Showing papers in "Fems Yeast Research in 2011"

Intracellular characterization of aerobic glucose metabolism in seven yeast species by 13C flux analysis and metabolomics.

Abstract: Key distinguishing characteristics of yeast glucose metabolism are the relative proportions of fermentation and respiration. Crabtree-positive yeast species exhibit a respirofermentative metabolism, whereas aerobic species respire fully without secretion of fermentation byproducts. Physiological data suggest a gradual transition in different species between these two states. Here, we investigate whether this gradual transition also occurs at the intracellular level by quantifying the intracellular metabolism of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces exiguus, Kluyveromyces thermotolerans, Yarrowia lipolytica, Pichia angusta and Candida rugosa by 13C-flux analysis and metabolomics. Different from the extracellular physiology, the intracellular fluxes through the tricarboxylic acid cycle fall into two classes where the aerobic species exhibit much higher respiratory fluxes at otherwise similar glycolytic fluxes. More generally, we found the intracellular metabolite concentrations to be primarily species-specific. The sole exception of a metabolite-flux correlation in a species-overarching manner was found for fructose-1,6-bisphosphate and dihydroxyacetone-phosphate, indicating a conservation of the functional properties around these two metabolites.

Batch and continuous culture‐based selection strategies for acetic acid tolerance in xylose‐fermenting Saccharomyces cerevisiae

Abstract: Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two evolutionary engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L(-1) : (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L(-1) acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g(-1) biomass h(-1) . After storage of samples from both selection experiments at -80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L(-1) acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol.

Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids

Abstract: The increasing incidence of hospital-acquired infections caused by drug-resistant pathogens, host toxicity, the poor efficacy of drugs and high treatment costs has drawn attention to the potential of natural products as antifungals in mucocutaneous infections and combinational therapies. Moreover, cellular and subcellular targets for these compounds may provide better options for the development of novel antifungal therapies. Eugenol, methyl eugenol and estragole are phenylpropanoids found in essential oil. They are known to possess pharmacological properties including antimicrobial activity. Induction of oxidative stress characterized by elevated levels of free radicals and an impaired antioxidant defence system is implicated as a possible mechanism of cell death. An insight into the mechanism of action was gained by propidium iodide cell sorting and oxidative stress response to test compounds in Candida albicans. The extent of lipid peroxidation (LPO) of cytoplasmic membranes was estimated to confirm a state of oxidative stress. Activity levels of primary defence enzymes and glutathione were thus further determined. Whereas these compounds cause fungal cell death by disrupting membrane integrity at minimum inhibitory concentrations (MIC), sub-MIC doses of these compounds significantly impair the defence system in C. albicans. The study has implications for understanding microbial cell death caused by essential oil components eliciting oxidative stress in Candida. The formation of membrane lesions by these phenylpropanoids thus appears to be the result of free radical cascade-mediated LPO.

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

Abstract: The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.

Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

Abstract: The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production.

Astaxanthin biosynthesis is enhanced by high carotenogenic gene expression and decrease of fatty acids and ergosterol in a Phaffia rhodozyma mutant strain.

Abstract: An astaxanthin-overproducing (∼1000 μg g(-1)) strain of Phaffia rhodozyma, termed MK19, was established through 1-methyl-3-nitro-1-nitrosoguanidine and Co60 mutagenesis from wild-type JCM9042 (merely 35-67 μg g(-1)). The total fatty acid content of MK19 was much lower than that of the wild type. Possible causes of the astaxanthin increase were studied at the gene expression level. The expression of the carotenogenic genes crtE, crtI, pbs, and ast, which are responsible for astaxanthin biosynthesis from geranylgeranyl pyrophosphate, was highly induced at the mRNA level, leading to excessive astaxanthin accumulation. In contrast, transcription levels of the genes (hmgs, hmgr, idi, mvk, mpd, fps), responsible for the initial steps in the terpenoid pathway, were essentially the same in wild type and MK19. Although fatty acid and total ergosterol content were reduced by 40-70 mg g(-1) and 760.3 μg g(-1) , respectively, in MK19 as compared with the wild type, but the transcription levels of rate-limiting genes in fatty acid and ergosterol pathways such as acc and sqs were similar. Because fatty acids and ergosterol are two branch pathways of astaxanthin biosynthesis in P. rhodozyma, our findings indicate that enhancement of astaxanthin in MK19 results from decreased fatty acid and ergosterol biosynthesis, leading to precursor accumulation, and transfer to the astaxanthin pathway. Strengthening of the mevalonate pathway is suggested as a promising metabolic engineering approach for further astaxanthin enhancement in MK19.

