal activity for PfABF62a and PfABF62b were measured at 40°C and at pH ranging between 2.6 and 4.5. Arabinan and arabinoxylan, but no other sugars or polymers were found to augment the thermal transition of the three enzymes by 3-5°C as measured by differential scanning fluorimetry. Finally, enzymatic hydrolysis fingerprints of heteroxylans allowed concluding that the mode of action of the GH62 enzymes from this fungal species was to remove arabinofuranosyl residues linked in position O-2 and O-3 of substituted xylose units in arabinoxylan chains.

Investigation of Aspergillus fumigatus biofilm formation by various "omics" approaches.

In the lung, Aspergillus fumigatus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix called biofilm (BF). This extracellular matrix embeds and glues hyphae together and protects the fungus from an outside hostile environment. This extracellular matrix is absent in fungal colonies grown under classical liquid shake conditions (PL), which were historically used to understand A. fumigatus pathobiology. Recent works have shown that the fungus in this aerial grown BF-like state exhibits reduced susceptibility to antifungal drugs and undergoes major metabolic changes that are thought to be associated to virulence. These differences in pathological and physiological characteristics between BF and liquid shake conditions suggest that the PL condition is a poor in vitro disease model. In the laboratory, A. fumigatus mycelium embedded by the extracellular matrix can be produced in vitro in aerial condition using an agar-based medium. To provide a global and accurate understanding of A. fumigatus in vitro BF growth, we utilized microarray, RNA-sequencing, and proteomic analysis to compare the global gene and protein expression profiles of A. fumigatus grown under BF and PL conditions. In this review, we will present the different signatures obtained with these three "omics" methods. We will discuss the advantages and limitations of each method and their complementarity.

Xylosylation as an effective means for reducing yeast growth inhibition by 2-phenylethanol.

Microbial bioproduction processes of 2-phenylethanol, an important rose-like flavor and fragrance compound that occurs naturally in the essential oils of many flowers and plants, are hindered by the growth inhibition it exerts towards the producing microorganism, mainly yeast. We show here for the first time that glycosylation of 2-phenylethanol with xylose increased the inhibitory concentration inducing 50% decrease of the yeast Saccharomyces cerevisiae strain BY4741 growth rate (IC50 ) from 14 mM (1.71 g/L) 2-phenylethanol (2PE) to 100 mM (25.5 g/L) 2-phenylethyl ß-D-xylopyranoside (X-2PE). More interestingly, the IC10 was only 3 mM (0.37 g/L) for 2PE and 86 mM (21.9 g/L) for X-2PE. Xylosylation of 2-phenylethanol can offer therefore an effective means for reducing yeast growth inhibition in microbial bioproduction processes of this important flavor and fragrance compound.

1,3-Propanediol production in a two-step process fermentation from renewable feedstock.

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l(-1)) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l(-1) of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l(-1), leading to a final 1,3-propanediol concentration of 25.5 g l(-1), a productivity of 0.16 g l(-1) h(-1) and 1,3-propanediol yields of 0.56 g g(-1) glycerol and 0.24 g g(-1) sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l(-1), 27.4 g l(-1) of glycerol were produced, leading to 14.6 g l(-1) of 1.3-propanediol and similar values of productivity, 0.15 g l(-1) h(-1), and overall yield, 0.26 g g(-1) sugar.

An atomic force microscopy analysis of yeast mutants defective in cell wall architecture.

Yeast cells are surrounded by a thick cell wall, the composition and structure of which have been characterized by biochemical and genetic methods. In this study, we used atomic force microscopy (AFM) to visualize the cell surface topography and to determine cell wall nanomechanical properties of yeast mutants defective in cell wall architecture. While all mutants investigated showed some alteration in cell surface topography, this alteration was particularly salient in mutants defective in beta-glucan elongation (gas1), chitin synthesis (chs3) and cross-linkages between chitin and beta-glucan (crh1crh2). In addition, these alterations in surface topology were accompanied by increased roughness of the cell. From force-indentation curves, the Young's modulus was determined, as it gives a measure of the elasticity of the cell wall. A value of approximately 1.6 MPa was obtained for the cell walls of the wild-type strain in exponential and stationary phases of growth. The same value was measured in a mnn9 mutant defective in protein mannosylation, and was two-fold reduced in a mutant with reduced beta-glucan (fks1Delta and knr4Delta), only in the stationary phase of growth. In contrast, the elasticity was dramatically reduced in mutants defective in chitin synthesis (chs3Delta), beta-glucan elongation (gas1Delta) and, even more remarkably, in a crh1Deltacrh2Delta mutant defective in the enzymes that catalyse cross-linkages of chitin to beta-glucan. Taken together, these results provide direct physical evidence that the nanomechanical properties of the yeast cell wall are mainly dependent on cross-links and cell wall remodelling, rather than on cell wall composition or thickness.

An approach to the study of gene expression in hepatocarcinogenesis initiation.

In carcinogenesis, determination of gene and protein expression profiles is important for prevention and treatment. Caffeic acid phenethyl ester (CAPE) in a single dose administered before carcinogenic initiation induced by diethylnitrosamine (DEN) prevents the appearance of preneoplastic lesions. On the basis of this approach, the main purpose of this work was to compare the gene expression profiles induced by DEN or a previously administered single dose of CAPE. Using a modified hepatocarcinogenesis-resistant hepatocyte model, male Fischer-344 rats were administered with one intraperitoneal dose of CAPE (20 mg/kg) 12 hours before DEN administration (200 mg/kg). Livers were removed and processed for microarray analysis and reverse transcription-polymerase chain reaction 12 hours after CAPE dosing and 24 hours after DEN administration with or without CAPE. CAPE alone did not alter the expression profile. DEN treatment modified the expression of 665 genes, and CAPE plus DEN induced changes in 1371 genes. DEN treatment increased the expression of genes associated with oxidative stress such as glutathione reductase, genes involved in cell cycle regulation including p53, and modified cytochrome P450. CAPE plus DEN diminished the expression of cytochrome involved in DEN bioactivation such as CYP2B1 as well as the expression of regulators of oxidative stress such as glutathione reductase, GST-kappa and GST-theta, and cell cycle regulators such as p53. Using CAPE as a tool, we uncovered new approaches for studying the altered expression of reactive genes and identifying proteins that will help to propose well-sustained and concrete hypothesis of DEN mechanism of hepatocarcinogenesis initiation.

Exposure to high static or pulsed magnetic fields does not affect cellular processes in the yeast Saccharomyces cerevisiae.

We report results of a study of the effects of strong static (up to 16 T for 8 h) and pulsed (up to 55 T single-shot and 4 x 20 T repeated shots) magnetic fields on Saccharomyces cerevisiae cultures in the exponential phase of growth. In contrast to previous reports restricted to only a limited number of cellular parameters, we have examined a wide variety of cellular processes: genome-scale gene expression, proteome profile, cell viability, morphology, and growth, metabolic and fermentation activity after magnetic field exposure. None of these cellular activities were impaired in response to static or pulsed magnetic field exposure. Our results confirm and extend previous reports on the absence of magnetic field effects on yeast and support the hypothesis that magnetic fields have no impact on the transcriptional machinery and on the integrity of unicellular biological systems.

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.

BACKGROUND: Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. RESULTS: From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. CONCLUSION: In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.