Saccharomyces Cerevisiae BY4741 Research Articles

Saccharomyces cerevisiae yeast cells as a test system for assessing Zeocin toxicity

Having unique genetic machinery and a high degree of conservation with higher eukaryotes, the yeast Saccharomyces cerevisiae is recognised as a smart experimental system for studying the modes of chemical toxicity. The present study was undertaken to elucidate the changes in the intracellular redox homeostasis and key macromolecule structure following exposure to Zeocin. Cell populations of logarithmic, quiescent (Q) and non-quiescent (NQ) cells of Saccharomyces cerevisiae BY4741 were used as a model to examine the cytotoxic effect of this radiomimetic. The levels of endogenous ROS, oxidized lipids, carbonylated proteins, and glutathione were analysed after treatment with Zeocin (IC50). An increase in ROS production and respectively increased oxidative stress was detected in all three types of cell populations, with the highest degree being observed in proliferating S. cerevisiae BY4741 cells. The stress response of both proliferating and stationary phase (Q and NQ) cells to Zeocin included an overexpression of glutathione. The quiescent cells also showed very low DNA susceptibility to high Zeocin concentration (100–300 µg/ml), presented as no induced double-strand breaks (DSBs) in the macromolecule. Based on our research it could be concluded that the cellular physiological state is a critical factor determining the resistance to environmental stress with Q cells being the most robust.

Integrative Gene Expression and Metabolic Analysis Tool IgemRNA.

Genome-scale metabolic modeling is widely used to study the impact of metabolism on the phenotype of different organisms. While substrate modeling reflects the potential distribution of carbon and other chemical elements within the model, the additional use of omics data, e.g., transcriptome, has implications when researching the genotype–phenotype responses to environmental changes. Several algorithms for transcriptome analysis using genome-scale metabolic modeling have been proposed. Still, they are restricted to specific objectives and conditions and lack flexibility, have software compatibility issues, and require advanced user skills. We classified previously published algorithms, summarized transcriptome pre-processing, integration, and analysis methods, and implemented them in the newly developed transcriptome analysis tool IgemRNA, which (1) has a user-friendly graphical interface, (2) tackles compatibility issues by combining previous data input and pre-processing algorithms in MATLAB, and (3) introduces novel algorithms for the automatic comparison of different transcriptome datasets with or without Cobra Toolbox 3.0 optimization algorithms. We used publicly available transcriptome datasets from Saccharomyces cerevisiae BY4741 and H4-S47D strains for validation. We found that IgemRNA provides a means for transcriptome and environmental data validation on biochemical network topology since the biomass function varies for different phenotypes. Our tool can detect problematic reaction constraints.

Cell surface display of palladium binding peptide on Saccharomyces cerevisiae EBY100 cells using the a-agglutinin anchor system developed for the biosorption of Pd (II)

Platinum group metals (PGMs) are concentrated in the earth’s core and mantle and have low natural abundances. Due to the limited availability of PGMs; their recovery and reuse from waste solutions is economically attractive. Conventional methods used for the removal of PGMs from effluents include precipitation, ion exchange, and reverse osmosis; these methods have many shortfalls and this had led to a search for new technologies for the removal of metals from effluents, one such technology is biosorption. The primary objective of this research project was to develop a genetically engineered Saccharomyces cerevisiae EBY100 strain for the biosorption of Pd(II) in aqueous solutions. The palladium binding peptide was successfully expressed on Saccharomyces cerevisiae EBY100 cells by means of cell surface display using the a-agglutinin anchor system; this was confirmed by means of DNA sequencing, flow cytometry and fluorescence microscopy. The maximum adsorption capacity of genetically transformed Saccharomyces cerevisiae EBY100 cells for Pd(II) was 125 mg/g.

