QTL mapping reveals novel genes and mechanisms underlying variations in H2S production during alcoholic fermentation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae requirement for reduced sulfur to synthesize methionine and cysteine during alcoholic fermentation, is mainly fulfilled through the sulfur assimilation pathway. Saccharomyces cerevisiae reduces sulfate into sulfur dioxide (SO2) and sulfide (H2S), whose overproduction is a major issue in winemaking, due to its negative impact on wine aroma. The amount of H2S produced is highly strain-specific and also depends on SO2 concentration, often added to grape must. Applying a bulk segregant analysis to a 96-strain-progeny derived from two strains with different abilities to produce H2S, and comparing allelic frequencies along the genome of pools of segregants producing contrasting H2S quantities, we identified two causative regions involved in H2S production in the presence of SO2. A functional genetic analysis allowed the identification of variants in four genes able to impact H2S formation, viz; ZWF1, ZRT2, SNR2, and YLR125W, and involved in functions and pathways not associated with sulfur metabolism until now. These data point out that, in wine fermentation conditions, redox status, and zinc homeostasis are linked to H2S formation while providing new insights into the regulation of H2S production, and a new vision of the interplay between the sulfur assimilation pathway and cell metabolism.

Identification of a Glycosylated Fraction Involved in Mushroom Off-Flavors in Grapes: Influence of Gray Rot, Powdery Mildew and Crustomyces subabruptus.

An organoleptic defect, termed fresh mushroom off-flavor and mainly caused by the C8 compounds 1-octen-3-one, 3-octanol and 1-octen-3-ol, has been identified in wines and spirits since the 2000s. The aim of this work was to identify the presence of glycosidic precursors of these C8 compounds and to evaluate the influence of different molds on the glycosylated fractions of three grape varieties. Must samples contaminated by molds (gray rot, powdery mildew and Crustomyces subabruptus) and three levels of attack severity (from healthy to 10-15%) were studied. After a ß-glycosidase treatment on Meunier and Pinot noir musts contaminated by Crustomyces subabruptus, 1-octen-3-one, 1-octen-3-ol and 3-octanol were identified by GC-MS, proving the existence of glycosidic fractions in the musts. A Pinot noir must contaminated by Crustomyces subabruptus displayed a 230% increase in the glycosylated fraction responsible for 1-octen-3-one in comparison with an uncontaminated sample. Powdery mildew did not appear to affect the levels of the studied glycosidic fractions in Chardonnay musts. Gray rot on Meunier and Pinot noir musts had opposite effects depending on glycoside type, decreasing the 1-octen-3-one fraction and increasing the 1-octen-3-ol fraction.

Identification of new Saccharomyces cerevisiae variants of the MET2 and SKP2 genes controlling the sulfur assimilation pathway and the production of undesirable sulfur compounds during alcoholic fermentation.

BACKGROUND: Wine yeasts can produce undesirable sulfur compounds during alcoholic fermentation, such as SO2 and H2S, in variable amounts depending mostly on the yeast strain but also on the conditions. However, although sulfur metabolism has been widely studied, some of the genetic determinants of differences in sulfite and/or sulfide production between wine yeast strains remain to be identified. In this study, we used an integrated approach to decipher the genetic determinants of variation in the production of undesirable sulfur compounds. RESULTS: We examined the kinetics of SO2 production by two parental strains, one high and one low sulfite producer. These strains displayed similar production profiles but only the high-sulfite producer strain continued to produce SO2 in the stationary phase. Transcriptomic analysis revealed that the low-sulfite producer strain overexpressed genes of the sulfur assimilation pathway, which is the mark of a lower flux through the pathway consistent with a lower intracellular concentration in cysteine. A QTL mapping strategy then enabled us to identify MET2 and SKP2 as the genes responsible for these phenotypic differences between strains and we identified new variants of these genes in the low-sulfite producer strain. MET2 influences the availability of a metabolic intermediate, O-acetylhomoserine, whereas SKP2 affects the activity of a key enzyme of the sulfur assimilation branch of the pathway, the APS kinase, encoded by MET14. Furthermore, these genes also affected the production of propanol and acetaldehyde. These pleiotropic effects are probably linked to the influence of these genes on interconnected pathways and to the chemical reactivity of sulfite with other metabolites. CONCLUSIONS: This study provides new insight into the regulation of sulfur metabolism in wine yeasts and identifies variants of MET2 and SKP2 genes, that control the activity of both branches of the sulfur amino acid synthesis pathway and modulate sulfite/sulfide production and other related phenotypes. These results provide novel targets for the improvement of wine yeast strains.

