Influence of essential inorganic elements on flavour formation during yeast fermentation.

The relative concentration of available inorganic elements is critical for yeast growth and metabolism and has potential to be a tool leading to directed yeast flavour formation during fermentation. This study investigates the influence of essential inorganic elements during alcoholic fermentation of brewers wort, fermented using three independent yeast strains, Saccharomyces pastorianus W34/70, and Saccharomyces cerevisiae strains M2 and NCYC2592 under a range of conditions replicated for each yeast strain. 10 treatments were applied: 1 control and 9 inorganic supplementations: standard brewers wort, ammonia-nitrogen, inorganic phosphate, potassium, magnesium, copper, zinc, iron, manganese and a composite mixture, Twenty-five chemical markers were evaluated by HPLC (ethanol, glycerol), and GC-MS (aroma). There was a significant change in volatile aroma compounds during fermentation, which was more prominent when supplemented with ammonia nitrogen, inorganic phosphate, potassium or magnesium (P < 0.05). Heavy metal ions mostly had a negative effect on the flavour formation.

Closely related budding yeast species respond to different ecological signals for spore activation.

Spore activation is one of the most important developmental decisions in fungi as it initiates the transition from dormant and stress-resistant cells to vegetative cells. Because in many species mating follows spore activation and germination, signals that trigger this developmental transition can also contribute to species reproductive barriers. Here, we examine the biochemical signals triggering spore activation in a natural species complex of budding yeast, Saccharomyces paradoxus (lineages SpA, SpB, SpC and SpC*). We first demonstrate that we can quantitatively monitor spore activation in these closely related lineages. Second, we dissect the composition of culture media to identify components necessary and/or sufficient to activate spores in the four lineages. We show that, contrary to expectation, glucose is necessary but not sufficient to trigger spore activation. We also show that two of the North American lineages (SpC and SpC*) diverge from the other North American (SpB) and European (SpA) lineages in terms of germination signal as their spore activation requires inorganic phosphate. Our results show that the way budding yeast interpret environmental conditions during spore activation diverged among closely related and incipient species, which means that it may play a role in their ecological differentiation and reproductive isolation. TAKE AWAY: Sensing of multiple compounds allows spore activation in non-domesticated budding yeast. Spore activation cues differ among Saccharomyces paradoxus lineages. Dextrose and phosphate signal activation in SpC and SpC* spores.

Compromised chitin synthesis in lager yeast affects its Congo red resistance and release of mannoproteins from the cells.

A mutant lager strain resistant to the cell wall-perturbing agent Congo red (CR) was isolated and the genetic alterations underlying CR resistance were investigated by whole genome sequencing. The parental lager strain was found to contain three distinct Saccharomyces cerevisiae (Sc)-type CHS6 (CHitin Synthase-related 6) alleles, two of which have one or two nonsense mutations in the open reading frame, leaving only one functional allele, whereas the functional allele was missing in the isolated CR-resistant strain. On the other hand, the Saccharomyces eubayanus-type CHS6 alleles shared by both the parental and mutant strains appeared to contribute poorly to chitin synthase-activating function. Therefore, the CR resistance of the mutant strain was attributable to the overall compromised activity of CHS6 gene products. The CR-resistant mutant cells exhibited less chitin production on the cell surface and smaller amounts of mannoprotein release into the medium. All these traits, in addition to the CR resistance, were complemented by the functional ScCHS6 gene. It is of great interest whether the frequent nonsense mutations found in ScCHS6 open reading frame in lager yeast strains are a consequence of the domestication process of lager yeast.

Elevated minimum inhibitory concentrations to antifungal drugs prevail in 14 rare species of candidemia-causing Saccharomycotina yeasts.