Comparative metabolite profiling of wild type and thermo-tolerant mutant of Saccharomyces cerevisiae

Simultaneous saccharification and fermentation (SSF) is a process of enzymatic saccharification and yeast fermentation used for producing lignocellulosic ethanol, and is performed at high temperature for effective enzymatic saccharification. Thermo-tolerant yeast is needed for high ethanol yield from yeast fermentation. A thermo-tolerant mutant yeast, SSK2, derived from Saccharomyces cerevisiae BY4741, has been generated previously. However, the thermo-tolerance mechanisms in SSK2 remain unclear. In this study, to investigate the change of metabolisms in thermo-tolerant mutant, metabolomics was performed based on gas chromatography/time-of-flight mass spectrometry (GC/TOF MS). In total, 91 intracellular metabolites were identified by GC/TOF MS; the metabolite profiles of BY4741 and SSK2, cultivated at 30 °C and 42 °C, were clearly discriminated by principal component analysis (PCA) and hierarchical clustering analysis (HCA). The metabolomics results indicated that the metabolism of amino acids, cell wall and membrane, and fatty acids were changed by heat stress and were key factors in yeast thermo-tolerance. In this study, metabolomics was successfully exploited to reveal the changes of metabolisms in the thermo-tolerant yeast, and these metabolic changes could be used as the clues for future development of thermo-tolerant yeasts.

Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

Functional characterization and expression profile of microsomal FAD2 and FAD3 genes involved in linoleic and α-linolenic acid production in Leucas cephalotes.

The online version contains supplementary material available at 10.1007/s12298-021-01016-z.

Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization

High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV–Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.

Anhydrobiosis in yeast: role of cortical endoplasmic reticulum protein Ist2 in Saccharomyces cerevisiae cells during dehydration and subsequent rehydration.

Two Saccharomyces cerevisiae strains, BY4741 and BY4741-derived strain lacking the IST2 gene (ist2Δ), were used to characterise the possible role of cortical endoplasmic reticulum (ER) protein Ist2 upon cell dehydration and subsequent rehydration. For the first time, we show that not only protein components of the plasma membrane (PM), but also at least one ER membrane protein (Ist2) play an important role in the maintenance of the viability of yeast cells during dehydration and subsequent rehydration. The low viability of the mutant strain ist2∆ upon dehydration-rehydration stress was related to the lack of Ist2 protein in the ER. We revealed that the PM of ist2∆ strain is not able to completely restore its molecular organisation during reactivation from the state of anhydrobiosis. As the result, the permeability of the PM remains high regardless of the type of reactivation (rapid or gradual rehydration). We conclude that ER protein Ist2 plays an important role in ensuring the stability of molecular organisation and functionality of the PM during dehydration-rehydration stress. These results indicate an important role of ER-PM interactions during cells transition into the state of anhydrobiosis and the subsequent restoration of their physiological activities.

Transcriptomic analysis of nonylphenol effect on Saccharomyces cerevisiae.

Nonylphenol (NP) is a bioaccumulative environmental estrogen that is widely used as a nonionic surfactant. We have previously examined short-term effects of NP on yeast cells using microarray technology. In the present study, we investigated the adaptive response of Saccharomyces cerevisiae BY4742 cells to NP exposure by analyzing genome-wide transcriptional profiles using RNA-sequencing. We used 2 mg/L NP concentration for 40 days of exposure. Gene expression analysis showed that a total of 948 genes were differentially expressed. Of these, 834 genes were downregulated, while 114 genes were significantly upregulated. GO enrichment analysis revealed that 369 GO terms were significantly affected by NP exposure. Further analysis showed that many of the differentially expressed genes were associated with oxidative phosphorylation, iron and copper acquisition, autophagy, pleiotropic drug resistance and cell cycle progression related processes such as DNA and mismatch repair, chromosome segregation, spindle checkpoint activity, and kinetochore organization. Overall, these results provide considerable information and a comprehensive understanding of the adaptive response to NP exposure at the gene expression level.

Evaluating Young's Modulus of Single Yeast Cells Based on Compression Using an Atomic Force Microscope with a Flat Tip.

