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Aims: We undertook to improve an industrial Saccharomyces cerevisiae strain by derepressing it for maltose utilization in the presence of high glucose concentrations. Methods and Results: A mutant was obtained from an industrial S. cerevisiae strain following random UV mutagenesis and selection on maltose/5-thioglucose medium. The mutant acquired the ability to utilize glucose simultaneously with maltose and possibly also sucrose and galactose. Aerobic sugar metabolism was still largely fermentative, but an enhanced respirative metabolism resulted in a 31% higher biomass yield on glucose. Kinetic characterization of glucose transport in the mutant revealed the predominance of the high-affinity component. Northern blot analysis showed that the mutant strain expresses only the HXT6/7 gene irrespective of the glucose concentration in the medium, indicating a severe deregulation in the induction/repression pathways modulating HXT gene expression. Interestingly, maltose-grown cells of the mutant display inverse diauxy in a glucose/maltose mixture, preferring maltose to glucose. Conclusion: In the mutant here reported, the glucose transport step seems to be uncoupled from downstream regulation, because it seems to be unable to sense abundant glucose, via both repression and induction pathways. Significance and Impact of the Study: We report here the isolation of a S. cerevisiae mutant with a novel derepressed phenotype, potentially interesting for the industrial fermentation of mixed sugar substrates.

A previous culture-dependent survey of phylloplane yeasts from selected Mediterranean plants showed that a few species were present in high densities in almost all leaf samples, regardless of the plant type, location or sampling season. However, a few species appeared to be restricted to Cistus albidus leaves, namely Cryptococcus cistialbidi. Here, we describe a culture-independent FISH assay to detect and quantify whole yeast cells in leaf washings. After optimization, the technique was used to check the apparent association between C. albidus leaves and C. cistialbidi and the abundance and ubiquity of other basidiomycetous yeast species such as Erythrobasidium hasegawianum and Sporobolomyces spp. in leaf samples from this and other neighboring plants (Acer monspessulanum and Quercus faginea). No yeast cells were detected in Pistacia lentiscus leaf samples. We were also able to demonstrate that three phylloplane yeasts (C. cistialbidi, E. hasegawianum and Sporobolomyces spp.) appeared to be log-normally distributed among individual C. albidus leaves. The log-normal distribution has important implications for the quantification of phylloplane yeasts based on the washing and plating of bulk leaf samples, which will tend to overestimate the size of the respective populations and become an error source in yeast surveys or related biocontrol studies.

The glucose/xylose facilitator Gxf1 from Candida intermedia was expressed in the recombinant xylose-fermenting Saccharomyces cerevisiae strain TMB 3057. The new strain, TMB 3411, displayed approximately two times lower K (m) for xylose transport compared to a control strain not expressing Gxf1. In aerobic batch cultivation, the specific growth rate was significantly higher at low xylose concentration, 4 g/L, when Gxf1 was expressed, whereas it remained unchanged at high xylose concentration, 40 g/L. Similarly, in aerobic-xylose-limited chemostat culture, the Gxf1-expressing strain consumed more xylose than the control strain at low dilution rates (low xylose concentration), whereas the situation was reversed at higher dilution rates (high xylose concentration). Also, under anaerobic conditions, the Gxf1-expressing strain showed faster xylose uptake and ethanol formation at low substrate concentrations. The results are discussed in relation to previous observations, which suggested that transport controlled xylose utilization in recombinant xylose-utilizing S. cerevisiae only at low xylose concentrations.

Microbiology, 154 Two glucose/xylose transporter genes from Candida intermedia were recently cloned and characterized: GXF1, which encodes a glucose/xylose facilitator; and GXS1, which encodes a glucose/xylose proton symporter. Here we report the functional expression of these transporters in Saccharomyces cerevisiae. While Gxf1p seems to be fully functional in S. cerevisiae, the symporter Gxs1p exhibits very low glucose/xylose transport activity, which could not be ascribed to insufficient production of the protein or incorrect subcellular localization. In addition, coexpression of glucose/xylose facilitators with Gxs1p strongly reduced GXS1 mRNA levels, and consequently symport activity, in glucose-grown, but not in ethanol-grown, cells. The observed decrease in GXS1 transcript levels seems to be related to an enhanced glucose influx mediated by glucose facilitator protein(s), and not to a specific interaction between Gxs1p and other transporters. We found GXS1 mRNA levels to be severely reduced as a result of glucose addition, and we show that this effect takes place at the level of GXS1 mRNA stability. Our results suggest that a decrease in mRNAs encoding high-affinity/active sugar transport systems may be a widespread and conserved mechanism in yeasts, limiting expression of these proteins whenever their activity is dispensable.

