Growth Of Yeast Cells Research Articles

Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

BackgroundSaccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown.ResultsIn this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance.ConclusionTranslation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

Citral and geraniol induce necrotic and apoptotic cell death on Saccharomyces cerevisiae.

Essential oils and their main components, monoterpenes, have been proven to be important alternatives for the control of pathogenic and spoiling microorganisms, but the mode of action of these compounds is poorly understood. This work aimed to determine the mode of action of citral and geraniol on the model yeast Saccharomyces cerevisiae using a flow cytometry approach. Exponentially growing yeast cells were treated with different concentrations of citral and geraniol for 3h, and evaluated for cell wall susceptibility to glucanase, membrane integrity, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, and metacaspase activity. Results provide strong evidence that citral and geraniol acute fungicidal activity against Saccharomyces cells involves the loss of membrane and cell wall integrity resulting in a dose-dependent apoptotic/necrotic cell death. However, yeast cells that escape this first cell membrane disruption, particularly evident on sub-lethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular ROS. The deleted mutant on the yca1 gene showed high tolerance to citral and geraniol.

A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia.

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

Myristic Acid Derived Sophorolipid: Efficient Synthesis and Enhanced Antibacterial Activity.

Microbial glycolipids are one of the most interesting alternatives to chemical-based surfactants as they exhibit improved biodegradability and less toxicity. However, their potential has been limited because of specificity of the yeast toward fatty acids having a carbon 16 or carbon 18 chain. This study focuses on sophorolipid (SL) production by the yeast Starmerella bombicola using myristic acid, a medium-chain carbon-14 fatty acid that has not been used as a substrate for SL production. The production was optimized for inoculum size and lipophilic substrate concentration. Furthermore, we also studied the effect of medium-chain fatty acid on yeast cell growth and optimized the process for excellent yield. The myristic acid SL (MASL) so synthesized consisted of mono- and diacetylated forms with preferential glycosylation at the methyl end group, as determined by high-resolution mass spectrometry. Individual congeners of the crude mixture were separated using dry column chromatography and then structurally characterized by mass spectrometry. The synthesized MASL was also shown to have promising surface tension, lowering abilities with a low CMC of 14 mg/L. The SL derived from myristic acid exhibited superior antibacterial activity as compared to SL derived from oleic acid. MASL was also found to be more potent against Gram-positive organisms as compared to Gram-negative organisms. This work, therefore, demonstrates successful synthesis of myristic acid-derived SL and its superior antibacterial activity, establishing a promising future for this biosurfactant.

Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora.

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

Characterization of constitutive ER-phagy of excess membrane proteins.

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

Antioxidant Capacities of Traditionally Formulated Thai Herbal Decoction and Its Effect on Cell Growth Using Saccharomyces cerevisiae Model

Polyherbal tea is widely consumed for rejuvenating of health in Thailand. Among them are Ya-Tri-Phigut (YTP), Ya-Tri-Kasornmas (YTK), Ya-Plook-Fithat (YPF), and Ya-Benjakul (YB). In addition, they have been listed as essential medicines with several others in the National List of Essential Medicines, Thailand. To ascertain their nutraceutical potentials, aqueous extracts of these herbal formulations were screened for their antioxidant activities. Extracts obtained from YTP showed higher yield (8.70 %) than YPF (6.40 %), YTK (6.30 %) and YB (6.30 %) (P <0.05). YTP also showed better antioxidant capacities than other tested formulations (P <0.05). Thus, it was used to test for its ability to support yeast cell. The extract (YTP) supported the yeast cell growth than the standard used called cryptotanshinone. Thus, YTP formulated herbal tonic can serve as a rich nutraceutical agent to combat oxidative stress and health related diseases.

Yeast Cellular Stress: Impacts on Bioethanol Production

Bioethanol is the largest biotechnology product and the most dominant biofuel globally. Saccharomyces cerevisiae is the most favored microorganism employed for its industrial production. However, obtaining maximum yields from an ethanol fermentation remains a technical challenge, since cellular stresses detrimentally impact on the efficiency of yeast cell growth and metabolism. Ethanol fermentation stresses potentially include osmotic, chaotropic, oxidative, and heat stress, as well as shifts in pH. Well-developed stress responses and tolerance mechanisms make S. cerevisiae industrious, with bioprocessing techniques also being deployed at industrial scale for the optimization of fermentation parameters and the effective management of inhibition issues. Overlap exists between yeast responses to different forms of stress. This review outlines yeast fermentation stresses and known mechanisms conferring stress tolerance, with their further elucidation and improvement possessing the potential to improve fermentation efficiency.