Antifungal susceptibility profiles of rare Saccharomycotina yeasts remain missing, even though an increase in prevalence of such rare Candida species was reported in candidemia. Majority of these rare yeast species carry intrinsic resistances against at least one antifungal compound. Some species are known to be cross-resistant (against multiple drugs of the same drug class) or even multi-drug resistant (against multiple drugs of different drug classes). We performed antifungal susceptibility testing (AFST) according to EUCAST broth microdilution for 14 rare species (Clavispora lusitaniae, Candida intermedia, Candida auris, Diutina rugosa, Wickerhamiella pararugosa, Yarrowia lipolytica, Pichia norvegensis, Candida nivariensis, Kluyveromyces marxianus, Wickerhamomyces anomalus, Candida palmioleophila, Meyerozyma guilliermondii, Meyerozyma caribbica, and Debaryomyces hansenii) known to cause candidemia. In total, 234 isolates were tested for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, micafungin, and caspofungin. Amphothericin B had the broadest efficiency against the 14 tested rare yeast species, while high minimum inhibitory concentrations (MICs) against azole drugs and echinocandins were common. Voriconazole was the most efficient azole drug. Multidrug resistance was observed for the species C. auris and K. marxianus. Multidrug resistant individual isolates were found for Y. lipolytica and M. caribbica. In conclusion, the observed high MIC values of the rare Saccharomycotina species tested limit antifungal treatment options, complicating the management of such infections.

Effect of sulfite addition and pied de cuve inoculation on the microbial communities and sensory profiles of Chardonnay wines: dominance of indigenous Saccharomyces uvarum at a commercial winery.

The microbial consortium of wine fermentations is highly dependent upon winemaking decisions made at crush, including the decision to inoculate and the decision to add sulfur dioxide (SO2) to the must. To investigate this, Chardonnay grape juice was subjected to two inoculation treatments (uninoculated and pied de cuve inoculation) as well as two SO2 addition concentrations (0 and 40 mg/L). The bacterial communities, fungal communities and Saccharomyces populations were monitored throughout fermentation using culture-dependent and culture-independent techniques. After fermentation, the wines were evaluated by a panel of experts. When no SO2 was added, the wines underwent alcoholic fermentation and malolactic fermentation simultaneously. Tatumella bacteria were present in significant numbers, but only in the fermentations to which no SO2 was added, and were likely responsible for the malolactic fermentation observed in these treatments. All fermentations were dominated by a genetically diverse indigenous population of Saccharomyces uvarum, the highest diversity of S. uvarum strains to be identified to date; 150 unique strains were identified, with differences in strain composition as a result of SO2 addition. This is the first report of indigenous S. uvarum strains dominating and completing fermentations at a commercial winery in North America.

Survival and stability of Lactobacillus fermentum and Wickerhamomyces anomalus strains upon lyophilisation with different cryoprotectant agents.

The stability of microorganisms along the time is important for allowing their industrial use as starter agents, improving fermentation processes. This study aimed to evaluate the survival and maintenance of the cell viability of the lactic acid bacteria Lactobacillus fermentum IAL 4541 and the yeast Wickerhamomyces anomalus IAL 4533, both isolated from wheat sourdough, after lyophilisation with different cryoprotectant and storage at room temperature along a year. Treatments involved adding control solution (S1 = 0.1% peptone water), and four cryoprotectant solutions S2 (10% sucrose), S3 (5% trehalose), S4 (10% skim milk powder) and S5 (10% skim milk powder plus 5% sodium glutamate) to the microbial cells previously of freeze drying processing. To verify the effect of lyophilisation on the number of microbial cells recovered, microbiological analyses were performed and cell viability was calculated before and after lyophilisation and regularly during a storage period of 365 days at room temperature. Viability after freeze-drying was influenced by the cryoprotectant agent employed, as well the microbial stability conferred along the storage. Differences on the microorganism response to some protectors were observed between the lactic acid bacteria and the yeast evaluated. W. anomalus was more affected by absence of cryoprotectant (S1) during freeze drying processing, but this microorganism was more stable than L. fermentum along the storage without the presence of protectant agents. For L. fermentum, S5 was the best protectant, allowing the recovering of 100% of the bacterial cells after lyophilisation and 87% of cell viability was observed after one year storage, followed by S4 (96 and 74%, respectively). S4 and S5 were the best protectant to W. anomalus (viability >80% after 1 year), but no increase in the yeast cell viability was conferred by addition of glutamate (S5) to skim milk. After 1 year of storage, trehalose was much more effective on protection of the yeast than bacteria (72% and 7% of viability, respectively). S2 was the less protectant agent among the tested, and their effectiveness was higher in L. fermentum (allowing 14% of cell recovering up to 120 days of storage) if compared to W. anomalus (25% of viability until 90 days of storage). Our results demonstrate that freeze-drying is a realistic technology for the stability and maintenance of the potential sourdough starter L. fermentum and W. anomalus for long time; however, the choice of cryoprotectant will influence the process effectiveness.