Identification of the UDP‐glucosyltransferase gene UGTA1, responsible for the first glucosylation step in the sophorolipid biosynthetic pathway of Candida bombicola ATCC 22214

Abstract: Candida bombicola ATCC 22214 is applied commercially for the production of sophorolipids from renewable resources such as vegetable oils or waste streams. Although much research has been performed on optimization of fermentation conditions and on the influence of feed source and process parameters on sophorolipid structures and yields, the metabolic pathway of these important bioproducts remains unclear. Here, we identify a glucosyltransferase gene UGTA1 and show that the gene product is responsible for the first glucosylation step in the biosynthetic pathway of sophorolipids. Moreover, we provide evidence that the second glucosylation step is catalysed by a different glucosyltransferase that acts independently from the first. Therefore, the biosynthesis of sophorolipids by C. bombicola involves two glucosyltransferases that act in a stepwise manner. The UGTA1 gene described here is the first identified gene with a clear function in sophorolipid production by this economically important yeast.

Alkaline stress triggers an immediate calcium fluctuation in Candida albicans mediated by Rim101p and Crz1p transcription factors

Abstract: In the human fungal pathogen Candida albicans, environmental pH has profound effects on morphogenesis and response to extracellular pH is clearly relevant to the pathogenicity of this fungus Yeast cells have evolved a complex network of mechanisms in response to the environmental pH and they often require the integration of the Rim101 and calcineurin/Crz1 signaling pathways Ca(2+) burst is a common cellular response when cells are exposed to environmental stresses; therefore, in this study, we asked whether it follows the same case under alkaline stress and whether this calcium change is regulated by Rim101p and Crz1p We confirmed the calcium influx was activated by KOH stimuli using a flow cytometry-based method, but it was obviously abolished in cells lacking MID1 or CCH1 We also found that alkaline pH-induced activation of the PHO89 promoter was blocked without the same gene; moreover, the response was Crz1p- and Rim101p-dependent Finally, we investigated the regulation role of Rim101p and Crz1p in calcium influx through MID1, CCH1 and YVC1 genes, which were all downregulated in rim101Δ/Δ and crz1Δ/Δ mutants The important role of calcium influx in the alkaline stress response and its regulation suggested a potential integration effect of Rim101 and Crz1 signaling pathways in C albicans

Alcohol‐based quorum sensing plays a role in adhesion and sliding motility of the yeast Debaryomyces hansenii

Abstract: The yeast Debaryomyces hansenii was investigated for its production of alcohol-based quorum sensing (QS) molecules including the aromatic alcohols phenylethanol, tyrosol, tryptophol and the aliphatic alcohol farnesol. Debaryomyces hansenii produced phenylethanol and tyrosol, which were primarily detected from the end of exponential phase indicating that they are potential QS molecules in D. hansenii as previously shown for other yeast species. Yields of phenylethanol and tyrosol produced by D. hansenii were, however, lower than those produced by Candida albicans and Saccharomyces cerevisiae and varied with growth conditions such as the availability of aromatic amino acids, ammonium sulphate, NaCl, pH and temperature. Tryptophol was only produced in the presence of tryptophane, whereas farnesol in general was not detectable. Especially, the type strain of D. hansenii (CBS767) had good adhesion and sliding motility abilities, which seemed to be related to a higher hydrophobicity of the cell surface of D. hansenii (CBS767) rather than the ability to form pseudomycelium. Addition of phenylethanol, tyrosol, tryptophol and farnesol was found to influence both adhesion and sliding motility of D. hansenii.

Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species.

Abstract: At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets

The proteome analysis of oleaginous yeast Lipomyces starkeyi

Abstract: Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional μRPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts.

A multiplex set of species‐specific primers for rapid identification of members of the genus Saccharomyces

Abstract: The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens.

Cellular responses of Saccharomyces cerevisiae at near-zero growth rates : transcriptome analysis of anaerobic retentostat cultures

Abstract: Extremely low specific growth rates (below 0.01 h−1) represent a largely unexplored area of microbial physiology. In this study, anaerobic, glucose-limited retentostats were used to analyse physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. While quiescence is typically investigated as a result of carbon starvation, cells in retentostat are fed by small, but continuous carbon and energy supply. Yeast cells cultivated near-zero specific growth rates, while metabolically active, exhibited characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in exit from the cell cycle into G0. Unexpectedly, analysis of transcriptome data from retentostat and chemostat cultures showed, as specific growth rate was decreased, that quiescence-related transcriptional responses were already set in at specific growth rates above 0.025 h−1. These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal. Furthermore, cells in retentostat do not (or hardly) divide while remaining metabolically active, which emulates the physiological status of metazoan post-mitotic cells. We propose retentostat as a powerful cultivation tool to investigate chronological ageing-related processes.

Sequential deletion of Pichia pastoris genes by a self-excisable cassette

Abstract: A rapid and convenient method is presented for unmarked gene deletions in Pichia pastoris. Cre/mutated lox system, Zeocin(®) (Invitrogen) resistance marker and homologous arms were spliced together by fusion PCR to generate the gene disruption cassettes (homologous region-lox71-Cre-ZeoR-lox66-homologous region), which could be integrated into the P. pastoris genome via homologous recombination. After transferring double-cross-over recombinants to methanol induction medium, transient expression of Cre recombinase caused the recombination of lox71-Cre-ZeoR-lox66 fragment into a double-mutant lox72 site, thus excising the Cre-ZeoR cassette from the P. pastoris genome. As the double-mutant lox72 site displays strongly reduced binding affinity for Cre recombinase, this method could be used sequentially to disrupt P. pastoris genes without introducing selectable markers. The effectiveness of this strategy was verified by introducing both single and double gene deletions into the P. pastoris genome.

Candida glabrata Pwp7p and Aed1p are required for adherence to human endothelial cells.

Abstract: Candida glabrata owes its success as a pathogen, in part, to a large repertoire of adhesins present on the cell surface. Our current knowledge of C. glabrata adhesins and their role in the interaction between host and pathogen is limited to work with only a single family of epithelial adhesins (Epa proteins). Here, we report on the identification and characterization of a family of glycosylphosphatidylinositol-anchored cell wall proteins in C. glabrata. These proteins are absent in both Saccharomyces cerevisiae and Candida albicans, suggesting that C. glabrata has evolved different mechanism(s) for interaction with host cells. In the current study, we present data on the characterization of Pwp7p (PA14 domain containing Wall Protein) and Aed1p (Adherence to Endothelial cells) of this family in the interaction of C. glabrata with human umbilical vein endothelial cells. The deletion of C. glabrata genes PWP7 and AED1 results in a significant reduction in adherence to endothelial cells compared with the wild-type parent. These data indicate that C. glabrata utilizes these proteins for adherence to endothelial cells in vitro.

Differential carotenoid production and gene expression in Xanthophyllomyces dendrorhous grown in a nonfermentable carbon source

Abstract: Xanthophyllomyces dendrorhous is a basidiomycetous yeast of considerable biotechnological interest because it synthesizes astaxanthin as its main carotenoid. The carotenoid production increases when it is grown using nonfermentable compounds as the sole carbon source. This work analyzes the expression of the carotenogenic genes and their relationship with the amount and types of carotenoids produced when X. dendrorhous is grown using a nonfermentable (succinate) or a fermentable carbon source (glucose). When X. dendrorhous is grown in succinate, carotenoid production is approximately three times higher than when it is grown in glucose. Moreover, carotenoid biosynthesis occurs at the start of the growth cycle when X. dendrorhous is grown in succinate, whereas when it is grown in glucose, carotenoids are produced at the end of the exponential phase. Additionally, we observed that some carotenogenic genes, such as alternative transcripts of crtYB and crtI, are differentially expressed when the yeast is grown in these carbon sources; other genes, such as crtS, exhibit a similar pattern of expression. Our data indicate that transcriptional regulation is not sufficient to explain the differences in carotenoid production between the two culture conditions, indicating that additional regulatory mechanisms may be operating in the carotenogenic pathway of X. dendrorhous.

