Growth Of Yeast Cells Research Articles

Biochemical Characterization of Recombinant Enterococcus faecalis EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against Candida albicans.

Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV88-His6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV88-His6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV88-His6, we revealed that EntV88-His6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV88-His6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro. The combination of EntV88-His6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast

BackgroundHuman hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication–coupled epigenetic inheritance process, and chromatin stability.ResultsBy comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability.ConclusionOur findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.

Separate hydrolysis and fermentation of softwood bark pretreated with 2-naphthol by steam explosion

Background2-Naphthol, a carbocation scavenger, is known to mitigate lignin condensation during the acidic processing of lignocellulosic biomass, which may benefit downstream processing of the resulting materials. Consequently, various raw materials have demonstrated improved enzymatic saccharification yields for substrates pretreated through autohydrolysis and dilute acid hydrolysis in the presence of 2-naphthol. However, 2-naphthol is toxic to ethanol-producing organisms, which may hinder its potential application. Little is known about the implications of 2-naphthol in combination with the pretreatment of softwood bark during continuous steam explosion in an industrially scalable system.ResultsThe 2-naphthol-pretreated softwood bark was examined through spectroscopic techniques and subjected to separate hydrolysis and fermentation along with a reference excluding the scavenger and a detoxified sample washed with ethanol. The extractions of the pretreated materials with water resulted in a lower aromatic content in the extracts and stronger FTIR signals, possibly related to guaiacyl lignin, in the nonextractable residue when 2-naphthol was used during pretreatment. In addition, cyclohexane/acetone (9:1) extraction revealed the presence of pristine 2-naphthol in the extracts and increased aromatic content of the nonextractable residue detectable by NMR for the scavenger-pretreated materials. Whole-slurry enzymatic saccharification at 12% solids loading revealed that elevated saccharification recoveries after 48 h could not be achieved with the help of the scavenger. Glucose concentrations of 16.9 (reference) and 15.8 g/l (2-naphthol) could be obtained after 48 h of hydrolysis. However, increased inhibition during fermentation of the scavenger-pretreated hydrolysate, indicated by yeast cell growth, was slight and could be entirely overcome by the detoxification stage. The ethanol yields from fermentable sugars after 24 h were 0.45 (reference), 0.45 (2-naphthol), and 0.49 g/g (2-naphthol, detoxified).ConclusionThe carbocation scavenger 2-naphthol did not increase the saccharification yield of softwood bark pretreated in an industrially scalable system for continuous steam explosion. On the other hand, it was shown that the scavenger's inhibitory effects on fermenting microorganisms can be overcome by controlling the pretreatment conditions to avoid cross-inhibition or detoxifying the substrates through ethanol washing. This study underlines the need to jointly optimize all the main processing steps.

Ethanol removal by vacuum-assisted gas stripping: Influence of operating conditions

One way to overcome the inhibitory effects caused by ethanol on yeast cell growth is the use of extractive fermentation, whereby ethanol is removed from the fermentation broth as it is produced. The present work investigates ethanol removal from solution by vacuum-assisted gas stripping, which is a promising method for enhancing performance, compared to conventional gas stripping. Evaluation was made of the effects of carbon dioxide flow rate (ϕCO2), temperature (T), and pressure (P) on ethanol removal performance. Bench-scale assays were performed using a 2-L bubble column containing 10 % v/v ethanol solution, with monitoring of the gas and liquid phases by FT-MIR spectroscopy. The ethanol entrainment factor (FE) increased with ϕCO2 and temperature, but decreased at higher pressure. Only ϕCO2 had significant and positive effects on the concentration factor (FC) and selectivity (αE/W), within the operating ranges of the variables studied. An ethanol removal model was obtained that provided an accurate description of the process behavior, with good agreement between the experimental and simulated data. In addition, the energy requirement for ethanol removal by vacuum-assisted gas stripping in the bioreactor was lower than for the conventional stripping process.