Two yeasts, Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012, which show rapid growth on L-arabinose and very high rates of L-arabinose uptake on screening, were selected for characterization of L-arabinose transport and the first steps of intracellular L-arabinose metabolism. The kinetics of L-arabinose uptake revealed at least two transport systems with distinct substrate affinities, specificities, functional mechanisms and regulatory properties. The L-arabinose catabolic pathway proposed for filamentous fungi also seems to operate in the yeasts studied. The kinetic parameters of the initial L-arabinose-metabolizing enzymes were determined. Reductases were found to be mostly NADPH-dependent, whereas NAD was the preferred cofactor of dehydrogenases. The differences found between the two yeasts agree with the higher efficiency of L-arabinose metabolism in C. arabinofermentans. This is the first full account of the initial steps of L-arabinose catabolism in yeast including the biochemical characterization of a specific L-arabinose transporter.

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

L-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to D-glucose and D-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from D-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated--arabitol from L-arabinose and xylitol from D-xylose. Different L-arabinose concentrations and oxygen conditions were tested to better understand L-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from L-arabinose (and also xylitol from D-xylose) than C. arabinofermentans PYCC 5603(T). In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex L-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in L-arabinose-assimilating yeasts.

A total of 214 members of the sugar porter (SP) family (TC 2.A.1.1) from eight hemiascomycetous yeasts: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Ashbya (Eremothecium) gossypii, Debaryomyces hansenii, Yarrowia lipolytica, Candida albicans and Pichia stipitis, were identified. The yeast SPs were classified in 13 different phylogenetic clusters. Specific sugar substrates could be allocated to nine phylogenetic clusters, including two novel TC clusters that are specific to fungi, i.e. the glycerol:H+ symporter (2.A.1.1.38) and the high-affinity glucose transporter (2.A.1.1.39). Four phylogenetic clusters are identified by the preliminary fifth number Z23, Z24, Z25 and Z26 and the substrates of their members remain undetermined. The amplification of the SP clusters across the Hemiascomycetes reflects adaptation to specific carbon and energy sources available in the habitat of each yeast species.

Recently a new habitat for microbial life has been discovered at the base of polythermal glaciers. In ice from these subglacial environments so far only non-photosynthetic bacterial communities were discovered, but no eukaryotic microorganisms. We found high numbers of yeast cells, amounting to a maximum of 4,000 CFU ml(-1) of melt ice, in four different high Arctic glaciers. Twenty-two distinct species were isolated, including two new yeast species. Basidiomycetes predominated, among which Cryptococcus liquefaciens was the dominant species (ca. 90% of total). Other frequently occurring species were Cryptococcus albidus, Cryptococcus magnus, Cryptococcus saitoi and Rhodotorula mucilaginosa. The dominant yeast species were psychrotolerant, halotolerant, freeze-thaw resistant, unable to form mycelium, relatively small-sized and able to utilize a wide range of carbon and nitrogen sources. This is the first report on the presence of yeast populations in subglacial ice.

A survey of epiphytic yeasts on leaves of selected Mediterranean plant species collected at the 'Arrábida Natural Park' (Portugal) yielded about 850 isolates, mostly of basidiomycetous affinity. Amongst the basidiomycetes, 35 strains showed the following characteristics: production of orange-coloured colonies, ability to produce starch-like compounds, assimilation of D-glucuronic acid and/or inositol, inability to utilize nitrate, and formation of ballistoconidia by many of the isolates. This group of yeasts was assigned to the Tremellales lineage of the Hymenomycetes and was further characterised using a combination of conventional phenotypic identification tests with molecular methods, namely PCR fingerprinting and rDNA sequencing. Eight additional strains presumptively identified as Bullera armeniaca, B. crocea or Cryptococcus hungaricus were also studied. Twenty-eight strains could be assigned to or were phylogenetically related to recognised species of Dioszegia in the 'Luteolus clade', but the 15 remaining strains belonged to other clades within the Tremellales. Ten phylloplane isolates were identified as Dioszegia hungarica, one as D. aurantiaca, another as D. crocea and three others were ascribed to the recently described species D. zsoltii. Seven novel species, viz. Cryptococcus amylolyticus, C. armeniacus, C. cistialbidi, Dioszegia buhagiarii, D. catarinonii, D. fristingensis and D. takashimae, are proposed for the remaining strains that did not correspond to any of the hitherto recognised species.

Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1 ) shares 90% and 54% identity with MAL31 and AGT1 , respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the α-glucoside transporters described so far.

Seven phylloplane yeast strains that were collected in the Arrábida Natural Park, Portugal, and identified preliminarily as Bullera alba, the anamorphic stage of Bulleromyces albus, were investigated. In contrast to Bulleromyces albus, these isolates produced a brownish pigment when grown on potato dextrose agar. The pigment caused darkening of the cultures and diffused into the culture medium. Mating studies revealed that the Arrábida isolates did not react with the different mating types of Bulleromyces albus, but were sexually compatible with them and produced mycelium with clamp connections, haustoria and transversally septate basidia that ejected the basidiospores. Various taxonomic criteria that were evaluated during the present study and comparison with other sexual taxa of the Tremellales indicated that this teleomorph should be classified in a novel genus. Therefore, Auriculibuller fuscus gen. nov., sp. nov. (type strain, PYCC 5690T=CBS 9648T) is proposed. In addition, during the course of this investigation, a member of a novel Bullera species, Bullera japonica sp. nov. (type strain, PYCC 4534T=CBS 2013T), was found among collection isolates that were identified formerly as Bullera alba. In molecular phylogenetic analysis of the D1/D2 domains of the 26S rDNA and the internal transcribed spacer region, the two taxa were found to be closely related, but distinct at the species level.

Two new species of the ascosporic yeast genus Metschnikowia were isolated from nectaries and associated muscoid flies of flowers from the common milkweed (Asclepias syriaca) in North America, and are described as Metschnikowia vanudenii [type strain=PYCC 4650(T)=CBS 9134(T)=NRRL Y-27243(T)=UWO(PS) 86A4.1(T)] and Metschnikowia lachancei [type strain=PYCC 4605(T)=CBS 9131(T)=NRRL Y-27242(T)=UWO(PS) 7ASB2.3(T)]. As with the previously described Metschnikowia gruessii, M. vanudenii has vegetative cells with an 'aeroplane' or cross-like configuration, produces ovoid chlamydospores and forms ellipsoidopedunculate asci with two acicular ascospores. Metschnikowia lachancei is distinguished from other Metschnikowia species by formation of club-shaped asci with 1-2 thick clavate ascospores. The phylogenetic positions of the proposed new species within Metschnikowia were determined from sequence analysis of the D1/D2 domain of 26S rDNA. The new species show low nuclear DNA relatedness with neighbouring taxa.

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.

The extent to which the transport of glucose across the plasma membrane of the yeast Saccharomyces bayanus controls the glycolytic flux was determined. The magnitude of control was quantified by measuring the effect of small changes in the activity of the glucose transport system on the rate of glucose consumption. Two effectors were used to modulate the activity of glucose transport: (i) maltose, a competitive inhibitor of the glucose transport system in S. bayanus (as well as in Saccharomyces cerevisiae) and (ii) extracellular glucose, the substrate of the glucose transport system. Two approaches were followed to derive from the experimental data the flux control coefficient of glucose transport on the glycolytic flux: (i) direct comparison of the steady-state glycolytic flux with the zero trans-influx of glucose and (ii) comparison of the change in glycolytic flux with the concomitant change in calculated glucose transport activity on variation of the extracellular glucose concentration. Both these approaches demonstrated that in cells of S. bayanus grown on glucose and harvested at the point of glucose exhaustion, a high proportion of the control of the glycolytic flux resides in the transport of glucose across the plasma membrane.

Summary Eighty five strains representing the four sibling species currently recognized within the Saccharomyces sensu stricto group were tested for the capacity to transport fructose by an active proton symport. Active transport of fructose was not detected in the strains of Sacch. cerevisiae and Sacch. paradoxus , whereas it was found in the strains assigned to Sacch. bayanus and Sacch. pastorianus . The maximum temperature for growth (T max ) of the latter two species ranged from 30°C to 34°C and from 28°C to 35°C respectively, at variance with the ranges of T max values of Sacch. cerevisiae (35 δC–43 δC, with one exception showing a lower T max ) and Sacch. paradoxus (35 °C–41 °C). The identity of the strains has been defined by DNA/DNA reassociation studies and their genome size estimated from the kinetics of DNA renaturation. The data support earlier reports of the intraspecific heterogeneity of Sacch. cerevisiae and of close relationships between species. This work represents an extension of our initial investigation of sibling Saccharomyces species. While showing that active fructose transport is a suitable systematic character for rapid differentiation of the two subgroups within Saccharomyces sensu stricto , useful differential tests to be applied within each subgroup were not found.