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae.

Abstract: This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Abstract: Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.

Some biological features of Candida albicans mutants for genes coding fungal proteins containing the CFEM domain

Abstract: Several biological features of Candida albicans genes (PGA10, RBT5 and CSA1) coding for putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as common in several fungal extracellular membrane (CFEM) are described. The deletion of these genes resulted in a cascade of pleiotropic effects. Thus, mutant strains exhibited higher cell surface hydrophobicity levels and an increased ability to bind to inert or biological substrates. Confocal scanning laser microscopy using concanavalin A-Alexafluor 488 (which binds to mannose and glucose residues) and FUN-1 (a cytoplasmic fluorescent probe for cell viability) dyes showed that mutant strains formed thinner and more fragile biofilms. These apparently contained lower quantities of extracellular matrix material and less metabolically active cells than their parental strain counterpart, although the relative percentage of mycelial forms was similar in all cases. The cell surface of C. albicans strains harbouring deletions for genes coding CFEM-domain proteins appeared to be severely altered according to atomic force microscopy observations. Assessment of the relative gene expression within individual C. albicans cells revealed that CFEM-coding genes were upregulated in mycelium, although these genes were shown not to affect virulence in animal models. Overall, this study has demonstrated that CFEM domain protein-encoding genes are pleiotropic, influencing cell surface characteristics and biofilm formation.

Drug-resistant cassettes for the efficient transformation of Candida guilliermondii wild-type strains.

Abstract: Candida guilliermondii is an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains an interesting biotechnological model for the industrial production of value-added metabolites. The recent whole-genome sequencing of the C. guilliermondii ATCC 6260 reference strain provides an interesting resource for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering. In the present study, we designed an efficient transformation system for C. guilliermondii wild-type strains using both nourseothricin- and hygromycin B-resistant markers. To demonstrate the potential of these drug-resistant cassettes, we carried out the disruption and the complementation of the C. guilliermondii FCY1 gene (which encodes cytosine deaminase) known to be associated with flucytosine sensitivity in yeast. These two new dominant selectable markers represent powerful tools to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.

Candida albicans--a pre-whole genome duplication yeast--is predominantly aerobic and a poor ethanol producer.

Abstract: Yeast species belonging to the lineage that underwent the whole genome duplication (WGD), and including Saccharomyces cerevisiae, can grow under anaerobiosis and accumulate ethanol in the presence of glucose and oxygen. The pre-WGD yeasts, which branched from the S. cerevisiae lineage just prior to the WGD event, including Kluyveromyces lactis, are more dependent on oxygen and do not accumulate large amounts of ethanol in the presence of excess oxygen. Yeasts that belong to the so-called 'lower branches' of the yeast phylogenetic tree and diverged from S. cerevisiae more than 200 million years ago, have so far not been thoroughly investigated for their physiology and carbon metabolism. We have hereby studied several isolates of Candida albicans and Debaryomyces hansenii for their dependence on oxygen. C. albicans grew very poorly at oxygen concentration below 1 p.p.m. and D. hansenii could not grow at all. In aerobic batch cultivations C. albicans exhibited a predominately aerobic metabolism, accumulating only small amounts of ethanol (0.01-0.09 g g(-1) glucose). Apparently, C. albicans and several other pre-WGD yeasts still exhibit the original traits of the yeast progenitor: poor accumulation of ethanol under aerobic conditions and strong dependence on the presence of oxygen. (Less)

