Genomic and metabolic instability during long-term fermentation of an industrial Saccharomyces cerevisiae strain engineered for C5 sugar utilization.

The genetic stability and metabolic robustness of production strains is one of the key criteria for the production of bio-based products by microbial fermentation on an industrial scale. These criteria were here explored in an industrial ethanol-producer strain of Saccharomyces cerevisiae able to co-ferment D-xylose and L-arabinose with glucose through the chromosomal integration of several copies of pivotal genes for the use of these pentose (C5) sugars. Using batch sequential cultures in a controlled bioreactor that mimics long-term fermentation in an industrial setting, this strain was found to exhibit significant fluctuations in D-xylose and L-arabinose consumption as early as the 50th generation and beyond. These fluctuations seem not related to the few low-consumption C5 sugar clones that appeared throughout the sequential batch cultures at a frequency lower than 1.5% and that were due to the reduction in the number of copies of transgenes coding for C5 sugar assimilation enzymes. Also, subpopulations enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work highlighted the existence of genetic and metabolic instabilities in an industrial yeast which, although modest in our conditions, could be more deleterious in harsher industrial conditions, leading to reduced production performance.

Rapid and Accurate Diagnosis of Dermatophyte Infections Using the DendrisCHIP® Technology.

Dermatophytosis is a superficial fungal infection with an ever-increasing number of patients. Culture-based mycology remains the most commonly used diagnosis, but it takes around four weeks to identify the causative agent. Therefore, routine clinical laboratories need rapid, high throughput, and accurate species-specific analytical methods for diagnosis and therapeutic management. Based on these requirements, we investigated the feasibility of DendrisCHIP® technology as an innovative molecular diagnostic method for the identification of a subset of 13 pathogens potentially responsible for dermatophytosis infections in clinical samples. This technology is based on DNA microarray, which potentially enables the detection and discrimination of several germs in a single sample. A major originality of DendrisCHIP® technology is the use of a decision algorithm for probability presence or absence of pathogens based on machine learning methods. In this study, the diagnosis of dermatophyte infection was carried out on more than 284 isolates by conventional microbial culture and DendrisCHIP®DP, which correspond to the DendrisCHIP® carrying oligoprobes of the targeted pathogens implicated in dermatophytosis. While convergence ranging from 75 to 86% depending on the sampling procedure was obtained with both methods, the DendrisCHIP®DP proved to identify more isolates with pathogens that escaped the culture method. These results were confirmed at 86% by a third method, which was either a specific RT-PCR or genome sequencing. In addition, diagnostic results with DendrisCHIP®DP can be obtained within a day. This faster and more accurate identification of fungal pathogens with DendrisCHIP®DP enables the clinician to quickly and successfully implement appropriate antifungal treatment to prevent the spread and elimination of dermatophyte infection. Taken together, these results demonstrate that this technology is a very promising method for routine diagnosis of dermatophytosis.

Progress advances in the production of bio-sourced methionine and its hydroxyl analogues.

The essential sulphur-containing amino acid, methionine, is becoming a mass-commodity product with an annual production that exceeded 1,500,000 tons in 2018. This amino acid is today almost exclusively produced by chemical process from fossil resources. The environmental problems caused by this industrial process, and the expected scarcity of oil resources in the coming years, have recently accelerated the development of bioprocesses for producing methionine from renewable carbon feedstock. After a brief description of the chemical process and the techno-economic context that still justify the production of methionine by petrochemical processes, this review will present the current state of the art of biobased alternatives aiming at a sustainable production of this amino acid and its hydroxyl analogues from renewable carbon feedstock. In particular, this review will focus on three bio-based processes, namely a purely fermentative process based on the metabolic engineering of the natural methionine pathway, a mixed process combining the production of the O-acetyl/O-succinyl homoserine intermediate of this pathway by fermentation followed by an enzyme-based conversion of this intermediate into L-methionine and lately, a hybrid process in which the non-natural chemical synthon, 2,4-dihydroxybutyric acid, obtained by fermentation of sugars is converted by chemo-catalysis into hydroxyl methionine analogues. The industrial potential of these three bioprocesses, as well as the major technical and economic obstacles that remain to be overcome to reach industrial maturity are discussed. This review concludes by bringing up the assets of these bioprocesses to meet the challenge of the "green transition", with the accomplishment of the objective "zero carbon" by 2050 and how they can be part of a model of Bioeconomy enhancing local resources.

Genomic, transcriptomic, and metabolic characterization of 2-Phenylethanol-resistant Saccharomyces cerevisiae obtained by evolutionary engineering.

