Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae

Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura⁺ phenotype. Finally, selection for Ura^? cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has ‘popped out’ the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.     

The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells

ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol Δ⁵ desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. The ERG3 gene has been mapped on chromosome XII, 13·7 centimorgans from GAL2 toward SPT8. The essentially of the gene is dependent on the conditions used for the cultivation of the mutants. Insertionally inactivated mutants of ERG3 fail to grow without ‘sparking’ levels of Δ⁵ sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol.     

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.     

Induced expression of bacterial β-glucosidase activity in saccharomyces

The bglA gene which encodes a β-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the yeast CYC-GAL promoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at the URA3 locus, or multiple integrations at the RDN1 locus. Integrative transformation at RDN1 yielded genetically stable clones with a high level of β-glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of β-glucosidase activity in different conditions of growth was assayed. While per-cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression in Saccharomyces of β-glucosidase activity is interesting for the development of strains able to ferment β-glycosidic sugars (i.e. cellobiose and lactose). From another point of view, the bglA product proved to be a convenient reporter enzyme to monitor heterologous gene expression.     

Isolation and characterization of Candida kefyr orotidine‐5′‐phosphate decarboxylase (URA3) gene

Candida kefyr is a common yeast species that can be found in fermented milk and cheeses. As a first step to developing a gene transfer system for C. kefyr, the orotidine‐5′‐phosphate decarboxylase (URA3) gene was cloned, using degenerate PCR and genome walking. The uninterrupted open reading frame of the C. kefyr URA3 gene spans 801 bp, corresponding to 267 amino acid residues. The functionality of the gene was confirmed by complementation of ura3 auxotrophs of C. albicans and Saccharomyces cerevisiae. Phylogenetic analysis of the deduced amino acid sequence indicated that it shares a high degree of homology with other Candida URA3 homologues. The GenBank Accession No. of the C. kefyr URA3 gene is FJ914763. Copyright © 2009 John Wiley & Sons, Ltd.     

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single‐stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice‐derived P. antarctica strain GB‐4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine‐5′‐phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB‐4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR‐amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil‐auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 μg DNA and a gene‐targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.     

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556

The URA3 gene, coding for orotidine-5′-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29·3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identifies of 81% and 88%, respectively. Using contour-clamped homogeneous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.     

Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.     

pMPY-ZAP: A reusable polymerase chain reaction-directed gene disruption cassette for Saccharomyces cerevisiae

Gene disruption is an important method for genetic analysis in Saccharomyces cerevisiae. We have designed a polymerase chain reaction-directed gene disruption cassette that allows rapid disruption of genes in S. cerevisiae without previously cloning them. In addition, this cassette allows recycling of URA3, generating gene disruptions without the permanent loss of the ura3 marker. An indefinite number of disruptions can therefore be made in the same strain.     

Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303‐1a

We report the construction of Saccharomyces cerevisiae strains isogenic to W303‐1a that are designed to allow efficient genetic analysis. To facilitate the generation of null alleles of target genes by PCR‐mediated gene disruption, we constructed designer deletion alleles of the ARG4, TRP1 and URA3 genes. In addition, a single pair of oligonucleotide primers were designed that can be used to amplify any of several marker genes for use in PCR‐mediated gene disruption. A new version of the ‘reusable’ hisG‐URA3‐hisG cassette was constructed for use in PCR‐mediated gene disruption. Finally, to facilitate the formation of isogenic diploids by selection, we constructed strains that contain combinations of wild‐type alleles of ADE2, HIS3, LEU2, TRP1 and URA3. Copyright © 1999 John Wiley & Sons, Ltd.     

Isolation and sequence analysis of the orotidine-5'-phosphate decarboxylase gene (URA3) of Candida utilis. Comparison with the OMP decarboxylase gene family

The URA3 gene of Candida utilis encoding orotidine-5′-phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation. The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine-5′-phosphate decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession Number Y12660. © 1998 John Wiley & Sons, Ltd.     

YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae

The genetics of flocculation in the yeast Saccharomyces cerevisiae are poorly understood despite the importance of this property for strains used in industry. To be able to study the regulation of flocculation in yeast, one of the genes involved, FLO1, has been partially cloned. The identity of the gene was confirmed by the non-flocculent phenotype of cells in which the C-terminal part of the gene had been replaced by the URA3 gene. Southern blots and genetic crosses showed that the URA3 gene had integrated at the expected position on chromosome I. A region of approximately 2 kb in the middle of the FLO1 gene was consistently deleted during propagation in Escherichia coli and could not be isolated. Plasmids containing the incomplete gene, however, were still able to cause weak flocculation in a nonflocculent strain. The 3′ end of the FLO1 gene was localized at approximately 24 kb from the right end of chromosome I, 20 kb centromere-proximal to PHO11. Most of the newly isolated chromosome I sequences also hybridized to chromosome VIII DNA, thus extending the homology between the right end of chromosome I and chromosome VIII to approximately 28 kb.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.     

ERG1, encoding squalene epoxidase,is located on the right arm of chromosome VII of Saccharomyces cerevisiae

The ERG1 gene of Saccharomyces cerevisiae encodes squalene epoxidase, a key enzyme in the ergosterol pathway. ERG1 is an essential gene. Disruption of the gene with URA3 results in a lethal phenotype when cells are grown under aerobic conditions, even in the presence of ergosterol. However, cells are viable in the presence of ergosterol under anaerobic growth conditions during which ergosterol is taken up by cells. Physical and genetic mapping data reveal that ERG1 is located on the right arm of chromosome VII proximal to QCR9 at a distance of 14·6 cM from ADE3.     

Cloning of orotidine-5′-phosphate decarboxylase (URA3) gene from sourdough yeast Candida milleri CBS 8195

The nucleotide sequence of the URA3 gene encoding orotidine-5′-phosphate decarboxylase (OMPDCase) in sourdough yeast Candida milleri CBS 8195 was determined by degenerate PCR and genome walking. Sequence analysis revealed the presence of an openreading frame of 810 bp, encoding 269 amino acid residue protein with the highest identity to the OMPDCase of the yeast Saccharomyces cerevisiae. Phylogenetic analysis of deduced amino acid sequence revealed that it shares a high degree of identity with other yeast OMPDCases. The cloned URA3 gene successfully complemented the ura3 mutation in S. cerevisiae, indicating that it encodes a functional OMPDCase in C. millieri CBS 8195.     

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5⁺ or Escherichia coli kan^r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.