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Background The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant. Results The sequencing of strain LS3 revealed that the nuclear genome of A. adeninivorans is 11.8 Mb long and consists of four chromosomes with regional centromeres. Its closest sequenced relative is Yarrowia lipolytica, although mean conservation of orthologs is low. With 914 introns within 6116 genes, A. adeninivorans is one of the most intron-rich hemiascomycetes sequenced to date. Several large species-specific families appear to result from multiple rounds of segmental duplications of tandem gene arrays, a novel mechanism not yet described in yeasts. An analysis of the genome and its transcriptome revealed enzymes with biotechnological potential, such as two extracellular tannases (Atan1p and Atan2p) of the tannic-acid catabolic route, and a new pathway for the assimilation of n-butanol via butyric aldehyde and butyric acid. Conclusions The high-quality genome of this species that diverged early in Saccharomycotina will allow further fundamental studies on comparative genomics, evolution and phylogenetics. Protein components of different pathways for carbon and nitrogen source utilization were identified, which so far has remained unexplored in yeast, offering clues for further biotechnological developments. In the course of identifying alternative microorganisms for biotechnological interest, A. adeninivorans has already proved its strengthened competitiveness as a promising cell factory for many more applications. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica is a genetically tractable yeast species that has become an attractive model for analyses of lipid metabolism, due to its oleaginous nature. We investigated the regulation and evolution of lipid metabolism in non-Saccharomycetaceae yeasts, by carrying out a comparative physiological analysis of eight species recently assigned to the Yarrowia clade: Candida alimentaria, Y. deformans, C. galli, C. hispaniensis, C. hollandica, C. oslonensis, C. phangngensis and Y. yakushimensis. We compared the abilities of type strains of these species to grow on 31 non hydrophobic (sugars and other carbohydrate compounds) and 13 hydrophobic (triglycerides, alkanes and free fatty acids) carbon sources. Limited phenotypic diversity was observed in terms of the range of substrates used and, in the case of short-chain fatty acids, their toxicity. We assessed the oleaginous nature of these species, by evaluating their ability to store and to synthesize lipids. The mean lipid content of cells grown on oleic acid differed considerably between species, ranging from 30% of cell dry weight in C. oslonensis to 67% in C. hispaniensis. Lipid synthesis in cells grown on glucose resulted in the accumulation of C18:1 (n-9) as the major compound in most species, except for C. alimentaria and Y. yakushimensis, which accumulated principally C18:2(n-6), and C. hispaniensis, which accumulated both C16:0 and C18:1(n-9). Thus, all species of the clade were oleaginous, but they presented specific patterns of growth, lipid synthesis and storage, and therefore constitute good models for the comparative analysis of lipid metabolism in this basal yeast clade. [ABSTRACT FROM AUTHOR]

Candida alimentaria, Candida deformans, Candida galli, and Candida phangngensis have been recently reported to be the close relatives of Yarrowia lipolytica. To explore this clade of yeasts, we sequenced the mitochondrial genome (mt DNA) of these four species and compared it with the mt DNA of Y. lipolytica. The five mt DNAs exhibit a similar architecture and a high level of similarity of protein coding sequences. Genome sizes are variable, ranging from 28 017 bp in C. phangngensis to 48 508 bp in C. galli, mainly because of the variations in intron size and number. All introns are of group I, except for a group II intron inserted in the cob gene of a single species, C. galli. Putative endonuclease coding sequences were present in most group I introns, but also twice as free-standing ORFs in C. galli. Phylogenetic relationships of the five species were explored using protein alignments. No close relative of the Yarrowia clade could be identified, but protein and rRNA gene orders were partially conserved in the mt DNA of Candida salmanticensis. [ABSTRACT FROM AUTHOR]

The intergenic spacer rDNA amplification and AluI fingerprinting (IGSAF) method detected four distinct groups among 170 Debaryomyces hansenii strains: D. hansenii var. hansenii; Candida famata var. famata; D. hansenii var. fabryi and C. famata var. flareri. IGS sequence comparison of representative strains showed that D. hansenii var. hansenii and C. famata var. famata belonged to one species, whereas D. hansenii var. fabryi and C. famata var. flareri belonged to two different ones. This confirmed the following three species recently reinstated: D. hansenii (=C. famata), Debaryomyces fabryi and Debaryomyces subglobosus (= Candida flareri). Accordingly, growth at 37 °C may no longer be used to differentiate D. hansenii from D. fabryi. Riboflavin production is more specific for D. fabryi and D. subglobosus strains. IGSAF identified all the other 17 species of the genus Debaryomyces, six of them sharing with D. hansenii an rRNA gene unit harbouring two 5S rRNA genes. The phylogenetic tree established with IGS sequences was congruent with the one based on ACT1, GPD1 and COX2 sequences depicting a distinct D. hansenii clade close to the D. subglobosus, Debaryomyces prosopidis and D. fabryi clade. Description of Debaryomyces vietnamensis sp. nov. (type strain CBS 10535T, MUCL 51648T), closely related to Debaryomyces nepalensis is given. [ABSTRACT FROM AUTHOR]

