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Saccharomycopsis yeasts are natural organic sulfur auxotrophs due to lack of genes required for the uptake and assimilation of sulfate/sulfite. Starvation for methionine induces a shift to a predatory, mycoparasitic life strategy that is unique amongst ascomycetous yeasts. Similar to fungal plant pathogens that separated from Saccharomycopsis more than 400 million years ago, a specialized infection structure called penetration peg is used for prey cell invasion. Penetration pegs are highly enriched with chitin. Here we demonstrate that an ancient and conserved MAP kinase signaling pathway regulates penetration peg formation and successful predation in the predator yeast S. schoenii. Deletion of the MAP kinase gene SsKIL1, a homolog of the Saccharomyces cerevisiae ScKSS1/ScFUS3 and the rice blast Magnaporthe oryzae MoPMK1 genes, as well as deletion of the transcription factor SsSTE12 generate non-pathogenic mutants that fail to form penetration pegs. Comparative global transcriptome analyses using RNAseq indicate loss of the SsKil1-SsSte12-dependent predation response in the mutant strains, while a methionine starvation response is still executed. Within the promoter sequences of genes upregulated during predation we identified a cis-regulatory element similar to the ScSte12 pheromone response element. Our results indicate that, re-routing MAP-kinase signaling by re-wiring Ste12 transcriptional control towards predation specific genes contributed to the parallel evolution of this predacious behaviour in predator yeasts. Consequently, we found that SsSTE12 is dispensable for mating. Author summary: Saccharomycopsis yeasts represent a unique yeast genus of predator yeasts that attack and kill fungal prey cells. This necrotrophic mycoparasitism is initiated upon starvation, preferably for organic sulphur/methionine as predator yeasts are methionine auxotrophs. We have characterized morphological and cellular features of predation using time lapse fluorescence microscopy. This indicated prey sensing, chitin enrichment of penetration pegs used in the attack of prey cells and coordination of predation with the cell cycle resulting in cessation of daughter cell growth and a block in mitosis during predation. A predator yeast attack from the onset of penetration peg formation to the disappearance of the nuclear GFP-signal in the Saccharomyces cerevisiae prey took ~25 minutes. We identified the MAP kinase KIL1 and its putative target STE12 as key virulence genes that govern penetration peg formation. Comparative transcriptomics of mutant strains with the wild type identified specific hunger and predation responses and associated genes. Predation genes were found to harbor a conserved DNA element in their promoters. These findings can aid in deepening our understanding of necrotrophic predator-prey interactions. [ABSTRACT FROM AUTHOR]

Despite the increasing number of publications on non-conventional yeasts (NCYs), many areas in this field remain poorly understood, making the examination of these strains important for determining their potential in wine fermentations. The amino acid metabolic pathways involved, particularly the catabolic Ehrlich pathway but also anabolic pathways such as the leucine biosynthesis pathway, are crucial for producing high-value aroma compounds that contribute to the final flavour of wine. We examined the potential use of Saccharomycopsis fermentans in wine fermentations. We selected mutant strains resistant to the toxic compound trifluoro-leucine (TFL), verified mutations in the SfLEU4 gene, and characterized the ability of the resulting strains to contribute to fermentation bouquets. Resistance to TFL relieves feedback inhibition in the leucine biosynthesis pathway and resulted in increased leucine biosynthesis. Concomitantly, the S. fermentans TFL-resistant mutants generated increased amounts of isoamyl alcohol and isovalerate during wine fermentation. Selection of TFL-resistant strains thus provides a generally applicable strategy for the improvement in NCYs and their utilization in co-fermentation processes for different grape must varieties. [ABSTRACT FROM AUTHOR]