Responses of Saccharomyces cerevisiae to nitrogen starvation in wine alcoholic fermentation.

Nitrogen is an important nutrient in alcoholic fermentation because its starvation affects both fermentation kinetics and the formation of yeast metabolites. In most alcoholic fermentations, yeasts have to ferment in nitrogen-starved conditions, which requires modifications of cell functions to maintain a high sugar flux and enable cell survival for long periods in stressful conditions. In this review, we present an overview of our current understanding of the responses of the wine yeast Saccharomyces cerevisiae to variations of nitrogen availability. Adaptation to nitrogen starvation involves changes in the activity of signaling pathways such as target of rapamycin (TOR) and nitrogen catabolite repression (NCR), which are important for the remodeling of gene expression and the establishment of stress responses. Upon starvation, protein degradation pathways involving autophagy and the proteasome play a major role in nitrogen recycling and the adjustment of cellular activity. Recent progress in the understanding of the role of these mechanisms should enable advances in fermentation management and the design of novel targets for the selection or improvement of yeast strains.

Deciphering regulatory variation of THI genes in alcoholic fermentation indicate an impact of Thi3p on PDC1 expression.

BACKGROUND: Thiamine availability is involved in glycolytic flux and fermentation efficiency. A deficiency of this vitamin may be responsible for sluggish fermentations in wine making. Therefore, both thiamine uptake and de novo synthesis could have key roles in fermentation processes. Thiamine biosynthesis is regulated in response to thiamine availability and is coordinated by the thiamine sensor Thi3p, which activates Pdc2p and Thi2p. We used a genetic approach to identify quantitative trait loci (QTLs) in wine yeast and we discovered that a set of thiamine genes displayed expression-QTL on a common locus, which contains the thiamine regulator THI3. RESULTS: We deciphered here the source of these regulatory variations of the THI and PDC genes. We showed that alteration of THI3 results in reduced expression of the genes involved in thiamine biosynthesis (THI11/12/13 and THI74) and increased expression of the pyruvate decarboxylase gene PDC1. Functional analysis of the allelic effect of THI3 confirmed the control of the THI and PDC1 genes. We observed, however, only a small effect of the THI3 on fermentation kinetics. We demonstrated that the expression levels of several THI genes are correlated with fermentation rate, suggesting that decarboxylation activity could drive gene expression through a modulation of thiamine content. Our data also reveals a new role of Thi3p in the regulation of the main pyruvate decarboxylase gene, PDC1. CONCLUSIONS: This highlights a switch from PDC1 to PDC5 gene expression during thiamine deficiency, which may improve the thiamine affinity or conservation during the enzymatic reaction. In addition, we observed that the lab allele of THI3 and of the thiamin transporter THI7 have diverged from the original alleles, consistent with an adaptation of lab strains to rich media containing an excess of thiamine.

A genetic approach of wine yeast fermentation capacity in nitrogen-starvation reveals the key role of nitrogen signaling.