Magnetic field platform for experiments on well-mixed and spatially structured microbial populations

Magnetic fields have been shown to affect sensing, migration, and navigation in living organisms. However, the effects of magnetic fields on microorganisms largely remain to be elucidated. We develop an open-source, 3D-printed magnetic field exposure device to perform experiments on well-mixed and spatially structured microbial populations. This device is designed in AutoCAD, modeled in COMSOL, and validated using a Gaussmeter and experiments on the budding yeast Saccharomyces cerevisiae. We find that static magnetic field exposure slows the spatially structured expansion of yeast mats that expand in two dimensions, but not yeast mats that expand in three dimensions, across the surface of semi-solid yeast extract-peptone-dextrose agar media. We also find that magnetic fields do not affect the growth of planktonic yeast cells in well-mixed liquid yeast extract-peptone-dextrose media. This study provides an adaptable device for performing controlled magnetic field experiments on microbes and advances our understanding of the effects of magnetic fields on fungi.

The role of ion homeostasis in adaptation and tolerance to acetic acid stress in yeasts.

Maintenance of asymmetric ion concentrations across cellular membranes is crucial for proper yeast cellular function. Disruptions of these ionic gradients can significantly impact membrane electrochemical potential and the balance of other ions, particularly under stressful conditions such as exposure to acetic acid. This weak acid, ubiquitous to both yeast metabolism and industrial processes, is a major inhibitor of yeast cell growth in industrial settings and a key determinant of host colonization by pathogenic yeast. Acetic acid toxicity depends on medium composition, especially on the pH (H+ concentration), but also on other ions' concentrations. Regulation of ion fluxes is essential for effective yeast response and adaptation to acetic acid stress. However, the intricate interplay among ion balancing systems and stress response mechanisms still presents significant knowledge gaps. This review offers a comprehensive overview of the mechanisms governing ion homeostasis, including H+, K+, Zn2+, Fe2+/3+, and acetate, in the context of acetic acid toxicity, adaptation, and tolerance. While focus is given on Saccharomyces cerevisiae due to its extensive physiological characterization, insights are also provided for biotechnologically and clinically relevant yeast species whenever available.

Regulation of the PFK1 gene on the interspecies microbial competition behavior of Saccharomyces cerevisiae

Saccharomyces cerevisiae is a widely used strain for ethanol fermentation; meanwhile, efficient utilization of glucose could effectively promote ethanol production. The PFK1 gene is a key gene for intracellular glucose metabolism in S. cerevisiae. Our previous work suggested that although deletion of the PFK1 gene could confer higher oxidative tolerance to S. cerevisiae cells, the PFK1Δ strain was prone to contamination by other microorganisms. High interspecies microbial competition ability is vital for the growth and survival of microorganisms in co-cultures. The result of our previous studies hinted us a reasonable logic that the EMP (i.e., the Embden-Meyerhof-Parnas pathway, the glycolytic pathway) key gene PFK1 could be involved in regulating interspecies competitiveness of S. cerevisiae through the regulation of glucose utilization and ethanol production efficiency. The results suggest that under 2% and 5% glucose, the PFK1Δ strain showed slower growth than the S288c wild-type and TDH1Δ strains in the lag and exponential growth stages, but realized higher growth in the stationary stage. However, relative high supplement of glucose (10%) eliminated this phenomenon, suggesting the importance of glucose in the regulation of PFK1 in yeast cell growth. Furthermore, during the lag growth phase, the PFK1Δ strain displayed a decelerated glucose consumption rate (P < 0.05). The expression levels of the HXT2, HXT5, and HXT6 genes decreased by approximately 0.5-fold (P < 0.05) and the expression level of the ZWF1 exhibited a onefold increase in the PFK1Δ strain compared to that in the S. cerevisiae S288c wild-type strain (P < 0.05).These findings suggested that the PFK1 inhibited the uptake and utilization of intracellular glucose by yeast cells, resulting in a higher amount of residual glucose in the medium for the PFK1Δ strain to utilize for growth during the reverse overshoot stage in the stationary phase. The results presented here also indicated the potential of ethanol as a defensive weapon against S. cerevisiae. The lower ethanol yield in the early stage of the PFK1Δ strain (P < 0.001) and the decreased expression levels of the PDC5 and PDC6 (P < 0.05), which led to slower growth, resulted in the strain being less competitive than the wild-type strain when co-cultured with Escherichia coli. The lower interspecies competitiveness of the PFK1Δ strain further promoted the growth of co-cultured E. coli, which in turn activated the ethanol production efficiency of the PFK1Δ strain to antagonize it from E. coli at the stationary stage. The results presented clarified the regulation of the PFK1 gene on the growth and interspecies microbial competition behavior of S. cerevisiae and would help us to understand the microbial interactions between S. cerevisiae and other microorganisms.Key points• PFK1Δ strain could realize reverse growth overshoot at the stationary stage• PFK1 deletion decreased ethanol yield and interspecific competitiveness• Proportion of E. coli in co-culture affected ethanol yield capacity of yeast cells

A Matlab-based application for quantification of yeast cell growth on solid media.