In this research, atomic force microscopy (AFM) with a flat tip cantilever is utilized to measure Young's modulus of a whole yeast cell (Saccharomyces cerevisiae BY4741). The results acquired from AFM are similar to those obtained using a microfluidic chip compression system. The mechanical properties of single yeast cells are important parameters which can be examined using AFM. Conventional studies apply AFM with a sharp cantilever tip to indent the cell and measure the force-indentation curve, from which Young's modulus can be calculated. However, sharp tips introduce problems because the shape variation can lead to a different result and cannot represent the stiffness of the whole cell. It can lead to a lack of broader meaning when evaluating Young's modulus of yeast cells. In this report, we confirm the differences in results obtained when measuring the compression of a poly(dimethylsiloxane) bead using a commercial sharp tip versus a unique flat tip. The flat tip effectively avoids tip-derived errors, so we use this method to compress whole yeast cells and generate a force–deformation curve. We believe our proposed method is effective for evaluating Young's modulus of whole yeast cells.

Antioxidant or pro-oxidant? The effects of boron compounds on Saccharomyces cerevisiae BY4741 strain

Boron is one of the most important elements with its indisputable biological importance and widespread use. The most studied derivatives of the boron element are boric acid and its salts. In this article, we searched the effects of boric acid and its lithium salt, lithium metaborate, on enzymatic defense system, cell damage, and cell surface morphology of Saccharomyces cerevisiae BY4741 strain. It was found that while all studied concentrations of boric acid showed toxicity against the yeast, even the highest studied concentration of lithium metaborate could not effectively inhibit cell viability. In addition, we observed reverse effect of lithium metaborate depend on its concentration on yeast cell proliferation and metabolic activity. As a defense mechanism, superoxide dismutase and glutathione S-transferase activities were significantly induced in yeast cells treated with boric acid. But these inductions could not protect cells from boric acid induced lipid peroxidation. It was determined that glutathione S-transferase was the only enzyme induced after lithium metaborate treatment. Finally, we visualized the signs of features of necrotic and early apoptotic mechanisms in yeast cells treated with boric acid and lithium metaborate, respectively, which should be investigated with further studies.

Engineering of membrane phospholipid component enhances salt stress tolerance in Saccharomyces cerevisiae.

To increase the growth of industrial strains under environmental stress, the Saccharomyces cerevisiae BY4741 salt-tolerant strain Y00 that tolerates 1.2 M NaCl was cultured through nitroguanidine mutagenesis. The metabolomics and transcription data of Y00 were compared with those of the wild-type strain BY4741. The comparison identified two genes related to salt stress tolerance, cds1 and cho1. Modular assembly of cds1 and cho1 redistributed the membrane phospholipid component and decreased the ratio of anionic-to-zwitterionic phospholipid in strain Y03 that showed the highest salt tolerance. Therefore, significantly increased membrane potential and membrane integrity helped strain Y03 to resist salt stress (1.2 M NaCl). This study provides an effective membrane engineering strategy to enhance salt stress tolerance.

Expression of flagellin at yeast surface increases biocontrol efficiency of yeast cells against postharvest disease of tomato caused by Botrytis cinerea

Abstract In this study, bacterial flagellin was expressed at the cell surface of Saccharomyces cerevisiae EBY100 via yeast surface display system. The effect of the yeast on controlling the postharvest decay of tomato fruit caused by Botrytis cinerea and the possible mechanisms were investigated. The results showed that the yeast expressing flagellin at cell surface could significantly induce disease resistance against B. cinerea in tomato wounds. The genes involved in biosynthesis of salicylic acid and jasmonic acid, and plant defense were markedly up-regulated in tomato wounds by the yeast strain expressing flagellin. In addition, application of the yeast strain significantly induced the superoxide radical generation in tobacco leaves and tomato fruit. These findings suggest that expressing flagellin at yeast cell surface may be an effective strategy to increase the biocontrol efficiency of antagonist yeast against disease in postharvest fruit.

The Anti-Aging Potential of Neohesperidin and Its Synergistic Effects with Other Citrus Flavonoids in Extending Chronological Lifespan of Saccharomyces Cerevisiae BY4742.