BACKGROUND: In conditions of nitrogen limitation, Saccharomyces cerevisiae strains differ in their fermentation capacities, due to differences in their nitrogen requirements. The mechanisms ensuring the maintenance of glycolytic flux in these conditions are unknown. We investigated the genetic basis of these differences, by studying quantitative trait loci (QTL) in a population of 133 individuals from the F2 segregant population generated from a cross between two strains with different nitrogen requirements for efficient fermentation. RESULTS: By comparing two bulks of segregants with low and high nitrogen requirements, we detected four regions making a quantitative contribution to these traits. We identified four polymorphic genes, in three of these four regions, for which involvement in the phenotype was validated by hemizygote comparison. The functions of the four validated genes, GCN1, MDS3, ARG81 and BIO3, relate to key roles in nitrogen metabolism and signaling, helping to maintain fermentation performance. CONCLUSIONS: This study reveals that differences in nitrogen requirement between yeast strains results from a complex allelic combination. The identification of three genes involved in sensing and signaling nitrogen and specially one from the TOR pathway as affecting nitrogen requirements suggests a role for this pathway in regulating the fermentation rate in starvation through unknown mechanisms linking nitrogen signaling to glycolytic flux.

Assessing the mechanisms responsible for differences between nitrogen requirements of saccharomyces cerevisiae wine yeasts in alcoholic fermentation.

Nitrogen is an essential nutrient for Saccharomyces cerevisiae wine yeasts during alcoholic fermentation, and its abundance determines the fermentation rate and duration. The capacity to ferment under conditions of nitrogen deficiency differs between yeasts. A characterization of the nitrogen requirements of a set of 23 strains revealed large differences in their fermentative performances under nitrogen deficiency, and these differences reflect the nitrogen requirements of the strains. We selected and compared two groups of strains, one with low nitrogen requirements (LNRs) and the other with high nitrogen requirements (HNRs). A comparison of various physiological traits indicated that the differences are not related to the ability to store nitrogen or the protein content. No differences in protein synthesis activity were detected between strains with different nitrogen requirements. Transcriptomic analysis revealed expression patterns specific to each of the two groups of strains, with an overexpression of stress genes in HNR strains and a stronger expression of biosynthetic genes in LNR strains. Our data suggest that differences in glycolytic flux may originate from variations in nitrogen sensing and signaling under conditions of starvation.

Oxidative stress response and nitrogen utilization are strongly variable in Saccharomyces cerevisiae wine strains with different fermentation performances.

We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

Copper-based grape pest management has impacted wine aroma.

Despite the high energetic cost of the reduction of sulfate to H2S, required for the synthesis of sulfur-containing amino acids, some wine Saccharomyces cerevisiae strains have been reported to produce excessive amounts of H2S during alcoholic fermentation, which is detrimental to wine quality. Surprisingly, in the presence of sulfite, used as a preservative, wine strains produce more H2S than wild (oak) or wine velum (flor) isolates during fermentation. Since copper resistance caused by the amplification of the sulfur rich protein Cup1p is a specific adaptation trait of wine strains, we analyzed the link between copper resistance mechanism, sulfur metabolism and H2S production. We show that a higher content of copper in the must increases the production of H2S, and that SO2 increases the resistance to copper. Using a set of 51 strains we observed a positive and then negative relation between the number of copies of CUP1 and H2S production during fermentation. This complex pattern could be mimicked using a multicopy plasmid carrying CUP1, confirming the relation between copper resistance and H2S production. The massive use of copper for vine sanitary management has led to the selection of resistant strains at the cost of a metabolic tradeoff: the overproduction of H2S, resulting in a decrease in wine quality.

Differential adaptation to multi-stressed conditions of wine fermentation revealed by variations in yeast regulatory networks.