Hybrid formation and introgressions had a profound impact on fermentative yeasts domesticated for beer, wine and cider fermentations. Here we provide a comparative genomic analysis of a British cider yeast isolate (E1) and characterize its fermentation properties. E1 has a Saccharomyces uvarum genome into which ~102 kb of S. eubayanus DNA were introgressed that replaced the endogenous homologous 55 genes of chromosome XIV between YNL182C and YNL239W. Sequence analyses indicated that the DNA donor was either a lager yeast or a yet unidentified S. eubayanus ancestor. Interestingly, a second introgression event added ~66 kb of DNA from Torulaspora microellipsoides to the left telomere of SuCHRX. This region bears high similarity with the previously described region C introgression in the wine yeast EC1118. Within this region FOT1 and FOT2 encode two oligopeptide transporters that promote improved nitrogen uptake from grape must in E1, as was reported for EC1118. Comparative laboratory scale grape must fermentations between the E1 and EC1118 indicated beneficial traits of faster consumption of total sugars and higher glycerol production but low acetic acid and reduced ethanol content. Importantly, the cider yeast strain produced high levels of fruity ester, including phenylethyl and isoamyl acetate. [ABSTRACT FROM AUTHOR]

Kveik are consortia of yeast used for farmhouse ale production in Western Norway. Yeast strains derived from these mixtures are known, for example, for their high fermentation rate, thermotolerance, lack of phenolic off flavor production (POF-) and strong flocculation phenotype. In this study, we used five single cell yeast isolates from different Kveik yeasts, analyzed their fermentation and flavor production, and compared it with a typical yeast used in distilleries using 20 °C and 28 °C as the fermentation temperatures. One of the isolates, Kveik No 3, showed an impairment of maltotriose utilization and thus a reduced ethanol yield. Kveik fermentations for spirit production often harbor bacteria for flavor enrichment. We sought to improve Kveik fermentations with non-conventional yeasts (NCY). To this end we co-fermented Kveik isolates with Hanseniaspora uvarum, Meyerozyma guilliermondii and Pichia kudriavzevii using 5:1 ratios (Kveik vs. NCY) at 20 °C. The combinations of Kveik No 1 with P. kudriavzevii and Kveik No 1 with Hanseniaspora uvarum showed substantially increased amounts of specific volatile aroma compounds that were previously identified in the NCYs. Our results indicate that Kveik isolates appear to be suitable for co-fermentations with certain NCY to enhance beer or spirit fermentations, increasing the potential of these yeasts for beverage productions. [ABSTRACT FROM AUTHOR]

Microorganisms need to sense and adapt to fluctuations in the environmental pH. In fungal species, this response is mediated by the conserved pacC/RIM101 pathway. In Aspergillus nidulans, PacC activates alkaline-expressed genes and represses acid-controlled genes in response to alkaline pH and has important functions in regulating growth and conidia formation. In Saccharomyces cerevisiae, the PacC homolog Rim101 is required for adaptation to extracellular pH and to regulate transcription of IME1, the Initiator of MEiosis. S. cerevisiae rim101 mutants are defective in sporulation. In Ashbya gossypii, a filamentous fungus belonging to the family of Saccharomycetaceae, little is known about the role of pH in regulating growth and sporulation. Here, we deleted the AgRIM101 homolog (AFR190C). Our analyses show that Rim101 is important for growth and essential for sporulation at alkaline pH in A. gossypii. Acidic liquid sporulation media were alkalinized by sporulating strains, while the high pH of alkaline media (starting pH = 8.6) was reduced to a pH ~ 7.5 by these strains. However, Agrim101 mutants were unable to sporulate in alkaline media and failed to reduce the initial high pH, while they were capable of sporulation in acidic liquid media in which they increased the pH like the wild type. [ABSTRACT FROM AUTHOR]