The industrial use of starter cultures containing a consortium of different strains from the same species is nowadays seen as a possible strategy to enhance the organoleptic complexity of wines. To assess the relative contribution of each strain to the final product it is essential to quantify population evolution during the wine fermentation process, which requires strain-specific methods to identify and differentiate each strain. In the present study, a molecular method based on analysis of the polymorphisms exhibited by the PCR-amplification of the delta regions of three Saccharomyces cerevisiae strains was developed. A set of three pairs of primers (delta1-delta2, delta12-delta2, delta12-delta21) was used for each strain, and analysis of the resulting polymorphism patterns showed that the delta12-delta2 primer pair exhibited the highest resolution and discriminatory power. Thus, this pair of primers was selected to monitor the population evolution of a laboratory-scale wine fermentation performed in synthetic grape juice that was inoculated with similar amounts of each strain. The results showed that all strains grew together during the exponential growth phase (2-3 days) and maintained high cell density values (10(6)-10(7) cfu ml(-1)) throughout the stationary growth phase without significantly changing their relative population proportion, thus indicating that each strain can influence the chemical composition and final flavor of wine, albeit at different levels. This study also showed that PCR-amplification of DNA delta sequences of S. cerevisiae strains is a reproducible, strain-specific and simple method that can be used successfully to monitor yeast strain population dynamics during wine fermentations.

A previous culture-dependent survey of phylloplane yeasts from selected Mediterranean plants showed that a few species were present in high densities in almost all leaf samples, regardless of the plant type, location or sampling season. However, a few species appeared to be restricted to Cistus albidus leaves, namely Cryptococcus cistialbidi. Here, we describe a culture-independent FISH assay to detect and quantify whole yeast cells in leaf washings. After optimization, the technique was used to check the apparent association between C. albidus leaves and C. cistialbidi and the abundance and ubiquity of other basidiomycetous yeast species such as Erythrobasidium hasegawianum and Sporobolomyces spp. in leaf samples from this and other neighboring plants (Acer monspessulanum and Quercus faginea). No yeast cells were detected in Pistacia lentiscus leaf samples. We were also able to demonstrate that three phylloplane yeasts (C. cistialbidi, E. hasegawianum and Sporobolomyces spp.) appeared to be log-normally distributed among individual C. albidus leaves. The log-normal distribution has important implications for the quantification of phylloplane yeasts based on the washing and plating of bulk leaf samples, which will tend to overestimate the size of the respective populations and become an error source in yeast surveys or related biocontrol studies.

Lactic acid-grown cells of a strain of Kluyveromyces marxianus transported D- and L-lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5.4: Vmax = 1.00 (+/- 0.13) mmol h-1 per g dry weight and Ks = 0.42 (+/- 0.08) mM. Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids. The latter, a non-metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH. The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species. Lactate uptake was not accompanied by the simultaneous uptake of protons, potassium ions or sodium ions excluding symport mechanisms. Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism. It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.

Candida arabinofermentans PYCC 5603 T and Pichia guilliermondii PYCC 3012 were shown to grow well on l -arabinose, albeit exhibiting distinct features that justify an in-depth comparative study of their respective pentose catabolism. Carbon-13 labeling experiments coupled with in vivo nuclear magnetic resonance (NMR) spectroscopy were used to investigate l -arabinose metabolism in these yeasts, thereby complementing recently reported physiological and enzymatic data. The label supplied in l -[2- 13 C]arabinose to nongrowing cells, under aerobic conditions, was found on C-1 and C-2 of arabitol and ribitol, on C-2 of xylitol, and on C-1, C-2, and C-3 of trehalose. The detection of labeled arabitol and xylitol constitutes additional evidence for the operation in yeast of the redox catabolic pathway, which is widespread among filamentous fungi. Furthermore, labeling at position C-1 of trehalose and arabitol demonstrates that glucose-6-phosphate is recycled through the oxidative pentose phosphate pathway (PPP). This result was interpreted as a metabolic strategy to regenerate NADPH, the cofactor essential for sustaining l -arabinose catabolism at the level of l -arabinose reductase and l -xylulose reductase. Moreover, the observed synthesis of d -arabitol and ribitol provides a route with which to supply NAD + under oxygen-limiting conditions. In P. guilliermondii PYCC 3012, the strong accumulation of l -arabitol (intracellular concentration of up to 0.4 M) during aerobic l -arabinose metabolism indicates the existence of a bottleneck at the level of l -arabitol 4-dehydrogenase. This report provides the first experimental evidence for a link between l -arabinose metabolism in fungi and the oxidative branch of the PPP and suggests rational guidelines for the design of strategies for the production of new and efficient l -arabinose-fermenting yeasts.

