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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]

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]

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]

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]

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]