Ashbya gossypii is a filamentous ascomycete belonging to the yeast family of Saccharomycetaceae. At the end of its growth phase Ashbya generates abundant amounts of riboflavin and spores that form within sporangia derived from fragmented cellular compartments of hyphae. The length of spores differs within species of the genus. Needle-shaped Ashbya spores aggregate via terminal filaments. A. gossypii is a homothallic fungus which may possess a and α mating types. However, the solo-MATa type strain is self-fertile and sporulates abundantly apparently without the need of prior mating. The central components required for the regulation of sporulation, encoded by IME1, IME2, IME4, KAR4, are conserved with Saccharomyces cerevisiae. Nutrient depletion generates a strong positive signal for sporulation via the cAMP-PKA pathway and SOK2, which is also essential for sporulation. Strong inhibitors of sporulation besides mutations in the central regulatory genes are the addition of exogenous cAMP or the overexpression of the mating type gene MATα2. Sporulation has been dissected using gene-function analyses and global RNA-seq transcriptomics. This revealed a role of Msn2/4, another potential PKA-target, for spore wall formation and a key dual role of the protein A kinase Tpk2 at the onset of sporulation as well as for breaking the dormancy of spores to initiate germination. Recent work has provided an overview of ascus development, regulation of sporulation and spore maturation. This will be summarized in the current review with a focus on the central regulatory genes. Current research and open questions will also be discussed. [ABSTRACT FROM AUTHOR]

Yeasts are unicellular fungi that harbour a large biodiversity of thousands of species, of which particularly ascomycetous yeasts are instrumental to human food and beverage production. There is already a large body of evidence showing that insects play an important role for yeast ecology, for their dispersal to newhabitats and for breeding and overwintering opportunities. Here,we sought to investigate a potential role of the terrestrial snails Cepaea hortensis and C. nemoralis, which in Europe are often found in associationwith human settlements and gardens, in yeast ecology. Surprisingly, even in a relatively limited culture-dependent sampling size of over 150 isolates,we found a variety of yeast genera, including species frequently isolated from grape must such as Hanseniaspora,Metschnikowia,Meyerozyma and Pichia in snail excrements. We typed the isolates using standard ITS-PCR-sequencing, sequenced the genomes of three non-conventional yeasts H. uvarum,Meyerozyma guilliermondii and P. kudriavzevii and characterized the fermentation performance of these three strains in grape must highlighting their potential to contribute to novel beverage fermentations. Aggravatingly, however,we also retrieved several human fungal pathogen isolates from snail excrements belonging to the Candida clade, namely Ca. glabrata and Ca. lusitaniae. Overall, our results indicate that diverse yeasts can utilise snails as taxis for dispersal. This courier service may be largely non-selective and thus depend on the diet available to the snails. [ABSTRACT FROM AUTHOR]

The fermentation industry is known to be very conservative, relying on traditional yeast management. Yet, in the modern fast-paced world, change comes about in facets such as climate change altering the quality and quantity of harvests, changes due to government regulations e.g., the use of pesticides or SO2, the need to become more sustainable, and of course by changes in consumer preferences. As a silent companion of the fermentation industry, the wine yeast Saccharomyces cerevisiae has followed mankind through millennia, changing from a Kulturfolger, into a domesticated species for the production of bread, beer, and wine and further on into a platform strain for the production of biofuels, enzymes, flavors, or pharmaceuticals. This success story is based on the ‘awesome power of yeast genetics’. Central to this is the very efficient homologous recombination (HR) machinery of S. cerevisiae that allows highly-specific genome edits. This microsurgery tool is so reliable that yeast has put a generally recognized as safe (GRAS) label onto itself and entrusted to itself the life-changing decision of mating type-switching. Later, yeast became its own genome editor, interpreted as domestication events, to adapt to harsh fermentation conditions. In biotechnology, yeast HR has been used with tremendous success over the last 40 years. Here we discuss several types of yeast genome edits then focus on HR and its inherent potential for evolving novel wine yeast strains and styles relevant for changing markets. [ABSTRACT FROM AUTHOR]

