A family of vectors that facilitate transposon and insertional mutagenesis of cloned genes in yeast

This report describes two sets of plasmid vectors that facilitate the identification of regions of complementation in cloned genomic inserts via transposon or insertional mutagenesis. The first set contains ARS-H4 CEN6, a yeast selectable nutritional marker (HIS3, TRP1 or URA3), and neo for selection in Escherichia coli. These plasmids lack the Tn3 transposition immunity region present in pBR322 derived vectors, and are permissive recipients for Tn3 transposon mutagenesis. The second family of plasmids described facilitate gene disruption procedures performed in vitro. These vectors carry disruption cassettes that contain different yeast selectable markers (HIS3, LEU2, TRP1 or URA3) adjacent to the Tn5 neo gene. These genes can be excised as a cassette on a common restriction fragment and introduced into any desired restriction site with selection for kanamycin resistance.     

Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster

Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 μm-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2^fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.     

Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration

Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these “ICE” vectors, for “Integrating after CEN Excision.” The ICE series was created by introducing a “rare-cutter” NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.     

Cloning an Autonomous Replication Sequence from Aspergillus oryzae

DNA sequence from Aspergillus oryzae genome which, confers on pOK-1 the ability to replicate autonomously in Saccharomyces cerevisiae is described. A hybrid plasmid pOK-1 was constructed from pBR325 and YEp13 fragment containing Leu2⁺ gene. Asp. oryzae DNA and pOK-1 were cleaved with BamHI, ligated and introduced into Escherichia coli. About 600 recombinant plasmids were obtained. One of them transforms S. cerevisiae SHY3 to Leu2⁺ with middle efficiency. The plasmid designated as pSB1–2 contained two BamHI fragments (4.3 kb and 4.1 kb) of Asp. oryzae DNA.     

The 2μm plasmid of laboratory yeast strains is a type-1/type-2 hybrid

Industrial yeast strains carry one of two homeologous 2μm plasmids designated as type-1 or type-2. The 2μm plasmid, Scp1, found in common laboratory strains of Saccharomyces cerevisiae is considered a type-2 plasmid, since the ori, STB, RAF and REP1 loci and intergenic sequences of the right-unique region of Scp1 are homologous to the corresponding loci in industrial strain type-2 plasmids. However, within both its 599 bp inverted repeats Scp1 has 142-bp sequences homologous to the bakers' yeast type-1 plasmid. DNA sequence analyses and oligonucleotide hybridizations indicate that the 142-bp insertion in Scp1 was probably due to homeologous recombination between type-1 and type-2 plasmids. These results suggest that some of the plasmid and chromosomal sequence polymorphisms seen in laboratory yeast strains result from homeologous recombination in their ancestral breeding stock.     

In vivo cloning by homologous recombination in yeast using a two-plasmid-based system

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient ‘recombination in vivo’ technique. Two sets of vectors were developed. The first set, called ‘expression vectors’, contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a ‘transfer vector’ which is compatible with the second set of E. coli–yeast shuttle vectors. This set of ‘recombination vectors’ contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 μm replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis. The sequence presented (Figure 1a) has been entered in the EMBL data library under Accession Number Z48747.     

Selectable marker replacement in Saccharomyces cerevisiae

Selectable markers integrated by the ‘gamma’ deletion method (Sikorski and Hieter, 1989) can be efficiently replaced in vivo with other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that all transformants that exhibited the expected phenotypes (loss of the original prototrophy and gain of the alternate prototrophy) resulted from homologous recombination between plasmid sequences at the target locus. This technique involves no plasmid construction and greatly facilitates the generation of yeast cells containing multiple gene disruptions.     

Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions

Analysis of the DNA sequences of new members of the Saccharomyces cerevisiae MEL1-MEL10 gene family showed high homology between the members. The MEL gene family, α-galactosidase-coding sequences, have diverged into two groups; one consisting of MEL1 and MEL2 and the other of MEL3-MEL10. In two S. cerevisiae strains containing five or seven MEL genes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes. The sequence homology and the abrupt change to sequence heterogeneity at the centromere-proximal 3′ end of the MEL genes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences. We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function. The new open reading frame (Accession number Z37509) is located in the 3′ non-coding region of MEL3-MEL10 genes in opposite orientation to the MEL genes (Accession numbers Z37508, Z37510, Z37511). Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed efficiently if at all.     

Patulin biodegradation by marine yeast Kodameae ohmeri

Patulin contamination of fruit- and vegetable-based products had become a major challenge for the food industry. Biological methods of patulin control can play an important role due to their safety and high efficiency. In this study, a strain of marine yeast with high patulin degradation ability was screened. The yeast was identified as Kodameae ohmeri by the BioLog identification system and partial 26S rRNA gene sequencing. The degradation products of patulin were identified as (E)- and (Z)-ascladiol through HPLC and LC-TOF/MS. High patulin tolerance at 100 μg ml^–1 and a high degradation rate at 35°C at a pH between 3 and 6 indicates the potential application of K. ohmeri for patulin detoxification of apple-derived products.     

