Application of the Cre-loxP system for multiple gene disruption in the yeast kluyveromyces marxianus

The yeast Kluyveromyces marxianus presents several interesting features that make this species a promising industrial yeast for the production of several compounds. In order to take full advantage of this yeast and its particular properties, proper tools for gene disruption and metabolic engineering are needed. The Cre-loxP system is a very versatile tool that allows for gene marker rescue, resulting in mutant strains free of exogenous selective markers, which is a very important aspect for industrial application. As the Cre-loxP system works in some non-conventional yeasts, namely Kluyveromyces lactis, we wished to know whether it also works in K. marxianus. Here, we report the validation of this system in K. marxianus CBS 6556, by disrupting two copies of the LAC4 gene, which encodes a β-galactosidase activity.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - (CAPES), Brazil;Agência de Inovação - projecto UMINHO/POCI-Zimlac/BI/2/0

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains

Citation: Roggenkamp E, Giersch RM, Wedeman E, Eaton M, Turnquist E, Schrock MN, Alkotami L, Jirakittisonthon T, Schluter-Pascua SE, Bayne GH, Wasko C, Halloran M and Finnigan GC (2017) CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains. Front. Microbiol. 8:1773. doi: 10.3389/fmicb.2017.01773Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and “integration cassettes” have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 “gene drive” system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome

Nuclear Pore Behaviour in Interphase and "Open" Mitosis of Ustilago maydis

This work presents findings on dynamic nuclear pore behaviour in interphase nuclei and in the “open” mitosis in Ustilago maydis. Proteins likely to function in the nuclear pore complexes (NPCs) are identified in the U. maydis genome by bioinformatic search. Of these, five nucleoporins are tagged with GFP and observed microscopically for their localization in interphase and mitosis. One of the stably incorporated nucleoporins, Nup107, is analyzed in more detail. Nup107 appears to be essential in U. maydis, with depletion of the protein causing NPC clustering at the nuclear envelope. The initial observation reveals that NPCs in U. maydis are motile. The movement of NPCs in interphase nuclei has been reported in budding yeast, but a detailed analysis of the underlying mechanism and of the biological significance of the phenomenon is missing. In contrast to the studies in budding yeast, which assumed diffusion as cause for NPC motility, this work in U. maydis finds directed motility of NPCs. The directed motility is energy-dependent and proceeds in two motility types. Type 1 depends on the microtubule cytoskeleton. Dynein is the driving force behind MINUS end directed movement. NPC type 2 motility is abolished when active transcription is inhibited, but cannot be directly correlated with chromatin movement. FRAP experiments demonstrate that type 1 motility is necessary to equally distribute NPCs across the nuclear envelope. Equal distribution is required to ensure efficient protein expression of a reporter from an inducible promoter. Following the GFP-tagged nucleoporins from different parts of the NPC structure into mitosis, it appears that NPCs are still assembled in prophase. A tendency of NPCs to accumulate at the tip of the elongated prophase is observed, but the function of NPCs in chromosome migration appears doubtful. At the end of prophase, NPCs disassemble into subcomplexes and disperse in different locations, with the Nup107-160 subcomplex associating with chromatin in metaphase. Complete nuclear envelope (NE) removal is not necessary for NPC disassembly and association of Nup107 with DNA. In contrast to findings in vertebrates, the Nup107-160 subcomplex does not associate with kinetochores in metaphase. In anaphase, the Nup107-160 subcomplex shifts its position to the leading outside edges of the chromatin. Nucleoporins accumulate at the NEs in telophase in a step-wise manner, and nuclear import starts after all nucleoporins investigated are assembled at the nuclei. Inhibitor studies to investigate the role of the cytoskeleton cannot fully explain the involvement of actin and microtubules in NE reassembly. The results from these observations on NPC motility in interphase nuclei open a new view on basic principles of ensuring efficient protein expression. The findings on NPC disassembly in mitosis place U. maydis close to the vertebrate situation, suggesting a more ancient origin for the “open” mitosis

Stress resistance and ageing in Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, can be used as a model in which the processes behind ageing can be investigated. Yeast life span can be determined in two ways, i) the number of buds produced by an actively dividing mother cell can be counted as a measure of a yeast's budding life span, ii) the viability over time of cells arrested in GO can be recorded as a measure of yeast's chronological life span. As is the case for Drosophila and C. elegans increasing the cellular stress resistance and antioxidant scavenging capabilities of yeast extends the chronological life span. By increasing the stress resistance of cerevisiae through the overactivation of the heat shock response resulting from defects in the Hsp90 chaperone the chronological ageing of GO arrested cells was extended. The budding potential of these cells however was not increased. Extensions to the chronological lifespan of yeast adapted torespiratory growth was achieved with the overexpression of the superoxide dismutase enzymes, Cu,Zn-Sod and Mn-Sod, as well as catalase. A two-fold extension to chronological lifespan extension was observed in cells with increased Cu,Zn-Sod activity. In this strain levels of free radicals are low and the onset of total cellular protein oxidation, which coincides with a dramatic reduction in cellular viability, is delayed. The generation of free radicals during respiration is therefore a limiting factor for longevity. Over expressions of the free radical scavenging enzymes did not increase the budding potential of yeast cells. Despite the benefits for chronological survival gained from the overexpression of Mn-Sod, a disruption in mitochondrial morphology and inheritance in this strain leads to a reduced number of buds a mother cell could produce. A future goal for yeast ageing research is the identification of novel pathways involved in the determination of lifespan. The final part of this study included the development of a system by which the Euroscarf collection of deleted yeast strains can be screened for long lived mutants

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

The Role of CLU1 in Maintaining Mitochondrial Genome Stability and Morphology in S. cerevisiae

Mitochondrial genome maintenance is essential for the normal function of the cell. Mitochondrial DNA (mtDNA) is located in the matrix, where it is in close proximity to the electron transport chain, which is within the inner mitochondrial membrane. During oxidative phosphorylation, the electron transport chain produces reactive oxygen species (ROS) that may damage the DNA and contribute to mutations within the genome. Mutations in the mitochondrial genome have long been hypothesized as a contributor to diseases, especially those of the neuromuscular system. Mitochondrial mutations have also been linked to some types of cancer, programmed cell death, and aging in humans. The ability to repair this damage is integral for cells to maintain proper fw1ction and longevity. S. cerevisiae is a facultative anaerobe that can grow in the absence of respiration under specific growth conditions, although mitochondria are still required for viability. The lab used a yeast two-hybrid assay with the known mitochondrial protein, Ilv5p, to isolate genes involved in the organization, repair, and recombination of mtDNA. The lab has identified the Clu1p in this screen. Clu1p function was previously found to be required for proper mitochondrial morphology and distribution (1). My thesis research has focused on creating clu1Δ strains and performing fluctuation analysis assays using different reporters that measure specific mitochondrial events. Initial characterization of CLU1 has shown that loss of Clu1p leads to an increased loss of mitochondrial function which may occur through various events, such as point mutations, recombinations or deletions, and DNA polymerase slippage. Microscopy has supported previous reports indicating that a clu1Δ strain displays a clustering phenotype (Fields et al. 1998). This deletion strain exhibits a branched mitochondrial network that is localized to one side within the yeast cell. These data provide evidence that Clu1p plays a central role in mitochondrial genome stability and morphology

Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5’ and 3’ untranslated region (UTR) and without introducing any extraneous unwanted “scar” sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells.United States. National Institutes of Health (NS087557)American Parkinson Disease Association, Inc