Quantitative assessment of growth and survival is a suitable technique in studying biochemical, genetic and physiological processes in the cells. The budding yeast Saccharomyces cerevisiae is one of the most widely used eukaryotic model organisms for studying cellular mechanisms and processes in evolutionarily distant species, including humans. Yeast growth can be evaluated on both liquid and solid media by measuring cell suspension turbidity and colony forming units, respectively. Several software tools utilizing different parameters have been proposed to quantify yeast growth on solid media. Here, we developed a Matlab-based application which provides a rapid and robust quantitative yeast growth analysis from spot plating assay. Spot plating assay is a typical procedure to evaluate yeast growth in low-throughput laboratory settings, including growth on different nutrient sources or treatment with specific stressors. The app has a one-step installation process, a self-explanatory interface and shorter analysis steps compared with previous established methods, providing a useful tool for both expert and non-expert yeast researchers.

Adsorption of Zearalenone by Aureobasidium pullulans Autolyzed Biomass Preparation and Its Detoxification Properties in Cultures of Saccharomyces cerevisiae Yeast.

Different preventive strategies are needed to minimize the intake risks of mycotoxins, including zearalenone (ZEN). The aim of this study was to determine the ZEN adsorption ability of an autolyzed biomass preparation of polymorphic yeast Aureobasidium pullulans A.p.-3. The evaluation of the antitoxic properties of the preparation was also performed in relation to Saccharomyces cerevisiae yeast (ATCC 2366, ATCC 7090 and ATCC 9763) used as a model cell exposed to a toxic ZEN dose. The preparation at a dose of 5 mg/mL showed the adsorption of ZEN present in model systems at concentrations between 1 μg/mL to 100 μg/mL. The highest degree of adsorption was established for ZEN concentrations of 1 μg/mL and 5 μg/mL, becoming limited at higher doses of the toxin. Based on the Langmuir model of adsorption isotherms, the predicted maximum ZEN adsorption was approx. 190 µg/mL, regardless of pH. The growth of three strains of S. cerevisiae yeast cells in the medium with ZEN at concentrations within the range of 1.56 μg/mL-100 μg/mL was analyzed to determine the minimum inhibitory concentration. The growth of all tested strains was especially limited by high doses of ZEN, i.e., 50 and 100 μg/mL. The protective effect of the tested preparation was noted in relation to yeast cells exposed to toxic 100 μg/mL ZEN doses. The highest yeast cell growth (app. 36% percentage) was noted for a S. cerevisiae ATCC 9763 strain compared to the medium with ZEN but without preparation. More detailed tests determining the antitoxic mechanisms of the A. pullulans preparation are planned in the future, including cell culture bioassays and animal digestive tract models.

Metabolomic analysis of hydroxycinnamic acid inhibition on Saccharomyces cerevisiae.

