Journal: FEMS yeast research
We aim in this study to provide levels of susceptibility of 162 bloodstream isolates of non-Candida albicans and non-C. tropicalis species from a sentinel program conducted in 11 hospitals in Brazil. Additionally, we compared the broth microdilution (BMD) method of the European Committee of Susceptibility Testing (EUCAST) with Clinical Laboratory Standards Institute (CLSI) BMD method for fluconazole, itraconazole, voriconazole, and amphotericin B. The study included 103 C. parapsilosis, 38 C. glabrata, 8 C. orthopsilosis, and 7 C. krusei isolates, and single isolates of Pichia anomala, C. famata, C. lusitaniae, C. kefyr, C. guilliermondii, and C. metapsilosis. Of note, we observed cross-resistance between fluconazole and voriconazole for two isolates being one C. parapsilosis and one C. glabrata. Good essential agreement (EA) was observed between the EUCAST and the CLSI results for C. parapsilosis and for fluconazole, itraconazole, voriconazole, and amphotericin B, respectively: 98%, 99%, 98%, and 97%. Otherwise, for C. glabrata, the EA for fluconazole was 84.2% and for voriconazole 89.4%. Because data from Brazil are scarce, our results contribute to the consolidation of the database of candidemia agents and monitoring of trends in the profile of drug resistance.
Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.g. six gene deletions) in a single transformation step by transforming up to three gRNA expression plasmids simultaneously. The method is not only suitable for gene deletion but is also applicable for in vivo site directed mutagenesis and integration of multiple DNA fragments in a single locus. In all cases, the strain transformed with the gRNA expression plasmids was equipped with a genomic integration of Spcas9, leading to strong and constitutive expression of SpCas9. The protocols detailed here have been streamlined to be executed by virtually any yeast molecular geneticist.
The origin of modern fruits brought to microbial communities an abundant source of rich food based on simple sugars. Yeasts, especially Saccharomyces cerevisiae, usually become the predominant group in these niches. One of the most prominent and unique features and likely a winning trait of these yeasts is their ability to rapidly convert sugars to ethanol at both anaerobic and aerobic conditions. Why, when and how did yeast remodel their carbon metabolism to be able to accumulate ethanol under aerobic conditions and at the expense of decreasing biomass production? We hereby review the recent data on the carbon metabolism in Saccharomycetaceae species, and attempt to reconstruct the ancient environment, which could promote the evolution of alcoholic fermentation. We speculate that the first step towards the so-called alcoholic fermentation lifestyle was the exploration of anaerobic niches resulting in an increased metabolic capacity to degrade sugar to ethanol. The strengthened glycolytic flow had in parallel a beneficial effect on the microbial competition outcome, and later evolved as a “new” tool promoting the yeast competition ability under aerobic conditions. The basic aerobic alcoholic fermentation ability was subsequently “upgraded” in several lineages by evolving additional regulatory steps, like glucose repression in the S. cerevisiae clade, to achieve a more precise metabolic control. This article is protected by copyright. All rights reserved.
ABC-transporters with broad substrate specificity are responsible for pathogenic yeast resistance to antifungal compounds. Here we asked whether highly hydrophobic chemicals with delocalized positive charge can be used to overcome the resistance. Such molecules efficiently penetrate the plasma membrane and accumulate inside the cells. We reasoned that these properties can convert an active efflux of the compounds into a futile cycle thus interfering with the extrusion of the antibiotics. To test this we studied the effects of several alkylated rhodamines on the drug resistance of yeastSaccharomyces cerevisiae We found that octylrhodamine synergetically increases toxicity of Pd5p substrate - clotrimazole, while the others were less effective. Next, we compared the contributions of three major pleiotropic ABC-transporters (Pdr5p, Yor1p, Snq2p) on the accumulation of the alkylated rhodamines. While all of the tested compounds were extruded by Pdr5p, Yor1p and Snq2p showed narrower substrate specificity. Interestingly, among the tested alkylated rhodamines, inactivation of Pdr5p had a strongest effect on the accumulation of octylrhodamine inside the cells, which is consistent with the fact that clotrimazole is a substrate of Pdr5p. As alkylated rhodamines were shown to be non-toxic on mice, our study makes them potential components of pharmacological antifungal compositions.
Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study CRISPR/SpCas9 was used to delete the 21 hexose transporters in a S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, ‘genetically unaltered’ hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.
Historians of the future may well describe 2018 as the year that the world’s first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer’s indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the biosafety, bioethics and regulatory aspects of their pioneering work. This article presents a shared vision of constructing a synthetic eukaryotic genome in a safe model organism by using novel concepts and advanced technologies. This multidisciplinary and collaborative project is conducted under a sound governance structure that does not only respect the scientific achievements and lessons from the past, but that is also focussed on leading the present and helping to secure a brighter future for all.
Humans have acted as vectors for species and expanded their ranges since at least the dawn of agriculture. While relatively well characterized for macrofauna and macroflora, the extent and dynamics of human-aided microbial dispersal is poorly described. We studied the role which humans have played in manipulating the distribution of Saccharomyces cerevisiae, one of the world’s most important microbes, using whole genome sequencing. We include 52 strains representative of the diversity in New Zealand to the global set of genomes for this species. Phylogenomic approaches show an exclusively European origin of the New Zealand population, with a minimum of ten founder events mostly taking place over the last 1,000 years. Our results show that humans have expanded the range of S. cerevisiae and transported it to New Zealand where it was not previously present, where it has now become established in vineyards, but radiation to native forests appears limited.
Interspecies hybridization is an important evolutionary mechanism in yeasts. The genus Zygosaccharomyces in particular contains numerous hybrid strains and/or species. Here, we investigated the genome of Zygosaccharomyces strain MT15, an isolate from Maotai-flavor Chinese liquor fermentation. We found that it is an interspecies hybrid and identified it as Z. pseudobailii. The Z. bailii species complex consists of three species: Z. bailii, which is not a hybrid and whose 10 Mb genome is designated ‘A’, and two hybrid species Z. parabailii (‘AB’ genome, 20 Mb) and Z. pseudobailii (‘AC’ genome, 20 Mb). The A, B and C subgenomes are all approximately 7-10% different from one another in nucleotide sequence, and are derived from three different parental species. Despite being hybrids, Z. pseudobailii and Z. parabailii are capable of mating and sporulating. We previously showed that Z. parabailii regained fertility when one copy of its MAT locus became broken into two parts, causing the allodiploid hybrid to behave as a haploid gamete. In Z. pseudobailii, we find that a very similar process occurred after hybridization, when a deletion of 1.5 kb inactivated one of the two copies of its MAT locus. The half-sibling species Z. parabailii and Z. pseudobailii therefore went through remarkably parallel but independent steps to regain fertility after they were formed by separate interspecies hybridizations.
Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilization of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilization of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose-xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HKX2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L-1 glucose and 10 g L-1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.
Prions are conformationally flexible proteins capable of adopting a native state and a spectrum of alternative states associated with a change in the function of the protein. These alternative states are prone to assemble into amyloid aggregates, which provide a structure for self-replication and transmission of the underlying conformer and thereby the emergence of a new phenotype. Amyloid appearance is a rare event in vivo, regulated by both the aggregation propensity of prion proteins and their cellular environment. How these forces normally intersect to suppress amyloid appearance and the ways in which these restrictions can be bypassed to create protein-only phenotypes remain poorly understood. The most widely studied and perhaps most experimentally tractable system to explore the mechanisms regulating amyloid appearance is the [PIN+] prion of Saccharomyces cerevisiae. [PIN+] is required for the appearance of the amyloid state for both native yeast proteins and for human proteins expressed in yeast. These observations suggest that [PIN+] facilitates the bypass of amyloid regulatory mechanisms by other proteins in vivo. Several models of prion appearance are compatible with current observations, highlighting the complexity of the process and the questions that must be resolved to gain greater insight into the mechanisms regulating these events.