Biodiversity in sulfur metabolism in hemiascomycetous yeasts

Abstract: The evolution of the metabolism of sulfur compounds among yeast species was investigated. Differences between species were observed in the cysteine biosynthesis pathway. Most yeast species possess two pathways leading to cysteine production, the transsulfuration pathway and the O-acetyl-serine (OAS) pathway, with the exception of Saccharomyces cerevisiae and Candida glabrata, which only display the transsulfuration pathway, and Schizosaccharomyces pombe, which only have the OAS pathway. An examination of the components of the regulatory network in the different species shows that it is conserved in all the species analyzed, as its central component Met4p was shown to keep its functional domains and its partners were present. The analysis of the presence of genes involved in the catabolic pathway shows that it is evolutionarily conserved in the sulfur metabolism and leads us to propose a role for two gene families which appeared to be highly conserved. This survey has provided ways to understand the diversity of sulfur metabolism products among yeast species through the reconstruction of these pathways. This diversity could account for the difference in metabolic potentialities of the species with a biotechnological interest.

Yeast prions: could they be exaptations? The URE2/[URE3] system in Kluyveromyces lactis.

Abstract: We examined aspects of the URE2/[URE3] prion system in Kluyveromyces lactis, which lies on a different evolutionary branch from Saccharomyces. We first analysed the polymorphism of the prion-forming domain in 38 strains. Considerable differences were found between these two genera, with little variation within K. lactis. We then analysed the regulatory function of Ure2p, using a deletion of URE2. We assessed the deregulation of two reporter genes: DAL5 and GDH2. Both were derepressed in the mutant strain, as in Saccharomyces. Finally, we tried to obtain the [URE3] prion from K. lactis. Despite the use of many different experimental conditions, we were unable to obtain a prion from Ure2p. This finding calls into question the extent to which the prion form of Ure2p may be considered an evolutionary adaptation, instead suggesting that an exaptation phenomenon may be more likely than a continuous selection history.

Production of the UVB-absorbing compound mycosporine–glutaminol–glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Abstract: The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.

An alkaline β‐glucosidase isolated from an olive brine strain of Wickerhamomyces anomalus

Abstract: An efficient β-glucosidase (βG)-producing strain, Wickerhamomyces anomalus BS81, was isolated from naturally fermented olive brine and identified based on PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene. The hydrolytic activity of the βG had an optimum pH of 8.5 and an optimum temperature of 35 °C. The enzyme had high substrate specificity and high catalytic efficiency (Km 0.99 mM, Vmax 14 U g−1 of cells) for p-nitrophenyl-β-d-glucopyranoside. The enzyme was activated by increasing concentrations of NaCl, with maximum activity at 150 g L−1 NaCl. Although βGs have been purified and characterized from several other sources, the W. anomalusβG is unique among βGs because its relative maximum activity occurs at alkaline pH and 35 °C. Moreover, the yeast strain has esterase activity that acts synergistically with βG to degrade oleuropein to debitter table olives and olive oil.

Ribosomal DNA sequence polymorphism and the delineation of two ascosporic yeast species: Metschnikowia agaves and Starmerella bombicola.

Abstract: The relationship between mating success and sequence divergence in the internal transcribed spacer (ITS)/5.8S-D1/D2 rDNA region was examined in isolates tentatively assigned to Metschnikowia agaves and Starmerella bombicola. Both species are haplontic and heterothallic, such that the formation of mature asci can be used as a measure of genetic compatibility. Parsimony haplotype network analysis and mating success confirmed that all known isolates of M. agaves are conspecific. The previously reported D1/D2 polymorphism of five substitutions was not corroborated; the maximum divergence observed between any two strains was three substitutions, four with ITS. Of 39 putative S. bombicola strains, 36 formed an ITS-D1/D2 haplotype network using the 95% criterion. Thirty-five strains could mate with one or more compatible partner. The excluded strains did not mate. Mature asci arose from crosses between individuals differing by as many as five, but not six or seven substitutions in the D1/D2 domain. All strains capable of mating formed mature asci with at least one partner and all network members could be linked to another member by three or fewer substitutions. These results support the use of sequence divergence as a criterion for species delineation, but caution against describing poorly sampled species solely on the basis of that criterion.