Ferulic acid (FA) and p-coumaric acid (p-CA) are hydroxycinnamic acid inhibitors that are mainly produced during the pretreatment of lignocellulose. To date, the inhibitory mechanism of hydroxycinnamic acid compounds on Saccharomyces cerevisiae has not been fully elucidated. In this study, liquid chromatography-mass spectrometry (LC-MS) and scanning electron microscopy (SEM) were used to investigate the changes in S. cerevisiae cells treated with FA and p-CA. In this experiment, the control group was denoted as group CK, the FA-treated group was denoted as group F, and the p-CA-treated group was denoted as group P. One hundred different metabolites in group F and group CK and 92 different metabolites in group P and group CK were selected and introduced to metaboanalyst, respectively. A total of 38 metabolic pathways were enriched in S. cerevisiae under FA stress, and 27 metabolic pathways were enriched in S. cerevisiae under p-CA stress as identified through Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis. The differential metabolites involved included S-adenosine methionine, L-arginine, and cysteine, which were significantly downregulated, and acetyl-CoA, L-glutamic acid, and L-threonine, which were significantly upregulated. Analysis of differential metabolic pathways showed that the differentially expressed metabolites were mainly related to amino acid metabolism, nucleotide metabolism, fatty acid degradation, and the tricarboxylic acid cycle (TCA). Under the stress of FA and p-CA, the metabolism of some amino acids was blocked, which disturbed the redox balance in the cells and destroyed the synthesis of most proteins, which was the main reason for the inhibition of yeast cell growth. This study provided a strong scientific reference to improve the durability of S. cerevisiae against hydroxycinnamic acid inhibitors. KEY POINTS: • Morphological changes of S. cerevisiae cells under inhibitors stress were observed. • Changes of the metabolites in S. cerevisiae cells were explored by metabolomics. • One of the inhibitory effects on yeast is due to changes in the metabolic network.

A kinetic model of iron trafficking in growing Saccharomyces cerevisiae cells; applying mathematical methods to minimize the problem of sparse data and generate viable autoregulatory mechanisms.

Iron is an essential transition metal for all eukaryotic cells, and its trafficking throughout the cell is highly regulated. However, the overall cellular mechanism of regulation is poorly understood despite knowing many of the molecular players involved. Here, an ordinary-differential-equations (ODE) based kinetic model of iron trafficking within a growing yeast cell was developed that included autoregulation. The 9-reaction 8-component in-silico cell model was solved under both steady-state and time-dependent dynamical conditions. The ODE for each component included a dilution term due to cell growth. Conserved rate relationships were obtained from the null space of the stoichiometric matrix, and the reduced-row-echelon-form was used to distinguish independent from dependent rates. Independent rates were determined from experimentally estimated component concentrations, cell growth rates, and the literature. Simple rate-law expressions were assumed, allowing rate-constants for each reaction to be estimated. Continuous Heaviside logistical functions were used to regulate rate-constants. These functions acted like valves, opening or closing depending on component "sensor" concentrations. Two cellular regulatory mechanisms were selected from 134,217,728 possibilities using a novel approach involving 6 mathematically-defined filters. Three cellular states were analyzed including healthy wild-type cells, iron-deficient wild-type cells, and a frataxin-deficient strain of cells characterizing the disease Friedreich's Ataxia. The model was stable toward limited perturbations, as determined by the eigenvalues of Jacobian matrices. Autoregulation allowed healthy cells to transition to the diseased state when triggered by a mutation in frataxin, and to the iron-deficient state when cells are placed in iron-deficient growth medium. The in-silico phenotypes observed during these transitions were similar to those observed experimentally. The model also predicted the observed effects of hypoxia on the diseased condition. A similar approach could be used to solve ODE-based kinetic models associated with other biochemical processes operating within growing cells.

STUDY OF THE PROLIFERATIVE ACTIVITY OF SACCHAROMYCES CEREVISIAE IN YEAST BIOTECHNOLOGY AND PHYSICO-CHEMICAL METHODS OF ITS DETERMINATION