Characterization of MEL genes in the genus Zygosaccharomyces

We cloned and sequenced a Zygosaccharomyces cidri MEL gene with a view to investigating the structure and regulation of yeast MEL genes. The amino acid sequence deduced from the nucleotide sequence showed 78·6% and 78·2% similarity to Saccharomyces cerevisiae and Saccharomyces pastorianus α-galactosidases, respectively. The expression of the MEL gene in several Zygosaccharomyces strains was induced by galactose. An electrophoretic karyotype of several Zygosaccharomyces species was obtained using contour-clamped electric field gel electrophoresis. The minimum number of chromosomes was five for Z. cidri, six for Z. fermentati, three for Z. florentinus, and four for Z. microellipsoides. The sizes of the chromosomes were generally larger than those of S. cerevisiae, the smallest containing approximately 0·4 megabase. The MEL gene was located, using the Z. cidri MEL gene as a probe, on the largest chromosome of the Z. cidri strains. In addition, a smaller chromosome (600 kb) in Z. cidri strain CBS4575 showed hybridization to the homologous MEL probe. This chromosome was absent in Z. cidri strain CBS5666. The probe hybridized to the largest chromosome of Mel⁺ Z. fermentati strains but failed to hybridize to any chromosome of Mel⁺ Z. mrakii or Z. florentinus strains. These results suggest the existence of a polymorphic MEL gene family in the yeast Zygosaccharomyces. The sequence has been deposited in the EMBL Data Library under Accession Number L24957.     

Non‐homologous end joining‐mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination‐based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non‐homologous end‐joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C‐terminal‐truncated non‐functional ura3 selection marker and the truncated region were PCR‐amplified separately, mixed and directly used for the transformation. URA3⁺ transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C‐terminal encoding sequence that could restore the function of a truncated non‐functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR‐amplified target DNAs and C‐terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ‐based cloning and recombinant DNA construction. The one‐step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.     

Sequences of Saccharomyces cerevisiae 2 μm DNA improving plasmid partitioning in Hansenula polymorpha

Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 μm DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 μm DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 μm STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24–28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region. © 1998 John Wiley & Sons, Ltd.     

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3‐1042 is a thermotolerant yeast strain suitable for high‐temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 × 10⁶ transformants/µg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3‐1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non‐homologous end‐joining (NHEJ) pathway. In yeast lacking KU70, high‐frequency non‐homologous gene integration was abolished and the Kmku70 mutants showed 82–95% homologous gene targeting efficiencies using homologous sequences of 40–1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid‐free gene manipulations in K. marxianus. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ‐based plasmids with a choice of three inducible promoters, P_GAL1, P_CUP1 and P_ADH2. These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA‐mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid‐based and integrated chromosomal cassettes, and compared to that of the glycolytic P_PGK1 promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P_PGK1, P_TEF1 and P_HXT7‐391. Copyright © 2012 John Wiley & Sons, Ltd.     

Recombination in yeast based on six base pairs of homologous sequences: Structural instability in two sets of isomeric model expression plasmids

Multicopy episomal yeast (Saccharomyces cerevisiae) plasmids are frequently employed in research and industrial production despite their known limited structural and segregational stability. Employing a set of six yEGFP3 model expression plasmids (identical in size but differing in the arrangement of their functional sequences, joined via six base pair SacI sequences), we used back transformation of total DNA extracted from yeast transformants into Escherichia coli to detect potential plasmid rearrangements. This approach revealed deletions, translocations, duplications, and flippings of functional sequences in our plasmids based on homologous recombination between the SacI sequences. To extend our findings, we assembled and analysed in the same way a corresponding plasmid set of six isoforms expressing the antibacterial insect peptide defensin A. In 833 individual amp^R clones (both sets combined), we traced 28 cases (3.4%) with precise structural changes. However, the frequency in one isoform in the pIFC4.13X series, pIFC4.131, was particularly high with 18.5% (15 out of 81 clones), indicating that the architecture of this plasmid is unfavourable to the host. With an increased sensitivity, a Polymerase Chain Reaction (PCR) approach revealed further structural changes in at least half of the isoforms of each set. The changes are considered the consequence of homologous recombination events involving the SacI sequences in a random fashion. The frequency of plasmid alterations is the product of selection and counterselection seemingly favouring or disfavouring certain structures. Although no sole architectural arrangement stuck out as being particularly stable, we were able to determine with our approach unfavourable sequence associations that should be avoided.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae

Combinatorial genetic libraries are powerful tools for diversifying and optimizing biomolecules. The process of library assembly is a major limiting factor for library complexity and quality. Gap repair by homologous recombination in Saccharomyces cerevisiae can facilitate in vivo assembly of DNA fragments sharing short patches of sequence homology, thereby supporting generation of high‐complexity libraries without compromising fidelity. In this study, we have optimized the ordered assembly of three DNA fragments into a gapped vector by in vivo homologous recombination. Assembly is achieved by co‐transformation of the DNA fragments and the gapped vector, using a modified lithium acetate protocol. The optimal gap‐repair efficiency is found at a 1:80 molar ratio of gapped vector to each of the three fragments. We measured gap‐repair efficiency in different genetic backgrounds and observed increased efficiency in mutants carrying a deletion of the SGS1 helicase‐encoding gene. Using our experimental conditions, a gap‐repair efficiency of > 10⁶ plasmid‐harbouring colonies/µg gapped vector DNA is obtained in a single transformation, with a recombination fidelity > 90%. Copyright © 2012 John Wiley & Sons, Ltd.     

Mutational analysis of the gene for Schizosaccharomyces pombe RNase MRP RNA,mrp1, using plasmid shuffle by counterselection on canavanine

Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leu1, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid–plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.