BACKGROUND: Variation of gene expression can lead to phenotypic variation and have therefore been assumed to contribute the diversity of wine yeast (Saccharomyces cerevisiae) properties. However, the molecular bases of this variation of gene expression are unknown. We addressed these questions by carrying out an integrated genetical-genomic study in fermentation conditions. We report here quantitative trait loci (QTL) mapping based on expression profiling in a segregating population generated by a cross between a derivative of the popular wine strain EC1118 and the laboratory strain S288c. RESULTS: Most of the fermentation traits studied appeared to be under multi-allelic control. We mapped five phenotypic QTLs and 1465 expression QTLs. Several expression QTLs overlapped in hotspots. Among the linkages unraveled here, several were associated with metabolic processes essential for wine fermentation such as glucose sensing or nitrogen and vitamin metabolism. Variations affecting the regulation of drug detoxification and export (TPO1, PDR12 or QDR2) were linked to variation in four genes encoding transcription factors (PDR8, WAR1, YRR1 and HAP1). We demonstrated that the allelic variation of WAR1 and TPO1 affected sorbic and octanoic acid resistance, respectively. Moreover, analysis of the transcription factors phylogeny suggests they evolved with a specific adaptation of the strains to wine fermentation conditions. Unexpectedly, we found that the variation of fermentation rates was associated with a partial disomy of chromosome 16. This disomy resulted from the well known 8-16 translocation. CONCLUSIONS: This large data set made it possible to decipher the effects of genetic variation on gene expression during fermentation and certain wine fermentation properties. Our findings shed a new light on the adaptation mechanisms required by yeast to cope with the multiple stresses generated by wine fermentation. In this context, the detoxification and export systems appear to be of particular importance, probably due to nitrogen starvation. Furthermore, we show that the well characterized 8-16 translocation located in SSU1, which is associated with sulfite resistance, can lead to a partial chromosomic amplification in the progeny of strains that carry it, greatly improving fermentation kinetics. This amplification has been detected among other wine yeasts.

Evolution of Markers Involved in the Fresh Mushroom Off-Flavor in Wine During Alcoholic Fermentation.

The fresh mushroom off-flavor (FMOff) is due to several C8 compounds such as 1-octen-3-one, 1-octen-3-ol and 1-hydroxyoctan-3-one, among others. Recently, glycosidic precursors of some FMOff compounds have been identified in grape musts, but the evolution of such compounds during alcoholic fermentation (AF) remains poorly studied. Therefore, the aim of this work was to monitor both FMOff glycosidic precursors and volatile compounds during AF by comparing healthy and Crustomyces subabruptus-contaminated musts. For the first time, glycosidic analysis revealed the presence of 1-hydroxyoctan-3-one glycosides in the laboratory-contaminated musts, together with other FMOff glycosidic fractions already described in the literature. During AF, the FMOff glycosidic fraction decreased, even more in the case of 1-hydroxyoctan-3-one precursors. For the volatile FMOff compounds, their evolutions were both compound- and matrix-dependent except for 1-hydroxyoctan-3-one, which seemed to reach an identical threshold concentration in wine regardless of its initial level in contaminated musts.

QTL mapping of the production of wine aroma compounds by yeast.

BACKGROUND: Wine aroma results from the combination of numerous volatile compounds, some produced by yeast and others produced in the grapes and further metabolized by yeast. However, little is known about the consequences of the genetic variation of yeast on the production of these volatile metabolites, or on the metabolic pathways involved in the metabolism of grape compounds. As a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of 44 compounds by a population of 30 segregants from a cross between a laboratory strain and an industrial strain genotyped at high density. RESULTS: We detected eight genomic regions explaining the diversity concerning 15 compounds, some produced de novo by yeast, such as nerolidol, ethyl esters and phenyl ethanol, and others derived from grape compounds such as citronellol, and cis-rose oxide. In three of these eight regions, we identified genes involved in the phenotype. Hemizygote comparison allowed the attribution of differences in the production of nerolidol and 2-phenyl ethanol to the PDR8 and ABZ1 genes, respectively. Deletion of a PLB2 gene confirmed its involvement in the production of ethyl esters. A comparison of allelic variants of PDR8 and ABZ1 in a set of available sequences revealed that both genes present a higher than expected number of non-synonymous mutations indicating possible balancing selection. CONCLUSIONS: This study illustrates the value of QTL analysis for the analysis of metabolic traits, and in particular the production of wine aromas. It also identifies the particular role of the PDR8 gene in the production of farnesyldiphosphate derivatives, of ABZ1 in the production of numerous compounds and of PLB2 in ethyl ester synthesis. This work also provides a basis for elucidating the metabolism of various grape compounds, such as citronellol and cis-rose oxide.

A sulphite-inducible form of the sulphite efflux gene SSU1 in a Saccharomyces cerevisiae wine yeast.