2-Phenylethanol is an aromatic compound commonly used in the food, cosmetic, and pharmaceutical industries. Due to increasing demand for natural products by consumers, the production of this flavor by microbial fermentation is gaining interest, as a sustainable alternative to chemical synthesis or expensive plant extraction, both processes relying on the use of fossil resources. However, the drawback of the fermentation process is the high toxicity of 2-phenylethanol to the producing microorganism. The aim of this study was to obtain a 2-phenylethanol-resistant Saccharomyces cerevisiae strain by in vivo evolutionary engineering and characterize the adapted yeast at the genomic, transcriptomic and metabolic levels. For this purpose, the tolerance to 2-phenylethanol was developed by gradually increasing the concentration of this flavor compound through successive batch cultivations, leading to an adapted strain that could tolerate 3.4 g/L of 2-phenylethanol, which was about 3-times better than the reference strain. Genome sequencing of the adapted strain identified point mutations in several genes, notably in HOG1 that encodes the Mitogen-Activated Kinase of the high-osmolarity signaling pathway. As this mutation is localized in the phosphorylation lip of this protein, it likely resulted in a hyperactive protein kinase. Transcriptomic analysis of the adapted strain supported this suggestion by revealing a large set of upregulated stress-responsive genes that could be explained in great part by HOG1-dependent activation of the Msn2/Msn4 transcription factor. Another relevant mutation was found in PDE2 encoding the low affinity cAMP phosphodiesterase, the missense mutation of which may lead to hyperactivation of this enzyme and thereby enhance the stressful state of the 2-phenylethanol adapted strain. In addition, the mutation in CRH1 that encodes a chitin transglycosylase implicated in cell wall remodeling could account for the increased resistance of the adapted strain to the cell wall-degrading enzyme lyticase. Finally, the potent upregulation of ALD3 and ALD4 encoding NAD+ -dependent aldehyde dehydrogenase together with the observed phenylacetate resistance of the evolved strain suggest a resistance mechanism involving conversion of 2-phenylethanol into phenylacetaldehyde and phenylacetate implicating these dehydrogenases.

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein.

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.

Tuning the expression of the bacterial relBE toxin-antitoxin system in Saccharomyces cerevisiae allows characterizing the subsequent growth inhibition.

The bacterial toxin-antitoxin systems are each composed of a toxin, which severely inhibits bacterial cells growth, and a specific neutralizing antitoxin. Some toxin-antitoxin systems are functional when expressed in the yeast Saccharomyces cerevisiae. For instance, the expression of the relE toxin gene leads to a strong growth defect in yeast, whereas the expression of the relB antitoxin gene restores growth. Nevertheless, there is no available data regarding the required expression levels of each component of the relBE system leading to these growth phenotypes, neither their effects on cell viability. Here we used a double inducible plasmid-based system to independently modulate the relative amounts of relB and relE, and performed growth and gene expression analyses. These results allow us to correlate growth phenotypes to the expression levels of the toxin and the antitoxin, and to determine the levels necessary to observe either a strong growth inhibition or a normal growth. We also showed that the relE expression produces cell cycle progression defect without affecting cell viability. These results provide a detailed characterization of the functioning of the relBE system in S. cerevisiae, and open applicative perspectives of yeast growth control by bacterial toxin-antitoxin systems.

Screening for Volatile α-Unsaturated Ester-Producing Yeasts from the Feces of Wild Animals in South Africa.

α-unsaturated esters are fruity-aromatic compounds which are largely spread in the volatilome of many different fruits, but they are rarely found in the volatilome of yeasts. The yeast S. suaveolens has been recently shown to produce relatively high amounts of α-unsaturated esters and it appears to be an interesting model for the production of these compounds. This study aimed to isolate new α-unsaturated ester-producing yeasts by focusing on strains displaying a similar metabolism to S. suaveolens. While the production of α-unsaturated esters by S. suaveolens is believed to be closely related to its ability to grow on media containing branched-chain amino acids (isoleucine, leucine and valine) as the sole carbon source (ILV+ phenotype), in this study, an original screening method was developed that selects for yeast strains displaying ILV+ phenotypes and is able to produce α-unsaturated esters. Among the 119 yeast strains isolated from the feces of 42 different South African wild animal species, 43 isolates showed the ILV+ phenotype, among which 12 strains were able to produce α-unsaturated esters. Two interesting α-unsaturated esters were detected in two freshly isolated strains, both identified as Galactomyces candidus. These new esters were detected neither in the volatilome of the reference strain S. suaveolens, nor in any other yeast species previously studied for their aroma production. This work demonstrated the efficiency of an original method to rapidly screen for α-unsaturated ester-producing yeasts. In addition, it demonstrated that wild animal feces are interesting resources to isolate novel strains producing compounds with original aromas.