The incomplete consumption of sugar resulting from stuck wine fermentation is associated with important economic losses. One of the solutions to this serious problem consists of reinoculating the brew with a yeast starter culture that is both alcohol tolerant and a vigorous fructose fermenter. The present work aimed to select yeast strains capable of restarting stuck wine fermentations, and identify key parameters that contribute to the efficiency of the strains. Commercial and non-commercial Saccharomyces wine strains were tested, as well as strains of the fermentative non-Saccharomyces species Zygosaccharomyces bailii and Torulaspora delbrueckii. Although the latter species were shown to be more resistant to a combination of ethanol- and acetic-acid-induced cell death, commercial Saccharomyces cerevisiae strains were the most efficient fructose consumers in medium simulating a stuck fermentation. Stationary-phase S. cerevisiae cells performed better than inocula prepared from exponentially growing cultures, which correlates with the higher resistance to ethanol of non-growing populations. Stationary-phase cells pre-adapted to ethanol did not improve fructose consumption rates; this was in contrast to exponential-phase cells that benefited from prior incubation in ethanol-containing medium. Notably, a correlation was observed between yeast fructose consumption capacity and glucose (or fructose) transport. Our results challenge the current belief that ethanol tolerance, expressed in terms of cell viability, is a reliable criterion for the selection of yeast strains to restart stuck fermentations. Instead, this capacity seems to be based on sugar transport and its resistance to ethanol. In an attempt to further improve cell viability in the presence of high ethanol concentrations, hybrid strains of T. delbrueckii and S. cerevisiae were produced, and they showed high potential as restarter strains. The present work opens perspectives for the application of innovative strategies in the wine-making industry., Agência de Inovação (AdI) program POCI2010/2.3, project PARFERM, for its funding grant

The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment.

Two yeasts, Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012, which show rapid growth on L-arabinose and very high rates of L-arabinose uptake on screening, were selected for characterization of L-arabinose transport and the first steps of intracellular L-arabinose metabolism. The kinetics of L-arabinose uptake revealed at least two transport systems with distinct substrate affinities, specificities, functional mechanisms and regulatory properties. The L-arabinose catabolic pathway proposed for filamentous fungi also seems to operate in the yeasts studied. The kinetic parameters of the initial L-arabinose-metabolizing enzymes were determined. Reductases were found to be mostly NADPH-dependent, whereas NAD was the preferred cofactor of dehydrogenases. The differences found between the two yeasts agree with the higher efficiency of L-arabinose metabolism in C. arabinofermentans. This is the first full account of the initial steps of L-arabinose catabolism in yeast including the biochemical characterization of a specific L-arabinose transporter.

The highly osmo- and cryotolerant yeast species Torulaspora delbrueckii is an important case study among the non-Saccharomyces yeast species. The strain T delbrueckii PYCC 532 1, isolated from traditional corn and rye bread dough in northern Portugal, is considered particularly interesting for the baking industry. This paper reports the sugar utilization patterns of this strain, using media with glucose, maltose and sucrose, alone or in mixtures. Kinetics of growth, biomass and ethanol yields, fermentation and respiration rates, hydrolase activities and sugar uptake rates were used to infer the potential applied relevance of this yeast in comparison to a conventional baker's strain of Saccharomyces cerevisiae. The results showed that both maltase and maltose transport in T delbrueckii were subject to glucose repression and maltose induction, whereas invertase was subject to glucose control but not dependent on sucrose induction. A comparative analysis of specific sugar consumption rates and transport capacities suggests that the transport step limits both glucose and maltose metabolism. Specific rates of CO2 production and O-2 consumption showed a significantly higher contribution of respiration to the overall metabolism in T delbrueckii than in S. cerevisiae. This was reflected in the biomass yields from batch cultures and could represent an asset for the large-scale production of the former species. This work contributes to a better understanding of the physiology of a non-conventional yeast species, with a view to the full exploitation of T delbrueckii by the baking industry., This work was partially funded by Agência de Inovação (AdI) program POCI2010/2.3, project ‘PARFERM’. C. A.-A. and A. P. were supported by PhD fellowships from PRAXIS XXI – BD/21543/99 and BD/13282/2003, respectively (Fundação para a Ciência e para a Tecnologia, Portugal)., info:eu-repo/semantics/publishedVersion

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose–proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose–H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide–H+ symporters encompassing proteins from several yeasts and filamentous fungi.

