scholarly journals A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

2015 ◽  
Vol 14 (8) ◽  
pp. 804-816 ◽  
Author(s):  
Daniel Solis-Escalante ◽  
Niels G. A. Kuijpers ◽  
Nuria Barrajon-Simancas ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Systems Approach ◽  
Gene Dosage ◽  
Physiological Role ◽  
Industrial Applications ◽  
Yeast Genome ◽  
Small Scale ◽  
Genetic Redundancy ◽  
Minimal Set ◽  
Content Type

ABSTRACTAs a result of ancestral whole-genome and small-scale duplication events, the genomes ofSaccharomyces cerevisiaeand many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of theSaccharomyceslineage, the duplication of glycolytic genes is purported to have played an important role leading toS. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modernS. cerevisiaestrains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic ofS. cerevisiaeand essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community.

scholarly journals Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Anna K. Wronska ◽  
Meinske P. Haak ◽  
Ellen Geraats ◽  
Eva Bruins Slot ◽  
Marcel van den Broek ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
De Novo ◽  
Laboratory Strain ◽  
Optimal Growth ◽  
Industrial Applications ◽  
Fast Growth ◽  
Growth Media ◽  
Free Medium ◽  
Content Type

ABSTRACT Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiae BIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae. This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiae. IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae. Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

scholarly journals EngineeringKluyveromyces marxianusas a Robust Synthetic Biology Platform Host

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Paul Cernak ◽  
Raissa Estrela ◽  
Snigdha Poddar ◽  
Jeffrey M. Skerker ◽  
Ya-Fang Cheng ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Kluyveromyces Marxianus ◽  
Lipid Production ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Human Society ◽  
Content Type ◽  
Renewable Chemicals

ABSTRACTThroughout history, the yeastSaccharomyces cerevisiaehas played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However,S. cerevisiaehas proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeastKluyveromyces marxianusto create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates ofK. marxianuscan be made heterothallic for sexual crossing. By breeding two of these mating-type engineeredK. marxianusstrains, we combined three complex traits—thermotolerance, lipid production, and facile transformation with exogenous DNA—into a single host. The ability to crossK. marxianusstrains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering ofK. marxianusisolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establishK. marxianusas a synthetic biology platform comparable toS. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCEThe yeastKluyveromyces marxianusgrows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeastSaccharomyces cerevisiaein industrial applications. Here, we describe genetic tools for genome editing and breedingK. marxianusstrains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to usingK. marxianusas a versatile synthetic biology platform organism for industrial applications.

scholarly journals A systems approach for forward and reverse logistics design

2016 ◽  
Vol 27 (3) ◽  
pp. 947-971 ◽  
Author(s):  
Ayham A.M. Jaaron ◽  
Chris Backhouse
Keyword(s):  
Case Studies ◽  
Design Process ◽  
Reverse Logistics ◽  
Systems Approach ◽  
Industrial Applications ◽  
Three Dimensions ◽  
Content Type ◽  
Information Demand ◽  
Logistics Services ◽  
Exploratory Case Studies

Purpose There is significant potential for adding value by involving customer in the design process and delivery of logistic services. In order to add value to the overall logistic system, the purpose of this paper is to apply an integrated systems approach for the design of forward and reverse logistics services in order to build a self-organising service that can maximise efficiencies and in particular reduce reverse logistics costs. Design/methodology/approach Two exploratory case studies were conducted in the logistics systems of housing repair and maintenance sector in the UK. Data were collected using semi-structured interviews, observations, and documented evidence. Findings The findings of the cross-case analysis suggests that systems approach expressed as the Vanguard Method (Seddon, 2008) has a direct impact on enhancing forward logistics performance and reducing reverse product flows by nourishing three dimensions for learning from demand-driven analysis; capturing customer clean information, demand predictability and categorisation, and failure demand analysis. Research limitations/implications Findings from exploratory case studies cannot be easily generalised. Hence, further case studies are needed to enrich the findings, and to facilitate their industrial applications. Further, the paper explores the utilisation of the Vanguard Method only in the area of housing repairs and maintenance logistics services. It would be valuable for future studies to further investigate the utilisation of the Vanguard Method in other logistics services settings. Originality/value The paper demonstrates an important dynamics of how logistics services can incorporate customer demands into the logistics design process.

scholarly journals Physiological and Transcriptional Responses of Anaerobic Chemostat Cultures of Saccharomyces cerevisiae Subjected to Diurnal Temperature Cycles

2014 ◽  
Vol 80 (14) ◽  
pp. 4433-4449 ◽  
Author(s):  
Marit Hebly ◽  
Dick de Ridder ◽  
Erik A. F. de Hulster ◽  
Pilar de la Torre Cortes ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Growth Rate ◽  
Temperature Cycling ◽  
Yeast Genome ◽  
Cycle Phase ◽  
Content Type ◽  
Diurnal Temperature ◽  
Temperature Cycles ◽  
Chemostat Cultures