The DendrisCHIP® Technology as a New, Rapid and Reliable Molecular Method for the Diagnosis of Osteoarticular Infections.

Osteoarticular infections are major disabling diseases that can occur after orthopedic implant surgery in patients. The management of these infections is very complex and painful, requiring surgical intervention in combination with long-term antibiotic treatment. Therefore, early and accurate diagnosis of the causal pathogens is essential before formulating chemotherapeutic regimens. Although culture-based microbiology remains the most common diagnosis of osteoarticular infections, its regular failure to identify the causative pathogen as well as its long-term modus operandi motivates the development of rapid, accurate, and sufficiently comprehensive bacterial species-specific diagnostics that must be easy to use by routine clinical laboratories. Based on these criteria, we reported on the feasibility of our DendrisCHIP® technology using DendrisCHIP®OA as an innovative molecular diagnostic method to diagnose pathogen bacteria implicated in osteoarticular infections. This technology is based on the principle of microarrays in which the hybridization signals between oligoprobes and complementary labeled DNA fragments from isolates queries a database of hybridization signatures corresponding to a list of pre-established bacteria implicated in osteoarticular infections by a decision algorithm based on machine learning methods. In this way, this technology combines the advantages of a PCR-based method and next-generation sequencing (NGS) while reducing the limitations and constraints of the two latter technologies. On the one hand, DendrisCHIP®OA is more comprehensive than multiplex PCR tests as it is able to detect many more germs on a single sample. On the other hand, this method is not affected by the large number of nonclinically relevant bacteria or false positives that characterize NGS, as our DendrisCHIP®OA has been designed to date to target only a subset of 20 bacteria potentially responsible for osteoarticular infections. DendrisCHIP®OA has been compared with microbial culture on more than 300 isolates and a 40% discrepancy between the two methods was found, which could be due in part but not solely to the absence or poor identification of germs detected by microbial culture. We also demonstrated the reliability of our technology in correctly identifying bacteria in isolates by showing a convergence (i.e., same bacteria identified) with NGS superior to 55% while this convergence was only 32% between NGS and microbial culture data. Finally, we showed that our technology can provide a diagnostic result in less than one day (technically, 5 h), which is comparatively faster and less labor intensive than microbial cultures and NGS.

Selection by UV Mutagenesis and Physiological Characterization of Mutant Strains of the Yeast Saprochaete suaveolens (Former Geotrichum fragrans) with Higher Capacity to Produce Flavor Compounds.

Yeast volatile organic compounds (VOCs), i.e. low molecular weight organic acids, alcohols and esters, are considered as potential and sustainable sources of natural aromas that can replace commonly used artificial flavors in food and other industrial sectors. Although research generally focuses on the yeast Saccharomyces cerevisiae, other so-called unconventional yeasts (NCY) are beginning to attract the attention of researchers, particularly for their ability to produce alternative panels of VOCs. With this respect, a Saprochaete suaveolens strain isolated from dragon fruit in Reunion Island was shown to produce α-unsaturated esters from branched-chain amino acids (BCAAs) such as isobutyl, isoamyl or ethyl tiglate, which are rarely found in other yeasts strains. Given that ß-oxidation allows the growth of S. suaveolens on BCAAs as sole carbon source, we developped a method based on UV mutagenesis to generate mutants that can no longer grow on BCAAs, while redirecting the carbon flow towards esterification of α-unsaturated esters. Among the 15,000 clones generated through UV irradiation, we identified nine clones unable to grow on BCAAs with one of them able to produce eight times more VOCs as compared to the wild-type strain. This higher production of α-unsaturated esters in this mutant strain coincided with an almost complete loss of enoyl-CoA hydratase activity of the ß-oxidation pathways and with a twofold increase of acyl-CoA hydrolase with not significant changes in the enzymes of the Ehrlich pathway. Moreover, from our knowledge, it constituted the first example of VOCs enhancement in a microbial strain by UV mutagenesis.

A Two-step Strategy for High-Value-Added Utilization of Rapeseed Meal by Concurrent Improvement of Phenolic Extraction and Protein Conversion for Microbial Iturin A Production.