All laboratories dealing with microbes have to develop a strain maintenance regime. While lyophilization based on freeze-drying may be feasible for large stock centers, laboratories around the world rely on cryopreservation and freezing of stocks at -80 °C. Keeping stocks at these low temperatures requires investments of several thousand kW/h per year. We have kept yeast stocks for several decades at room temperature on agar slants in glass reagent tubes covered with vaspar and sealed with cotton plugs. They were part of the Geisenheim Yeast Breeding Center stock collection that was started in the 19th century, well before -80 °C refrigeration technology was invented. Of these stocks, 60 tubes were analyzed and around one-third of them could be regrown. The strains were typed by sequencing of rDNA PCR fragments. Based on BlastN analyses, twelve of the strains could be assigned to Saccharomyces cerevisiae, two to S. kudriavzevii, and the others to Meyerozyma and Candida. The strains were used in white wine fermentations and compared to standard wine yeasts Uvaferm/GHM (Geisenheim) and Lalvin EC1118. Even with added nitrogen, the strains exhibited diverse fermentation curves. Post-fermentation aroma analyses and the determination of residual sugar and organic acid concentrations indicated that some strains harbor interesting flavor characteristics, surpassing current standard yeast strains. Thus, old strain collections bear treasures for direct use either in wine fermentations or for incorporation in yeast breeding programs aimed at improving modern wine yeasts. Furthermore, this provides evidence that low-cost/long-term culture maintenance at zero-emission levels is feasible. [ABSTRACT FROM AUTHOR]

Pathogenic yeasts and fungi are an increasing global healthcare burden, but discovery of novel antifungal agents is slow. The mycoparasitic yeast Saccharomycopsis schoenii was recently demonstrated to be able to kill the emerging multi-drug resistant yeast pathogen Candida auris. However, the molecular mechanisms involved in the predatory activity of S. schoenii have not been explored. To this end, we de novo sequenced, assembled and annotated a draft genome of S. schoenii. Using proteomics, we confirmed that Saccharomycopsis yeasts have reassigned the CTG codon and translate CTG into serine instead of leucine. Further, we confirmed an absence of all genes from the sulfate assimilation pathway in the genome of S. schoenii, and detected the expansion of several gene families, including aspartic proteases. Using Saccharomyces cerevisiae as a model prey cell, we honed in on the timing and nutritional conditions under which S. schoenii kills prey cells. We found that a general nutrition limitation, not a specific methionine deficiency, triggered predatory activity. Nevertheless, by means of genome-wide transcriptome analysis we observed dramatic responses to methionine deprivation, which were alleviated when S. cerevisiae was available as prey, and therefore postulate that S. schoenii acquired methionine from its prey cells. During predation, both proteomic and transcriptomic analyses revealed that S. schoenii highly upregulated and translated aspartic protease genes, probably used to break down prey cell walls. With these fundamental insights into the predatory behavior of S. schoenii, we open up for further exploitation of this yeast as a biocontrol yeast and/or source for novel antifungal agents. [ABSTRACT FROM AUTHOR]

Non- Saccharomyces species have been recognized for their beneficial contribution to fermented food and beverages based on their volatile compound formation and their ability to ferment glucose into ethanol. At the end of fermentation brewer's yeast flocculate which provides an easy means of separation of yeasts from green beer. Flocculation in Saccharomyces cerevisiae requires a set of flocculation genes. These FLO -genes, FLO1 , FLO5 , FLO9 , FLO10 , and FLO11 , are located at telomeres and transcription of these adhesins is regulated by Flo8 and Mss11. Here, we show that Saccharomycopsis fermentans , an ascomycete yeast distantly related to S. cerevisiae , possesses a very large FLO/ALS -like Sequence (FAS) family encompassing 34 genes. Fas proteins are variable in size and divergent in sequence and show similarity to the Flo1/5/9 family. Fas proteins show the general build with a signal peptide, an N-terminal carbohydrate binding PA14 domain, a central region differing by the number of repeats and a C-terminus with a consensus sequence for GPI-anchor attachment. Like FLO genes in S. cerevisiae , FAS genes are mostly telomeric with several paralogs at each telomere. We term such genes that share evolutionary conserved telomere localization " telologs " and provide several other examples. Adhesin expression in S. cerevisiae and filamentation in Candida albicans is regulated by Flo8 and Mss11. In Saccharomycopsis we identified only a single protein with similarity to Flo8 based on sequence similarity and the presence of a LisH domain. [ABSTRACT FROM AUTHOR]