Sulphite is widely used as a preservative in foods and beverages for its antimicrobial and antioxidant activities, particularly in winemaking where SO(2) is frequently added. Thus, sulphite resistance mechanisms have been extensively studied in the fermenting yeast Saccharomyces cerevisiae. Sulphite detoxification, involving a plasma membrane protein encoded by the SSU1 gene, is the most efficient resistance mechanism in S. cerevisiae. In this study, we characterized the unusual expression pattern of SSU1 in the wine strain 71B. We provide, for the first time, evidence of SSU1 induction by sulphite. The study of SSU1 expression during fermentation and in different growth conditions showed that sulphite is the main regulator of SSU1 expression, explaining its specific pattern. Combining analyses of gene expression and growth behaviour in response to sulphite, we found that 71B displayed unique behavioural patterns in response to sulphite pre-adaptation that may be explained by changes in SSU1 expression. Examination of the genomic organization of the SSU1 locus and sequencing of the region revealed three different alleles in 71B, two of which corresponded to translocated VIII-XVI forms. The lack of differences between promoter regions suggests that this inducible SSU1 expression pattern is due to modification of regulatory/signalling pathways.

New insights into {gamma}-aminobutyric acid catabolism: Evidence for {gamma}-hydroxybutyric acid and polyhydroxybutyrate synthesis in Saccharomyces cerevisiae.

The gamma-aminobutyrate (GABA) shunt, an alternative route for the conversion of alpha-ketoglutarate to succinate, involves the glutamate decarboxylase Gad1p, the GABA transaminase Uga1p and the succinate semialdehyde dehydrogenase Uga2p. This pathway has been extensively described in plants and animals, but its function in yeast remains unclear. We show that the flux through Gad1p is insignificant during fermentation in rich sugar-containing medium, excluding a role for this pathway in redox homeostasis under anaerobic conditions or sugar stress. However, we found that up to 4 g of exogenous GABA/liter was efficiently consumed by yeast. We studied the fate of this consumed GABA. Most was converted into succinate, with a reaction yield of 0.7 mol/mol. We also showed that a large proportion of GABA was stored within cells, indicating a possible role for this molecule in stress tolerance mechanisms or nitrogen storage. Furthermore, based on enzymatic and metabolic evidence, we identified an alternative route for GABA catabolism, involving the reduction of succinate-semialdehyde into gamma-hydroxybutyric acid and the polymerization of gamma-hydroxybutyric acid to form poly-(3-hydroxybutyric acid-co-4-hydroxybutyric acid). This study provides the first demonstration of a native route for the formation of this polymer in yeast. Our findings shed new light on the GABA pathway and open up new opportunities for industrial applications.

Relief from nitrogen starvation entails quick unexpected down-regulation of glycolytic/lipid metabolism genes in enological Saccharomyces cerevisiae.

Nitrogen composition of the grape must has an impact on yeast growth and fermentation kinetics as well as on the organoleptic properties of the final product. In some technological processes, such as white wine/rosé winemaking, the yeast-assimilable nitrogen content is sometimes insufficient to cover yeast requirements, which can lead to slow or sluggish fermentations. Growth is nevertheless quickly restored upon relief from nutrient starvation, e.g. through the addition of ammonium nitrogen, allowing fermentation completion. The aim of this study was to determine how nitrogen repletion affected the transcriptional response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour after nitrogen addition. We found almost 4800 genes induced or repressed, sometimes within minutes after nutrient changes. Some of these responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, and genes related to the retrograde response (RTG) specific to the tricarboxylic acid (TCA) cycle and nitrogen catabolite repression (NCR). Some unexpected transcriptional responses concerned all the glycolytic genes, carbohydrate metabolism and TCA cycle-related genes that were down-regulated, as well as genes from the lipid metabolism.

Different genetic responses to oenological conditions between a flocculent wine yeast and its FLO5 deleted strain: Insights from the transcriptome.