The impact of mixed amino acids supplementation on Torulaspora delbrueckii growth and volatile compound modulation in soy whey alcohol fermentation

Soy (tofu) whey is a liquid side stream generated from tofu production and is often discarded as waste after it is generated. Direct disposal of soy whey can result in environmental issue in the long run. Soy whey has been previously successfully fermented using different types of wine yeasts, but the yeast available nitrogen (YAN) was found to be deficient. In this study, the soy whey YAN was estimated to be approximately 45.9 mg N/L. A mixture of four amino acids (valine, leucine, isoleucine and phenylalanine) was added into soy whey at a total concentration of +40, +80, +120 and +160 mg N/L and fermented with Torulaspora delbrueckii Biodiva for a period of 10 days. Increasing amino acid supplementation did not affect the yeast cell growth, but it sped up the sugar utilization proportionally. Increasing amino acid supplementation resulted in lower organic acid production and higher glycerol production. Amino acid supplementation also enhances the production rate of higher alcohols; increasing amount of higher alcohols and their respective esters were obtained with increasing amount of amino acid supplementation. However, higher levels of amino acid supplementation (particularly at +160 mg N/L sample) resulted in higher residual nitrogen contents which may lead to microbial instability. Supplementation of 120 mg N/L of amino acids was found to be the optimum concentration to enhance the metabolism of the yeast without leaving a high residual amino acid content. Therefore, with proper control of the amino acid addition dosage, the usage of mixed amino acid supplementation may be a strategy to regulate the fermentation kinetics and volatile compound modulation in soy whey alcohol fermentation.

Microdroplet enabled cultivation of single yeast cells correlates with bulk growth and reveals subpopulation phenomena.

Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.

Effects of Moringa oleifera leaf extracts and its bioactive compound gallic acid on reducing toxicities of heavy metals and metalloid in Saccharomyces cerevisiae

Moringa oleifera leaf extract is rich in antioxidants and has high potential for use to alleviate metal toxicity. Previously, we have reported the roles of aqueous M. oleifera leaf extract in mitigating intracellular cadmium (Cd) accumulation and Cd-induced oxidative stress. In this study, we investigated the protective role of aqueous and/or ethanolic M. oleifera leaf extracts (AMOLE and/or EMOLE) against other metal(loid)s in the eukaryotic model Saccharomyces cerevisiae. Our results show that only the AMOLE remarkably promoted the growth of yeast cells grown in the presence of arsenite (As(III)), Cd, nickel (Ni), and lead (Pb). Although the AMOLE contained lower amount of total phenolic and flavonoid contents and displayed lower DPPH scavenging capacity than the EMOLE, both AMOLE and EMOLE had the same capacity for reducing intracellular ROS levels in yeast cells exposed to As(III), Cd, Ni, and Pb. Moreover, the AMOLE was more effective than the EMOLE in inhibiting intracellular accumulation of these toxic metal(loid)s. In addition, we found that gallic acid, one of important phenolic constituents present in both extracts, could protect yeast cells against As(III) toxicity, likely through its role in decreasing As(III) accumulation and As(III)-induced ROS production. Furthermore, the hydroxyl and carboxyl groups of gallic acid appear to play a critical role in chelating As(III). The present study suggests the promising applications of the AMOLE (and also gallic acid) as protective agents against hazardous metal(loid)s.