The whole MATA cassette from Yarrowia lipolytica, a dimorphic fungus, was replaced by the URA3 gene through a double homologous recombination. This MAT-less strain lost its mate capacity with A or B Y. lipolytica strains. Introduction of polymerase chain reaction-synthesized idiomorph MATB in a null strain of A locus by double homologous recombination gave rise to a “transsexual” B strain. Mating capacity of this engineered mutant was assayed using Y. lipolytica strains of either A or B mating type. Mating took place only with an A strain, demonstrating the MATB idiomorph functionality in a MATA phenotype. Our data suggest that specific downstream genes are responsible for the final A or B phenotypes present in all Y. lipolytica cells, independent of their MAT idiomorph phenotype. [ABSTRACT FROM AUTHOR]

Abstract: Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species. [Copyright &y& Elsevier]

The production of heterologous proteins is a research field of high interest, with both academic and commercial applications. Yeasts offer a number of advantages as host systems, and, among them, Yarrowia lipolytica appears as one of the most attractive. This non-conventional dimorphic yeast exhibits a remarkable regularity of performance in the efficient secretion of various heterologous proteins. This review presents the main characteristics of Y. lipolytica, and the genetic and molecular tools available in this yeast. A particular emphasis is given to newly developed tools such as efficient promoters, a non-homologous integration method, and an amplification system using defective selection markers. A table recapitulates the 42 heterologous proteins produced until now in Y. lipolytica. A few relevant examples are exposed in more detail, in order to illustrate some peculiar points of the Y. lipolytica physiology, and to offer a comparison with other production systems. This amount of data demonstrates the global reliability and versatility of Y. lipolytica as a host for heterologous production. [Copyright &y& Elsevier]

Homoisocitric dehydrogenase (EC 1.1.1.155) has been purified 525-fold from the yeast Saccharomycopsis lipolytica with a yield of 25%. The preparation was judged to be homogeneous by electrophoresis under denaturing and non-denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000. In the presence of homoisocitric acid, a higher molecular weight was observed. suggesting a dimeric structure for the native enzyme. Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (lys9 and lys10). Lys10 mutants displayed NAD-reducing activity, whereas lys9 mutants retained some carboxylating activity. Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps. [ABSTRACT FROM AUTHOR]

Abstract: Whatever their abundance in genomes, spliceosomal introns are the signature of eukaryotic genes. The sequence of Saccharomyces cerevisiae, achieved fifteen years ago, revealed that this yeast has very few introns, but conserved intron boundaries typical for an intron definition mechanism. With the improvement and the development of new sequencing technologies, yeast genomes have been extensively sequenced during the last decade. We took advantage of this plethora of data to compile and assess the intron content of the protein-coding genes of 13 genomes representative of the evolution of hemiascomycetous yeasts. We first observed that intron paucity is a general rule and that the fastest evolving genomes tend to lose their introns more rapidly (e.g. S. cerevisiae versus Yarrowia lipolytica). Noticeable differences were also confirmed for 5’ splice sites and branch point sites (BP) as well as for the relative position of the BP. These changes seemed to be correlated with the lineage specific evolution of splicing factors. [Copyright &y& Elsevier]

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being non-competitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed. [ABSTRACT FROM AUTHOR]

Abstract: In fungi, ambient pH sensing relies on the conserved Rim101 signalling pathway. All components of the pathway have been shown to be functionally conserved in the pathogenic yeast Candida albicans except for Rim9p which, in other fungi, has been suggested to be involved in this process. Here we report that, in C. albicans, the RIM9 homologue is required for Rim-dependent signalling. We also show that overexpressing Vps32p, an endosomal component required for transduction of the pH signal, does not bypass defects in upstream components such as Rim9p, Rim21p and Rim8p. [Copyright &y& Elsevier]

The article discusses a study on the yeast Yarrowia lipotica and its ambient signalling involving YlRim23p/PalC. The author explains that a Ylrim9p/Pal C mutants are similar to Aspergillus nidulans pall mutants that contains less phenotype compared to other mutants. similar to Aspergillus nidulans pall mutants. It also mentions that in Saccharomyces cerevisiae and Candida albicans, rim9 mutants exhibit a tight Rim phenotype. The authors present that the Y.lipolyticva PalC orthologue is required to the alkaline pH response.