Fungi produce a variety of volatile organic compounds (VOCs) during their primary and secondary metabolism. In the beverage industry, these volatiles contribute to the the flavor and aroma profile of the final products. We evaluated the fermentation ability and aroma profiles of non-conventional yeasts that have been associated with various food sources. A total of 60 strains were analyzed with regard to their fermentation and flavor profile. Species belonging to the genera Candida, Pichia and Wickerhamomyces separated best from lager yeast strains according to a principal component analysis taking alcohol and ester production into account. The speed of fermentation and sugar utilization were analysed for these strains. Volatile aroma-compound formation was assayed via gas chromatography. Several strains produced substantially higher amounts of aroma alcohols and esters compared to the lager yeast strain Weihenstephan 34/70. Consequently, co-fermentation of this lager yeast strain with a Wickerhamomyces anomalus strain generated an increased fruity-flavour profile. This demonstrates that mixed fermentations utilizing non-Saccharomyces cerevisiae biodiversity can enhance the flavour profiles of fermented beverages. [ABSTRACT FROM AUTHOR]

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth. [ABSTRACT FROM AUTHOR]

Oxide-derived copper (OD-Cu) electrodes exhibit unprecedented CO reduction performance towards liquid fuels, producing ethanol and acetate with >50 % Faradaic efficiency at −0.3 V (vs. RHE). By using static headspace-gas chromatography for liquid phase analysis, we identify acetaldehyde as a minor product and key intermediate in the electroreduction of CO to ethanol on OD-Cu electrodes. Acetaldehyde is produced with a Faradaic efficiency of ≈5 % at −0.33 V (vs. RHE). We show that acetaldehyde forms at low steady-state concentrations, and that free acetaldehyde is difficult to detect in alkaline solutions using NMR spectroscopy, requiring alternative methods for detection and quantification. Our results represent an important step towards understanding the CO reduction mechanism on OD-Cu electrodes. [ABSTRACT FROM AUTHOR]

Aroma alcohols of fermented food and beverages are derived from fungal amino acids catabolism via the Ehrlich pathway. This linear pathway consists of three enzymatic reactions to form fusel alcohols. Regulation of some of the enzymes occurs on the transcriptional level via Aro80. The riboflavin overproducer Ashbya gossypii produces strong fruity flavours in contrast to its much less aromatic relative Eremothecium cymbalariae. Genome comparisons indicated that A. gossypii harbors genes for aromatic amino acid catabolism ( ARO8a, ARO8b, ARO10, and ARO80) while E. cymbalariae only encodes ARO8a and thus lacks major components of aromatic amino acid catabolism. Volatile compound ( VOC) analysis showed that both Eremothecium species produce large amounts of isoamyl alcohol while A. gossypii also produces high levels of 2-phenylethanol. Deletion of the A. gossypii ARO-genes did not confer any growth deficiencies. However, A. gossypii ARO-mutants (except Agaro8a) were strongly impaired in aroma production, particularly in the production of the rose flavour 2-phenylethanol. Conversely, overexpression of ARO80 via the Ag TEF1 promoter resulted in 50% increase in VOC production. Together these data indicate that A. gossypii is a very potent flavour producer and that amongst the non- Saccharomyces biodiversity strains can be identified that could provide positive sensory properties to fermented beverages. [ABSTRACT FROM AUTHOR]

The genus Eremothecium belongs to the Saccharomyces complex of pre-whole-genome duplication (WGD) yeasts and contains both dimorphic and filamentous species. We established the 9.1-Mb draft genome of Eremothecium coryli, which encodes 4,682 genes, 186 tRNA genes, and harbors several Ty3 transposons as well as more than 60 remnants of transposition events (LTRs). The initial de novo assembly resulted in 19 scaffolds, which were assembled based on synteny to other Eremothecium genomes into six chromosomes. Interestingly, we identified eight E. coryli loci that bear centromeres in the closely related species E. cymbalariae. Two of these E. coryli loci, CEN1 and CEN8, however, lack conserved DNA elements and did not convey centromere function in a plasmid stability assay. Correspondingly, using a comparative genomics approach we identified two telomere-to-telomere fusion events in E. coryli as the cause of chromosome number reduction from eight to six chromosomes. Finally, with the genome sequences of E. coryli, E. cymbalariae, and Ashbya gossypii a reconstruction of three complete chromosomes of an Eremothecium ancestor revealed that E. coryli is more syntenic to this ancestor than the other Eremothecium species. [ABSTRACT FROM PUBLISHER]

