Construction of recombinant sake yeast containing a dominant FAS2 mutation without extraneous sequences by a two-step gene replacement protocol

A novel two-step gene replacement protocol was developed to construct a recombinant industrial yeast free of bacterial and drug-resistant marker sequences. A yeast strain exhibiting cerulenin resistance conferred by a dominant mutation of FAS2 was previously shown to produce high levels of a flavor component of Japanese sake. A N- and C-terminally truncated portion of the mutant FAS2 gene was subcloned to an integrating plasmid containing an aureobasidin A-resistant transformation marker and a galactose-inducible growth inhibitory sequence (GAL10p::GIN11). The plasmid was targeted into the chromosomal FAS2 locus of sake yeast Kyokai no. 7, resulting in a tandem repeat of inactive FAS2 sequences surrounding the integrated plasmid sequences. Cells containing the integrated plasmid were unable to grow on galactose medium due to the inhibitory effect of GAL10p::GIN11. This growth inhibition allowed efficient counter-selection for cells that had undergone homologous recombination between the FAS2 repeats by their growth on galactose medium. This recombination event resulted in loss of the integrated plasmid sequences and the resulting strains should contain a single copy of either wild-type or cerulenin-resistant FAS2. The selected cerulenin-resistant strains produced approximately 3.7-fold more ethyl caproate, a flavor component, than the Kyokai no. 7 strain. Southern blot and sequence analyses confirmed the presence of the FAS2 mutation and the absence of integrated plasmid sequences in the genome of the selected strain. This gene replacement method provides a straightforward approach for the construction of recombinant industrial yeasts free of undesirable DNA sequences.     

Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast

Counter-selection is a useful gene manipulation technique for repeated gene disruptions, gene shufflings and gene replacements in yeasts. We developed a novel counter-selection system using a galactose-inducible growth inhibitory sequence (Kawahata et al.1999. Yeast 15: 1-10). This counter-selection marker, named GAL10p-GIN11, has several advantages over previous counter-selection markers, i.e. use of an inexpensive galactose medium for counter-selection, combined use with any transformation markers for gene introduction, and no requirement of specific mutations in the host strains. The GIN11 sequence, which is a part of an X-element of the subtelomeric regions, contained a conserved autonomously replicating sequence, causing the possibility of inefficient chromosomal integration. We isolated GIN11 mutants that lost the replication activity but retained the growth-inhibitory effect when overexpressed. A mutant GIN11M86 sequence was selected and fused to the CUP1 promoter for the counter-selection on a copper-containing medium. The GALp-GIN11M86 and the CUPp-GIN11M86 were used for constructing sets of integrating plasmids containing auxotrophic markers involving HIS3, TRP1, LEU2, URA3 or ADE2, or a drug-resistant marker PGKp-YAP1. In addition, a set of gene disruption cassettes that contained each of the auxotrophic markers and the GALp-GIN11M86, which were flanked by direct repeats of a hisG sequence, were constructed. The counter-selectable integrating plasmids and the gene disruption cassettes can allow the markers to be used repeatedly for yeast gene manipulations.     

On the level of plasmid-bearing cells in transformed cultures of Saccharomyces cerevisiae.

LC1, a YIP5-derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10-20% or 30-50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hybridization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.     

The pYC plasmids, a series of cassette-based yeast plasmid vectors providing means of counter-selection

A series of 24 general-purpose yeast plasmid vectors has been constructed. The plasmid series is composed of inter-replaceable cassettes, allowing for easy interconversion of plasmid types. In addition to the usual replication origins, selectable markers and multiple cloning sites (MCS), cassettes dedicated to counter-selection have been constructed. A pair of unique 8 bp restriction enzyme recognition sites flank each type of cassette, FseI in the case of yeast replication origins, AscI in the case of selectable markers, PacI in the case of counter-selectable markers and NotI in the case of the MCS. Thus, any given cassette can be replaced by another cassette of the same type, facilitating interconversion of any given plasmid from one type to another, even after the insertion of DNA into the MCS. Hence, the plasmids have been named pYC for 'yeast cassettes'. The cassettes consist of either NONE, CEN4/ARS or 2micro as replication origin, either URA3, MET2-CA (Lg-MET2) or the G418 resistance gene (the apt1 gene from bacterial transposon Tn903, encoding aminoglycoside phosphotransferase) as selectable markers, either NONE, PMET25-PKA3 or PCHA1-PKA3 as counter-selectable marker, and the MCS, containing recognition sites for AflII, AvrII, BspEI, PmeI, SacII, SalI, SunI, BamHI, EcoRI, HindIII, KpnI, MluI, NarI and SacI (of which the seven first are unique in all plasmids). The counter-selectable markers consist of the PKA3 gene under control of the conditional MET25 or CHA1 promoters. At activating conditions these promoters express the PKA3 gene at toxic levels, facilitating easy selection for loss of plasmid or 'loop-out' of plasmid DNA sequence after genomic integration.     