Rapeseed meal (RSM) is a major by-product of oil extraction from rapeseed, consists mainly of proteins and phenolic compounds. The use of RSM as protein feedstock for microbial fermentation is always hampered by phenolic compounds, which have antioxidant property with health-promoting benefits but inhibit bacterial growth. However, there is still not any good process that simultaneously improve extraction efficiency of phenolic compounds with conversion efficiency of protein residue into microbial production. Here we established a two-step strategy including fungal pretreatment followed by extraction of phenolic compounds. This could not only increase extraction efficiency and antioxidant property of phenolic compounds by about 2-fold, but also improve conversion efficiency of protein residue into iturin A production by Bacillus amyloliquefaciens CX-20 by about 33%. The antioxidant and antibacterial activities of phenolic extracts were influenced by both total phenolic content and profile, while microbial feedstock value of residue was greatly improved because protein content was increased by ∼5% and phenolic content was decreased by ∼60%. Moreover, this two-step process resulted in isolating more proteins from RSM, bringing iturin A production to 1.95 g/L. In conclusion, high-value-added and graded utilization of phenolic extract and protein residue from RSM with zero waste is realized by a two-step strategy, which combines both benefits of fungal pretreatment and phenolic extraction procedures.

FLO11, a Developmental Gene Conferring Impressive Adaptive Plasticity to the Yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has a remarkable ability to adapt its lifestyle to fluctuating or hostile environmental conditions. This adaptation most often involves morphological changes such as pseudofilaments, biofilm formation, or cell aggregation in the form of flocs. A prerequisite for these phenotypic changes is the ability to self-adhere and to adhere to abiotic surfaces. This ability is conferred by specialized surface proteins called flocculins, which are encoded by the FLO genes family in this yeast species. This mini-review focuses on the flocculin encoded by FLO11, which differs significantly from other flocculins in domain sequence and mode of genetic and epigenetic regulation, giving it an impressive plasticity that enables yeast cells to swiftly adapt to hostile environments or into new ecological niches. Furthermore, the common features of Flo11p with those of adhesins from pathogenic yeasts make FLO11 a good model to study the molecular mechanism underlying cell adhesion and biofilm formation, which are part of the initial step leading to fungal infections.

The dual role of amyloid-ß-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae.

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein.

Impact of down-stream processing on functional properties of yeasts and the implications on gut health of Atlantic salmon (Salmo salar).

Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

Insights on the Control of Yeast Single-Cell Growth Variability by Members of the Trehalose Phosphate Synthase (TPS) Complex.

Single-cell variability of growth is a biological phenomenon that has attracted growing interest in recent years. Important progress has been made in the knowledge of the origin of cell-to-cell heterogeneity of growth, especially in microbial cells. To better understand the origins of such heterogeneity at the single-cell level, we developed a new methodological pipeline that coupled cytometry-based cell sorting with automatized microscopy and image analysis to score the growth rate of thousands of single cells. This allowed investigating the influence of the initial amount of proteins of interest on the subsequent growth of the microcolony. As a preliminary step to validate this experimental setup, we referred to previous findings in yeast where the expression level of Tsl1, a member of the Trehalose Phosphate Synthase (TPS) complex, negatively correlated with cell division rate. We unfortunately could not find any influence of the initial TSL1 expression level on the growth rate of the microcolonies. We also analyzed the effect of the natural variations of trehalose-6-phosphate synthase (TPS1) expression on cell-to-cell growth heterogeneity, but we did not find any correlation. However, due to the already known altered growth of the tps1Δ mutants, we tested this strain at the single-cell level on a permissive carbon source. This mutant showed an outstanding lack of reproducibility of growth rate distributions as compared to the wild-type strain, with variable proportions of non-growing cells between cultivations and more heterogeneous microcolonies in terms of individual growth rates. Interestingly, this variable behavior at the single-cell level was reminiscent to the high variability that is also stochastically suffered at the population level when cultivating this tps1Δ strain, even when using controlled bioreactors.

Evaluation of mixed-fermentation of Saccharomyces cerevisiae with Saprochaete suaveolens to produce natural fruity beer from industrial wort.