Species‐specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces

Abstract: Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.

Coexpression of α-l-arabinofuranosidase and β-glucosidase in Saccharomyces cerevisiae

Abstract: Monoterpenes are important aroma compounds in grape varieties such as Muscat, Gewurztraminer and Riesling, and are present as either odourless, glycosidically bound complexes or free aromatic monoterpenes. Commercial enzymes can be used to release the monoterpenes, but they commonly consist of crude extracts that often have unwanted and unpredictable side-effects on wine aroma. This project aims to address these problems by the expression and secretion of the Aspergillus awamoriα-l-arabinofuranosidase in combination with either the β-glucosidases from Saccharomycopsis fibuligera or from Aspergillus kawachii in the industrial yeast Saccharomyces cerevisiae VIN13. The concentration of five monoterpenes was monitored throughout alcoholic fermentation of Gewurztraminer grapes. The recombinant yeast strains that caused an early boost in the geraniol concentration led to a reduction in the final geraniol levels due to the downregulation of the sterol biosynthetic pathway. Monoterpene concentrations were also analysed 9 and 38 days after racking and the performance of the VB2 and VAB2 recombinant strains was similar, and in many cases, better than that of a commercial enzyme used in the same experiment. The results were backed by sensorial analysis, with the panel preferring the aroma of the wines produced by the VAB2 strain.

Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris

Abstract: A DNA fragment containing the mature human interleukin (IL)-6 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-6 production were identified, secreting as much as 280 mg L−1 rhIL-6 after 4 days of induction by methanol. The rhIL-6 was purified by PEG-8000 precipitation, followed by DEAE anion exchange and Sephadex G-75 gel filtration, yielding over 95% pure rhIL-6 at about 170 mg L−1. Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20 908.85 Da, which is close to the mass calculated from the sequence of the protein. Functional analysis of the purified rhIL-6 using the lymphocyte proliferation assay by an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazoliumbromide] method demonstrated a specific activity that is at least fivefold higher than the commercial rhIL-6 produced in Escherichia coli. In summary, the experimental procedure we have reported here allows us to obtain a large amount of rhIL-6 from P. pastoris suitable for subsequent biophysical studies.

Dimorphic cycle in Candida citri sp. nov., a novel yeast species isolated from rotting fruit in Borneo.

Abstract: Five dimorphic yeast strains were isolated from rotting lime fruits in Borneo. The sequences of the D1/D2 domains of the 26S rRNA genes, the internal transcribed spacer (ITS) chromosomal regions and the 18S rRNA genes were identical in the isolates and differed from the corresponding sequences of all known yeast species. Based on the sequence differences (12–15% in the D1/D2 domain) from the closest relatives and the different pattern of taxonomic traits, the new isolates are assigned the status of a new species, for which the name Candida citri sp. nov. is proposed. Its type strain is 11-469T, which has been deposited in Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 11858T, Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 29-181-1T and the National Collection of Agricultural and Industrial Microorganisms (Budapest, Hungary) as NCAIM Y.01978T. MycoBank number: MB 519100. The GenBank accession numbers for nucleotide sequences of its D1/D2 domain, ITS and 18S regions are [HM803241][1], [HM803242][2] and [HM803243][3], respectively. Candida citri produces invasive mycelium composed of true septate hyphae that grow towards nutrient-rich parts of the medium and develop large vacuoles at the nongrowing ends of their cells. The hyphae produce blastoconidia, which can establish satellite yeast colonies in the invaded solid substrate. [1]: /lookup/external-ref?link_type=GEN&access_num=info%3Addbj-embl-genbank%2FHM803241&atom=%2Ffemsyr%2F11%2F2%2F202.atom [2]: /lookup/external-ref?link_type=GEN&access_num=ddbj-embl-genbank%2FHM803242&atom=%2Ffemsyr%2F11%2F2%2F202.atom [3]: /lookup/external-ref?link_type=GEN&access_num=ddbj-embl-genbank%2FHM803243&atom=%2Ffemsyr%2F11%2F2%2F202.atom