Traditional flavor analysis relies on gas chromatography coupled to mass spectrometry (GC-MS) methods. Here we describe an indirect method coupling volatile compound formation to an ARO9-promoter-LacZ reporter gene. The resulting β-galactosidase activity correlated well with headspace solid phase micro extraction (HS/SPME) GC-MS data particularly with respect to the formation of rose flavor. This tool enables large-scale screening of yeast strains and their progeny to identify the most flavor active strains. [ABSTRACT FROM AUTHOR]

Abstract: Ashbya gossypii is a natural overproducer of riboflavin. Overproduction of riboflavin can be induced by environmental stress, e.g. nutritional or oxidative stress. The Yap-protein family has a well-documented role in stress response. Particularly, Yap1 has a major role in directing the oxidative stress responses. The A. gossypii YAP-family consists of only three genes in contrast to its closest relative Eremothecium cymbalariae, which has four YAP-homologs. Gene order at Eremothecium YAP-loci is conserved with the reconstructed yeast ancestor. AgYap1p is unique amongst Yap-homologs as it lacks the cysteine-rich domains (CRDs). AgYAP1 expression is inducible and GFP-AgYap1 localizes to the nucleus. Agyap1 mutants displayed higher sensitivity against oxidative stress – H2O2 and menadione - and are strongly reduced in riboflavin production. High levels of cAMP, which also reduce riboflavin production, show a synergistic effect on this sensitivity. AgYAP1 and a chimera of AgYAP1 (with the DNA-binding domain) and ScYAP1 (with the CRDs) can both complement the Scyap1 oxidative stress sensitivity. This suggests that the DNA-binding sites of ScYap1 are conserved in A. gossypii. Expression of AgRIB4, which contains three putative Yap1-binding sites, assayed via a lacZ-reporter gene was strongly reduced in an Agyap1 mutant suggesting a direct involvement of AgYap1 in riboflavin production. Furthermore, our data show that application of H2O2 stress leads to an increase in riboflavin production in a Yap1-dependent manner. [Copyright &y& Elsevier]

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Postgenomic gene-function analyses with Candida albicans are hindered by its constitutive diploidy and the lack of a sexual cycle. Rapid generation of mutant strains can be achieved using PCR-based techniques for directed gene alterations. Here, we report the analyses of nine C. albicans genes that encode Src Homology 3-domain proteins. Phenotypic analyses included the potential of the mutants to form hyphal filaments, maintain a polarized actin cytoskeleton or the ability to generate large vacuoles in the germ cells and in subapical compartments. The C. albicans homologs of the Saccharomyces cerevisiae BBC1, BOI2, BUD14, FUS1, HSE1, PIN3, RVS167, RVS167-2 and SHO1 were all found to be nonessential. Deletion of RVS167 resulted in a strain with a decreased ability to form hyphal filaments. The number of cortical actin patches was increased in Δ rvs167 strains and their distribution was depolarized in both mother and daughter yeast cells and along the hyphae during filamentous growth stages. Polarization of patches could be restored upon reintroduction of the wild-type gene. Deletion of BOI2 was found to generate a defect in vacuolar fusion in hyphae. In contrast to a deletion in the Δ wal1 gene, Δ boi2 cells formed abundant hyphae, indicating that fragmented vacuoles do not inhibit filamentation. Placing BOI2 under control of the MAL2-promoter allowed the regulation of this phenotype depending on the growth conditions. [ABSTRACT FROM AUTHOR]