The anti-aging activity of many plant flavonoids, as well as their mechanisms of action, have been explored in the current literature. However, the studies on the synergistic effects between the different flavonoid compounds were quite limited in previous reports. In this study, by using a high throughput assay, we tested the synergistic effects between different citrus flavonoids throughout the yeast’s chronological lifespan (CLS). We studied the effect of four flavonoid compounds including naringin, hesperedin, hesperitin, neohesperidin, as well as their different combinations on the CLS of the yeast strain BY4742. Their ROS scavenging ability, in vitro antioxidant activity and the influence on the extracellular pH were also tested. The results showed that neohesperidin extended the yeast’s CLS in a concentration-dependent manner. Especially, we found that neohesperidin showed great potential in extending CLS of budding yeast individually or synergistically with hesperetin. The neohesperidin exhibited the strongest function in decreasing the reactive oxygen species (ROS) accumulation in yeast. These findings clearly indicated that neohesperidin is potentially an anti-aging citrus flavonoid, and its synergistic effect with other flavonoids on yeast’s CLS will be an interesting subject for future research of the anti-aging function of citrus fruits.

Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides.

Three nickel-binding peptides were screened from a phage peptide library and displayed separately or in combination with surface-engineered Saccharomyces cerevisiae EBY100. The sorption of nickel ions on the surface of yeast cells increased with the increasing number of nickel Ni(II)-binding peptides displayed. The combined expression of the three peptides by EBY100/pYD1-N123 demonstrated the highest sorption of Ni(II) (2.603 ± 0.004 g g−1, dry weight) and an enhanced sorption capacity of 60.15%, compared to S. cerevisiae EBY100. An orthogonal test for yeast immobilization was designed. A maximum sorption capability of 68.62% was observed for a treatment at 25°C with 2.0% calcium chloride and 3.0% sodium alginate.

Identification and characterization of putative Aeromonas spp. T3SS effectors.

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate Aeromonas gene families are initially selected using Hidden Markov Model (HMM) profile searches against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. This approach identified 21 Aeromonas T3SS likely effector families, of which 8 represent known or characterized effectors, while the remaining 13 have not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.

Optical and impedimetric study of genetically modified cells for diclofenac sensing

Abstract. Whole-cell biosensors, based on genetically modified yeast cells, were employed to detect anthropogenic micropollutants (e.g. drugs). Specific stimuli, e.g. traces of drugs, lead to the induction of fluorescence in the respective cells. Receptors of the cells detect specific signal molecules and induce the formation of fluorescent proteins. In this work, genetically modified cells of the yeast Saccharomyces cerevisiae BY4741 were confined in a four-chamber microfluidic cell, providing an optical monitoring of the cell behaviour and their supply with the nutrients. The measurements of the time-dependent fluorescence intensity were performed with different concentrations of the drug diclofenac, and the sensitivity of yeast cells to diclofenac was demonstrated. Cell viability was monitored by simultaneous impedance recording.