Discovery of novel Lactobacillus plantarum co-existence-associated influencing factor(s) on Saccharomyces cerevisiae fermentation performance

The food fermentation performed by Saccharomyces cerevisiae sometimes needs the co-existence of or is occasionally contaminated by other microorganisms. Among these microorganisms, the co-existed lactic acid bacteria (LAB) could inhibit S. cerevisiae growth through secreting organic acids and competing for viable space and nutrients. However, besides these three known major inhibition factors, are there any other unknown influence(s) of LAB on S. cerevisiae? In this study, a “reeling-off”-based design was performed to discover the potential novel LAB co-existence-associated inhibition factor(s) on S. cerevisiae fermentation performance illustrated by the case of Lactobacillus plantarum. After excluding the previously reported major influence factors one by one, L. plantarum ATCC8014 could still inhibit yeast biomass. Such results suggested that there might really exist such previously un-detected factor(s) functioning in inhibiting yeast cell growth. Furthermore, it was discovered that the L. plantarum cell wall could inhibit S. cerevisiae growth through direct contact. Interestingly, such direct contact could also improve ethanol tolerance of S. cerevisiae and ethanol production efficiency per OD. These results would strengthen our understanding of microbial interactions during food fermentation.

Chemo-Radiative Stress of Plasma as a Modulator of Charge-Dependent Nanodiamond Cytotoxicity

Efficient and selective internalization of nanoscale diamonds (also termed nanodiamonds, NDs) by living cells is of fundamental importance for their bionanotechnological applications. The biocompatibility of NDs is well established and has been suggested to arise from the limited membrane perturbation during their cellular translocation. However, the latter may be affected when cells are subjected to external stress. This study shows that the oxidative stress generated by atmospheric pressure cold plasmas (APCP) alters cell sensitivity to NDs, and their cytotoxicity profile. Both positively and negatively charged NDs are nontoxic to cells, here Saccharomyces cerevisiae and human cell lines, i.e., near-normal human mammary epithelial cells (MCF-10A) and breast cancer cells (MDA-MB-468 and T47D), unless the APCP stress is introduced. A brief exposure of the cells to APCP leads to a significant increase in their ND affinity (uptake and/or surface attachment) and intracellular ROS accumulation, particularly for positively charged NDs and both yeast and cancer cells. A concomitant decrease in cell viability and yeast cell growth, reflected by longer lag phases and lower cell density after 24 h of incubation, demonstrates a considerably enhanced ND toxicity to these cells. These results suggest that chemo-radiative stress, such as that produced by plasma, may influence the toxicity of nanoparticles to different cells, with specificity achieved through controlling particle charges. Moreover, since oxidative stress is not only associated with the use of APCP but can arise unintentionally within an organism and/or in the environment, these findings may have broader implications for the use of nontoxic nanoparticles in bionanotechnology in general.

Modulating Wine Aromatic Amino Acid Catabolites by Using Torulaspora delbrueckii in Sequentially Inoculated Fermentations or Saccharomyces cerevisiae Alone.

Yeasts are the key microorganisms that transform grape juice into wine, and nitrogen is an essential nutrient able to affect yeast cell growth, fermentation kinetics and wine quality. In this work, we focused on the intra- and extracellular metabolomic changes of three aromatic amino acids (tryptophan, tyrosine, and phenylalanine) during alcoholic fermentation of two grape musts by two Saccharomyces cerevisiae strains and the sequential inoculation of Torulaspora delbrueckii with Saccharomyces cerevisiae. An UPLC-MS/MS method was used to monitor 33 metabolites, and 26 of them were detected in the extracellular samples and 8 were detected in the intracellular ones. The results indicate that the most intensive metabolomic changes occurred during the logarithm cellular growth phase and that pure S. cerevisiae fermentations produced higher amounts of N-acetyl derivatives of tryptophan and tyrosine and the off-odour molecule 2-aminoacetophenone. The sequentially inoculated fermentations showed a slower evolution and a higher production of metabolites linked to the well-known plant hormone indole acetic acid (auxin). Finally, the production of sulfonated tryptophol during must fermentation was confirmed, which also may explain the bitter taste of wines produced by Torulaspora delbrueckii co-fermentations, while sulfonated indole carboxylic acid was detected for the first time in such an experimental design.