Synthesis of chitin Je novo from glucose involves a linear pathway in Saccharomyces cerevisiae. Several of the pathway genes, including GNA1, are essential. Genes for chitin catabolism are absent in S. cerevisiae. Therefore, S. cerevisiae cannot use chitin as a carbon source. Chitin is the second most abundant polysaccharide after cellulose and consists of N-acetylglucosamine (GlcNAc) moieties. Here, we have generated S. cerevisiae strains that are able to use GlcNAc as a carbon source by expressing four Candida albicans genes (NAG3 or its NAG4 paralog, NAGS, NAG2, and NAG1) encoding a GlcNAc permease, a GlcNAc kinase, a GlcNAc-6-phosphate deacetylase, and a glucosamine-6-phosphate deaminase, respectively. Expression of NAG3 and NAGS or NAG4 and NAG5 in 5. cerevisiae resulted in strains in which the otherwise-essential ScGNA1 could be deleted. These strains required the presence of GlcNAc in the medium, indicating that uptake of GlcNAc and its phosphorylation were achieved. Expression of all four NAG genes produced strains that could use GlcNAc as the sole carbon source for growth. Utilization of a GlcNAc catabolic pathway for bioethanol production using these strains was tested. However, fermentation was slow and yielded only minor amounts of ethanol (approximately 3.0 g/liter), suggesting that fructose-6-phosphate produced from G/cNAc under these conditions is largely consumed to maintain cellular functions and promote growth. Our results present the first step toward tapping a novel, renewable carbon source for biofuel production. [ABSTRACT FROM AUTHOR]

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Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence. [ABSTRACT FROM AUTHOR]

Analysis of the Ashbya gossypii Wiskott­Aldrich syndrome-like gene AgWALI indicates that it is required for the maintenance of polarized hyphal growth. Growth and organelle dynamics of the wild type and of wal1 and other mutant strains were monitored by in vivo (fluorescence) time-lapse microscopy. Loss of WAL1 led to slow growth and defects in polarized growth that produced swellings in subapicl regions, whereas formation of hyphal tips and dichotomous tip branching occurred as in the wild-type. Few actin cables in Agwal1 cells were found to insert into the hyphal tip, but specific clustering of cortical actin patches was observed in subapical regions of hyphal tips instead of at the hyphal apex. Distribution and movement of vacuoles was observed in vivo using FM4-64. In the wild type and in the slowly growing mutant strains bem2 and cla4, which lack a Rho-GTPase-activating protein and a PAK kinase, respectively, early endosomes appeared in the hyphal tip, whereas very few early endosomes and small vacuoles were found in the wal1 mutant hyphal tips, thus linking the cortical patch defect of wal1 hyphae with the distribution of endosomes. Vivid movement of vacuoles seen the in the wild type and in the bem2 mutant in subapical regions was largely reduced in the wal1 and cla4 mutants. The tubular structure of mitochondria (as visualized by DIOC6 in vivo) was similar in the wild type and the wal1 mutant, although wal1 mitochondria appeared to be larger. Interestingly, mitochondria were found to insert into the hyphal tips in both strains. Our results indicate a function for Wal1p in filamentous fungi in coordinating actin patch distribution with polarized hyphal tip growth. [ABSTRACT FROM AUTHOR]

Cytokinesis is the ultimate step of a cell cycle resulting in the generation of two progeny. Failure of correct cell division may be lethal for both, mother and daughter cells, and thus such a process must be tightly regulated with other events of the cell cyle. Differing solutions to the same problem have been developed in bacteria and plants while cytokinesis in animal and fungal cells is highly similar and requires a contractile ring containing actomyosin. Cytokinesis in fungi can be viewed as a three-stage process: (i) selection of a division site, (ii) orderly assembly of protein complexes, and finally (iii) dynamic events that lead to a constriction of the contractile ring and septum construction. Elaborate mechanisms known as the Mitotic Exit Network (MEN) and the Septation Initiation Network (SIN) have evolved to link these events, particularly the final steps of cytokinesis, with nuclear division. The purpose of this review was to discuss the latest developments in the fungal field and to describe the central known players required for key steps on the road to cell division. Differences in the cytokinesis of yeast-like fungi that result in complete cell separation in contrast to septation which leads to the compartmentalization of fungal hyphae are highlighted. [Copyright &y& Elsevier]