Fruity beers can be promoted through production of flavoring compounds during fermentation by partial replacement of brewing yeast by non-conventional-yeasts with high aroma production abilities. We evaluated here the use of a wild Saprochaete suaveolens strain, producing atypical aroma compounds, to produce new natural fruity beer, while keeping classical production conditions used in brewing industry. S. suaveolens was inoculated as starter of culture during beer fermentation and the fermentation performance was evaluated through measurement of several physicochemical parameters. The aroma profile of the engineered beers was monitored using HS-SPME GC/MS. The results showed that high fruity aroma and low-ethanol content beers were obtained through single-fermentation using S. suaveolens. We also demonstrated that during mixed-fermentation, S. suaveolens maintained high metabolic activity and allowed production of beer enriched with fruity aroma. Production of high or low ethanol content fruity beer could be achieved by varying the composition of the starter of culture.

Evaluation of Filamentous Fungi and Yeasts for the Biodegradation of Sugarcane Distillery Wastewater.

Sugarcane Distillery Spent Wash (DSW) is among the most pollutant industrial effluents, generally characterized by high Chemical Oxygen Demand (COD), high mineral matters and acidic pH, causing strong environmental impacts. Bioremediation is considered to be a good and cheap alternative to DSW treatment. In this study, 37 strains of yeasts and filamentous fungi were performed to assess their potential to significantly reduce four parameters characterizing the organic load of vinasses (COD, pH, minerals and OD475nm). In all cases, a pH increase (until a final pH higher than 8.5, being an increase superior to 3.5 units, as compared to initial pH) and a COD and minerals removal could be observed, respectively (until 76.53% using Aspergillus terreus var. africanus and 77.57% using Aspergillus niger). Depending on the microorganism, the OD475nm could decrease (generally when filamentous fungi were used) or increase (generally when yeasts were used). Among the strains tested, the species from Aspergillus and Trametes genus offered the best results in the depollution of DSW. Concomitant with the pollutant load removal, fungal biomass, with yields exceeding 20 g·L-1, was produced.

Engineering microbial pathways for production of bio-based chemicals from lignocellulosic sugars: current status and perspectives.

Lignocellulose is the most abundant biomass on earth with an annual production of about 2 × 1011 tons. It is an inedible renewable carbonaceous resource that is very rich in pentose and hexose sugars. The ability of microorganisms to use lignocellulosic sugars can be exploited for the production of biofuels and chemicals, and their concurrent biotechnological processes could advantageously replace petrochemicals' processes in a medium to long term, sustaining the emerging of a new economy based on bio-based products from renewable carbon sources. One of the major issues to reach this objective is to rewire the microbial metabolism to optimally configure conversion of these lignocellulosic-derived sugars into bio-based products in a sustainable and competitive manner. Systems' metabolic engineering encompassing synthetic biology and evolutionary engineering appears to be the most promising scientific and technological approaches to meet this challenge. In this review, we examine the most recent advances and strategies to redesign natural and to implement non-natural pathways in microbial metabolic framework for the assimilation and conversion of pentose and hexose sugars derived from lignocellulosic material into industrial relevant chemical compounds leading to maximal yield, titer and productivity. These include glycolic, glutaric, mesaconic and 3,4-dihydroxybutyric acid as organic acids, monoethylene glycol, 1,4-butanediol and 1,2,4-butanetriol, as alcohols. We also discuss the big challenges that still remain to enable microbial processes to become industrially attractive and economically profitable.

SIR2 Expression Noise Can Generate Heterogeneity in Viability but Does Not Affect Cell-to-Cell Epigenetic Silencing of Subtelomeric URA3 in Yeast.

Chromatin structure clearly modulates gene expression noise, but the reverse influence has never been investigated, namely how the cell-to-cell expression heterogeneity of chromatin modifiers may generate variable rates of epigenetic modification. Sir2 is a well-characterized histone deacetylase of the Sirtuin family. It strongly influences chromatin silencing, especially at telomeres, subtelomeres and rDNA. This ability to influence epigenetic landscapes makes it a good model to study the largely unexplored interplay between gene expression noise and other epigenetic processes leading to phenotypic diversification. Here, we addressed this question by investigating whether noise in the expression of SIR2 was associated with cell-to-cell heterogeneity in the frequency of epigenetic silencing at subtelomeres in Saccharomyces cerevisiae Using cell sorting to isolate subpopulations with various expression levels, we found that heterogeneity in the cellular concentration of Sir2 does not lead to heterogeneity in the epigenetic silencing of subtelomeric URA3 between these subpopulations. We also noticed that SIR2 expression noise can generate cell-to-cell variability in viability, with lower levels being associated with better viability. This work shows that SIR2 expression fluctuations are not sufficient to generate cell-to-cell heterogeneity in the epigenetic silencing of URA3 at subtelomeres in Saccharomyces cerevisiae but can strongly affect cellular viability.