Sacharomyces cerevisiae as a screening tool to assess cathinones’ toxicity

Introduction: Novel psychoactive substances (NPS) are sold on Internet under the appearance of herbal blends or bath salts as an alternative to cannabis, khat and ephedra. Synthetic cathinones are artificial mimetics of khat and like methamphetamine are indirect agonists of dopamine, serotonin and noradrenaline receptors [1]. Ninety-seven fatal cases of intoxication by ingestion of mephedrone (4-methylmethcathinone), one of the types of synthetic cathinone derivatives, were reported on literature since 2010 [2]. New synthetic cathinones emerges in the street market and internet on a regular base and the properties of this substances change regularly, due to modification on the molecules, to avoid legislation. This makes almost impossible to characterize synthetic cathinones toxicological profiles on a short time scale, mostly due to the typical time-consuming experimental assays. In here we propose the use of Saccharomyces cerevisiae, which has been proven an invaluable model to study the fundamental molecular mechanisms involved in several human diseases [3], growth curves in the presence of synthetic cathinones to get an indicative measure of its toxicity. Materials and methods: BY4741 yeast cells were diluted to a standardized OD600 nm =2×10−3 (∼1.67×10-5 cells/mL) in YNB medium (Yeast Nitrogen Base w/o Amino Acids 0.67%, glucose 2%, required amino acids) and grown at 30 °C. Buphedrone was acquired from the best source available. The cytotoxic potential of synthetic cathinones was assessed by monitoring the growth of Saccharomyces cerevisiae BY4741 in the presence and absence of substances. Yeast growth curves (O.D.600nm versus growth time) were compared. Results: Figure 1 shows that yeast growth in the presence of 75 mM cat is lower when compared to the growth curve in the absence of the substance. These preliminary results points that cathinones inhibits yeast growth. Figure 1. Growth curves results in Saccharomyces cerevisiae BY4741 in the presence of 75 mM of buphedrone. Discussion and conclusions: Saccharomyces cerevisiae has been used previously to test the toxicity of pharmacological drugs. Our results show that yeast is sensitive to cathinones suggesting that this system has potential to assess the possible toxicity of these legal highs. This rapid and inexpensive methodology may allow high throughput screening of cathinones’ toxicity, which might help the legislators to schedule NPS avoiding expected public health problems.

Expression and enzymatic characterization of rice α-galactosidase II displayed on yeast cell surface

Abstract A yeast (Saccharomyces cerevisiae EBY100) surface displayed rice α-galactosidase II (YSD rice α-Gal II) was constructed. The optimum temperature and pH of YSD rice α-Gal II were 45 °C and 4.0–5.5, respectively. Ascorbic acid significantly increased the expression level of YSD rice α-Gal II by 5.7-fold, compared to control, indicating its importance in the production and application of YSD rice α-Gal II. Meanwhile, protective agents including Fe3+, EDTA, urea, and l -arginine also enhanced YSD rice α-Gal II activity by a certain extent (15–78%). Furthermore, YSD rice α-Gal II exhibited higher thermal, pH, and storage stability, and remarkable hydrolytic activity toward defatted soybean meal and guar gum. Hence, YSD rice α-Gal II has great potential for degradation of the non-digestible and flatulence-causing oligo- or poly-saccharides and could be a commendable candidate for extensive application in food and feed industries.

Heterologous Biosynthesis of the Fungal Sesquiterpene Trichodermol in Saccharomyces cerevisiae

Trichodermol, a fungal sesquiterpene derived from the farnesyl diphosphate pathway, is the biosynthetic precursor for trichodermin, a member of the trichothecene class of fungal toxins produced mainly by the genera of Trichoderma and Fusarium. Trichodermin is a promising candidate for the development of fungicides and antitumor agents due to its significant antifungal and cytotoxic effects. It can also serve as a scaffold to generate new congeners for structure-activity relationship (SAR) study. We reconstructed the biosynthetic pathway of trichodermol in Saccharomyces cerevisiae BY4741, and investigated the effect of produced trichodermol on the host by de novo RNA sequencing (RNA-Seq) and quantitative Real-time PCR analyses. Co-expression of pESC::FgTRI5 using plasmid pLLeu-tHMGR-UPC2.1 led to trichodiene production of 683 μg L-1, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L-1. Subsequent expression of the codon-optimized cytochrome P450 monooxygenase encoding genes, TaTRI4 and TaTRI11, resulted in trichodermol, with an estimated titer of 252 μg L-1 at shake flask level. RNA-Seq and qPCR analyses revealed that the produced trichodermol downregulated the expression of the genes involved in ergosterol biosynthesis, but significantly upregulated the expression of PDR5 related to membrane transport pathway in S. cerevisiae. Collectively, we achieved the first heterologous biosynthesis of trichodermol by reconstructing its biosynthetic pathway in yeast, and the reconstructed pathway will serve as a platform to generate trichodermin analogs as potential candidates for agrochemicals and anticancer agents through further optimizations.