Antioxidant properties of the Lebanese plant Iris x germanica L. crude extracts and antagonism of chlorpromazine toxicity on Saccharomyces cerevisiae

Iris x germanica L., which belongs to the Iridaceae family, has been reported in the literature for its antioxidant properties in acellular chemical-antioxidant assays. Chlorpromazine (CPZ) is an antipsychotic drug known to cause adverse reactions in humans. Oxidative stress is among the main mechanisms by which CPZ exerts its toxicity in animal cell models as well as in the yeast Saccharomyces cerevisiae. In this study we investigated the protective effects of I. germanica L. crude extracts against CPZ toxicity. We demonstrated that methanolic extracts from rhizome (R-M), leaf (L-M) and flower (Fl-M) had potent antioxidant activity by scavenging the free radical DPPH, with half-maximal effective concentrations (EC50) 193, 107, and 174 µg/mL, respectively. R-M, L-M and Fl-M at doses up to 1000 µg/mL, didn’t affect yeast cell growth. In addition, we demonstrated for the first time that L-M at 1000 µg/mL and R-M at all tested doses counteracted CPZ toxicity, probably by promoting yeast cell antioxidant agents. The R-M capacity to counteract CPZ toxicity was lost in the yeast strain mutant in catalase-encoding gene (Cta1), while strains mutant in Sod2, Skn7 and Rap1 showed mild or full R-M-induced protective effect against CPZ toxicity. Our results demonstrated that I. germanica L. R-M extract counteracted CPZ toxicity in the yeast cell model. Further studies are planned to isolate the involved bioactive compounds and identify the involved genes and the antioxidant agents.

The Genome-Wide Mutation Shows the Importance of Cell Wall Integrity in Growth of the Psychrophilic Yeast Metschnikowia australis W7-5 at Different Temperatures.

In this study, it was found that a Cre/loxP system could be successfully used as a tool for editing the genome of the psychrophilic yeast Metschnikowia australis W7-5 isolated from Antarctica. The deletion and over-expression of the TPS1 gene for trehalose biosynthesis, the GSY gene for glycogen biosynthesis, and the GPD1 and GPP genes for glycerol biosynthesis had no influence on cell growth of the mutants and transformants compared to cell growth of their wild-type strain M. australis W7-5, indicating that trehalose, glycogen, and glycerol had no function in growth of the psychrophilic yeast at different temperatures. However, removal of the SLT2 gene encoding the mitogen-activated protein kinase in the cell wall integrity (CWI) signaling pathway and the SWI4 and SWI6 genes encoding the transcriptional activators Swi4/6 had the crucial influence on cell growth of the psychrophilic yeast at the low temperature, especially at 25 °C and expression of the genes related to cell wall and lipid biosynthesis. Therefore, the cell wall could play an important role in growth of the psychrophilic yeast at different temperatures and biosynthesis of cell wall was actively regulated by the CWI signaling pathway. This was the first time to show that the genome of the psychrophilic yeast was successfully edited and the molecular evidences were obtained to elucidate mechanisms of low temperature growth of the psychrophilic yeast from Antarctica.

Deletion of a small, secreted and cysteine-rich protein Cpl1 leads to increased invasive growth of Cryptococcus neoformans into nutrient agar

Invasive growth of yeast cells into nutrient agar is induced by different stresses and contributes to the survival of yeast cells under several adverse conditions. The mechanism of invasive growth of Saccharomyces cerevisiae has been extensively investigated. However, there is very little information about the mechanism of invasive growth of another human pathogen yeast Cryptococcus neoformans. Here, we report that deletion of a small and secreted cysteine-rich protein Cpl1 in C. neoformans JEC21 leads to increased adhesive and invasive growth into nutrient agar. The increased adhesive and invasive growth does not depend on the only known adhesion protein Cfl1 and its main controller Znf2. Cpl1Δ accumulates significantly higher level of intracellular labile zinc ion, leading to increased glucose uptake, higher level of mitochondrial membrane potential, ATP and Reactive Oxygen Species(ROS) production. Higher level of ROS activates Snf1, leading to invasive growth of Cpl1Δ. Three cysteine residues at the N-terminals of the cysteine-rich domain controls the increased invasive growth under nutrient sufficient conditions. This is the first report that a small and secreted cysteine-rich protein negatively regulates invasive growth of C. neoformans through regulating the intracellular labile zinc ion level. The function of this cysteine-rich domain was systematically investigated by site-directed mutagenensis in C. neoformans. The work contributes to understanding the function of this protein family and the invasive growth mechanism in C. neoformans.