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.

Thus far it has been considered that hypersaline natural brines which are subjected to extreme solar heating, do not contain nonmelanized yeast populations. Nevertheless we have isolated yeasts in eight different salterns worldwide, as well as from the Dead Sea, Enriquillo Lake (Dominican Republic) and the Great Salt Lake (Utah). Among the isolates obtained from hypersaline waters, Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica and Candida parapsilosis are known contaminants of low water activity food, whereas Rhodosporidium sphaerocarpum, R. babjevae, Rhodotorula laryngis, Trichosporon mucoides, and a new species resembling C. glabrata were not known for their halotolerance and were identified for the first time in hypersaline habitats. Moreover, the ascomycetous yeast Metschnikowia bicuspidata, known to be a parasite of the brine shrimp, was isolated as a free-living form from the Great Salt Lake brine. In water rich in magnesium chloride (bitterns) from the La Trinitat salterns (Spain), two new species provisionally named C. atmosphaerica – like and P. philogaea – like were discovered. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species., Fundação para a Ciência e Tecnologia (FCT) through a pluriannual contract with Centro de Ciências do Ambiente (CCA), Universidade do Minho and PARIPIPI-Project A with Estação Vitivinícola Nacional

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.

Saccharomyces cerevisiae transports fructose through a facilitated diffusion system common to other hexoses and mediated by the Hxt proteins. The related species S. pastorianus (carlsbergensis) and S. bayanus produce, in addition, a specific fructose/H+ symporter. We have previously cloned a gene (FSY1) encoding the active fructose symporter from S. pastorianus PYCC 4457. Expression of Fsy1p in a S. cerevisiae mutant (hxt-null) devoid of the facilitated diffusion system allows growth on fructose but not on glucose. Here we present results concerning the regulation of Fsy1p expression, both in S. pastorianus and S. bayanus, where it occurs naturally, and in suitably engineered S. cerevisiae transformants. To that purpose, we made use of both Northern blot analysis and a Fsy1p–GFP fusion protein. The expression of Fsy1p is strongly regulated by both the carbon source and its concentration in the growth medium. In S. pastorianus, as well as in S. bayanus, very low concentrations of either fructose or glucose induced expression but higher sugar concentrations prevented transcription of the gene. Glucose was considerably more effective than fructose in repressing FSY1 expression. Proper regulation of the gene in S. cerevisiae seems to be exquisitely dependent on sugar transport. Analysis of Fsy1 expression in S. cerevisiae mutants shows that repression is mainly dependent on Mig1p, the final effector of the main glucose repression pathway. Interestingly, Mig1p also seems to mediate repression of FSY1 expression by high maltose concentrations. Copyright © 2004 John Wiley & Sons, Ltd.

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455 T . The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria , Bacteria ) with the phylogenetically distantly related organisms Pirellula sp. strain 1 ( Planctomycetes , Bacteria ) and Metallosphaera sedula ( Crenarchaeota , Archaea ) and the 18S rRNA accessibility of Saccharomyces cerevisiae ( Eucarya ). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli , 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3′ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae .

This report presents the description of a new heterothallic Rhodosporidium species, R. azoricum sp. nov. The new species is based on two strains previously identified as Rhodotorula glutinis, which were isolated from soil in São Miguel island, Azores, Portugal. Evidence that the two strains were conspecific and distinct from Rhodotorula glutinis was obtained in DNA fingerprinting experiments using the microsatellite-primed PCR approach (MSP-PCR) and the primers M13 and (GTG)5. In order to determine the phylogenetic position of the new species, the nucleotide sequence of the D1/D2 region of the 26S rDNA was analysed and Rhodosporidium azoricum was found to belong to a cluster including R. fluviale, R. lusitaniae, Sporidiobolus microsporus, and S. ruineniae. The life cycle of R. azoricum was investigated and comparisons integrating physiological, morphological, and molecular data were made with related species.Key words: Rhodosporidium azoricum sp. nov., microsatellite-primed PCR, Rhodotorula glutinis, basidiomycetous yeasts.