Molybdenum disulfide nanosheets loaded with chitosan and silver nanoparticles effective antifungal activities: in vitro and in vivo.

A method was developed for the liquid exfoliation of molybdenum disulfide (MoS2) from its bulk materials using ultrasound in aqueous phase with the assistance of chitosan (CS) and silver nanoparticles (Ag NPs) for effective loading of a variety of therapeutic molecules. The characterization results revealed that the as-made chemically-exfoliated MoS2 nanosheets had an average thickness of ∼10 nm. The new material, CS and Ag NPs-modified MoS2 (MoS2-CS-Ag), showed highly effective antifungal activities against Saccharomyces uvarum and Aspergillus niger. The Ag NPs-loaded MoS2 nanosheets achieved outstanding antifungal effect by inhibiting fungal growth both in vitro and in vivo. Notably, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis of spore morphological changes caused by MoS2-CS-Ag treatment reveal that it may directly cause the cell death. The result of the analysis of the application of MoS2-CS-Ag as an antifungal for fruits also demonstrated the MoS2-CS-Ag protective properties against fungi. The excellent film-forming ability of CS has been shown to contribute to the effectiveness of MoS2-CS-Ag in preserving the freshness of fruits, exhibited in four chemical quality sections: Vc, total carbohydrate, weight loss, and titratable acidity in fruit preservation application assay. The present study reports a new and exciting insight in a multi-functional drug carrier for protecting postharvest fruit.

Fungal bio-sorption potential of chromium in Norkrans liquid medium by shake flask technique.

In this study, the myco-reduction potential of fungi isolated from soil was ascertained by Norkrans shake flask experiment contaminated with chromium(VI). Fungal tolerance assay and induced tolerance training of the fungi were also carried out. Aspergillus niger, Penicillium, and Saccharomyces strains were isolated from the soil samples using culture based technique. Norkrans samples were collected and analyzed for Cr(VI) concentration using diphenylcarbazide spectrophotometric method. Penicillium strain was observed to be most effect at Cr(VI) concentrations of 16.1 and 8.1 mg L-1 since it was able to reduce Cr(VI) more than Saccharomyces strain and A. niger on day 20. Bio-sorption kinetics for this study was better described by pseudo second order model while Langmuir isotherm model fitted better to the equilibrium data. There was virtually steady increase in fungal growth for all the treatments through-out the experimental period. Significant negative correlation (p < 0.05) was observed between fungal growth and Cr(VI) reduction rate. The results from the induced tolerance training showed that Penicillium had the highest tolerance index (TI) values at 18, 20, and 25 mg L-1 concentrations of Cr(VI) compared to A. niger and Saccharomyces strain. These results demonstrated that these fungi have the potential to bio-absorb Cr(VI) and if properly harnessed, could be used in place of conventional remediation technology to clean-up the Cr(VI) contaminant in the field.

Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

Ethanol addition on inactivation of Saccharomyces pastorianus by a two-stage system with low-pressure carbon dioxide microbubbles can accelerate the cell membrane injury.