The article discusses the solution of issues related to increasing the efficiency of technological processes and ensuring the release of high-quality finished products, relevant for production based on the use of S. cerevisiae yeast. The paper identifies major reasons for the need to regulate yeast culture metabolism, considers existing practice and proposes methods of changing its metabolic activity. The purpose of the research is to create the classification of the supplements / preparations from different origin to improve yeast vital activity by adjusting its culture medium composition with the use of complex hierarchical faceted method.&#x0D; The proliferative processes taking place in the yeast cell are quite easily regulated. Therefore, knowing the dependence between the specific conditions of the environment and certain aspects of the yeast culture's vital activity, it is possible to purposefully change its growth, development and metabolism. Creating and maintaining certain conditions for the cultivation of yeast allows you to control the course of the fermentation process.&#x0D; The influence of biologically active substances on the proliferative activity of S. cerevisiae cells was investigated. Concentrations were determined and the regulatory effect of succinic and folic acid preparations on the proliferative activity of yeast cells was proven. A comparative analysis of influence of different concentrations of exogenous substances on microorganisms culture growth was carried out. The most optimal concentrations of drugs for activation of yeast cell division were determined: succinic acid/ascorbic acid – 0.0015/0.0002 mg/ml; folic acid – 0.006 mg/ml.&#x0D; Specific growth rate of yeast during cultivation in 20 % sucrose solution with succinic/ascorbic and folic acid preparations was calculated. It was established that the highest specific rate of cell growth was observed in the complex preparation, which included three organic acids. It was established that the specific rate of yeast reproduction in the complex preparation increased by an average of 30–35 % relative to the control sample. Increase in specific growth rate by 20–25 % of yeast was also observed in the medium with folic acid, the addition of succinic/ascorbic acids did not significantly increase specific rate (by approximately 10 %) of the growth of the S. cerevisiae yeast culture. It has been established that biologically active substances have certain concentrations that activate cell division and contribute to their active development and critical limits at which the development and growth of yeast cells are inhibited.

Spontaneous Mutations in Saccharomyces cerevisiae mtDNA Increase Cell-to-Cell Variation in mtDNA Amount.

In a eukaryotic cell, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is usually maintained within a specific range. This suggests the presence of a negative feedback loop mechanism preventing extensive mtDNA replication and depletion. However, the experimental data on this hypothetical mechanism are limited. In this study, we suggested that deletions in mtDNA, known to increase mtDNA abundance, can disrupt this mechanism, and thus, increase cell-to-cell variance in the mtDNA copy numbers. To test this, we generated Saccharomyces cerevisiae rho- strains with large deletions in the mtDNA and rho0 strains depleted of mtDNA. Given that mtDNA contributes to the total DNA content of exponentially growing yeast cells, we showed that it can be quantified in individual cells by flow cytometry using the DNA-intercalating fluorescent dye SYTOX green. We found that the rho- mutations increased both the levels and cell-to-cell heterogeneity in the total DNA content of G1 and G2/M yeast cells, with no association with the cell size. Furthermore, the depletion of mtDNA in both the rho+ and rho- strains significantly decreased the SYTOX green signal variance. The high cell-to-cell heterogeneity of the mtDNA amount in the rho- strains suggests that mtDNA copy number regulation relies on full-length mtDNA, whereas the rho- mtDNAs partially escape this regulation.

Evolution of the Tóxicos en Levadura 63 (TL63) gene family in plants and functional characterization of Arabidopsis thaliana TL63 under oxidative stress.

TL63 orthologs were angiosperm specific and had undergone motifs loss and gain, and increased purifying selection. AtTL63 was involved in the response of yeast and Arabidopsis plants to oxidative stress. The Tóxicos en Levadura (TL) family, a class of E3 ubiquitin ligases with typical RING-H2 type zinc finger structure, plays a pivotal role in mediating physiological processes and responding to stress in plants. However, the evolution and function of TL63 remain unclear. In this study, TL63 homologs were dated roughly back to the origin of land plants and confirmed to have subjected to the gain and loss of motifs and increased purifying selection. Phylogenetic analysis displayed that 279 TL63s could be divided into four main clades (Clade A-D). Notably, the ancestral tandem TL40/41 cluster contributed to the expansion of modern Brassicaceae TL40/41. The substitution rate tests revealed that the TL63 lineage was evidently different from other lineages. The codon usage index exhibited that monocotyledons preferred to use not A3s and T3s, but C3s, G3s, CAI, CBI and Fop. Sequence analysis showed that the TL63 homologs had conserved TM and GLD motifs and RING-H2 domain whose key amino acid residues accounted for the high average abundance. Particularly, Arabidopsis thaliana TL63 (AtTL63) was located in the nuclei, cell membranes and peroxisomes and expressed universally and significantly throughout A. thaliana development. Under H2O2 treatment, low or moderate expression of the AtTL63 held beneficial effects on the growth and viability of yeast cells and the mutation or overexpression of the AtTL63 positively affected the growth of A. thaliana plants. In brief, this study could supply useful insight into the evolution of the plant TL63s and the AtTL63 functions under oxidative stress.