Commonly used protocols for the transformation of the dimorphic human fungal pathogen Candida albicans rely on established methods for the yeast Saccharomyces cerevisiae. With respect to transformation efficiency, however, there is a great difference between these two organisms when using the lithium acetate procedure. Here we present a modified version of this protocol for use with C. albicans. Among the different parameters tested, two turned out to be particularly relevant and, when combined, resulted in an up to 10-fold increase in transformation efficiency (400–500 integrative transformants) compared with previous protocols: first, adjusting the heat shock applied to the cells to 44 °C for C. albicans instead of 42 °C for S. cerevisiae and, second, treating C. albicans cells with lithium acetate in an overnight incubation instead of for 30 min as used for S. cerevisiae. With these modifications, the lithium acetate procedure becomes a very efficient and reliable tool for C. albicans transformation. [ABSTRACT FROM AUTHOR]

The evolution of multiple, independent and multispecific mating-type loci is a feature unique to homobasidiomycete fungi. To propose a model of evolution, data assembled for the wood-rotting fungus Schizophyllum commune were analyzed. In one mating-type locus, pheromone receptors and several pheromones are encoded which have been investigated in some detail and can be used to understand the ligand–receptor interactions and activation of signal transduction which are essential to sexual propagation. Previous models for the evolution of new alleles were complicated and involved three subsequent steps (without selectable phenotype) prior to the establishment of a new stable pheromone–receptor pair. This paper presents a model for the evolution of new specificities by recombination and selection that incorporates the multi-state receptor activation recently established for S. commune, explaining differential responses to different pheromones in one receptor molecule. The model takes into account the occurrence of multiple pheromone genes in each locus and unilateral nuclear donor/acceptor strains that may in nature act as steps in the evolution of new specificities. A second homobasidiomycete fungus, Coprinus cinereus, was similarly characterized at the molecular level. Data acquired in this system support the conclusion that the presented model can be generalized. [ABSTRACT FROM AUTHOR]

In filamentous ascomycetes hyphae are compartmentalized by septation in which the cytoplasm of the compartments are interconnected via septal pores. Thus, septation in filamentous fungi is different from cytokinesis in yeast like fungi. We have identified an Ashbya gossypii orthologue of the Saccharomyces cerevisiae CYK1 gene which belongs to the IQGAP-protein family. In contrast to S. cerevisiae disruption of AgCYK1 yields viable mutant strains that exhibit wildtype-like polarized hyphal growth rates. In the Agcyk1 mutant cortical actin patches localize to growing hyphal tips like wildtype, however, mutant hyphae are totally devoid of actin rings at presumptive septal sites. Septation in wildtype results in the formation of chitin rings. Agcyk1 mutant hyphae are aseptate and do not accumulate chitin in their cell walls. Agcyk1 mutant strains are completely asporogenous indicating that septation is essential for the formation of sporangia in A. gossypii. AgCyk1p-GFP localizes to sites of future septation as a ring prior to chitin depositioning. Furthermore, decrease in Cyk1p-ring diameter was found to be a prerequisite for the accumulation of chitin and septum formation. [Copyright &y& Elsevier]

The cold-tolerant yeast Saccharomyces cerevisiae is industrially useful for lager fermentation, high-quality wine, and frozen dough production. S. cerevisiae Cheongdo is a recent isolate from frozen peach samples which has a good fermentation performance at low temperatures and desirable flavor profiles. Here, phenotype microarray was used to investigate industrial potentials of S. cerevisiae Cheongdo using 192 carbon sources. Compared to commercial wine yeast S. cerevisiae EC1118, Cheongdo showed significantly different growth rates on 34 substrates. The principal component analysis of the results highlighted that the better growth of Cheongdo on galactose than on EC1118 was the most significant difference between the two strains. The intact GAL4 gene and the galactose fermentation performance at a low temperatures suggested that S. cerevisiae Cheongdo is a promising host for industrial fermentation rich in galactose, such as lactose and agarose. [ABSTRACT FROM AUTHOR]