The effect of ethanol on the inactivation of Saccharomyces pastorianus by a two-stage system with low-pressure carbon dioxide microbubbles (two-stage MBCO2 ) was investigated. Zero and >5 log reductions of S. pastorianus populations suspended in physiological saline (PS) containing 0% and 10% ethanol, respectively, occurred by the two-stage MBCO2 at a mixing vessel pressure of 1 MPa and a heating coil temperature of 40°C. Conversely, the detected number of surviving S. pastorianus cells in PS containing 5% ethanol was higher in yeast and mold agar (YMA, an optimum agar) than YMA with 2.5% sodium chloride, followed by yeast nitrogen base agar (YNBA, a minimum agar). The fluorescence polarization of S. pastorianus in PS containing 5% and 10% ethanol increased similarly with exposure time in the heating coil of two-stage MBCO2 and was correlated with the surviving cell number measured in YNBA. The intracellular pH (pHin ) of S. pastorianus in PS containing 5% ethanol decreased linearly with exposure time in the heating coil of two-stage MBCO2 . Also, the pHin -lowering of S. pastorianus in PS containing 10% ethanol was drastically caused by two-stage MBCO2 at 1 min exposure time in the heating coil but then stayed constant until 5 min, agreeing with the inactivation efficiency. Therefore, ethanol in S. pastorianus suspension was suggested to accelerate the cell membrane injury caused by two-stage MBCO2 . © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:282-286, 2018.

Enhancing expression of SSU1 genes in Saccharomyces uvarum leads to an increase in sulfite tolerance and a transcriptome profile change.

Saccharomyces uvarum is a good wine yeast species that may have great potential for the future. However, sulfur tolerance of most S. uvarum strains is very poor. In addition there is still little information about the SSU1 gene of S. uvarum, which encodes a putative transporter conferring sulfite tolerance. In order to analyze the function of the SSU1 gene, two expression vectors that contained different SSU1 genes were constructed and transferred into a sulfite-tolerant S. uvarum strain, A9. Then sulfite tolerance, SO2 production, and PCR, sequencing, RT-qPCR and transcriptome analyses were used to access the function of the S. uvarum SSU1 gene. Our results illustrated that enhancing expression of the SSU1 gene can promote sulfite resistance in S. uvarum, and an insertion fragment ahead of the additional SSU1 gene, as seen in some alleles, could affect the expression of other genes and the sulfite tolerance level of S. uvarum. This is the first report on enhancing the expression of the SSU1 gene of S. uvarum.

The interactive effect of fungicide residues and yeast assimilable nitrogen on fermentation kinetics and hydrogen sulfide production during cider fermentation.

BACKGROUND: Fungicide residues on fruit may adversely affect yeast during cider fermentation, leading to sluggish or stuck fermentation or the production of hydrogen sulfide (H2 S), which is an undesirable aroma compound. This phenomenon has been studied in grape fermentation but not in apple fermentation. Low nitrogen availability, which is characteristic of apples, may further exacerbate the effects of fungicides on yeast during fermentation. The present study explored the effects of three fungicides: elemental sulfur (S0 ) (known to result in increased H2 S in wine); fenbuconazole (used in orchards but not vineyards); and fludioxonil (used in post-harvest storage of apples). RESULTS: Only S0 led to increased H2 S production. Fenbuconazole (≥0.2 mg L-1 ) resulted in a decreased fermentation rate and increased residual sugar. An interactive effect of yeast assimilable nitrogen (YAN) concentration and fenbuconazole was observed such that increasing the YAN concentration alleviated the negative effects of fenbuconazole on fermentation kinetics. CONCLUSION: Cidermakers should be aware that residual fenbuconazole (as low as 0.2 mg L-1 ) in apple juice may lead to stuck fermentation, especially when the YAN concentration is below 250 mg L-1 . These results indicate that fermentation problems attributed to low YAN may be caused or exacerbated by additional factors such as fungicide residues, which have a greater impact on fermentation performance under low YAN conditions. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29±0.29% for cysteine to 3.65±0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.

Variation in Indole-3-Acetic Acid Production by Wild Saccharomyces cerevisiae and S. paradoxus Strains from Diverse Ecological Sources and Its Effect on Growth.

Phytohormone indole-3-acetic acid (IAA) is the most common naturally occurring and most thoroughly studied plant growth regulator. Microbial synthesis of IAA has long been known. Microbial IAA biosynthesis has been proposed as possibly occurring through multiple pathways, as has been proven in plants. However, the biosynthetic pathways of IAA and the ecological roles of IAA in yeast have not been widely studied. In this study, we investigated the variation in IAA production and its effect on the growth of Saccharomyces cerevisiae and its closest relative Saccharomyces paradoxus yeasts from diverse ecological sources. We found that almost all Saccharomyces yeasts produced IAA when cultured in medium supplemented with the primary precursor of IAA, L-tryptophan (L-Trp). However, when cultured in medium without L-Trp, IAA production was only detected in three strains. Furthermore, exogenous added IAA exerted stimulatory and inhibitory effects on yeast growth. Interestingly, a negative correlation was observed between the amount of IAA production in the yeast cultures and the IAA inhibition ratio of their growth.