Successful full-length genomic cloning and characterization of site-specific nick structures of Phytophthora endornaviruses 2 and 3 in yeast, Saccharomyces cerevisiae.

Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, Saccharomyces cerevisiae. These full-length cDNA sequences were ligated downstream of a constitutive expression promoter (TDH3) or a galactose-inducing promoter (GAL1) in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5'-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the GAL1 promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.

A 3D printed plastic frame deeply impacts yeast cell growth.

Solid State Fermentation (SSF) processes have been explored for yeast growth and protein and metabolites production. However, most of these processes lack standardization. In this work, we present a polylactic acid (PLA) 3D printed matrix that dramatically enhances yeast growth when embedded in liquid media compared to equivalent static cultures, and changes yeast expression patterns at the proteome level (data are available via ProteomeXchange with identifier PXD043759). Moreover, differences in sugar assimilation and ethanol production, as the main product of alcoholic fermentation, are observed. Our results suggest that these matrixes may be useful for a vast range of biotechnological applications based on yeast fermentation.

How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for invitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.

The Yeast Protein Kinase Sch9 Functions as a Central Nutrient-Responsive Hub That Calibrates Metabolic and Stress-Related Responses.

Yeast cells are equipped with different nutrient signaling pathways that enable them to sense the availability of various nutrients and adjust metabolism and growth accordingly. These pathways are part of an intricate network since most of them are cross-regulated and subject to feedback regulation at different levels. In yeast, a central role is played by Sch9, a protein kinase that functions as a proximal effector of the conserved growth-regulatory TORC1 complex to mediate information on the availability of free amino acids. However, recent studies established that Sch9 is more than a TORC1-effector as its activity is tuned by several other kinases. This allows Sch9 to function as an integrator that aligns different input signals to achieve accuracy in metabolic responses and stress-related molecular adaptations. In this review, we highlight the latest findings on the structure and regulation of Sch9, as well as its role as a nutrient-responsive hub that impacts on growth and longevity of yeast cells. Given that most key players impinging on Sch9 are well-conserved, we also discuss how studies on Sch9 can be instrumental to further elucidate mechanisms underpinning healthy aging in mammalians.

Bioethanol production from sugarcane molasses with supplemented nutrients by industrial yeast

Sugarcane molasses, a sugar industry by-product, is being used as a convenient source of carbon for microorganisms to produce bioethanol. The industrial yield of bioethanol derived from Saccharomyces cerevisiae decreases with increasing temperature in the summer season and it is a common issue in the tropical region. It is inevitable to boost the production of bioethanol on an industrial scale to make it commercially profitable. This study has been conducted to analyze and compare the effect of three supplements on boosting alcohol products including thiamine (vitamin B1), pyridoxine (vitamin B6), and D-biotin (vitamin H). Collected molasses were diluted to 20 Brix to make them consumable by yeast, and thiamine, pyridoxine, and biotin were added as 1 mg/l, 1 mg/l, and 0.44 µg/l, respectively. Results depicted that the addition of vitamins impacted very positively on increasing alcohol production, yeast growth, and dry yeast weight where the residual glucose concentration was further decreased. Despite an extended lag phase resulting from higher vitamin doses, there was a significant improvement observed in both alcohol generation and yeast cell growth. This study presents the optimal dosing of supplements in the fermentation broth as a strategy to enhance bioethanol production.

Regulation of fructose levels on carbon flow and metabolites in yeast during food fermentation.

In this study, the effects of fructose levels on yeast growth, metabolic pathways and products, and redox status were investigated by simulated dough medium. The results showed that yeast was subjected to oxidative stress and damage under both sugar-free and high-fructose conditions. Yeast has a strong ability to metabolize pentose phosphate, trehalose, and tricarboxylic acid under sugar-free conditions. In the high fructose environment, yeast preferentially produced trehalose and glycerol in the early stage and gradually increased the metabolism of pentose phosphate in the later stage. Compared with the low fructose concentration, yeast had stronger pentose phosphate and tricarboxylic acid cycle (TCA) metabolism to ensure nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP) content in higher fructose levels. Therefore, sugar-free and high fructose levels affected the growth of yeast cells and yeast responded to fructose levels by regulating the metabolic carbon flow of glycolysis, pentose phosphate, trehalose, and TCA.