ABSTRACTDiurnal temperature cycling is an intrinsic characteristic of many exposed microbial ecosystems. However, its influence on yeast physiology and the yeast transcriptome has not been studied in detail. In this study, 24-h sinusoidal temperature cycles, oscillating between 12°C and 30°C, were imposed on anaerobic, glucose-limited chemostat cultures ofSaccharomyces cerevisiae. After three diurnal temperature cycles (DTC), concentrations of glucose and extracellular metabolites as well as CO2production rates showed regular, reproducible circadian rhythms. DTC also led to waves of transcriptional activation and repression, which involved one-sixth of the yeast genome. A substantial fraction of these DTC-responsive genes appeared to respond primarily to changes in the glucose concentration. Elimination of known glucose-responsive genes revealed an overrepresentation of previously identified temperature-responsive genes as well as genes involved in the cell cycle andde novopurine biosynthesis. In-depth analysis demonstrated that DTC led to a partial synchronization of the cell cycle of the yeast populations in chemostat cultures, which was lost upon release from DTC. Comparison of DTC results with data from steady-state cultures showed that the 24-h DTC was sufficiently slow to allowS. cerevisiaechemostat cultures to acclimate their transcriptome and physiology at the DTC temperature maximum and to approach acclimation at the DTC temperature minimum. Furthermore, this comparison and literature data on growth rate-dependent cell cycle phase distribution indicated that cell cycle synchronization was most likely an effect of imposed fluctuations of the relative growth rate (μ/μmax) rather than a direct effect of temperature.

scholarly journals Force Sensitivity in Saccharomyces cerevisiae Flocculins

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Cho X. J. Chan ◽  
Sofiane El-Kirat-Chatel ◽  
Ivor G. Joseph ◽  
Desmond N. Jackson ◽  
Caleen B. Ramsook ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Shear Stress ◽  
Candida Albicans ◽  
Cell Surface ◽  
Industrial Applications ◽  
Amino Acid Sequences ◽  
Content Type ◽  
Fungal Adhesion ◽  
Flocculation Activity ◽  
Cellular Aggregates

ABSTRACT The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications. Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca2+, yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications.

scholarly journals Transcriptional Rewiring, Adaptation, and the Role of Gene Duplication in the Metabolism of Ethanol of Saccharomyces cerevisiae

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Beatriz Sabater-Muñoz ◽  
Florian Mattenberger ◽  
Mario A. Fares ◽  
Christina Toft
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Gene Duplication ◽  
Growth Parameters ◽  
Small Scale ◽  
Whole Genome ◽  
Duplicated Genes ◽  
Content Type ◽  
Transcriptional Rewiring

ABSTRACT Ethanol is the main by-product of yeast sugar fermentation that affects microbial growth parameters, being considered a dual molecule, a nutrient and a stressor. Previous works demonstrated that the budding yeast arose after an ancient hybridization process resulted in a tier of duplicated genes within its genome, many of them with implications in this ethanol “produce-accumulate-consume” strategy. The evolutionary link between ethanol production, consumption, and tolerance versus ploidy and stability of the hybrids is an ongoing debatable issue. The implication of ancestral duplicates in this metabolic rewiring, and how these duplicates differ transcriptionally, remains unsolved. Here, we study the transcriptomic adaptive signatures to ethanol as a nonfermentative carbon source to sustain clonal yeast growth by experimental evolution, emphasizing the role of duplicated genes in the adaptive process. As expected, ethanol was able to sustain growth but at a lower rate than glucose. Our results demonstrate that in asexual populations a complete transcriptomic rewiring was produced, strikingly by downregulation of duplicated genes, mainly whole-genome duplicates, whereas small-scale duplicates exhibited significant transcriptional divergence between copies. Overall, this study contributes to the understanding of evolution after gene duplication, linking transcriptional divergence with duplicates’ fate in a multigene trait as ethanol tolerance. IMPORTANCE Gene duplication events have been related with increasing biological complexity through the tree of life, but also with illnesses, including cancer. Early evolutionary theories indicated that duplicated genes could explore alternative functions due to relaxation of selective constraints in one of the copies, as the other remains as ancestral-function backup. In unicellular eukaryotes like yeasts, it has been demonstrated that the fate and persistence of duplicates depend on duplication mechanism (whole-genome or small-scale events), shaping their actual genomes. Although it has been shown that small-scale duplicates tend to innovate and whole-genome duplicates specialize in ancestral functions, the implication of duplicates’ transcriptional plasticity and transcriptional divergence on environmental and metabolic responses remains largely obscure. Here, by experimental adaptive evolution, we show that Saccharomyces cerevisiae is able to respond to metabolic stress (ethanol as nonfermentative carbon source) due to the persistence of duplicated genes. These duplicates respond by transcriptional rewiring, depending on their transcriptional background. Our results shed light on the mechanisms that determine the role of duplicates, and on their evolvability.

scholarly journals Whole-genome sequencing from the New Zealand Saccharomyces cerevisiae population reveals the genomic impacts of novel microbial range expansion

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter Higgins ◽  
Cooper A Grace ◽  
Soon A Lee ◽  
Matthew R Goddard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
New Zealand ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Range Expansion ◽  
Copy Number ◽  
Small Scale ◽  
Whole Genome ◽  
Copy Number Changes ◽  
The Impact