Lichen-forming fungus Caloplaca flavoruscens inhibits transcription factors and chromatin remodeling system in fungi.

Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus-derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and 39 extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations (IC25.5, IC25 and IC50, respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling.

Polygenic evolution of a sugar specialization trade-off in yeast.

The evolution of novel traits can involve many mutations scattered throughout the genome. Detecting and validating such a suite of alleles, particularly if they arose long ago, remains a key challenge in evolutionary genetics. Here we dissect an evolutionary trade-off of unprecedented genetic complexity between long-diverged species. When cultured in 1% glucose medium supplemented with galactose, Saccharomyces cerevisiae, but not S. bayanus or other Saccharomyces species, delayed commitment to galactose metabolism until glucose was exhausted. Promoters of seven galactose (GAL) metabolic genes from S. cerevisiae, when introduced together into S. bayanus, largely recapitulated the delay phenotype in 1% glucose-galactose medium, and most had partial effects when tested in isolation. Variation in GAL coding regions also contributed to the delay when tested individually in 1% glucose-galactose medium. When combined, S. cerevisiae GAL coding regions gave rise to profound growth defects in the S. bayanus background. In medium containing 2.5% glucose supplemented with galactose, wild-type S. cerevisiae repressed GAL gene expression and had a robust growth advantage relative to S. bayanus; transgenesis of S. cerevisiae GAL promoter alleles or GAL coding regions was sufficient for partial reconstruction of these phenotypes. S. cerevisiae GAL genes thus encode a regulatory program of slow induction and avid repression, and a fitness detriment during the glucose-galactose transition but a benefit when glucose is in excess. Together, these results make clear that genetic mapping of complex phenotypes is within reach, even in deeply diverged species.

Induced gene expression in industrial Saccharomyces pastorianus var. carlsbergensis TUM 34/70: evaluation of temperature and ethanol inducible native promoters.

Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions. Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts.

Dissecting the Genetic Basis of a Complex cis-Regulatory Adaptation.

Although single genes underlying several evolutionary adaptations have been identified, the genetic basis of complex, polygenic adaptations has been far more challenging to pinpoint. Here we report that the budding yeast Saccharomyces paradoxus has recently evolved resistance to citrinin, a naturally occurring mycotoxin. Applying a genome-wide test for selection on cis-regulation, we identified five genes involved in the citrinin response that are constitutively up-regulated in S. paradoxus. Four of these genes are necessary for resistance, and are also sufficient to increase the resistance of a sensitive strain when over-expressed. Moreover, cis-regulatory divergence in the promoters of these genes contributes to resistance, while exacting a cost in the absence of citrinin. Our results demonstrate how the subtle effects of individual regulatory elements can be combined, via natural selection, into a complex adaptation. Our approach can be applied to dissect the genetic basis of polygenic adaptations in a wide range of species.

Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation.

Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid.

Evolution of gene network activity by tuning the strength of negative-feedback regulation.

Despite the examples of protein evolution via mutations in coding sequences, we have very limited understanding on gene network evolution via changes in cis-regulatory elements. Using the galactose network as a model, here we show how the regulatory promoters of the network contribute to the evolved network activity between two yeast species. In Saccharomyces cerevisiae, we combinatorially replace all regulatory network promoters by their counterparts from Saccharomyces paradoxus, measure the resulting network inducibility profiles, and model the results. Lowering relative strength of GAL80-mediated negative feedback by replacing GAL80 promoter is necessary and sufficient to have high network inducibility levels as in S. paradoxus. This is achieved by increasing OFF-to-ON phenotypic switching rates. Competitions performed among strains with or without the GAL80 promoter replacement show strong relationships between network inducibility and fitness. Our results support the hypothesis that gene network activity can evolve by optimizing the strength of negative-feedback regulation.