High fidelity segregation of a YEp vector in [cir0] strains of the yeast Saccharomyces cerevisiae

YEp vectors carrying the GPD promoter and PGK terminator for constitutive expression showed high partition efficiencies specifically in [cir(0)] strains, when DNA fragments were inserted into the cloning site. These plasmids appeared to be more stable than yeast centromeric plasmids, being lost in less than 10(-2) cells after each generation, and more than 90% of the cells carried the plasmids after 20 generations of growth in the absence of selective pressure. The REP3 region was essential together with the GPD promoter and PGK terminator for plasmid equipartitioning in [cir0] strains. The present results suggest that this host-vector system would be useful for astute observation of the phenotype caused by gene overexpression, and for heterogeneous protein production using natural medium, because of efficient partitioning of the plasmid without selective pressure.     

Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae

Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast. Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid. Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0). Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss. Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function. These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast.     

Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.     

Possible gene interchange between plasmid and chromosome in yeast

Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.     

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.     

A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function

The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.     

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.     

New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination

New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on ‘split-marker’ recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large-scale applications such as one-step cloning of target sequence homologs and combinatorial use. Gene deletions or gap-repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap-repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I-SceI sites, which allow their subsequent elimination from the transformant without the need for counter-selection. The application of the ‘split-marker’ vectors to the analysis of a few open reading frames of chromosome XI is described.     

A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors.

We describe a set of replicative, integrative and single-stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha-complementation. The nucleotide sequence of each of them is completely known.     

New insights into galactose metabolism by Schizosaccharomyces pombe: isolation and characterization of a galactose-assimilating mutant

The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase δ variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7+), SPBPB2B2.12c (gal10+) and SPBPB2B2.13 (gal1+), homologous to Saccharomyces cerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7Δ strain, carrying an integrated ura4+ marker at the gal7+ locus, grew on 5-fluoroorotic acid (5-FOA)-containing medium. In contrast, the FG2-8 gal7Δ strain could not grow on 5-FOA medium. In addition, expression of gal7+, SPBPB2B2.13, gal10+ and gal1+ genes increased in the wild-type strain when carried on a vector, and these transformants grew on galactose medium. We suggest that gal7+, gal10+, and gal1+ are localized close to a chromosomal terminal repressed by gene silencing in S. pombe. In contrast, gene silencing was defective in the FG2-8 strain making galactose assimilation possible.     

Yeast/E. coli shuttle vectors with multiple unique restriction sites

Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously in Saccharomyces cerevisiae and in E. coli; (2) they contain the beta-lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of beta-galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these auxotrophic markers in yeast and E. coli; (7) both plasmids are retained with high frequency in yeast grown under non-selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for construction of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for autonomous replication in yeast. The derivative plasmids YIp351 and YIp352 can therefore be used to integrate specific sequences into yeast chromosomal DNA.     

Development of a transformation system for the yeast Yamadazyma (Pichia) ohmeri

This communication describes the development of genetic tools for the yeast Yamadazyma ohmeri. Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis. Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, including YoLEU2 and YoURA3 that were sequenced. Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation. Transformation with pBR322-based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per μg of DNA. In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome. Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild-type URA3 gene by an in vitro generated deletion. Sequences conferring extrachromosomal replication were isolated from Y. ohmeri DNA. Plasmids based on pBR322 carrying such an ARS and either selective markers transformed at 10⁴/μg and were shown to replicate freely in Y. ohmeri at an approximate copy number of 40. Unexpectedly, we observed that BS-SK^R derivatives carrying either YoLEU2 or YoURA3 but no Y. ohmeri ARS also replicated extrachromosomally. Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four-fold. The sequences are available for consultation under EMBL accession number Z35101 for YoLEU2 and Z35100 for YOURA3.     

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.