Abstract: Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae × S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto. [Copyright &y& Elsevier]

The article reports on the characterization of Ylvps mutations affecting the ESCRT-1 complex of the multivesicular body pathway in Yarrowia lipolytica. It shows that Ylvps23 and Ylvps28 mutations not only affect the endocytic pathway, as expected from the function of the proteins encoded by their homologues in Saccharomyces cerevisiae and Candida albicans, but also the mutant strains have a bona fide Rim phenotype, as described in the two other yeasts. The results suggest that Rim and Vps pathways cooperate in ambient pH signaling and that this relation is conserved across the full range of hemiascomycetous yeasts.

Abstract: Yeasts combine the ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. Yeasts thus provide attractive platforms for the production of recombinant proteins. Here, four important species are presented and compared: the methylotrophic Hansenula polymorpha and Pichia pastoris, distinguished by an increasingly large track record as industrial platforms, and the dimorphic species Arxula adeninivorans and Yarrrowia lipolytica, not yet established as industrial platforms, but demonstrating promising technological potential, as discussed in this article. [Copyright &y& Elsevier]

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1-independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/. [ABSTRACT FROM AUTHOR]

Examines mutagenesis in n-alkane-assimilating yeast Yarrowia lipolytica through the use of a short zeta-based mutagenesis cassette. Choice and construction of recipient strains; Isolation of tagged mutants; Phenotypic analysis of tagged mutants; Analysis of the insertion event and identification of the interrupted gene.

A total of 296 isolates of Saccharomyces cerevisiae sampled from naturally fermenting grape musts from various locations in Lebanon were typed by interdelta fingerprinting. Of these, 88 isolates were compared with oenological strains originating from various countries, using microsatellite characterization at six polymorphic loci. These approaches evidenced a large diversity of the natural oenological Lebanese flora over the territory as well as in individual spontaneous fermentations. Several cases of dominance and perenniality of isolates were observed in the same wineries, where fermentations appeared to involve lineages of sibling isolates. Our work thus evidenced a "winery effect" on strains' relatedness. Similarly, related or identical strains were also detected in vicinal wineries, suggesting strain circulation within small geographical areas and a further "vicinity effect". Moreover, and despite its diversity, the Lebanese flora seemed interrelated, on the basis of microsatellite loci analysis, in comparison to worldwide communities. We finally tested the ability of 21 indigenous strains to act as potential starters for winemaking. Seven of them passed our pre-selection scheme and two of them at least may be good candidates for use provided pilot-scale assays confirm their suitability. [ABSTRACT FROM AUTHOR]

In this article, the author discusses various issues concerning yeasts. It provides an overview of the article "Complementation Used to Clone a Human Homologue of the Fission Yeast Cell Cycle Control Gene cdc2," by Lee and Nurse, published in "Nature." Information on Saccharomyces cervisiae is provided, including research related to it. The author also provides a historical perspective on yeast.

Yarrowia lipolytica has been developed as a production host for a large variety of biotechnological applications. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPont's eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleum's single-cell protein product). The natural occurrence of the species in food, particularly cheese, other dairy products and meat, is a further argument supporting its safety. The species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions. The infections can be treated effectively by the use of regular antifungal drugs, and in some cases even disappeared spontaneously. Based on our assessment, we conclude that Y. lipolytica is a 'safe-to-use' organism. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence. [ABSTRACT FROM AUTHOR]

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus. [ABSTRACT FROM AUTHOR]

Background: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings: The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters. Conclusions/Significance: With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity. [ABSTRACT FROM AUTHOR]

Abstract: The outer layer of the Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity (SDS and Calcofluor White sensitivity) and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study. [Copyright &y& Elsevier]

Abstract: The Candida albicans gpi7/gpi7 null mutant strain (Δgpi7), which is affected in glycosylphosphatidylinositol (GPI) anchor biosynthesis, showed a reduced virulence following systemic infection of C57BL/6 mice. In vitro production of TNF-α, IL-6 and IL-1β by macrophages in response to Δgpi7 cells was significantly increased as compared to control (wild type GPI7/GPI7 and revertant gpi7/GPI7) cells; this probably contributes to the enhanced recruitment of neutrophils to the peritoneal cavity in response to Δgpi7 cells. Survival of knockout mice for Toll-like receptor (TLR) 2 and TLR4 following intravenous injection of Δgpi7 cells showed no significant differences as compared to C57BL/6 mice. In vitro production of TNF-α by macrophages and neutrophil recruitment were significantly inhibited in TLR2−/− mice in response to control yeast strains. Interestingly both TNF-α production and neutrophil recruitment in response to Δgpi7 were significantly increased in all three types of mice, with no differences among them, and laminarin failed to inhibit this increased production of TNF-α. These results indicate that the enhanced proinflammatory response to Δgpi7 does not involve recognition through TLR2, TLR4 nor dectin-1. Therefore, complete GPI anchors confer surface properties that are involved in modulation of cytokine production by macrophages in response to C. albicans. [Copyright &y& Elsevier]