An undescribed anamorphic yeast species of ascomycetous affinity, for which the name Candida tartarivorans is proposed, was isolated from dried wine lees in Portugal using a selective medium with L(+)-tartaric acid as the sole source of carbon and energy. The single isolate (IGC 4854T) showed the following characteristics: sympodial holoblastic conidiogenesis, absence of asci with ascospores, a negative colour reaction with Diazonium Blue B, production of elaborate pseudomycelium and ability to grow with inositol as sole source of carbon. Analysis of the physiological data pointed to a close relationship with other inositol-assimilating taxa, namely the genera Arxula, Stephanoascus, Sympodiomyces, Zygoascus and selected Candida species. Comparative analysis of the D1/D2 variable domain of the 26S rRNA gene of all available sequences for ascomycetous yeasts showed that strain IGC 4854T did not match with any other species in the database. The closest relative was Candida auringiensis Santa Maria, but the two species differed in 24 nucleotide positions. A description of the new species is given.

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)(-1)] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)(-1)]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1delta) in the PPP and deletion of trehalose 6-phosphate synthase (tps1delta) together with trehalose 6-phosphate phosphatase (tps2delta) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) - resulting in reduced enzyme activities - increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1delta) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)(-1). Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)(-1) (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)(-1). Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol.

The annual SMYTE meetings were launched in 1983 to join the international scientific community interested in solute membrane transport and related energetics in yeasts and to foster discussions about the most recent developments in this field. The 2001 meeting took place in Chania, Crete (Greece) at MAICh (Mediterranean Agronomic Institute of Chania), one of the four constituent Institutes of CIHEAM (International Centre for Advanced Mediterranean Agronomic Studies) and it was organised by George Diallinas (University of Athens) and Vicky Sophianopoulou (Institute of Biology, National Center for Scientific Research ‘Demokritos’, Athens). Due …

Fructose utilization in laboratory-scale sucrose adjunct brewers wort fermentations, using the brewing strainS. cerevisiae IGC 4261, is predicted by a mathematical model based on the kinetic parameters of the membrane transport proteins which affect fructose uptake into the cell. These include biphasic fructose transport via a proton symport and the constitutive hexose facilitated diffusion system, plus the competitive inhibitory effect that glucose has on this latter component. Also the non-competitive inhibitory effects of a) maltose on fructose uptake via its proton symport and b) ethanol on biphasic fructose transport are incorporated within the model, as well as the inoculum size.

Cellobiose-grown cells of Candida wickerhamii transported cellobiose as glucose by a glucose-proton symport after previous hydrolysis of the disaccharide by an exocellular β-glucosidase. Both the symport and the β-glucosidase were subject to glucose-induced repression and inactivation while glucose also acted as a competitive inhibitor of the enzyme (K i 0.3 mM). Under conditions of glucose repression glucose was transported by facilitated diffusion. Cellobiose acted as a competitive inhibitor of the latter (K i 75 mM) and is possibly a low-affinity substrate, while it inhibited non-competitively the glucoseproton symport (K i 80 mM). The affinity of cellobiose for the cell-bound β-glucosidase was much higher (K m 4.2 mM) than for the purified enzyme as reported by others (K m 67–225 mM). Ethanol reversibly inhibited the two glucose transport systems with exponential non-competitive kinetics. The minimum inhibitory concentrations were about 3% and 4% (w/v) for facilitated diffusion and proton symport while the respective exponential inhibition constants were 0.58 l mol-1 and 1.65 l mol-1. Ethanol affected the β-glucosidase in a complex way, a major effect was deviation from Michaelis-Menten kinetics for ethanol concentrations higher than 4% (w/v), the Hill coefficient increasing up to 1.8 at 6% (w/v) ethanol.

A survey was made of 81 starch-assimilating yeasts, representing 59 species and varieties, with respect to their capacity for the direct conversion of starch into SCP. The extent of starch conversion by the native amylases of the strains during exponential growth, expressed as yield on starch (final amount of dry biomass formed per unit mass of starch originally supplied), varied over a wide range (0.043–0.590) The highest yields were obtained with strains ofLipomyces starkeyi andL. kononenkoae which converted on the average respectively 84% and nearly 100% of the starch supplied. The rate of starch hydrolysis byL. kononenkoae did not limit its specific rate of growth and SCP production.

