Description: In the view of constantly increasing demand for cost-effective, low-energy and environmentally friendly industrial processes and household care products, the enzymes production occupies essential place in the field of biotechnology. Along with increasing demand for industrial and household care enzymes, the demand for heterologous expression platforms has also increased. Apart from conventional hosts ( Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris ), routinely used in heterologous proteins expression, the nonconventional ones have become more and more exploited in this field. Among the available yeast host systems, Yarrowia lipolytica appears as an attractive alternative. The aim of this study was to compare efficiency of two Yarrowia -based expression platforms, commercial Po1g-pYLSC and custom-made A18-pYLTEF, in expression of an insect-derived, raw-starch digesting alpha-amylase, to select the “champion” system for further studies on this valuable enzyme. Both expression platforms were compared with respect to copy number of the integrated expression cassette per transformed genome, and the recombinant strains performance (Po1g-pYLSC-derived 4.29 strain, and A18-pYLTEF-derived B9 strain) during batch bioreactor cultures. Our results demonstrate that the average number of integration events into the recipient's genome was comparable for both expression systems under investigation, but with varying distribution of the multicopy integrants; and the number of the recombinant gene copies was highly correlated with the acquired amylolytic activity of the strains. Due to severe susceptibility of the recombinant AMY1 polypeptide towards native proteases of the custom-made expression system, the final yield of the enzyme was substantially lower, when compared to the commercial Po1g-pYLSC (reaching the maximum level of 142.84 [AU/L]).

Description: Paracoccidioidomycosis is a systemic mycosis endemic to Latin America, with Paracoccidioides brasiliensis and P. lutzii being the causal agents of this disorder. Several issues have been raised in the hundred years since its discovery and in this article we discuss features of this fascinating fungal pathogen including its biology, eco-epidemiology and aspects of its pathogenicity. We also consider some of its virulence determinants, the most recent advances in the study of its metabolic pathways and the molecular and genetic research tools developed for this research. We also review the animal models used to study host-fungal interactions and how the host defense mechanisms against this pathogen work.

Description: Currently, research is being focussed on the industrial scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the un-dissociated form, which is lipophilic and can diffuse into the cell. There are no studies done on the impact of high extracellular concentration of fumaric acid in aerobic conditions on S. cerevisiae, which is a relevant issue to study for industrial scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose limited chemostat cultures at a cultivation pH of 3.0 (pH 〈 pK). Steady states were achieved with different extracellular levels of fumaric acid obtained by adding different amounts of fumaric acid to the feed media. The experiments were carried out with the wild type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-third of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for industrial scale production of fumaric acid are discussed.

Description: The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to basidiomycetous fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled reporter gene expression cassette consisted of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted into the binary vector pZPK. Hygromycin resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harboring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than fivefold and followed a decreasing sequence of PPGI , PPGK , PFBA , PTPI , and PGPD . This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals.

Description: Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, like limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series consisting of single-copy and multi-copy integrative, centromeric, and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism resulting in stable single- and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behavior was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with an improved copy number control.

Description: This study includes a screening system for future brewing yeasts focusing on non- Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, PCR fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off-flavor tests and trial fermentations. Trial fermentations were analyzed for extract reduction, pH drop, yeast concentration in a bulk of fluid, and fermentation by-products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavor and a beer of average ethanol content with a high volatile flavor compound concentration. Two other strains can possibly be used for pre-fermentation as a bio-flavoring agents for beers that have been post-fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavor-forming properties.

Description: With the advent of high-throughput technologies for sequencing, the complete description of the genetic variation that occurs in populations, also known as population genomics, is foreseeable but far from being reached. Explaining the forces that govern patterns of genetic variation is essential to elucidate the evolutionary history of species. Genetic variation results from a wide assortment of evolutionary forces, among which mutation, selection, recombination and drift play major roles in shaping genomes. In addition, exploring the genetic variation within a population also corresponds to the first step towards dissecting the genotype-phenotype relationship. In this context, yeast species are of particular interest because they represent a unique resource for studying the evolution of intraspecific genetic diversity in a phylum spanning a broad evolutionary scale. Here, we briefly review recent progress in yeast population genomics and provide some perspective on this rapidly evolving field. In fact, we truly believe that it is of interest to supplement comparative and early population genomic studies with the deep sequencing of more extensive sets of individuals from the same species. In parallel, it would be more than valuable to uncover the intraspecific variation of a large number of unexplored species, including those that are closely and more distantly related. Altogether, these data would enable substantially more powerful genomic scans for functional dissection.

Description: Candida parapsilosis is a common cause of invasive candidiasis especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans , relatively few have been developed for C. parapsilosis . In this study, we show that plasmids harboring green, yellow, and mCherry fluorescent protein sequences, previously developed for expression in C. albicans , can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis , which are typically prototrophic, because the plasmids include NAT1 , a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers that require the ability to directly visualize C. parapsilosis , for example in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models. This article is protected by copyright. All rights reserved.

Description: Electron microscopic examinations have demonstrated local modifications in the cell wall of the yeast Candida maltosa grown on hexadecane. In our earlier studies, these modified sites, observed in other yeasts grown on oil hydrocarbons, were conventionally called “canals”. The biochemical and cytochemical studies of C. maltosa have revealed a correlation between the formation of “canals” and decrease in the amount of cell wall polysaccharides: glucan and mannan. The ultrathin sections and surface replicas have shown that "canals" are destroyed by pronase, thus indicating that a significant proportion of their content is represented by proteins. This finding was compatible with our earlier data on the localization of oxidative enzymes in "canals" and possible participation of “canals” in the primary oxidation of hydrocarbons. A completely unexpected and intriguing phenomenon has been the appearance of “canals” in yeast C. maltosa under starvation conditions. Unlike the yeasts grown on hexadecane, mannan almost disappears in starving cells, while the quantity of glucan first decreases then restores its initial level. The role of "canals" in starving cells is unclear yet; it is assumed that they acquire exoenzymes involved in the utilization of products of cell lysis in the starving population. In the future, "canals" of starving cells will be studied in connection with their possible participation in apoptosis.

Description: Budding yeast Saccharomyces cerevisiae are able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this up-regulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids. The first one is up-regulation of PTR2 in a population of cells caused by amino acid secretion that accompanies peptide uptake. The second one is loss of Ptr2p activity due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.