Abstract: Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mgl−1 in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications. [Copyright &y& Elsevier]

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1Δ contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1Δ did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream β-1,2 mannosylation, mit1Δ represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators, mit1Δ was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1Δ was not able to escape macrophage lysis through down-regulation of the ERK½ phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced β-mannosylation of phosphopeptidomannan. Increased β-mannosylation of mannoproteins was observed under growth conditions that prevented the association of β-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating β-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of β-oligomannoses on the host response need to be defined. [ABSTRACT FROM AUTHOR]

Summary The pathogenic fungus Candida albicans commonly causes mucosal surface infections. In immunocompromised patients, C. albicans may penetrate into deeper tissue, enter the bloodstream and disseminate within the host causing life-threatening systemic infections. In order to elucidate how C. albicans responds to the challenge of a blood environment, we analysed the transcription profile of C. albicans cells exposed to human blood using genomic arrays and a cDNA subtraction protocol. By combining data obtained with these two methods, we were able to identify unique sets of different fungal genes specifically expressed at different stages of this model that mimics bloodstream infections. By removing host cells and incubation in plasma, we were also able to identify several genes in which the expression level was significantly influenced by the presence of these cells. Differentially expressed genes included those that are involved in the general stress response, antioxidative response, glyoxylate cycle as well as putative virulence attributes. These data point to possible mechanisms by which C. albicans ensures survival in the hostile environment of the blood and how the fungus may escape the bloodstream as an essential step in its systemic dissemination. [ABSTRACT FROM AUTHOR]

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml−1 in shake flasks, while a strain containing multiple integrations reached 2000 U ml−1 in shake flasks, 11 500 U ml−1 in batch and 90 500 U ml−1 in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml−1 in batch with a mono-copy lapA integrant and 28 000 mU ml−1 in fed batch with a multi-copy transformant. [ABSTRACT FROM AUTHOR]

Discusses evidence that the side chain added by Gpi7p cells participates in the proper anchoring of glycosylphosphatidylinositol (GPI)-anchoring proteins targeted to the cell wall in the yeast Saccharomyces cerevisiae and Candida albicans. Modification of the composition of the mutant cell wall; Release of cell wall proteins into the medium of gpi7 disrupted strains.

Summary Glycosylphosphatidylinositol (GPI)-anchored proteins are involved in cell wall integrity and cell–cell interactions. We disrupted the Candida albicans homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a GPI anchor-modifying activity. In the mutant and on solid media, the yeast-to-hyphae transition was blocked, whereas chlamydospore formation was enhanced. However, the morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis and cell shape were observed in both liquid and solid media. The cell wall structure was also modified in the mutants, as shown by hypersensitivity to Calcofluor white. In vitro and in vivo assays revealed that the mutant interacted with its host in a modified way, resulting in reduced virulence in mice and reduced survival in the gastrointestinal environment of mice. The mitogen-activated protein (MAP) kinase pathway of macrophages was downregulated by the wild-type cells but not by the ΔCagpi7 null strains. In agreement with this abnormal behaviour, mutant cells were more sensitive to the lytic action of macrophages. Our results indicate that a functional GPI anchor is required for full hyphal formation in C. albicans , and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences. [ABSTRACT FROM AUTHOR]

Assesses the genetic control of extracellular protease synthesis in yeast. Evaluation on the hydrogen ion concentration (pH) for the growth medium of yeast; Response of alkaline protease to pH; Identification of homologs for Yarrowia lipolytica.

Investigates the secretion vectors of Yarrowia lipolytica and Kluyveromyces lactis for the expression of heterologous proteins. Factors affecting the yield of secretion of soluble single-chain antibody fragment (scFv); Analysis of purified scFv; Capacities of the scFv protein.

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.alliicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42°C. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1- regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis. [ABSTRACT FROM AUTHOR]

Examines the development of gene-tagging approach to identify genes required for filamentous growth in Yarrowia lipolytica. Genetic determinants of morphogenesis in different yeasts; Analysis of genes involved in morphogenesis; Importance of Ras-cyclic pathway for hyphae formation.