A strain of the yeast Lipomyces kononenkoae which converted starch into SCP with a high yield, produced three extracellular amylases which were purified from the culture fluid by Ficoll concentration, dialysis, isopropanol precipitation and DE-cellulose chromatography: an α-amylase, a glucoamylase and a debranching transferase. The latter transferred α-1,6-glucosyl units from panose to glucose forming maltose and appeared to have some debranching activity on amylopectin. The α-amylase had the following properties: MW 38000 daltons; no effect of added calcium ions on activity; optimum temperature and pH for activity around 40°C and pH 5.5; ΔH‡ and ΔS‡ of heat inactivation 24360 cal mol−1 and 29.2 cal deg−1 mol−1; range of pH stability pH 4–6.5; pI=7.1; final low molecular weight products of starch hydrolysis, maltose and glucose; Km (40°C, pH 5.5) for starch 2.7 gl−1, for maltotriose 109 gl−1; uncompetitive inhibition by maltose with Ki (40°C, pH 5.5) 29.5 gl−1. The glucoamylase had the following properties: MW 81500 daltons; optimum temperature and pH for activity around 50°C and pH 4.5: ΔH‡ and ΔS‡ of heat inactivation 20400 cal mol−1 and 17.7 cal deg−1 mol−1; range of pH stability pH 4–6.5; pI=6.1; Km (30°C, pH 4.5) for soluble starch 16.2 gl−1, for maltose 0.36 gl−1, for p-nitrophenyl-α-D-glucoside 0.35 gl−1; competitive inhibition by glucose with Ki (30°C, pH 4.5) 4.7 gl−1.

The temperature profile of growth and death rates of a strain of Kluyveromyces marxianus var. marxianus was found to be essentially dissociative, with an optimum temperature for growth at around 38 °C and sustained exponential growth up to 43–44 °C. At temperatures near the maximum temperature for growth a second growth period was observed. This did not correspond to the second branch in the ARRHENIUS plots characteristic of associative profiles, since growth was not balanced and after a while the growth rate increased to values similar to those of the first period. The biomass yield on lactose was not dependent on the growth temperature, a behaviour typical of yeast strains with dissociative temperature profiles.

Candida wickerhamii IGC 3244 growing in glucose medium transported glucose by facilitated diffusion (at 25°C and pH 5, the K s value was 1.7 mM and the V max value was 1.6 mmol/h per g dry wt.), while cells grown under derepressed conditions produced a glucose proton symport (at 25°C and pH 5, the K s value was 0.18 mM and the V max value was 1.8–1.9 mmol/h per g dry wt.). In each case, the Lineweaver-Burk plot of initial uptake rates was linear, indicating the presence of a single system. In buffer with 2% glucose, the symport suffered catabolite inactivation while the facilitated diffusion system emerged concomitantly in such a way that the combined V max remained nearly constant. During the conversion process, the Linewaver-Burk plots were biphasic, indicating the transitory co-existence of the two systems. A model is proposed that envisions the proton symport as composed of the facilitated diffusion system in association with (an)other transport protein(s), the latter being sensitive to carbon catabolite repression and inactivation.

A strain of the starch-converting yeast Lipomyces kononenkoae produced, when grown on starch, a debranching enzyme that proved to be an isoamylase (glycogen 6-glucanohydrolase; E.C. 3.2.1.68). So far, only bacteria have been found to produce extracellular isoamylases. The yeast isoamylase enhanced β-amylolysis of amylopectin and glycogen and completely hydrolyzed these substrates into maltose when combined with a β-amylase but had no action on dextran or pullulan. By isopropanol precipitation and carboxymethyl cellulose chromatography, L. kononenkoae isoamylase was partially purified from the supernatant of cultures grown on a mineral medium with soluble starch. Optimum temperature and pH for activity of the isoamylase were 30°C and 5.6. The molecular weight was around 65,000, and the pI was at pH 4.7 to 4.8. The K m (30°C, pH 5.5) for soluble starch was 9 g liter −1 .

Les analogues du glucose, le 2-deoxyglucose et le D-glucosamine, suppriment la formation de l'endodextranase extracellulaire de la levure L.b. Bien que ces deux analogues sont utilises avec succes dans la separation selective des mutants derepressifs et hyperproductifs, le 2-deoxyglucose s'avere etre superieur au D-glucosamine en ce qui concerne la concentration effective et la limitation de la croissance supplementaire