Abstract Saccharomyces cerevisiae is extensively utilized for commercial fermentation, and is also an important biological model; however, its ecology has only recently begun to be understood. Through the use of whole-genome sequencing, the species has been characterized into a number of distinct subpopulations, defined by geographical ranges and industrial uses. Here, the whole-genome sequences of 104 New Zealand (NZ) S. cerevisiae strains, including 52 novel genomes, are analyzed alongside 450 published sequences derived from various global locations. The impact of S. cerevisiae novel range expansion into NZ was investigated and these analyses reveal the positioning of NZ strains as a subgroup to the predominantly European/wine clade. A number of genomic differences with the European group correlate with range expansion into NZ, including 18 highly enriched single-nucleotide polymorphism (SNPs) and novel Ty1/2 insertions. While it is not possible to categorically determine if any genetic differences are due to stochastic process or the operations of natural selection, we suggest that the observation of NZ-specific copy number increases of four sugar transporter genes in the HXT family may reasonably represent an adaptation in the NZ S. cerevisiae subpopulation, and this correlates with the observations of copy number changes during adaptation in small-scale experimental evolution studies.

scholarly journals Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 681-698 ◽  
Author(s):  
A J Link ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Physical Map ◽  
Single Copy ◽  
Restriction Endonucleases ◽  
Yeast Genome ◽  
Hybridization Probes ◽  
Copy Dna ◽  
Single Copy Dna

Abstract A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

Numerical Approaches for Modeling Gas–Solid Fluidized Bed Reactors: Comparison of Models and Application to Different Technical Problems

2019 ◽  
Vol 141 (7) ◽  
Author(s):  
Peter Ostermeier ◽  
Annelies Vandersickel ◽  
Stephan Gleis ◽  
Hartmut Spliethoff
Keyword(s):  
Calcium Carbonate ◽  
Size Distribution ◽  
Fluidized Bed ◽  
Fluid Model ◽  
Industrial Applications ◽  
Small Scale ◽  
Fluidized Bed Reactors ◽  
Discrete Phase Model ◽  
Technical Problems ◽  
Numerical Approaches

Gas–solid fluidized bed reactors play an important role in many industrial applications. Nevertheless, there is a lack of knowledge of the processes occurring inside the bed, which impedes proper design and upscaling. In this work, numerical approaches in the Eulerian and the Lagrangian framework are compared and applied in order to investigate internal fluidized bed phenomena. The considered system uses steam/air/nitrogen as fluidization gas, entering the three-dimensional geometry through a Tuyere nozzle distributor, and calcium oxide/corundum/calcium carbonate as solid bed material. In the two-fluid model (TFM) and the multifluid model (MFM), both gas and powder are modeled as Eulerian phases. The size distribution of the particles is approximated by one or more granular phases with corresponding mean diameters and a sphericity factor accounting for their nonspherical shape. The solid–solid and fluid–solid interactions are considered by incorporating the kinetic theory of granular flow (KTGF) and a drag model, which is modified by the aforementioned sphericity factor. The dense discrete phase model (DDPM) can be interpreted as a hybrid model, where the interactions are also modeled using the KTGF; however, the particles are clustered to parcels and tracked in a Lagrangian way, resulting in a more accurate and computational affordable resolution of the size distribution. In the computational fluid dynamics–discrete element method (CFD–DEM) approach, particle collisions are calculated using the DEM. Thereby, more detailed interparticulate phenomena (e.g., cohesion) can be assessed. The three approaches (TFM, DDPM, CFD–DEM) are evaluated in terms of grid- and time-independency as well as computational demand. The TFM and CFD–DEM models show qualitative accordance and are therefore applied for further investigations. The MFM (as a variation of the TFM) is applied in order to simulate hydrodynamics and heat transfer to immersed objects in a small-scale experimental test rig because the MFM can handle the required small computational cells. Corundum is used as a nearly monodisperse powder, being more suitable for Eulerian models, and air is used as fluidization gas. Simulation results are compared to experimental data in order to validate the approach. The CFD–DEM model is applied in order to predict mixing behavior and cohesion effects of a polydisperse calcium carbonate powder in a larger scale energy storage reactor.

scholarly journals Discovery of a Novel Class of Orally Active Antifungal β-1,3-d-Glucan Synthase Inhibitors

2011 ◽  
Vol 55 (11) ◽  
pp. 5099-5106 ◽  
Author(s):  
Scott S. Walker ◽  
Yiming Xu ◽  
Ilias Triantafyllou ◽  
Michelle F. Waldman ◽  
Cara Mendrick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Pharmacokinetic Data ◽  
Morphological Response ◽  
Glucan Synthase ◽  
Content Type ◽  
Safety Issues ◽  
Spontaneous Mutants ◽  
Orally Active

ABSTRACTThe echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibitedin vitroactivity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GSin vitro, and there was a strong correlation between enzyme inhibition andin vitroantifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants ofSaccharomyces cerevisiaewith reduced susceptibility to the piperazinyl-pyridazinones had substitutions inFKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model ofCandida glabratainfection.

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