Aluminum induces oxidative damage in Saccharomyces cerevisiae

The mechanism of aluminum toxicity was studied in the model cells of Saccharomyces cerevisiae. Cell growth of yeast was inhibited by aluminum. The spot assay showed that the mechanism of aluminum detoxification in yeast cells was different from that of heavy metal cadmium. After treatment with aluminum, intracellular levels of reactive oxygen species, protein carbonyl, and thiobarbituric acid reactive substances were dramatically increased. Meanwhile, the percentage of aluminum-treated cells permeable to propidium iodide was augmented significantly. These data demonstrated that aluminum toxicity was attributed to oxidative stress in yeast, and it induced oxidative damage by causing lipid peroxidation, injuring cell membrane integrity. Moreover, aluminum triggered the antioxidant defense system in the cells. Glutathione levels were found to be decreased, while activities of superoxide dismutase and catalase were increased after treatment with aluminum. Additionally, an oxidative-stress-related mutation sensitivity assay showed that aluminum-induced yeast oxidative stress was closely related to glutathione. These data demonstrated that the oxidative damage caused by aluminum was different from that of hydrogen peroxide, in yeast. Aluminum could cause DNA damage, and aluminum toxicity was associated with sulfhydryl groups, such as glutathione, while it was independent of YAP1.

A mechanism-aware and multiomic machine-learning pipeline characterizes yeast cell growth

Metabolic modeling and machine learning are key components in the emerging next generation of systems and synthetic biology tools, targeting the genotype-phenotype-environment relationship. Rather than being used in isolation, it is becoming clear that their value is maximized when they are combined. However, the potential of integrating these two frameworks for omic data augmentation and integration is largely unexplored. We propose, rigorously assess, and compare machine-learning-based data integration techniques, combining gene expression profiles with computationally generated metabolic flux data to predict yeast cell growth. To this end, we create strain-specific metabolic models for 1,143 Saccharomyces cerevisiae mutants and we test 27 machine-learning methods, incorporating state-of-the-art feature selection and multiview learning approaches. We propose a multiview neural network using fluxomic and transcriptomic data, showing that the former increases the predictive accuracy of the latter and reveals functional patterns that are not directly deducible from gene expression alone. We test the proposed neural network on a further 86 strains generated in a different experiment, therefore verifying its robustness to an additional independent dataset. Finally, we show that introducing mechanistic flux features improves the predictions also for knockout strains whose genes were not modeled in the metabolic reconstruction. Our results thus demonstrate that fusing experimental cues with in silico models, based on known biochemistry, can contribute with disjoint information toward biologically informed and interpretable machine learning. Overall, this study provides tools for understanding and manipulating complex phenotypes, increasing both the prediction accuracy and the extent of discernible mechanistic biological insights.

FDA Approved Drug Library Screening Identifies Robenidine as a Repositionable Antifungal.

Due to the increasing prevalence of pathogenic fungal infections, the emergence of antifungal resistant clinical isolates worldwide, and the limited arsenal of available antifungals, developing new antifungal strategies is imperative. In this study, we screened a library of 1068 FDA-approved drugs to identify hits that exhibit broad-spectrum antifungal activity. Robenidine, an anticoccidial agent which has been widely used to treat coccidian infections of poultry and rabbits, was identified in this screen. Physiological concentration of robenidine (8 μM) was able to significantly inhibit yeast cell growth, filamentation and biofilm formation of Candida albicans – the most extensively studied human fungal pathogen. Moreover, we observed a broad-spectrum antifungal activity of this compound against fluconazole resistant clinical isolates of C. albicans, as well as a wide range of other clinically relevant fungal pathogens. Intriguingly, robenidine-treated C. albicans cells were hypersensitive to diverse cell wall stressors, and analysis of the cell wall structure by transmission electron microscopy (TEM) showed that the cell wall was severely damaged by robenidine, implying that this compound may target the cell wall integrity signaling pathway. Indeed, upon robenidine treatment, we found a dose dependent increase in the phosphorylation of the cell wall integrity marker Mkc1, which was decreased after prolonged exposure. Finally, we provide evidence by RNA-seq and qPCR that Rlm1, the downstream transcription factor of Mkc1, may represent a potential target of robenidine. Therefore, our data suggest that robenidine, a FDA approved anti-coccidiosis drug, displays a promising and broadly effective antifungal strategy, and represents a potentially repositionable candidate for the treatment of fungal infections.