During winemaking Saccharomyces cerevisiae strains are exposed continuously to environmental changes and this microorganism responds modifying its transcriptional profile. Yeast flocculation is considered a social trait that allows the cells to escape from hostile conditions by sedimentation. This behaviour is based on the self-interaction of flocculins, proteins encoded by FLO family genes. These are considered responsible of the facultative-helping type cooperation and were designed as green-beard genes. In order to understand the role of flocculation to stress response, the genome wide expression analysis of a wine flocculent S. cerevisiae F6789A strain and its FLO5 deleted strain (F6789A-Δflo5) were determined, using DNA microarray technology. Results highlighted that F6789A strain showed a shorter lag phase in winemaking condition. The comparison of transcriptomic profiles underlined that, while F6789A-Δflo5 strain seemed engaged in the re-organization of the cell wall and in finding different adhesion ways, F6789A strain presented an up-regulation of genes involved in stress response and higher alcohol production.

Relief from nitrogen starvation triggers transient destabilization of glycolytic mRNAs in Saccharomyces cerevisiae cells.

Nitrogen replenishment of nitrogen-starved yeast cells resulted in substantial transcriptome changes. There was an unexplained rapid, transient down-regulation of glycolytic genes. This unexpected result prompted us to search for the factors controlling these changes, among which is the possible involvement of different nutrient-sensing pathways such as the TORC1 and cAMP/PKA pathways. To that end, the effects of various gene deletions or chemical blocking agents were tested by investigating the expression of PGK1, one of the glycolytic genes most affected after nitrogen replenishment. We report here that several factors affected glycolytic mRNA stability, among which were glucose sensing, protein elongation, nitrogen metabolism, and TOR signaling. Ammonium sensing was not involved in the response, but ammonium metabolism was required. Thus, our results suggest that, in the presence of glucose, carbon/nitrogen cross-talk is likely involved in the response to nitrogen upshift. Our data suggest that posttranscriptional control of glycolytic gene expression may be an important response to nitrogen replenishment.

A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation.

Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation.

FLO5 gene controls flocculation phenotype and adhesive properties in a Saccharomyces cerevisiae sparkling wine strain.

Flocculation is an important feature for yeast survival in adverse conditions. The natural diversity of flocculating genes in Saccharomyces cerevisiae can also be exploited in several biotechnological applications. Flocculation is mainly regulated by the expression of genes belonging to the FLO family. These genes have a similar function, but their specific contribution to flocculation ability is still unclear. In this study, the distribution of FLO1, FLO5 and FLO8 genes in four S. cerevisiae wine strains was investigated. Subsequently, both FLO1 and FLO5 genes were separately deleted in a flocculent S. cerevisiae wine strain. After gene disruption, flocculation ability and agar adhesion were evaluated. FLO1 and FLO5 genes inheritance was also monitored. All strains presented different lengths for FLO1 and FLO5 genes. Results confirm that in S. cerevisiae strain F6789, the FLO5 gene drives flocculation and influences adhesive properties. Flocculation ability monitoring after a cross with a non-flocculent strain revealed that FLO5 is the gene responsible for flocculation development.

SUT1-promoted sterol uptake involves the ABC transporter Aus1 and the mannoprotein Dan1 whose synergistic action is sufficient for this process.

Efficient sterol influx in the yeast Saccharomyces cerevisiae is restricted to anaerobiosis or to haem deficiency resulting from mutations. Constitutive expression of SUT1, an hypoxic gene encoding a transcriptional regulator, induces sterol uptake in aerobiosis. A genome-wide approach using DNA microarray was used to identify the mediators of SUT1 effects on aerobic sterol uptake. A total of 121 ORFs (open reading frames) were significantly and differentially expressed after SUT1 overexpression, 61 down-regulated and 60 up-regulated. Among these genes, the role of the putative ABC transporter (ATP-binding-cassette transporter) Aus1, and of the cell-wall mannoprotein Dan1, was characterized better. These two genes play an essential role in aerobic sterol uptake, since their deletion compromised the SUT1 effects, but individual overexpression of either of these genes in a wild-type background was not sufficient for this process. However, constitutive co-expression of AUS1 and DAN1 in a wild-type background resulted in sterol influx in aerobiosis. These results suggest that the corresponding proteins may act synergistically in vivo to promote sterol uptake.